There are some very good packages out there both commercial and GNU. I will run them down for you.
Gordon Gordon Couger gcouger-at-couger.com Stillwater, OK www.couger.com/gcouger 405 624-2855 GMT -6:00 ----- Original Message ----- } From: "Philip Oshel" {peoshel-at-facstaff.wisc.edu} To: {Microscopy-at-sparc5.microscopy.com} Sent: Thursday, August 31, 2000 2:01 PM
I have a product from Berkshire 800 242 7000 or 413 528 2602 or 2802 model LT00e166-R I got from a VCR tech. The are a Camious like swab that don't seem to leave lent. They do require more pressure on the glass but I have not had a scratch yet.
I used it to remove a very stubborn water stain on a prism that I couldn't touch with cotton and anything I had for a solvent. Isopropyl and this swab made it look easy.
Cotton has a considerable amount of oil on it as grown. Much of it is removed in processing but it still has some slight oil content that plus the fact it can pick up a static charge to a point that has to be seen to be believed make it problematic. If I was trying to use cotton so it wouldn't stick I would treat it with and anti static solution wash it in a couple of changes of hexane and use it in a high humidity environment.
A piece of lens tissue works well if you just twist it on a rough stick.\
Gordon Gordon Couger gcouger-at-couger.com Stillwater, OK www.couger.com/gcouger
} From: "David Knecht" {knecht-at-uconn.edu} } } I often use water first as a cleaner because often it is stuff } soluble in aqueous media that is dripped on the lens and often this } stuff is not soluble in organic solvents. Then I do an ethanol wash } to dry and remove organic soluble stuff. } } If I can add to the original question, I have had a great deal of } trouble cleaning lenses and especially CCD imagers due to residual } dust. I find that cotton swabs (at least those I have) leave an } enormous amount of residue behind that cannot be blown off easily. } Does anyone have a source of a more dust free equivalent to the } cotton swab? Dave } -- }
I have a question regarding artifacts in the myelin sheath of a peripheral nerve: Samples were immersionfixed in Karnovsky´s half-strength fixative(2% Glut, 2% PFA in 0.1M Cacodylate)en bloc stained with osmiumtetroxide and UAc and embedded in Araldite. The myelin sheaths show the following appearance: 1. The fine lammellar structure is focally disturbed, the layers appear weavy, wobbly ... 2. The outer rings are stained more intense than the inner rings (to the axon). 3. The myelin sheaths are de-rounded, but this seems to be not so grave and I noted this often in other pictures published. Maybe this is "normal"?
I would appreaciate any suggestions from you all to overcome this problem. Thanks in advance! Greetings, Michael
Michael Reiner University of Cologne, Germany Dept. of Anatomy I _______________________________________________________________________ 1.000.000 DM gewinnen - kostenlos tippen - http://millionenklick.web.de IhrName-at-web.de, 8MB Speicher, Verschluesselung - http://freemail.web.de
David Knecht asked: } If I can add to the original question, I have had a great deal of } trouble cleaning lenses and especially CCD imagers due to residual } dust. I find that cotton swabs (at least those I have) leave an } enormous amount of residue behind that cannot be blown off easily. } Does anyone have a source of a more dust free equivalent to the } cotton swab?
Kodak has several application notes covering various aspects of CCD sensors available on our web site:
Specifically, application note DS 00-009 has the recommended procedure for cleaning the coverglass on our sensors. Of course, we do not speak for other manufacturers. It is always safest to check with the manufacturer of your particular sensor for the recommended cleaning procedure.
DS 00-009 is available, in Adobe Acrobat format at: http://www.kodak.com/US/en/digital/pdf/ccdCoverGlass.pdf
The engineer who cleans our light microscopes tells me that CCD coverglasses tend to attract dust because of static charges. Good housekeeping practices and the use of dust covers minimizes the need for cleaning.
Best Regards,
John Minter Eastman Kodak Company Analytical Technology Division Rochester, NY 14650-2152 Phone: (716) 722-3407 FAX: (716) 477-7781 email: john.minter-at-kodak.com
We often see some disturbance of the myelin sheaths. According to a review chapter by Friedrich and Mugnaini: "The preservation of myelin sheaths presents a special problem. The membrane lamellae often separate from one another, even in otherwise well-preserved material. On the other hand, we have also encountered specimens whose myelin sheaths were reasonably well preserved, but in which the neuronal elements were poorly fixed. Apparently the myelin sheaths differ from other tissue components in their reaction to fixation and they may require special conditions for optimal preservation. Improvements in the technology of fixation may eventually eliminate this disparity, but for the present it seems sensible to discount the condition of myelin sheaths in evaluating fixed material unless the myelin sheath itself is the main target of the investigation." He also lists a "Procedure for Myelin"..
This is from a book chapter (unfortunately, the copy I have is missing the source reference) which may be a number of years old. Perhaps someone else has some new information on this.
Marie
Dr. Marie E. Cantino Dept. of Physiology and Neurobiology, U-2131 University of Connecticut Storrs, CT 06269-2131 Phone: 860-486-3588 Fax: 860-486-1936
agreed. in addition, one must be aware of the difference between the melting point and the temperature of mobility (tamman temperature). the tamman temp is about 0.52 bulk melting point of a metal in degrees K. liquid-like behavior of metal particles can occur at this point. the picture is further complicated by any support interactions. in the case of nanotubes, you are dealing with a basal plane ({0001} face) of graphite on which the metal particle is nucleated. all transition metals are very sensitive to such interfacial phenomenon, which will directly impact their nucleating/wetting/spreading behavior (see some of R. T. K. Baker's controlled atmosphere electron microscopy studies, j. catal.). for instance, evaporated gold will tend to nucleate on edges of graphite, but not on the basal plane. such behavior was and still is taken advantage of using gold decoration techniques (pioneered by g. r. hennig in the 60's). cool stuff!
paul
---------------- Paul E. Anderson Catalytic and Nanostructured Materials Laboratory Department of Chemistry 102 Hurtig Hall Northeastern University Boston, MA 02115 617 373 5909 FAX 617 373 8795 paanders-at-lynx.neu.edu
} If I can add to the original question, I have had a great deal of } trouble cleaning lenses and especially CCD imagers due to residual } dust. I find that cotton swabs (at least those I have) leave an } enormous amount of residue behind that cannot be blown off easily. } Does anyone have a source of a more dust free equivalent to the } cotton swab? Dave
Photographic Solutions makes a product called "Sensor Swab" http://www.photosol.com/swab.html for use with professional digital SLR cameras to clean CCD's. They are approved by Kodak for use with their DCS Camera line. They also make a high quality optics cleaner called Eclipse.
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I would like to suggest all those people who are working on the metallurgy of Mg alloys to refer that original paper published in English. I apologize that I don't have any reprints of that paper and I don't remember the formula either, since I've left there for a long time. Regards, Zhiping
} From: Milo Kral {m.kral-at-mech.canterbury.ac.nz} } To: Zhiping Luo {zhiping_luo-at-hotmail.com} } Subject: Re: Help with TEM of Mg alloy } Date: Fri, 01 Sep 2000 07:50:04 +1200 } } Just one more thing - could you send the formula for the electrolyte } and the polishing conditions to me via e-mail? } } Thanks } Milo } } } Dear Milo, } } } } To prepare TEM thin foil samples and also the samples for optical } } metallography, please refer a paper from our group (S.Q. Zhang, } } Acta } } Metall. Sinica, 1990, Vol. 3A, p.110), which contains details for } } the electrolytes. Those electrolytes worked well for all Mg alloys we } } studied. However, they should NOT be deposited after the experiment } } since they are explosive after some time especially at higher } } temperatures. } } } } Another way is just to use the conventional ion milling. I didn't } } detect any clear beam damage by the ion milling even at room } } temperature. } } } } In Mg-based alloys there are many complex intermetallics (Luo et } } al., Scripta Metall. Mater., 1993, Vol. 28, p. 1513) and I observed } } a number of new microstructures including the new Mg-Zn-rare earth } } quasicrystals by TEM. } } } } Good luck in your work. } } } } Zhiping Luo } }
} } } From: Milo Kral {m.kral-at-mech.canterbury.ac.nz} } } } To: Microscopy-at-sparc5.microscopy.com } } } Subject: Help with TEM of Mg alloy } } } Date: Thu, 31 Aug 2000 09:36:15 +1200 } } } } } } ----------------------------------------------------------------------- - } } } The Microscopy ListServer -- Sponsor: The Microscopy Society of } } } America
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Hi All, I have had a request to explore obtaining a fluorescent microscope with appropriate add-ons for doing quantitation of fluorescence. I know nothing about this and would appreciate advise on where to start looking. Would anyone who has such a system please contact me with general information about the system as well as as idea of cost. An investigator is writing a grant and would like to include such a system in the grant. Thanks, Debby
Debby Sherman, Manager Phone: 765-494-6666 Microscopy Center in Agriculture FAX: 765-494-5896 Dept. of Botany & Plant Pathology E-mail: sherman-at-btny.purdue.edu Purdue University 1057 Whistler Building West Lafayette, IN 47907-1057
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J. W. Robinson Mechanical and Materials Engineering University of Windsor Windsor, Ontario N9B 3P4
Phone : 519-253-4232, ext 2598
Does someone have an address or phone number for Al Bingham of Semoptics or someone else who does service on the Nanolab SEM's? Any help would be appreciated.
It is not clear to me what you mean exactly by imaging programs. Do you mean programs for acquiring images or programs for manipulating existing images. For the later purpose, the Corel Graphics Suite (includes Draw and Photo Paint) is very powerful and I use it often rather than the Adobe Photoshop and Illustrator programs. Caveat: I have not used these programs on the Linus platfrom myself.
Larry D. Ackerman Lily & Yuh Nung Jan Laboratories Howard Hughes Medical Institute UCSF, Box 0725, Rm U226 533 Parnassus Ave. San Francisco, CA 94143
Is there anyone in Southern California who does freeze fracture/freeze etch SEM? I would like to find a collaborator or even better a service lab that I can send samples to for FF/FE work.
Gregory M. Fahy 21st Century Medicine ________________________________________________________________ YOU'RE PAYING TOO MUCH FOR THE INTERNET! Juno now offers FREE Internet Access! Try it today - there's no risk! For your FREE software, visit: http://dl.www.juno.com/get/tagj.
I have a project which needs to see the preservation of testis and kidney morphology of rats. I want to set up a couple of ultrastructural criteria for this. Can any of you provide me some info about good classical reference books or literature. Any experience or suggestions are greatly appreciated.
Regards
Gang Ning
EM Facility Medical College of Wisconsin Milwaukee, WI 53045 414-456-8141 414-456-6517 (Fax)
Dear Dr. Robinson, There is a web site for Semoptics at: http://www.magma.ca/~semoptx/ and the phone and fax number listed there is the same as before: (613) 727-1698. At 10:47 AM 9/1/00 -0400, you wrote: } Phone : 519-253-4232, ext 2598 } } Does someone have an address or phone number for Al Bingham of } Semoptics or someone else who does service on the Nanolab SEM's? Any help } would be appreciated. } } Regards, Mary
Mary Mager Electron Microscopist Metals and Materials Engineering University of British Columbia 6350 Stores Road Vancouver, B.C. V6T 1Z4 CANADA tel: 604-822-5648 e-mail: mager-at-interchg.ubc.ca
Dear Dr. Robinson, There is a web site for Semoptics at: http://www.magma.ca/~semoptx/ and the phone and fax number listed there is the same as before: (613) 727-1698. At 10:47 AM 9/1/00 -0400, you wrote: } Phone : 519-253-4232, ext 2598 } } Does someone have an address or phone number for Al Bingham of } Semoptics or someone else who does service on the Nanolab SEM's? Any } help would be appreciated. }
Regards, Mary
Mary Mager Electron Microscopist Metals and Materials Engineering University of British Columbia 6350 Stores Road Vancouver, B.C. V6T 1Z4 CANADA tel: 604-822-5648 e-mail: mager-at-interchg.ubc.ca
Dear Dr. Robinson, There is a web site for Semoptics at: http://www.magma.ca/~semoptx/ and the phone and fax number listed there is the same as before: (613) 727-1698. At 10:47 AM 9/1/00 -0400, you wrote: } Phone : 519-253-4232, ext 2598 } } Does someone have an address or phone number for Al Bingham of } Semoptics or someone else who does service on the Nanolab SEM's? Any help } would be appreciated. }
Regards, Mary
Mary Mager Electron Microscopist Metals and Materials Engineering University of British Columbia 6350 Stores Road Vancouver, B.C. V6T 1Z4 CANADA tel: 604-822-5648 e-mail: mager-at-interchg.ubc.ca
emxray-at-uwindsor.ca-at-sparc5.microscopy.com wrote: } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } J. W. Robinson } Mechanical and Materials Engineering } University of Windsor } Windsor, Ontario } N9B 3P4 } } Phone : 519-253-4232, ext 2598 } } Does someone have an address or phone number for Al Bingham of } Semoptics or someone else who does service on the Nanolab SEM's? Any help } would be appreciated.
J. W., Give Derek Saunders a call. He's in Nepean, Ontario at Electron Optics Service, Inc. e-mail eos-at-magma.ca. I believe he bought all the remaining NanoLab stock and is very familiar with their equipment
Elliptical distortion in SAED ring patterns is successfully taken into account in the newest version of the free ProcessDiffraction program. Calibration of the eccentricity and direction of axes is performed and integration goes along ellipses (as contrasted to circles). The program converts your SAED ring pattern (measured in a TEM) into and XRD-like distribution of intensity as a function of scattering vector (proportional to the radius of the ring). d-values and intensities are measured and listed. Markers show positions of known phases. And more ...
Previous problems with missing Help were also corrected for. (There was a mismatch between the name of the installed Help-file and the name the program looked for as Help.) Now the Help works fine and the Help system explains with illustrations what is the program good for and how to use it.
The program can be downloaded FREE from http://www.mfa.kfki.hu/~labar/ProcDif.htm . Steps of installation are also explained there. I suggest to uninstall previous versions and install the newest (V1.1.0) version.
I hope you will find this program useful and easy to use. Any comments, suggestions are welcome.
János
Dr. Janos L. Labar Research Institute for Technical Physics and Materials Science H-1121 Budapest, Konkoly-Thege u. 29-33 Postal address: H-1525 Budapest-114, Po Box 49 Tel: (36)(1) 392-26-92 Fax: (36)(1) 275-49-96 Fax/phone: (36)(1) 395-92-32 home page: www.mfa.kfki.hu/~labar
Sorry just checking... Tried to post something the other day but it didn't work.
---------------------------- Dr. Maxim V. Sidorov TEM Applications Specialist Philips Electron Optics, Applications Laboratory Building AAE, Achtseweg Noord 5 5600 MD Eindhoven, the Netherlands
} Hello dear microscopists! } } I have a question regarding artifacts in the myelin sheath of a peripheral nerve: } Samples were immersionfixed in Karnovsky´s half-strength fixative(2% Glut, 2% PFA in 0.1M Cacodylate)en bloc stained with osmiumtetroxide and UAc and embedded in Araldite. } The myelin sheaths show the following appearance: } 1. The fine lammellar structure is focally disturbed, the layers appear weavy, wobbly ... } 2. The outer rings are stained more intense than the inner rings (to the axon). } 3. The myelin sheaths are de-rounded, but this seems to be not so grave and I noted this often in other pictures published. Maybe this is "normal"? } } I would appreaciate any suggestions from you all to overcome this problem. } Thanks in advance! } Greetings, } Michael } } } Michael Reiner } University of Cologne, Germany } Dept. of Anatomy I
Michael Reiner University of Cologne, Germany Dept. of Anatomy I _______________________________________________________________________ 1.000.000 DM gewinnen - kostenlos tippen - http://millionenklick.web.de IhrName-at-web.de, 8MB Speicher, Verschluesselung - http://freemail.web.de
Dear All: I wrote a piece of software which I believe would be of interest to the microscopy community. It's a highly interactive program which calculates/displays the contrast transfer function of TEMs. I know that there are similar programs floating around but ctfExplorer does not only 1d but also 2d calculations/display with 2-fold and 3-fold astigmatism imposed. There are other unique features to it. All parameters (defocus, voltage, Cs, etc) can be changed interactively.
DISCLAIMER: I do work for FEI/Philips Electron Optics. However, this software is not an official product of FEI and FEI is not responsible for its distribution/support. This software is freeware.
To the best of my knowledge, the software does not have any nasty bugs. It is tested under Windows 95/98 and Windows NT4.
Please give it a try. Please direct your suggestions and comments to maxsidorov-at-bigfoot.com I hope you'll find the software useful.
Here is the link: http://clik.to/ctfexplorer
Enjoy,
Max Sidorov --- TEM Applications Specialist FEI/Philips Electron Optics Eindhoven, The Netherlands e-mail: maxsidorov-at-bigfoot.com
(Sorry again for my previous "test" post. Apparently my e-mail program was not set up correctly.)
We have just recently received and had installed a JEOL 5900LV sem and although training for this instrument is only a week away, I would like to do some basic study on the low vacuum aspect of electron microscopy. I am very familiar with high vac operation of sem's in general but lack knowledge of the techniques and uses of low vac. Any pointers to web sites that have a basic to advanced discussion of this subject would be greatly appreciated.
Peter Sterling SEM Lab, Structural Materials Labs Materials and Processes Rocketdyne Division, The Boeing Company (818) 586-1434
Open Position for an Electron Microscopist at Northwestern University Medical School
We have an opening for an Electron Microscopy Technologist in the Cell Imaging Facility of Northwestern University Medical School, hosted within the Department of Cell and Molecular Biology. This is a multi-user facility equipped with modern microscopy instruments and available for researchers of the whole university. The responsibilities of this position include: EM technical service; Supervising facility's daily activities; training users (EM, confocal and deconvolution microscopy, microinjection, and digital image analysis); maintenance of equipment; helping with developing new microscopy techniques; ordering supplies, and repairs; instrument scheduling and billing, updating web page and helping with reports. The preferred qualifications include: bachelor's degree in life sciences or the equivalent combination of education, training and experience from which comparable skills can be acquired; The candidates need to have electron microscopy and ultramicrotomy experience. Experiences with fluorescence, confocal and deconvolution microscopy, microinjection, image analysis and computer skills are very desirable. Salary is commensurate with experience and education. Please send applications to Dr. Weiming Yu, Department of Cell and Molecular Biology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Detailed information about the Cell Imaging Facility can be found at http://www.basic.nwu.edu/corefacilities/cellimaging.html and http://www.cmb.nwu.edu/cif.html This is a full time position attached with full benefit package, which can be found at http://www.northwestern.edu/hr/benefits/
Weiming Yu, PhD Director, Cell Imaging Facility Northwestern University Medical School 303 E. Chicago Ave. W7-159 312/503-2841 fax:312/503-7912
Our admin people have decided to bar code all the equipment here, and need to have costs associated with the coding for security reasons. The following is a list of equipment that was either given to us, or we lost the paper trail and I'm having a hard time finding out prices for them. Prices and approximate dates of purchase, not estimated costs of present day value. If anyone has records of the same equipment and could give me a price and approximate purchase date that I could pass onto them, I would greatly appreciate it.
Edwards Coating System E306A Purchased in 1983 Reichert-Jung Ultracut E Microtome Sonicleaner Dawe Type 6443AE (rough price of a sonicator) LKB 2178 Knifemaker II Fisher slide warmer model 77 cat 12-594 Phillips EM-410 TEM
Thanks, Susan
Susan Carbyn Electron Microscopy Technician Atlantic Food and Horticulture Research Centre Agriculture and Agri-Food Canada 32 Main Street, Kentville, Nova Scotia B4N 1J5 Canada
Dear Listers, I'll be preparing samples of bacteria culture anaerobically and/or microanaerobically from sediments obtained at different bog sites in central Pa. I am looking for reference material for use with a class of graduate students. I expect to use conventional sample prep and an SEM to image Arthrobacter and Streptomyces. I am also looking for current literature on Aquaspirillum(magnetotactic bacteria). Rosemary
Rosemary Walsh, Manager The Electron Microscope Facility for the Life Sciences, A Shared Technology Facility, The Life Sciences Consortium 1 South FrearLab Penn State University University Park, PA 16802 (814) 865-0212 rw9-at-psu.edu http://www.lsc.psu.edu/stf/em/home.html
Peter Sterling wrote: } I am very familiar with high vac operation of sem's in general but lack } knowledge of the techniques and uses of low vac. Any pointers to web } sites that have a basic to advanced discussion of this subject would be } greatly appreciated.
I have a few pages about EDS in LVSEM and ESEM at the address:
http://www.risoe.dk/afm/news1new.htm
Best regards, Jørgen
***************************** J. B. Bilde-Soerensen Senior Research Scientist, Ph. D. Materials Research Department Risoe National Laboratory DK-4000 Roskilde Denmark
Dear all, Out of interest (i.e. I am not in a position to buy one right now), I am interested in people's opinion on ion beam thinners on the market today.
I have used a Gatan PIPS in the past and have been very satisfied with this including many of the features including the low angle milling and the ability to mount a TV camera to watch the milling in real time (great for hole detection on transparent ceramics that defeat auto-termination systems).
I know, however, that Gatan are not the only manufacturers of ion-beam thinning equipment and I am sure that their competitors must also have some decent machines. So, please tell me what else there is on the market (since I already know plenty about the Gatan PIPS), what specifications these machines have, and about your experiences with them. Also, comparisons between different makes/models of ion beam thinner would be interesting.
Looking forward to hearing from you.
Best wishes
===== Ian MacLaren Beijing Laboratory of Electron Microscopy Chinese Academy of Sciences, P.O. Box 2724 100080 Beijing, China Email: ian.maclaren-at-physics.org / ianmaclaren-at-hotmail.com Home page: http://members.tripod.co.uk/IanMacLaren/
____________________________________________________________ Do You Yahoo!? Get your free -at-yahoo.co.uk address at http://mail.yahoo.co.uk or your free -at-yahoo.ie address at http://mail.yahoo.ie
I am using Osmium to stain the binders in the coating layer. As you know, coated paper is composed of fibres (mechanical andchemical) and the coating layer (pigment minerals and binders). In order to try to predict how printing inks will interact with the paper surface during printing it is very important to characterise the coating layer. The quantification of the binders (styrene/butadiene latex) is thus important. How to do that?? I use to embedd paper samples in epoxy resin, take cross-section images in the SEM,BEI mode and use image analysis to quantify the binder distribution. However, it is very difficult to see any contrast between the binders in the coating layer and the epoxy resin. So, I stain the paper samples in Osmium before embedding in epoxy. The paper samples are placed in a staining chamber with a solution of 100 mg OsO4 in 10 ml water. The samples are not in contact with the solution in order to avoid structural changes in the paper cross-section. The samples are then vapour stained for 48 hrs. I get very nice results improving the contrast between the latex and the epoxy and also the mechanical (lighter) and chemical fibres. However, I have experienced a certain roughening of the paper surface due possibly to the water vapour inside the chamber. Now my question, how can I avoid this effect, Is it possible to dissolve OsO4 in another solvent?. I also tried to stain the samples with crystalline Osmium (without water), but the results are not so satisfactory.
Hi, Some time in the middle of the summer I put out a post seeking information on safe values for vacuum viewing port glass thicknesses, i.e., data on thickness of glass versus unsupported diameter for various glasses with atmospheric pressure on one side and vacuum on the other. Particular interest is in leaded glass and quartz and in small diameters in the range of 1 to 3 cm. I didn't get any responses but my guess is that a lot of list members were not monitoring the microscopy list as frequently as they normally would. So, with your indulgence, the question is reposted.
Thanks.
Don Marshall
Donald J. Marshall Relion Industries P.O. Box 12 Bedford, MA 01730 Ph: 781-275-4695 FAX: 781-271-0252 email dmrelion-at-world.std.com
Cathodoluminescence, mass spectroscopy, electron beam technology
"A weed is a flower out of place."
(Please note: Do not send email with attachments to this address. Instead, send it to donbarlen-at-aol.com. Thank you.)
I have not used a Gatan PIPS but here at Sheffield University SERC FEGTEM Facility (http://www.shef.ac.uk/uni/academic/D-H/eee/) we have both the Leica EMRES 100 and the Technoorg Linda IV3 with an additional low energy gun. The Leica is good for general fast milling - it is completely computer controlled, with video camera viewing and end point determination, on-board timer and, in our case, Peltier cooling of the sample holder. You can just set the thing going and leave it for an hour or two. The Technoorg Linda, though not computer controlled, is more versatile, having both the facility to use a low energy ion gun and liquid nitrogen cooling to the sample holder - topping up is the problem here. Neither company has a factory in the UK (Leica's instrument is made by Balzers in Lichtenstein and Technoorg Linda are Hungarian) but both are very helpful when it comes to problem solving. Leica have a UK based rep. - not very useful in China I don't suppose! My point is that they are willing to give every possible assistance by email or phone or, if necessary, a site visit.
Hope this helps.
Kind regards,
Alan Walker
********************************************* Alan Walker Dept of Electronic and Electrical Engineering University of Sheffield Mappin Street Sheffield S1 3JD United Kingdom
} Hi! } } I am using Osmium to stain the binders in the coating layer. As you know, } coated paper is composed of fibres (mechanical andchemical) and the } coating } layer (pigment minerals and binders). In order to try to predict how } printing inks will interact with the paper surface during printing it is } very important to characterise the coating layer. The quantification of } the binders (styrene/butadiene latex) is thus important. How to do } that?? I use to embedd paper samples in epoxy resin, take } cross-section images in the SEM,BEI mode and use image analysis } to quantify the binder distribution. However, it is very difficult to see } any } contrast between the binders in the coating layer and the epoxy resin. So, } I stain the paper samples in Osmium before embedding in epoxy. The paper } samples are placed in a staining chamber with a solution of 100 mg OsO4 in } 10 ml water. The samples are not in contact with the solution in order to } avoid } structural changes in the paper cross-section. The samples are then vapour } stained for 48 hrs. I get very nice results improving the contrast } between } the latex and the epoxy and also the mechanical (lighter) and chemical } fibres. However, I have experienced a certain roughening of the paper } surface due possibly to the water vapour inside the chamber. Now my } question, how can I avoid this effect, Is it possible to dissolve OsO4 in } another } solvent?. I also tried to stain the samples with crystalline Osmium } (without water), but the results are not so satisfactory. } } Any help will be appreciated. } } Gary.
Osmium will dissolve in several organic solvents, carbon tetrachloride comes to mind.
Why dissolve it at all? I suspect that osmium crystals in a sealed container would produce enough vapor to do the job. Gentle warming would speed things up.
Geoff -- ********************************************** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854 voice: (732)-235-4583; fax: -4029 mcauliff-at-umdnj.edu **********************************************
From root Wed Sep 6 08:47:07 2000 Return-Path: {Microscopy-request-at-sparc5.microscopy.com} Received: (from daemon-at-localhost) by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) id IAA22103 for dist-Microscopy; Wed, 6 Sep 2000 08:22:02 -0500 (CDT) Received: from no_more_spam.com (sparc5 [206.69.208.10]) by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) with SMTP id IAA22098 for "MicroscopyFilteredEmail-at-msa.microscopy.com"; Wed, 6 Sep 2000 08:21:31 -0500 (CDT) Received: from mailhub1.shef.ac.uk (mailhub1.shef.ac.uk [143.167.1.9]) by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) with ESMTP id IAA22089 for {Microscopy-at-msa.microscopy.com} ; Wed, 6 Sep 2000 08:21:20 -0500 (CDT) Received: from ridingwood.shef.ac.uk ([143.167.59.249]) by mailhub1.shef.ac.uk with esmtp (Exim 3.02 #2) id 13Wf36-0001E0-00 for Microscopy-at-MSA.Microscopy.Com; Wed, 06 Sep 2000 14:15:32 +0100 Received: from RIDINGWOOD/SpoolDir by ridingwood.shef.ac.uk (Mercury 1.46); 6 Sep 00 14:15:34 +0100 Received: from SpoolDir by RIDINGWOOD (Mercury 1.46); 6 Sep 00 14:15:31 +0100
Hi Ian,
I have not used a Gatan PIPS but here at Sheffield University SERC FEGTEM Facility (http://www.shef.ac.uk/uni/academic/D-H/eee/) we have both the Leica EMRES 100 and the Technoorg Linda IV3 with an additional low energy gun. The Leica is good for general fast milling - it is completely computer controlled, with video camera viewing and end point determination, on-board timer and, in our case, Peltier cooling of the sample holder. You can just set the thing going and leave it for an hour or two. The Technoorg Linda, though not computer controlled, is more versatile, having both the facility to use a low energy ion gun and liquid nitrogen cooling to the sample holder - topping up is the problem here. Neither company has a factory in the UK (Leica's instrument is made by Balzers in Lichtenstein and Technoorg Linda are Hungarian) but both are very helpful when it comes to problem solving. Leica have a UK based rep. - not very useful in China I don't suppose! My point is that they are willing to give every possible assistance by email or phone or, if necessary, a site visit.
Hope this helps.
Kind regards,
Alan Walker
********************************************* Alan Walker Dept of Electronic and Electrical Engineering University of Sheffield Mappin Street Sheffield S1 3JD United Kingdom
Have you tried using some other coating on your glass slides? Or putting the section on "Plus" slides (I use Fisher Scientific's, but I'm sure other companies have similar slides). I'm not sure the PLL is the best choice for this - I'd go with Plus slides or some "old-fashioned" type of subbing. I can send protocols if you are interested.
Good Luck!
Tamara Howard CSHL
On Wed, 6 Sep 2000, didier goux wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Dear Microscopist } } We persist to use Unicryl for cryo embedding (UV) while many problems. } } For in situ hybridization, the section of 3 mycron are put on polylysine } covered glasses } } this sections go away (floating) in SDS and SSC bath at 55 degres } } how could we "fixe" the sections on glasses? } } thank you for reply } } Didier } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } Didier Goux } CENTRE DE MICROSCOPIE ELECTRONIQUE } Université de CAEN Tel : (33) 02.31.56.58.13 } Campus I Fax : (33) 02.31.56.56.00 } Esplanade de la Paix 14032 CAEN CEDEX } mailto:didier.goux-at-unicaen.fr } } } } } } }
Does anyone know if the M&M 2000 proceedings have been mailed yet.?? Greg Gregory W. Erdos, Ph.D. Assistant Director, Biotechnology Program PO Box 110580 University of Florida Gainesville, FL 32611
You can probably go pretty thin with sapphire, given your small diameters and resulting pressures of less than 50 pounds. Most materials should work.
Call Melles Griot to ask about burst pressure and strength modulus for their products. That will help. Their contact information is:
E-mail: optics-at-irvine.mellesgriot.com
Call 1-800-835-2626 or 949-261-5600 and ask for the Optics Specialist
Fax 1-949-261-7790, attention Optics Specialist
Good luck,
Nathan Haese Lafayette, CA
From root Wed Sep 6 09:04:32 2000 Return-Path: {Microscopy-request-at-sparc5.microscopy.com} Received: (from daemon-at-localhost) by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) id IAA22147 for dist-Microscopy; Wed, 6 Sep 2000 08:25:33 -0500 (CDT) Received: from no_more_spam.com (sparc5 [206.69.208.10]) by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) with SMTP id IAA22139 for "MicroscopyFilteredEmail-at-msa.microscopy.com"; Wed, 6 Sep 2000 08:25:02 -0500 (CDT) Received: from furao2.ip.pt (furao.ip.pt [195.23.132.13]) by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) with SMTP id IAA22130 for {Microscopy-at-sparc5.microscopy.com} ; Wed, 6 Sep 2000 08:24:43 -0500 (CDT) Received: (qmail 6916 invoked by uid 1008); 6 Sep 2000 13:18:52 -0000 Received: from unknown (HELO mail2.ip.pt) (194.79.69.132) by furao2.ip.pt with SMTP; 6 Sep 2000 13:18:52 -0000 Received: from anatomip (195-23-160-192.nr.ip.pt [195.23.160.192]) by mail2.ip.pt with SMTP id OAA76579 for {Microscopy-at-MSA.Microscopy.Com} ; Wed, 6 Sep 2000 14:18:50 +0100 (WEST) Message-ID: {002501c01806$67f42da0$c0a017c3-at-anatomip}
I think that there is old work (From Afzelius?) in which osmium tetroxide was used in some organic solvent (carbon tetrachloride or chloroform?) to fix difficult specimens (eggs ?). You could check in old texts on TEM - like Pearse's "Histological Techniques for Electron Microscopy"
Dr. A.P. Alves de Matos Anatomo-Pathology Department Curry Cabral Hospital Lisbon
----- Original Message ----- } From: Gary Dietrich Chinga {garyc-at-stud.ntnu.no} To: ERIC {biology-at-ucla.edu} Cc: {microscopy-at-sparc5.microscopy.com} Sent: Wednesday, September 06, 2000 10:33 AM
They were mailed before the mtg. Contact Bill Bailey or the Springer guy: Herb Neimerow -at- 212-460-1686
Ron Anderson, IBM, Hopewell Jct., New York, USA. anderron-at-us.ibm.com
IBM Analytical Services; http://www.chips.ibm.com/services/asg
Didier, You can use treat glass with 1% silane in acetone to make it sticky. Marek.
At 9/6/00 Wednesday01:52 PM-0700, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Marek Malecki, M.D., Ph.D. Director and Principal Investigator
Molecular Imaging Laboratories University of California at San Diego address: 1500 Suite Bonner Hall, 9500 Gilman Drive, La Jolla, CA 92093-0368 phone: 8588223373 fax: 8588223715 pager: 8586161420 e.mail: mmm-at-ucsd.edu e.pager: 1620024619-at-alphapage.airtouch.com www site: http://mil.ucsd.edu/ ftp site: mil1.ucsd.edu
Workshop Objective This course will cover all aspects of pre-thinning and focus on final thinning via Tripod Polishing. Due to the limited class size and the extensive hands-on opportunities, this course is well suited to novices as well as advanced specimen preparation technicians. This course has been updated and expanded from past workshops to include pre-FIB preparation techniques, post-FIB Plasma Trimming as well as the latest techniques available in low energy ion milling, plasma cleaning and ion beam sputter deposition and etching for TEM and SEM samples.
The workshop will be held in San Clemente, CA on October 27-28, 2000. Please contact me directly for registration information.
David Henriks Vice President TEL: 800-728-2233 (toll free in the USA) South Bay Technology, Inc. +1-949-492-2600 1120 Via Callejon FAX: +1-949-492-1499 San Clemente, CA 92673 USA e-mail: henriks-at-southbaytech.com
Would anyone have for sale or trade-in a lens control system for JEOL's 1200EX?
Marek Malecki, M.D., Ph.D. Director and Principal Investigator
Molecular Imaging Laboratories University of California at San Diego address: 1500 Suite Bonner Hall, 9500 Gilman Drive, La Jolla, CA 92093-0368 phone: 8588223373 fax: 8588223715 pager: 8586161420 e.mail: mmm-at-ucsd.edu e.pager: 1620024619-at-alphapage.airtouch.com www site: http://mil.ucsd.edu/ ftp site: mil1.ucsd.edu
Dear Ian, I have had a VCR Group Ion Mill for several years now and have been very happy with it. It very seldom needs service, is easy for me to service if needed, does low-angle and atom thinning, has a liquid N2-cooled stage and an automatic terminator based on an ion gun, which works fine for transparent samples. My model is old now and the newer ones are computer controlled. The VCR Group is now part of South Bay Technologies. (www.southbay.com) At 09:04 AM 9/6/00 +0100, you wrote: } } Dear all, } Out of interest (i.e. I am not in a position to buy one right now), I am } interested in people's opinion on ion beam thinners on the market today. } } I have used a Gatan PIPS in the past and have been very satisfied with this } including many of the features including the low angle milling and the ability } to mount a TV camera to watch the milling in real time (great for hole } detection on transparent ceramics that defeat auto-termination systems). } } I know, however, that Gatan are not the only manufacturers of ion-beam thinning } equipment and I am sure that their competitors must also have some decent } machines. So, please tell me what else there is on the market (since I already } know plenty about the Gatan PIPS), what specifications these machines have, and } about your experiences with them. Also, comparisons between different } makes/models of ion beam thinner would be interesting. } } Looking forward to hearing from you. } } Best wishes } } ===== } Ian MacLaren } Beijing Laboratory of Electron Microscopy } Chinese Academy of Sciences, P.O. Box 2724 } 100080 Beijing, China } Email: ian.maclaren-at-physics.org / ianmaclaren-at-hotmail.com } Home page: http://members.tripod.co.uk/IanMacLaren/ } } ____________________________________________________________ } Do You Yahoo!? } Get your free -at-yahoo.co.uk address at http://mail.yahoo.co.uk } or your free -at-yahoo.ie address at http://mail.yahoo.ie } } Mary Mager Electron Microscopist Metals and Materials Engineering University of British Columbia 6350 Stores Road Vancouver, B.C. V6T 1Z4 CANADA tel: 604-822-5648 e-mail: mager-at-interchg.ubc.ca
There is a Siemens Elmiskop II TEM that has been mothballed and placed on display in the lobby of a lecture hall on the University of California, Davis, campus. Several years ago we taught a course and it was used as the student scope. Once decommissioned, the power supply and ht cable were given away to an EM service company and the console was placed in the lobby as an artifact of the early days of EM on the UC campus. The lecture hall now belongs to a department that has no interest in keeping the old scope on display and would like to dispose of it. Is there any interest out there in this device either for parts or for a display item? If not, it will be buried at the landfill.
Contact me off-list if you have any interest or suggestions for this scope.
Rick A. Harris, Director Microscopy and Imaging Facility Section of Molecular and Cellular Biology 1241 Life Sciences Addition University of California Davis, CA 530 752 2914 530 754 7536 fax http://katie.ucdavis.edu raharris-at-ucdavis.edu
Try the following company to get your answers: Larson Electronic Glass (Redwood City, CA) Tel. (650)369-6734. E-mail gls2mtl-at-juno.com . I am not aware of their web site.
Vitaly Feingold Scientific Instruments and Applications 2773 Heath Lane, Duluth GA 30096 (770)232-7785 ph. (770)232-1791 fax -----Original Message----- } From: donald j marshall {dmrelion-at-world.std.com} To: Microscopy-at-sparc5.microscopy.com {Microscopy-at-sparc5.microscopy.com}
I was wondering the same thing. I haven't received my copy as of 6-Sept-00.
Hope they are mailed shortly. Michelle Taurino Aventis Pharmaceuticals Senior Scientist Bioimaging and Molecular Histology Tel-908-231-3357 Fax-908-231-3962 e-mail: Michelle.Taurino-at-aventis.com
-----Original Message----- } From: Greg Erdos [mailto:gwe-at-biotech.ufl.edu] Sent: Wednesday, September 06, 2000 9:47 AM To: Microscopy-at-sparc5.microscopy.com
Does anyone know if the M&M 2000 proceedings have been mailed yet.?? Greg Gregory W. Erdos, Ph.D. Assistant Director, Biotechnology Program PO Box 110580 University of Florida Gainesville, FL 32611
No one in my group has received theirs either...has *anyone* received their Proceedings yet?
Larry
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Dr. Lawrence F. Allard Senior Research Staff Member High Temperature Materials Laboratory Oak Ridge National Laboratory 1 Bethel Valley Road Bldg. 4515, MS 6064 PO Box 2008 Oak Ridge, TN 37831-6064
I got mine right after returning from Philadelphia.
Randy
-----Original Message----- } From: Larry Allard [mailto:l2a-at-ornl.gov] Sent: Thursday, September 07, 2000 7:59 AM To: Microscopy-at-sparc5.microscopy.com
No one in my group has received theirs either...has *anyone* received their Proceedings yet?
Larry
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Dr. Lawrence F. Allard Senior Research Staff Member High Temperature Materials Laboratory Oak Ridge National Laboratory 1 Bethel Valley Road Bldg. 4515, MS 6064 PO Box 2008 Oak Ridge, TN 37831-6064
Responding to the message of {v04210101b5dd412fa34d-at-[128.219.47.130]} from Larry Allard {l2a-at-ornl.gov} : } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } No one in my group has received theirs either...has *anyone* received } their Proceedings yet? } } Larry }
I received mine very shortly after I returned, but it is possible that I was a special case :)
Thank you all for attending, and I know next year's program commitee will look forward to seeing you next year in Long Beach CA.
__________________ Stuart McKernan stuartm-at-tc.umn.edu Office:(612) 626-7594 IT Characterization Facility, University of Minnesota Desk:(612) 624-6009 100 Union Street S. E., Minneapolis, MN 55455 NEW-} Fax:(612) 625-5368
I am preparing turf grass samples for TEM. We will be examining the crowns and lead blades for infectious agents. Any tidbits on fixation and embedding of this tissue would be appreciated!
My starting point is this protocol, which has been used for wood samples. If it is overkill, please let me know.
Fix: 2% Glutaraldehyde in 50 mM NaCacodylate pH 7.2, with light vacuum for 24 hours Wash: 3X for 1 H each RT in buffer with mild agitation PostFix: 1% OsO4 in water Overnight at 4C Wash: 3X for 1H each at RT in water with mild agitation en bloc stain: saturated UA for 30 min with mild agitation Dehydration series: 25%, 50%, 75%, 100% Acetone, each 30 min with mild agitation 100% acetone + 1% DMP for 30 min with mild agitation -- 100% acetone + 1% DMP overnight with mild agitation 100% acetone + 1% DMP overnight with mild agitation Infiltration: 33% Spurr's in Acetone + 1% DMP for 2 H min with mild agitation 66% Spurr's in Acetone + 1% DMP for 2 H min with mild agitation 100 % Spurr's for 2H with mild agitation 100 % Spurr's Overnight with mild agitation 100 % Spurr's for 8-12 H with mild agitation 100 % Spurr's for 8-12 H with mild agitation Cast in fresh resin
Thanks, Sally
Sally Burns Center for Advanced Microscopy B5 Center for Integrated Plant Studies Michigan State University East Lansing, MI 48823 (517) 355-5004 burnssal-at-msu.edu
I checked with Springer on this. They received a list of 227 names to ship Proceedings to at the end of June. Shipment occurred about August 1. It is safe to assume that not all of them have reached their destinations yet. The form you filled out in the registration bulletin said it would take 4-6 weeks
HERE'S THE IMPORTANT PART: If you signed up as a full meeting registrant AFTER June 15th (or so) and elected to have the Proceedings shipped, Springer didn't get the labels yet. I called the Meeting Manager's office and, after a short chat, got a commitment that they would ship the additional labels overnight to Springer. Herb, at Springer, committed to getting the additional proceedings shipped early next week.
Ron Anderson MSA Pres-Elect
Ron Anderson, IBM, Hopewell Jct., New York, USA. anderron-at-us.ibm.com
IBM Analytical Services; http://www.chips.ibm.com/services/asg
Available with the Center for High Resolution Electron Microscopy at Arizona State University. Position is sponsored by major chemical company and primary focus of the research will be the development and application of environmental electron microscopy to industrially relevant catalyst systems. Areas of particular interest include the study of phase transformations under reaction environments, in situ polymerization, mobility and dynamic microstructural changes. Candidate will have a Ph.D. in material science, material physics, solid state chemistry or chemical engineering, with extensive experience in catalyst characterization by transmission electron microscopy. Experience in the areas of catalyst synthesis, testing and characterization is preferred. Please submit your resume together and the names of 3 referees to: Dr. Peter A. Crozier, Industrial Associates Program, Center for Solid State Science, Arizona State University, Tempe, AZ 85287-1704, Fax (480) 965-9004, email crozier-at-asu.edu.
John C. Wheatley Lab Manager Arizona State University Center for Solid State Science PSA-213 BOX 871704 Tempe, AZ 85287-1704
} Hi - } } I have a project which needs to see the preservation of testis and } kidney morphology of rats. I want to set up a couple of ultrastructural } criteria for this. Can any of you provide me some info about good } classical reference books or literature. Any experience or suggestions } are greatly appreciated. } } Regards } } Gang Ning } } EM Facility } Medical College of Wisconsin } Milwaukee, WI 53045 } 414-456-8141 } 414-456-6517 (Fax)
See "The ultrastructure of the rat renal medulla as observed after improved fixation methods" by S-O. Bohman. J. Ultrastruc. Res 47:329-360, 1974. Beautiful EMs. This paper deals with the medulla for the most part, renal cortex is not too difficult to fix well. Any edition of "The Kidney" by Brenner and Rector (see vol. 1) should provide good references. As far as testis goes, Martin Dym was a major researcher in that field long ago (20 years or so). A lit search for his name might be productive. Or look at the list of references at the end of the chapter on male reproductive system in any good histology text (Bloom and Fawcett, Ham [when Ham was still writing it]).
Geoff -- ********************************************** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854 voice: (732)-235-4583; fax: -4029 mcauliff-at-umdnj.edu **********************************************
Please excuse me if you've already received this message - I sent it yesterday to both servers, but received it from neither, so I think our system ate it on its way out :)
Tamara
---------- Forwarded message ----------
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I received my proceedings over 1 week ago....They were no doubt sent 3rd class so may take a number of weeks for all to be delivered depending on where you are in the country. Debby
--------------------------------------
No one in my group has received theirs either...has *anyone* received their Proceedings yet?
Larry
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Dr. Lawrence F. Allard Senior Research Staff Member High Temperature Materials Laboratory Oak Ridge National Laboratory 1 Bethel Valley Road Bldg. 4515, MS 6064 PO Box 2008 Oak Ridge, TN 37831-6064
Mine were here when I returned from the Conference.
Richard Shalvoy Arch Chemicals Cheshire, CT
-----Original Message----- } From: Larry Allard [mailto:l2a-at-ornl.gov] Sent: Thursday, September 07, 2000 8:59 AM To: Microscopy-at-sparc5.microscopy.com
No one in my group has received theirs either...has *anyone* received their Proceedings yet?
Larry
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Dr. Lawrence F. Allard Senior Research Staff Member High Temperature Materials Laboratory Oak Ridge National Laboratory 1 Bethel Valley Road Bldg. 4515, MS 6064 PO Box 2008 Oak Ridge, TN 37831-6064
I'm having a bit of trouble with Formvar film stability under the TEM beam at 60 kV. The films, attached to copper or nickel grids (cleaned with dilute HCl + acetone in sonicator same day films picked up) develop holes which grow over a few minutes time until eventually the entire film more or less disintegrates, collapses. I also noticed that initially, the films are often pulled away from the rim of the grid by about one square "hole" away.
I'm using new Formvar 15/95 resin powder and new bottle of ethylene dichloride solvent, mixing to 0.3% weight to volume. I use "Rite-On" glass slides, rinsed with ethanol to clean of dust and particulates and air dried, to cast the films. The films float off easily, and look very clean under the TEM. They were dried overnight prior to inspection. I take pains to use very clean glassware for mixing the Formvar solution.
Of course, I could caarbon coat them to provide stability, but that's extra work and I don't seem to have had this kind of problem before.
I barely recall some discussion here some time ago about how Formvar has "changed" due to different process of manufacturing, or whatever.
Any clues?
Thanks in advance.
Gib
Gib Ahlstrand Electron Optical Facility, University of Minnesota, Dept. Plant Pathology 495 Borlaug Hall, St. Paul, MN. USA. 55108 (612)625-8249 612-625-9728 FAX, giba-at-puccini.cdl.umn.edu http://biosci.umn.edu/MIC/consortium.html
First, a thank you to Mary for the kind words about our ion milling system. Just a quick correction, our website is actually at www.southbaytech.com. Hopefully this will slow down the number of calls we have received asking us why we are now selling DSL service!
Unfortunately, we do not have any information yet on the website about the XLA2000 ion mill so you will need to contact me directly on that. There is some information on the Technorg-Linda Low Energy Mill.
David Henriks Vice President TEL: 800-728-2233 (toll free in the USA) South Bay Technology, Inc. +1-949-492-2600 1120 Via Callejon FAX: +1-949-492-1499 San Clemente, CA 92673 USA e-mail: henriks-at-southbaytech.com
} } } } } Please visit us at http://www.southbaytech.com { { { { {
Manufacturers of precision sample preparation equipment and supplies for metallography, crystallography and electron microscopy.
Message text written by Mary Mager } ------------------------------------------------------------------------ The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Dear Ian, I have had a VCR Group Ion Mill for several years now and have been very happy with it. It very seldom needs service, is easy for me to service if needed, does low-angle and atom thinning, has a liquid N2-cooled stage and an automatic terminator based on an ion gun, which works fine for transparent samples. My model is old now and the newer ones are computer controlled. The VCR Group is now part of South Bay Technologies. (www.southbay.com) {
we routinely use formvar at a concentration of 0.5 - 0.7 %. however, you shurely are aware that the thickness of the film critically depends on the lowering speed of the fluid (slow = thin; fast = thick). if you can manage, coat with carbon afterwards, there are no disadvantages by doing so, only advantages. good luck peter
************************************************** please reply to: peter.heimann-at-biologie.uni-bielefeld.de ("E-MAIL")
Dr. Peter Heimann Developmental Biology & Molecular Pathology; W7-107 University of Bielefeld D 33501 Bielefeld / Germany phone: xx49(0)521-106-5628 / 5627 FAX : xx49(0)521-106-5654 WEB-Site: http://www.uni-bielefeld.de/biologie/Entwicklungsbiologie WEB-Site: http://www.uni-bielefeld.de/SFB549 ******************************************************
one of the best techniques to perfuse (testis, but brain and other organs as well) is published by: Forssmann, WG et al; 1977; Anat. Rec. 188; (3); 307- 314
for excellent testis-EM look for instance after author Holstein, A.F.. I for myself use the "forssmann"-technique on mouse testis with excellent results.
good luck
peter heimann ************************************************** please reply to: peter.heimann-at-biologie.uni-bielefeld.de ("E-MAIL")
Dr. Peter Heimann Developmental Biology & Molecular Pathology; W7-107 University of Bielefeld D 33501 Bielefeld / Germany phone: xx49(0)521-106-5628 / 5627 FAX : xx49(0)521-106-5654 WEB-Site: http://www.uni-bielefeld.de/biologie/Entwicklungsbiologie WEB-Site: http://www.uni-bielefeld.de/SFB549 ******************************************************
Here in Connecticut it is often difficult to make good formvar films in the summer, due to high humidity. The films are covered with holes of varying sizes, even when made in a dry bottles and with freshly opened solvent. I think this problem is caused by condensation of water on the glass surface, which is cooled significantly during evaporation of the solvent. It doesn't take much cooling to get condensation when the ambient humidity is 90%! Perhaps you have tiny pinholes in your films that are enlarging to become visible under the beam.
Marie
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Dr. Marie E. Cantino Dept. of Physiology and Neurobiology, U-2131 University of Connecticut Storrs, CT 06269-2131 Phone: 860-486-3588 Fax: 860-486-1936
Position Open -- Immediately Microscopy and Microanalysis Research Assistant
An immediate position is open for an SEM microscopist at the North Carolina State University Analytical Instrumentation Facility (AIF). Duties and responsibilities include: Operation and maintenance of SEM instrumentation and sample preparation and analysis equipment; scheduling of access to and oversight of the above instrumentation; user
training and assistance; assistance with the teaching of electron microscopy laboratory classes, and analysis of a wide variety of samples. Qualifications must include a BS or MS or equivalent experience in a materials related discipline along with one or more years hands on experience with SEM or related techniques. Required skills include: extensive hands on experience with SEM and related techniques and accessories (e.g. specimen preparation and associated analytical tools). Preferred qualifications include: teaching and user training; familiarity with modern electronics; and experience with vacuum systems. Please send resume and three letters of reference to: Phil Russell, Director; Analytical Instrumentation Facility; North Carolina State University; Box 7531, Room 318A EGRC; 1010 Main Campus Drive; Raleigh, NC 27695-7531 or email PRUSSELL-at-NCSU.EDU.
North Carolina State University is an Equal Opportunity and Affirmative Action Employer. ADA Accommodations: Phil Russell, prussell-at-ncsu.edu, 919-515-7501. In its commitment to diversity and equity, North Carolina
State University seeks applications from women, minorities, and persons with disabilities
Phillip E. Russell Professor, Materials Science and Engineering Director, Analytical Instrumentation Facility Box 7531, Room 318 EGRC 1010 Main Campus Drive North Carolina State University Raleigh, NC 27695-7531
You can slightly improve stability under the beam by increasing kV, using a thinner film (and finer mesh grids), using less beam current and better still irradiating the grid at very low powers (200x) for a couple of minutes. The brightness appears great at low powers but actual beam intensity is much greater at higher powers and the lower power irradiation stabilises the film. Ultimately you can carbon coat.
BUT WHY are you and most people who really want to use plain plastic films use Formvar??? Butvar is the strongest and most stable of plastic films and is much more stable under the beam than Formvar without carbon. EM techniques (for some obvious reasons) die hard. Butvar has been around for over twenty years but people keep on going back to the old Formvar references. Disclaimer: ProSciTech does not care if you buy Butvar or Formvar. Cheers Jim Darley ProSciTech Microscopy PLUS PO Box 111, Thuringowa QLD 4817 Australia Ph +61 7 4774 0370 Fax:+61 7 4789 2313 service-at-proscitech.com Great microscopy catalogue, 500 Links, MSDS, User Notes ABN: 99 724 136 560 www.proscitech.com
On Friday, September 08, 2000 7:22 AM, Gib Ahlstrand [SMTP:giba-at-puccini.cdl.umn.edu] wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Dear Microlisters, } } I'm having a bit of trouble with Formvar film stability under the TEM beam at } 60 } kV. The films, attached to copper or nickel grids (cleaned with dilute HCl + } acetone in sonicator same day films picked up) develop holes which grow over a } } few minutes time until eventually the entire film more or less disintegrates, } } collapses. I also noticed that initially, the films are often pulled away from } } the rim of the grid by about one square "hole" away. } } I'm using new Formvar 15/95 resin powder and new bottle of ethylene dichloride } } solvent, mixing to 0.3% weight to volume. I use "Rite-On" glass slides, rinsed } } with ethanol to clean of dust and particulates and air dried, to cast the } films. } The films float off easily, and look very clean under the TEM. They were dried } } overnight prior to inspection. I take pains to use very clean glassware for } mixing the Formvar solution. } } Of course, I could caarbon coat them to provide stability, but that's extra } work } and I don't seem to have had this kind of problem before. } } I barely recall some discussion here some time ago about how Formvar has } "changed" due to different process of manufacturing, or whatever. } } Any clues? } } Thanks in advance. } } Gib } } Gib Ahlstrand } Electron Optical Facility, University of Minnesota, Dept. Plant Pathology } 495 Borlaug Hall, St. Paul, MN. USA. 55108 (612)625-8249 } 612-625-9728 FAX, giba-at-puccini.cdl.umn.edu } http://biosci.umn.edu/MIC/consortium.html }
Is coating with carbon more work than remounting a specimen after you have destroyed it with your beam. Carbon coating is part of the procedure in my lab, and students know not to stick a grid into the scope without it. We observe many samples weekly at 120kV without destroying any. Think of all the contamination you are introducing into the specimen chamber when you "burn" your film and sample.
I have a BalTec RES 100 and I am extremely pleased with it. There are quite a few options available with it. Not only have I have prepared very good TEM samples with it, but I have used it for ion polishing and ion etching of SEM samples for our High Resolution FESEM. BalTec has a number of holders for SEM applications. In addition, I can use it to sputter deposit films onto samples. The newest guns that I just had put on ours our very stable and settle down very quickly. Whatever changes they made in the software and hardware dramatically improved the operation over the first generation guns. I really like saving recipes available that I can reuse in the programming mode. Although I don't have the opportunity to use it much on glass samples, the image processing for termination is probably the most sensitive method that I have used. There are a lot of options that the user can adjust for optimizing the optical termination through image processing. Alignment of the guns is extremely easy and accurate. Further, they stay aligned. If you get the ion deflection system, they have an automatic alignment system. I bought ours because of the Faraday cup termination that they have available. However, I hardly use it. With the camera system, it is easy to watch the screen to determine when it is finished. I have the remote control/monitoring capability but have not put the system on our LAN yet. I plan to do that very soon.
If you have a FESEM in the same lab as your TEM, you should consider what this mill could do for you.
PS Let me tell you that I like the PIPS also. It is very simple to use. It has one mode that is unique to it, the ability to mill on both top and bottom of the sample from one direction coming in from the perpendicular direction to the interface of a cross section. Reza Alani of Gatan has shown many examples of beautiful samples prepared this way. But, if I were to make the decision again, I would still go with the BalTec system. It is versatile and can be used in a high tech way as well as a routine way with technicians pumping through samples. BalTec has responded to my needs and have considered things that I have said in terms of their software updates. I have experimented with some sample preparation techniques for SEM that look very promising using this mill.
Although I do not have direct experience with the other ion mills on the market, I have talked extensively with their manufacturers and they all have some nice features. The low energy guns in the Technorg-Linda system designed by Barna should be carefully considered. My advice to you is to consider what you want the mill to do in your lab with your samples and go from there. You need to weigh what features are available on each and how they would apply to your samples. You also have to weigh experience with your users to the level of competency required for each of the mills out there.
I hope this helps.
-Scott
Scott D. Walck, Ph.D. PPG Industries, Inc. Glass Technology Center Guys Run Rd. (packages) P. O. Box 11472 (letters) Pittsburgh, PA 15238-0472
Walck-at-PPG.com
(412) 820-8651 (office) (412) 820-8161 (fax)
"The opinions expressed are those of Scott D. Walck and not of PPG Industries, Inc. nor of any PPG-associated companies." --
} -----Original Message----- } From: Ian MacLaren [mailto:maclariz-at-yahoo.co.uk] } Sent: Wednesday, September 06, 2000 4:05 AM } To: Microscopy-at-sparc5.microscopy.com } Subject: Modern ion beam thinners } } } -------------------------------------------------------------- } ---------- } The Microscopy ListServer -- Sponsor: The Microscopy Society } of America } To Subscribe/Unsubscribe -- Send Email to } ListServer-at-MSA.Microscopy.Com } On-Line Help } http://www.msa.microscopy.com/MicroscopyListse} rver/FAQ.html } } } -------------------------------------------------------------- } ---------. } } } Dear all, } Out of interest (i.e. I am not in a position to buy one right } now), I am } interested in people's opinion on ion beam thinners on the } market today. } } I have used a Gatan PIPS in the past and have been very } satisfied with this } including many of the features including the low angle } milling and the ability } to mount a TV camera to watch the milling in real time (great for hole } detection on transparent ceramics that defeat } auto-termination systems). } } I know, however, that Gatan are not the only manufacturers of } ion-beam thinning } equipment and I am sure that their competitors must also have } some decent } machines. So, please tell me what else there is on the } market (since I already } know plenty about the Gatan PIPS), what specifications these } machines have, and } about your experiences with them. Also, comparisons between different } makes/models of ion beam thinner would be interesting. } } Looking forward to hearing from you. } } Best wishes } } ===== } Ian MacLaren } Beijing Laboratory of Electron Microscopy } Chinese Academy of Sciences, P.O. Box 2724 } 100080 Beijing, China } Email: ian.maclaren-at-physics.org / ianmaclaren-at-hotmail.com } Home page: http://members.tripod.co.uk/IanMacLaren/ } } ____________________________________________________________ } Do You Yahoo!? } Get your free -at-yahoo.co.uk address at http://mail.yahoo.co.uk } or your free -at-yahoo.ie address at http://mail.yahoo.ie }
----- Original Message ----- } From: {keg-at-alpha3.csd.uwm.edu} To: {keg-at-csd.uwm.edu} Sent: Friday, September 08, 2000 9:23 AM
Hello Tamara and All, An unfortunate, although interesting predicament. There are three variables that come to mind which may affect the shipping strategy: temperature change, pressure change and time. I worry that leaving the samples in methanol/acetic acid and raising the temperature would lead to denaturation of protein structure due to overfixation. If the samples could be kept acceptably cool during transport this might help, but I'm not sure of the technical difficulties (and expense) associated with overseas transport on dry ice-I believe Fed Ex has pressurized cargo (which would slow the sublimation of the dry ice). If these samples could be sent via overnight express this would probably work. Otherwise, placing the samples in a buffer solution similar to that proposed for your incubation protocol(s) for shipping could be a viable approach. Adding some sodium azide to the buffer for transport to prevent bacterial contamination would be a good idea in my opinion. I haven't tried any of these approaches but perhaps someone with more experience than I will agree/disagree and you will be able to find a quasi-reliable approach to your problem. Good Luck! Cheers, Karl Garsha ----- Original Message ----- } From: Tamara Howard {howard-at-cshl.org} To: Microscopy Listserver {Microscopy-at-sparc5.microscopy.com} ; Histology listserver {histonet-at-pathology.swmed.edu} Sent: Thursday, September 07, 2000 11:18 AM
Hello, Used equipment can be auctioned off/bidded on at www.biobid.com. It seems like a nifty idea. Regards, Karl Garsha ----- Original Message ----- } From: MICHAEL DELANNOY {delannoy-at-mail.jhmi.edu} To: {microscopy-at-sparc5.microscopy.com} Sent: Tuesday, September 05, 2000 3:21 PM
All: does anyone in the North Texas area have a carbon coater capable of handling an 8-inch wafer? We need to get a wafer coated prior to some FIB edit and ours has not returned from the vendor. Please reply to me so we don't clutter up the listserver.
I have used Sparkle Window cleaner (its the purple one!) with great success but beware that there are big differences in window cleaner brands. good luck. tom
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
-- Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility
3 Tucker Hall Division of Biological Sciences University of Missouri Columbia, MO 65211-7400 (573)-882-4712 (voice) (573)-882-0123 (fax)
A postdoctoral position is available immediately in the area of Environmental Catalysis. The position will be part of the Institute for Environmental Catalysis (http://www.iec.nwu.edu) and will involve TEM, both HREM as well as diffraction (Direct Methods) on oxide surfaces as a function of gas treatment in collaboration with other faculty at Northwestern as well as Industry, using the unique UHV-HREM at Northwestern University (see http://www.numis.nwu.edu). A strong background in TEM is required, and expertise in UHV and associated techniques (e.g. XPS) is desirable.
Send applications (by email) to: Professor L. D. Marks Department of Materials Science and Engineering mailto:ldm3-at-apollo.numis.nwu.edu
Include a CV and the names of at least two referees.
Friday, September 29, 2000 University of Wisconsin Memorial Union South 227 North Randall Avenue Madison, WI 53715 (608) 263-2600
Admission is free with MMMS Membership. MMMS Membership is $10.00 - new members will be accepted at registration
Meeting Schedule
MORNING COMBINED SESSION, Room, Union South 9:45AM - 10:30AM Registration - Coffee, Juice, Pastries will be provided.
10:30 AM Opening remarks
10:35 AM Dr. Sue Welch, UW-Madison Department of Geology and Geophysics "The Biogeochemical Cycling in the Tennyson Mine" Co-authors: Matthias Labrenz, Anne Skatvold, Gregory K. Druschel, David A. Fowle, Tamara Thomsen-Ebert and Jill Banfield
11:35 AM Dr. Will Bigelow, Professor Emeritus of Materials Engineering, University of Michigan "The current state of vacuum science in microscopy, or: why it still takes so long to pump down to an operating vacuum"
AFTERNOON BIOEMPHASIS SESSION: Room , Union South
1:30 PM Hans Ris, Professor Emeritus, UW Madison. "3D Imaging of cell structures by high resolution , low voltage , field emission SEM"
2:00 PM Daryl Meyer, UW Madison "Multiple labeling for EM: Synthesis and use of colloidal nano-particles of different metal composition and shape."
2:30 PM Judith Croxdale, UW Madison "Leaf Surfaces, Microscopy and Thievery useful in Analysis".
3:00 PM Jayne Squirrell, UW Madison "Application of multiphoton microscopy to the study of embryo development".
AFTERNOON PHYSICAL SCIENCE EMPHASIS SESSION: Room , Union South
1:30 PM Carl Loper, UW-Madison Dept of Material Science & Engineering "Interaction of Pb and Ca and Its Effect on Graphite Morphology in Cast Irons" co-authors: Junyoung Park and John Fournelle
2:00 PM Reid Cooper, UW-Madison Dept of Material Science & Engineering "Redox Reactions in Silicate Melts and Glasses"
2:30 PM John H. Perepezko, UW-Madison Dept of Material Science & Engineering " Synthesis and Initial Crystallization of Amorphous Al Alloys" coauthors: R.I. Wu, R. Hebert and Z. Dong, University of Wisconsin-Madison
3:00 PM Thomas F. Kelly, UW-Madison Dept of Materials Sceince & Engineering, and Imago Scientific Instruments Corporation, Madison, WI "Local Electrode Atom Probes: Background and Application to Microelectronic Materials"
3:45 PM CHEESE, CHEESE, & ICE CREAM (remember this is Wisconsin) RECEPTION -- UNION SOUTH
4:45 PM MEETING ADJOURNS Have a SAFE ride home
Support of this meeting has been provided by: University of Wisconsin Department of Geology & Geophysics, The Biological & Biomaterials Preparation, Imaging, & Characterization Laboratory, The Advanced Microscopy Facility, and "Microscopy Today"
Getting to Madison:
Madison is located in south central Wisconsin and is accessible via several major highways. Madison is:
- 1 1/2 hour drive from Milwaukee (via Interstate 94) - 2 1/2 hour drive from Chicago (via Interstate 90) - 4 1/2 hour drive from Minneapolis/St. Paul (via Interstate 94) - 2 hour drive from Dubuque (via US 151)
Getting to UW-Madison:
- Take I-90 or US 151 to Hwy 12/18 - Follow Hwy 12/18 exit at Park Street (Exit 261 B) - Proceed north on Park Street about four miles to Regent Street and turn left. - Proceed on Regent to Randall St. and Turn Right. (See detail map.)
Below is a message from the family of Hildy Crowley. I thought it would be of interest to the many people who subscribe to this list why she signed off recently, and so abruptly. I asked her son if it would be OK to put it here. He thought it was a good idea and that it would be what she wanted as she loved EM so much. If you think it is appropriate I will let you post it. I know that even though I never met her, your listserver brought us close and that I will miss her.
Paul Webster
HILDY CROWLEY 29 December 1935 - 7 September 2000
After a long and difficult illness Hildegard Heinrich Crowley died quietly at home on September 7. Earlier in the week she had given her grandsons, ages 2 and 4, Batman slippers. The boys showed their Oma (German for Grandma) how much higher the slippers let them jump. A few days before that she had received the Morton D. Masur Distinguished Service Award from the Microscopy Society of America because, through the years her advice also had allowed many electron microscopists to jump a little higher. With Hildy's help anyone could jump a little higher.
Hildy was born in Stuttgart, Germany. Her mother, also Hildegard, died that day. She was raised for several years by aunts and a Grandmother in North German Pomerania, now a part of Poland. Her father then remarried and she joined him again. Throughout her life she had frightening wartime dreams from those days – bombings, straffings, government repression, malnutrition, and Allied tanks arriving in her town of Eslingen.
Hildy's father, a distinguished aeronautical engineer, was one of the German scientists brought to the US after the war. Only after many months could the family join him. Years later she spoke with joy of those early teen years in America, with plenty of food, nice teachers, and wonderful friends.
A superb student, Hildy also won prizes at the County Fair for cooking and sewing, and she was an attendant to the college Homecoming Queen; beauty contests still were "in" then. After college she taught junior high school science.
In 1962 Hildy married Tom Crowley, a young medical student. In 1963 she began working as an electron-microscopy technologist at the University of Minnesota. She was a perfectionist, and EM is a perfectionist's dream and nightmare. "I love solving problems", she said, and each EM problem solved exposed ten new ones. The couple's sons, Christopher and Devin, arrived in 1965 and 1967.
After several years of raising youngsters, Hildy returned to EM at the University of Colorado Health Sciences Center, then at the National Jewish Hospital, and for the last decade, at Denver University. Armed only with a Bachelor's degree, superb intellect, and dogged determination, Hildy became internationally recognized for solving technical problems in EM, focussing in recent years on immunocytochemistry. She was especially proud of the lab's display of technical expertise in two Comparative Neurology articles (July and September 2000), on which she was a prominent co-author.
Hildy and Tom had moved to Denver for its skiing. Again the technical perfectionist, she became expert in equipment, waxes, and edge care. Her skiing continued improving well into her 60's, when she still did the easier expert runs some 35 days each year.
If Hildy was great at EM, she was even greater at teaching it. Brilliant undergraduate students came to the lab for Honors Theses. Hildy taught them biology and EM, of course, but she also supported them through romantic crises, sometimes helped them find physicians or therapists for their physical or personal problems, and advised them about their parents' illnesses or malfeasances. She also fed them delicious home-made cakes at lab meetings. She at first was miffed when someone called her the "Lab Mom", until a student said plaintively, "I want you to be our Mom. You're so much better than my own Mom". She gave students level-headed, practical, grandmotherly, every-day advice; like her ancestors from the foggy, cold Baltic, she distrusted "that touchy-feely stuff". The students went off to fine medical or dental or graduate schools, but when they visited Denver they often took her to lunch. They invited her to attend, or to be in, their weddings; one came back to ski with her. After knowing her, they all jumped a little higher.
Hildy's greatest commitment and contribution, of course, was to her family. Their loss is too large to speak of here.
Hildy learned in June 1999 that her illness would be terminal, but she wanted that kept secret from most of her friends, who, at the Portland Microscopy Society meeting, thought her gift of a limo ride to a fancy restaurant was just a madcap moment. It was a farewell.
Being Hildy, she worked with Tom to design her own simple headstone, which was erected a couple of months ago. It says, "You shall live in what you love." She loved so much, and so many, and so well. Hildy, with love, farewell. Farewell Oma, Mom, Wife, Sister, Lab Mom, Scientist, Teacher, Mentor, Friend, Colleague. Our Batman slippers fit just right. You helped us all jump higher.
-------Tom
Hildy's funeral will be at the First Universalist Church, 4101 East Hampden Avenue, Denver, at 10 AM Tuesday, September 12. A graveside interment ceremony at Fairmount Cemetery will follow.
Paul Webster, Ph.D. Associate Scientist & Director Ahmanson Advanced Electron Microscopy & Imaging Center House Ear Institute 2100 West Third St. Los Angeles, CA 90057
I had no idea who she was, but I always liked reading her postings, and her last one was just as gracious as ever.
My sympathy to her family.
rtch
Ritchie Sims Phone : 64 9 3737599 ext 7713 Department of Geology Fax : 64 9 3737435 The University of Auckland email : r.sims-at-auckland.ac.nz Private Bag 92019 Auckland New Zealand
Hi Everybody! We have two Hitachii TEM's (H600 and H7000). We use liquid nitrogen to improve the vacuum (back trap) and to decrease contamination of the column (front trap). Recently, I have been told by the university safety officer that I can not use LN in the back trap (I and the users have to climb on a step ladder and lift LN container to fill the trap) because of the safety reasons. One of our users strained her back while filling the back trap and she has been on a sick leave for some time. They suggested to build a platform in the back of a microscope so it is more sturdy and safer. Have you ever had problems of that sort? The lab has been opened for 15 years and we have never had any accident with LN. Is the platform really safer? Will it interfere with the performance of the microscope, if it is metal one? Will performance of the scope be affected if I do not use LN in the back trap? Thanks for answers Dorota
I apologize in advance for what may well be a very sophomoric question.
I haven't paid attention to what may have been discussed regarding alternatives to Freon 113 as a final drying agent because I have scrupulously rationed my supply, ml by ml, for several years. Now I'm into my final 300 ml and must begin to search for a substitute since it has been my understanding that Freon 113 is no longer available. I only use very small volumes at a time and 4x500 ml bottles has lasted me nearly 10 years. Most of my specimens are cell layers or other small (less than 1 cubic mm) tissue samples for SEM.
Any suggestions and information about what is currently used as an alternative to critical point drying out of Freon 113 exchanged with liquid CO2 would be greatly appreciated.
Thanks,
Gerald Harrison Electron Microscope Facility Dental School Research University of Pennsylvania
We are looking for camera suggestions that will handle Low Light, Real time imaging(both for flurorescence and DIC) in the fluorescence microscope. Something in the $20K range or less for the system.
High resolution is required also. These images can be extremely dark. The user prefers also to be able to output the realtime imaging of cell growth to videotape as that has proved to be the most convenient in the past but is also interested in the "firewire" option and it's capabilities.
We realize that a compromise in some areas (likely the frame rate)will be required but high resolution during low light imaging is very important.
We would be particularly interested in hearing from others that have used a camera that would suit our application. We have commercial info for several models but would really like some user feedback.
Thanks,
Karen Rethoret York University Biology Department Toronto, Ontario 416-736-2100 x33289
I am very sad about the message that reached me today.
Unfortunatly, I knew Hildy only as a member of the listserver and by private emails. She was a source of wisdom in this community. I told her about technical problems and she repeated promptly. One day a letter from Denver reached me here in Germany with her protocol and I was so proud to receive it. Someone more or less anynomous cared for my problems, that impressed me much. I feel indeed that she was a very gentle person and I envy everybody who knew her personally.
My deeply sympathy to her family and her friends.
Michael
Michael Reiner University of Cologne, Germany Dept. of Anatomy I _______________________________________________________________________ 1.000.000 DM gewinnen - kostenlos tippen - http://millionenklick.web.de IhrName-at-web.de, 8MB Speicher, Verschluesselung - http://freemail.web.de
I have had concerns about this for some while. The difficulty isn't just with lifting something heavy (which is, most certainly, a major problem), but also that the heavy thing is inherently hazardous. If you are pouring liquid up above you, and you spill it, some will almost certainly splash on you. This is not good if the liquid is nitrogen (or any other cryogen, amongst other hazardous materials).
Using a step ladder is also not a very safe prodecure, and if you are leaning to one side to pour from a heavy dewar, it makes it all the more unstable.
I have built a wooden platform (well, I had the carpenters build it!) for the worst of our instruments. I used wood because it was much easier than designing and ordering a custom metal one, but there is no issue of interfering with the microscope. The arrangement works well. It is very simple - 2x4's and plywood, painted with floor paint. The carpenters designed it as they went along.
Another way of dealing with nitrogen filling in a safe way would be to use a pressurized tank, and fill the trap, x-ray detector or whatever through a pipe. The arrangement could be made portable. I have had in mind researching the cost of a system like this.
I wouldn't recommend an automatic fill system, though. The valves tend to stick in the open position, draining your liquid nitrogen supply and freezing (and possibly damaging) the items onto which the liquid spills. I'm talking from experience here!
Good luck!
Tony.
} Hi Everybody! } We have two Hitachii TEM's (H600 and H7000). We use liquid nitrogen } to improve the vacuum (back trap) and to decrease contamination of } the column (front trap). Recently, I have been told by the university } safety officer that I can not use LN in the back trap (I and the } users have to climb on a step ladder and lift LN container to fill } the trap) because of the safety reasons. One of our users strained } her back while filling the back trap and she has been on a sick } leave for some time. They suggested to build a platform in the back } of a microscope so it is more sturdy and safer. Have you ever had } problems of that sort? The lab has been opened for 15 years and } we have never had any accident with LN. Is the platform really safer? } Will it interfere with the performance of the microscope, if it is } metal one? Will performance of the scope be affected if I do not use } LN in the back trap? } Thanks for answers } Dorota }
** Anthony J. Garratt-Reed ** MIT Room # 13-1027 ** 77 Massachusetts Avenue ** Cambridge, MA 02139-4307 ** USA ** ** Phone: (+) 1-617-253-4622 ** Fax: (+) 1-617-258-6479 **
Dr. David Kirk has a Philips 300 TEM available for donation. The recipient will pay all moving charges. If interested, please contact him directly
David Kerk, Ph.D. Professor and Chair Department of Biology Point Loma Nazarene University 3900 Lomaland Dr San Diego, CA 92106 Ph: 619-849-2398 FAX: 619-849-2598 email: dkerk-at-ptloma.edu
This is the last posting, I promise, seeking a used (of course no objection to new) Probe Current Detector (ie Faraday Cup device) for a JEOL 840 or 6400.
Anyone got one pining away in a cupboard?
cheers
rtch
Ritchie Sims Phone : 64 9 3737599 ext 7713 Department of Geology Fax : 64 9 3737435 The University of Auckland email : r.sims-at-auckland.ac.nz Private Bag 92019 Auckland New Zealand
It seems my fears were unfounded as it relates to small volume laboratory use. It is still possible to obtain Freon 113, in modest amounts, for laboratory work.
Gerald Harrison Electron Microscope Facility Dental School Research University of Pennsylvania =================================
I buy F113 fro Re-Cycle & Re-Use Industries, Mansfield, TX
Try John Metz 817.477.5207
I have about 2 gallons in stock and use maybe a quart or so a year.
gary
At 10:55 AM 9/11/00, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Dear Dorota, I sympathize, since I have the H-800, which is the taller, 200 kV version of the H-600 and I have found that short people do have some problems filling the back trap. You are lucky you don't have the EDS, which is even higher up. My solution is to have a self-pressurized dispenser on the 50L liquid nitrogen dewar attached to a hose that can reach the back trap. Position the hose, turn on the liquid N2 slowly, close the tap when the trap is full and wait for the hose to soften before moving it. There is no reason a platform wouldn't work, so long as it is made of non-magnetic materials, such as plastic, aluminum or wood. At 01:49 PM 9/11/00 -0300, you wrote: } } Hi Everybody! } We have two Hitachii TEM's (H600 and H7000). We use liquid nitrogen } to improve the vacuum (back trap) and to decrease contamination of } the column (front trap). Recently, I have been told by the university } safety officer that I can not use LN in the back trap (I and the } users have to climb on a step ladder and lift LN container to fill } the trap) because of the safety reasons. One of our users strained } her back while filling the back trap and she has been on a sick } leave for some time. They suggested to build a platform in the back } of a microscope so it is more sturdy and safer. Have you ever had } problems of that sort? The lab has been opened for 15 years and } we have never had any accident with LN. Is the platform really safer? } Will it interfere with the performance of the microscope, if it is } metal one? Will performance of the scope be affected if I do not use } LN in the back trap? } Thanks for answers } Dorota } Regards, Mary
Mary Mager Electron Microscopist Metals and Materials Engineering University of British Columbia 6350 Stores Road Vancouver, B.C. V6T 1Z4 CANADA tel: 604-822-5648 e-mail: mager-at-interchg.ubc.ca
----- Original Message ----- } } I have had concerns about this for some while. The difficulty isn't just } with lifting something heavy (which is, most certainly, a major problem), } but also that the heavy thing is inherently hazardous. If you are pouring } liquid up above you, and you spill it, some will almost certainly splash on } you. This is not good if the liquid is nitrogen (or any other cryogen, } amongst other hazardous materials). } } Using a step ladder is also not a very safe prodecure, and if you are } leaning to one side to pour from a heavy dewar, it makes it all the more } unstable. } } I have built a wooden platform (well, I had the carpenters build it!) for } the worst of our instruments. I used wood because it was much easier than } designing and ordering a custom metal one, but there is no issue of } interfering with the microscope. The arrangement works well. It is very } simple - 2x4's and plywood, painted with floor paint. The carpenters } designed it as they went along. } } Another way of dealing with nitrogen filling in a safe way would be to use } a pressurized tank, and fill the trap, x-ray detector or whatever through a } pipe. The arrangement could be made portable. I have had in mind } researching the cost of a system like this.
Building a portable LN pump would be pretty simple. You need a lid for the LN container, a wet line to the bottom of the container, a very small heating element and a vapor vent to stop the transfer.
To use the pump LN fill the tank and put the lid on with the vapor valve open. The output of the wet line is connected and the vapor valve closed and the heater turned on. A 500 Ohm resistor and 6 volts should make a good enough heater. Keep the resistor very small so it will cool rapidly. To stop the transfer open the vapor valve and turn off the heater. Once the vapor valve is open the liquid should drain back down the wet line and everything should be ready to disconnect in a minute or so. The only problem is if you get into a siphon situation. If you do that you need a way to relieve the suction.
On vapor releif and suction releif valves I would not totaly rely on something that could freeze up but I would add a breakable vapor reief valve that a sharp rap with a stick would beak and shut the system down.
I have rigged a lot of pumps that work this way using about 4 psi. The original pump I built was for pumping diesel wiht propane and if your pressure was over 4 PSI you desolved propane in the diesel and vapor locked your injectors. I have used in in a lot of situations for moving liquids. I built one that went in bung of a 55 gallon barrel and was completly sealed except for the pressure releif valve that should be open at all time when the pump is not in use.
The head on this type pump should not be very great or the transfer rate is slow, you waste a lot of vapor when you vent and the pressure can get a higher than is safe. For low head pumping 4 psi will pump 50 gallons of deisel through a 1.5 inc hose 10 or 15 minutes at a 18 inch head. or 5 or 10 minutes with a -10 inch head.
Hello, Isn't there a better alternative to Freon 113? We use ethanol for critical point drying in our lab with fine results. Freon is bad for the ozone layer. I've copied below, some information from the US Environmental Protection Agency.
C. Abiotic Effects
Freon 113 moves slowly through the lower atmosphere into the stratosphere. Photodegradation of freon 113 in the upper atmosphere releases chlorine atoms which react with ozone. Stratospheric depletion of ozone increases the amount of ultraviolet-B radiation that reaches the earth's surface (U.S. EPA 1983). Increased, surface UV radiation can adversely affect human health and the environment.
\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\ Gordon Ante Vrdoljak Electron Microscope Lab ICQ 23243541 http://nature.berkeley.edu/~gvrdolja 26 Giannini Hall gvrdolja-at-nature.berkeley.edu UC Berkeley phone (510) 642-2085 Berkeley CA 94720-3330 fax (510) 643-6207 cell (510) 290-6793
On Mon, 11 Sep 2000, Gary Gaugler wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } I buy F113 fro Re-Cycle & Re-Use Industries, Mansfield, TX } } Try John Metz } 817.477.5207 } } I have about 2 gallons in stock and use maybe a quart or so a year. } } gary } } } At 10:55 AM 9/11/00, you wrote: } } ------------------------------------------------------------------------ } } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } -----------------------------------------------------------------------. } } } } } } Hello form the Univ. of Penn., } } } } I apologize in advance for what may well be a very sophomoric } } question. } } } } I haven't paid attention to what may have been discussed } } regarding alternatives to Freon 113 as a final drying agent because I } } have scrupulously rationed my supply, ml by ml, for several years. Now } } I'm into my final 300 ml and must begin to search for a substitute since } } it has been my understanding that Freon 113 is no longer available. I } } only use very small volumes at a time and 4x500 ml bottles has lasted me } } nearly 10 years. Most of my specimens are cell layers or other small } } (less than 1 cubic mm) tissue samples for SEM. } } } } Any suggestions and information about what is currently used as } } an alternative to critical point drying out of Freon 113 exchanged with } } liquid CO2 would be greatly appreciated. } } } } Thanks, } } } } Gerald Harrison } } Electron Microscope Facility } } Dental School Research } } University of Pennsylvania } } }
I have a memory lapse-can you remind me of the reagent used as an alternative to CPD for SEM;or do you know of/recommend any safe (not too toxic) alternative method to CPD?-any reply would be appreciated
Cameron
Mr. Cameron Hind Research Scientist Advanced Technologies group Baxter R & D Europe S.C.R.L. Rue du Progrès 7 1400 Nivelles Belgium
Hildy must indeed have been quite a remarkable person. I have benefited several times over recent years from her replies and advice. I note that I currently have fifteen of her messages saved. Three of those were in connection with my own recent enquiry about folds in semi-thin resin sections. She replied so quickly to additional mailings when asked about an additional point. Her knowledge of electron microscopy and specimen processing was very wide-ranging. She will be missed by this List.
Keith Ryan Marine Biological Association Plymouth UK
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Hi Everybody! } We have two Hitachii TEM's (H600 and H7000). We use liquid nitrogen } to improve the vacuum (back trap) and to decrease contamination of } the column (front trap). Recently, I have been told by the university } safety officer that I can not use LN in the back trap (I and the } users have to climb on a step ladder and lift LN container to fill } the trap) because of the safety reasons. One of our users strained } her back while filling the back trap and she has been on a sick } leave for some time. They suggested to build a platform in the back } of a microscope so it is more sturdy and safer. Have you ever had } problems of that sort? The lab has been opened for 15 years and } we have never had any accident with LN. Is the platform really safer? } Will it interfere with the performance of the microscope, if it is } metal one? Will performance of the scope be affected if I do not use } LN in the back trap? } Thanks for answers } Dorota }
---------------------------------------- Patton, David Email: David.Patton-at-uwe.ac.uk "University of the West of England"
I would add to Tony's discussion on a wooden platform, that a metal platform is not advisable in such proximity to an instrument that has both high voltage and running water. Should wire insulation crack over time due to spillage of LN2, and you get a water leak while standing on a metal platform, you won't be in a very good position! In fact, one lab in which I worked banned the use of all metal step stools in favor of wooden or plastic ones for such reasons.
On an aside, how large is the trap? Perhaps a smaller dewar for filling that particular one is in order. You could easily use a thermos sized dewar (1L) to fill that trap, especially if you are keeping it full on a regular basis.
HTH,
David Bell Scientist Electron Microscopy Lab Millipore Corporation 80 Ashby Road Bedford, MA 01730 (781) 533-2108
On Tue, 12 Sep 2000 08:53:03 -0500 Cameron Hind {hindc-at-baxter.com} wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } } Hi Listservers, } } I have a memory lapse-can you remind me of the reagent used as an } alternative to CPD for SEM;or do you know of/recommend any safe (not too } toxic) alternative method to CPD?-any reply would be appreciated } } Cameron } } Mr. Cameron Hind } Research Scientist } Advanced Technologies group } Baxter R & D Europe S.C.R.L. } Rue du Progrès 7 } 1400 Nivelles } Belgium } } Tel:+32 67 882 511 } Fax:+32 67 217 191 } e-mail: hindc-at-baxter.com } } } } } } } } } } } } } } } } } } } }
---------------------------------------- Patton, David Email: David.Patton-at-uwe.ac.uk "University of the West of England"
I gather there are not alot of people out there trying to immunolabel microsomes because I only received one response last time (Thank you Jan), but I'll post the latest problem in hopes that someone may have some insight. I am attempting to immunolabel a protein inside sheep liver microsomes. Jan Leunissen suggested I combine the microsome suspension with geletin and coat the inside of an eppendorf with the geletin/microsome film then preembedd label this film.
When I finished, I had very nice geletin interspersed with large areas of indistinct matter. I think these areas are where the microsomes were, before the labelling protocol disrupted them. I processed a small amount of the geletin/microsome from the same tubes before labelling and it looked very normal: geletin interspersed with large area of microsomes. Does anyone have any ideas on how to stabilize the microsomes, or where in the world they are going?
Thank you,
Michelle Peiffer ************************************************************* Electron Microscope Facility for the Life Sciences Penn State University Biotechnology Institute 001 South Frear Lab University Park PA 16802
I guess that you are thinking of Ted Pella's Peldri. This has not been available for some years. You could try to use a solvent drying method, which for some specimens works surprisingly well.
Place a double layer of filter paper in a glass Petrie dish. Add sufficient chloroform to well saturate the filter paper. Move fixed and dehydrated specimen from ethanol onto a microscope slide placed onto the filter paper. Close Petrie dish and store in refrigerator for two days until all solvent has evaporator. Don't open Petrie dish until the dish has warmed to at least to room temperature. Mount and coat specimen etc.
Disclaimer: we do not sell glass Petrie dishes, filter papers, chloroform or refrigerators. We do sell CPD and Freeze driers! Cheers Jim Darley ProSciTech Microscopy PLUS PO Box 111, Thuringowa QLD 4817 Australia Ph +61 7 4774 0370 Fax:+61 7 4789 2313 service-at-proscitech.com Great microscopy catalogue, 500 Links, MSDS, User Notes ABN: 99 724 136 560 www.proscitech.com
On Tuesday, September 12, 2000 11:53 PM, Cameron Hind [SMTP:hindc-at-baxter.com] wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } } Hi Listservers, } } I have a memory lapse-can you remind me of the reagent used as an } alternative to CPD for SEM;or do you know of/recommend any safe (not too } toxic) alternative method to CPD?-any reply would be appreciated } } Cameron } } Mr. Cameron Hind } Research Scientist } Advanced Technologies group } Baxter R & D Europe S.C.R.L. } Rue du Progres 7 } 1400 Nivelles } Belgium } } Tel:+32 67 882 511 } Fax:+32 67 217 191 } e-mail: hindc-at-baxter.com } } } } } } } } } } } } } } } } } } }
At 8:53 AM -0500 9/12/00, Cameron Hind wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Lee
Leona Cohen-Gould, M.S. Sr. Staff Associate Director, Electron Microscopy Core Facility Manager, Optical Microscopy Core Facility Joan & Sanford I. Weill Medical College of Cornell University voice (212)746-6146 fax (212)746-8175
Hi: The chemical you are looking for is called Hexamethyldisilazane (HMDS). You can find it in mist EM catalogues}
Cameron Hind wrote:
} Hi Listservers, } } I have a memory lapse-can you remind me of the reagent used as an } alternative to CPD for SEM;or do you know of/recommend any safe (not too } toxic) alternative method to CPD?-any reply would be appreciated } } Cameron } } Mr. Cameron Hind } Research Scientist } Advanced Technologies group } Baxter R & D Europe S.C.R.L. } Rue du Progrès 7 } 1400 Nivelles } Belgium } } Tel:+32 67 882 511 } Fax:+32 67 217 191 } e-mail: hindc-at-baxter.com
-- Regards, Gregory Rudomen Technical Specialist Electron Microscopy State University of New York at Stony Brook University Microscopy Imaging Center Stony Brook, NY 11794-8088 631-444-7372 Greg-at-umic.sunysb.edu ************************************************* Standard disclaimer: The opinions expressed in this communication are my own and do not necessarily reflect those of the University Microscopy Imaging Center. *************************************************
Karen, You may want to check out the Magnafire "firewire" system from Optronics. (www.optronics.com) Brett
Brett M. Connolly, Ph.D. Merck Research Laboratories Department of Pharmacology WP26A-3000 PO Box 4 West Point, PA 19486 Ph. 215-652-2501 FAX 215-652-2075 e-mail: brett_connolly-at-merck.com
} ---------- } From: Karen Rethoret[SMTP:rethoret-at-yorku.ca] } Sent: Monday, September 11, 2000 2:14 PM } To: Microscopy-at-sparc5.microscopy.com } Subject: Low Light Camera Required } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } } Hello, } } We are looking for camera suggestions that will handle Low Light, Real } time imaging(both for flurorescence and DIC) in the fluorescence } microscope. Something in the $20K range or less for the system. } } High resolution is required also. These images can be extremely dark. The } user prefers also to be able to output the realtime imaging of cell growth } } to videotape as that has proved to be the most convenient in the past but } is also interested in the "firewire" option and it's capabilities. } } We realize that a compromise in some areas (likely the frame rate)will be } required but high resolution during low light imaging is very important. } } We would be particularly interested in hearing from others that have used } a camera that would suit our application. We have commercial info for } several models but would really like some user feedback. } } Thanks, } } Karen Rethoret } York University } Biology Department } Toronto, Ontario } 416-736-2100 x33289 } } } }
Greetings, Several replies have mentioned this mysterious solvent for CPD, and that it works on some type of specimens. Could someone outline how this is used (infiltrate and then evap to air, or what?) and if there is any rhyme or reason about what kinds of specimens that work???
THanks, Tobias
} } } We use HMDS (Hexamethyldisilazane) from Sigma. } } Dave } } } On Tue, 12 Sep 2000 08:53:03 -0500 Cameron Hind } {hindc-at-baxter.com} wrote: } } } ------------------------------------------------------------------------ } } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } -----------------------------------------------------------------------. } } } } } } } } Hi Listservers, } } } } I have a memory lapse-can you remind me of the reagent used as an } } alternative to CPD for SEM;or do you know of/recommend any safe (not too } } toxic) alternative method to CPD?-any reply would be appreciated } } } } Cameron } } } } Mr. Cameron Hind } } Research Scientist } } Advanced Technologies group } } Baxter R & D Europe S.C.R.L. } } Rue du ProgrËs 7 } } 1400 Nivelles } } Belgium } } } } Tel:+32 67 882 511 } } Fax:+32 67 217 191 } } e-mail: hindc-at-baxter.com } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } ---------------------------------------- } Patton, David } Email: David.Patton-at-uwe.ac.uk } "University of the West of England"
Let me inform you, that the Proceedings of the "12th EUROPEAN CONGRESS ON ELECTRON MICROSCOPY" (EUREM12), Brno, Czech Republic, July 9 – 14, are available.
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"Better" is rather tough to define--at least in my application and use. First off, I use about F-113 perhaps six times a year and use about 20cc of it initially. I dehydrate in a sequential immersion in methanol, acetone, F-113. Each immersion is accomplished in a sealed vessel. When the F-113 has too much gunk on the surface, I skim it off, put it in a salvage container and continue using the F-113 until it is too low in volume to be useful. Eventually, I have a pretty full salvage container. This is sent back to the recycle place where they clean it and re-sell the F-113. Very little is lost.
An alternative is CPD. However, considering that F-113 costs about $100 per pound and a CPD setup is perhaps $15K plus a lot of hassle, CPD is not cost effective or time effective. If I did dehydration on a regular basis, I would certainly do CPD.
Recycling and containment makes F-113 a low cost and effective approach to dehydration. And I think the method I use is environmentally friendly. There are of course ways to abuse this material. I don't think that I am doing that.
gary g.
At 10:14 PM 9/11/00, you wrote:
} Hello, } Isn't there a better alternative to Freon 113? We use ethanol for } critical point drying in our lab with fine results. Freon is bad for the } ozone layer. I've copied below, some information from the US } Environmental Protection Agency. } } C. Abiotic Effects } } Freon 113 moves slowly through the lower atmosphere into the } stratosphere. Photodegradation of freon 113 in the upper atmosphere } releases chlorine atoms which react with ozone. Stratospheric } depletion of ozone increases the amount of ultraviolet-B radiation } that reaches the earth's surface (U.S. EPA 1983). Increased, } surface UV radiation can adversely affect human health and the } environment. } } \/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\ } Gordon Ante Vrdoljak Electron Microscope Lab } ICQ 23243541 http://nature.berkeley.edu/~gvrdolja 26 Giannini Hall } gvrdolja-at-nature.berkeley.edu UC Berkeley } phone (510) 642-2085 Berkeley CA 94720-3330 } fax (510) 643-6207 cell (510) 290-6793 } } On Mon, 11 Sep 2000, Gary Gaugler wrote: } } } ------------------------------------------------------------------------ } } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } -----------------------------------------------------------------------. } } } } } } I buy F113 fro Re-Cycle & Re-Use Industries, Mansfield, TX } } } } Try John Metz } } 817.477.5207 } } } } I have about 2 gallons in stock and use maybe a quart or so a year. } } } } gary } } } } } } At 10:55 AM 9/11/00, you wrote: } } } ------------------------------------------------------------------------ } } } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } } } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } } } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } -----------------------------------------------------------------------. } } } } } } } } } Hello form the Univ. of Penn., } } } } } } I apologize in advance for what may well be a very sophomoric } } } question. } } } } } } I haven't paid attention to what may have been discussed } } } regarding alternatives to Freon 113 as a final drying agent because I } } } have scrupulously rationed my supply, ml by ml, for several years. Now } } } I'm into my final 300 ml and must begin to search for a substitute since } } } it has been my understanding that Freon 113 is no longer available. I } } } only use very small volumes at a time and 4x500 ml bottles has lasted me } } } nearly 10 years. Most of my specimens are cell layers or other small } } } (less than 1 cubic mm) tissue samples for SEM. } } } } } } Any suggestions and information about what is currently used as } } } an alternative to critical point drying out of Freon 113 exchanged with } } } liquid CO2 would be greatly appreciated. } } } } } } Thanks, } } } } } } Gerald Harrison } } } Electron Microscope Facility } } } Dental School Research } } } University of Pennsylvania } } } } } }
I also have a Hitachi TEM (7000) and put liquid nitrogen in both the turbo molecular pump and the anticontamination trap. What I know for sure is that my Varian 880 vacuum ionization gauge usually reads about one torr better with the liquid nitrogen then with out it. As a generalization, I have very little astigmatism and would like to think that the anticontamination trap is working, but I really don't know that.
Tim
Timothy G. Schneider Director of Electron Microscopy Department of Pathology Room 229 Jefferson Hall Thomas Jefferson University 1020 Locust St. Philadelphia Pa. 19107 215-503-4798 work 610-613-8170 cellular timothy.schneider-at-mail.tju.edu
} I have a memory lapse-can you remind me of the reagent used as an } alternative to CPD for SEM
It is hexamethyldisilazane (HMDS).
Rob
Robin H Cross Director : EM Unit, Rhodes University, Grahamstown, South Africa R.Cross-at-ru.ac.za tel: +27 46 603 8168 - fax: +27 46 622 4377 http://www.ru.ac.za/emu/index.htm
** remember that ICEM-15 in 2002 is in Durban, South Africa **
Mr. Cameron Hind wrote: ============================================================= } I have a memory lapse-can you remind me of the reagent used as an } alternative to CPD for SEM;or do you know of/recommend any safe (not too } toxic) alternative method to CPD?-any reply would be appreciated ============================================================= The current material that is presently available, to my knowledge the only such material, is HMDS, or Hexamethyldisilazane, otherwise known as 1,1,1,3, 3,3 hexamethyldisilazane [CAS # 999-97-3].
More information about this material, which is offered by SPI Supplies and some of the other leading suppliers of chemicals and consumables to the EM world, can be found on URL http://www.2spi.com/catalog/chem/chem2a2.html
An opinion: Nothing has ever been developed in the way of a technique of sample preparation for SEM that is as good as critical point drying. Some years ago, Ted Pella offered a product called Pel-Dry, it was good, but not quite as good as actually doing it via CPD. HMDS, again, in my opinion, falls a bit short of what Pel-Dry used to do. But for someone without a CPD unit, wanting to prepare wet samples, the HMDS technique is lightyears better than air drying. I should also say that there are many people who use HMDS regularly, saying that for their samples, it is "good enough" either because of the lack of fine structure, or the very low magnifications they are using.
Disclaimer: SPI has offered for many years our own SPI-Chem HMDS and also a CPD unit.
Chuck
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I've been out of touch with EM of membranes and tissues for many many years, but as I recall acetone as a dehydration agent is rather harsh. After your fixation and cross-linking steps it may be that a more gently dehydration would help further preserve your plant tissue. I am definitely not up to speed on these preps and I am sure some others will respond, but I think I would try ethanol or another slightly less polar solvent for the early dehydration steps. I only say this from my (ancient) experience with acetone and its aggressive extraction behavior to plant tissue and photosynthetic membranes. Otherwise, I'd stick to your recipe.
Good Luck Brad Huggins BP Amoco
} ---------- } From: Sally Burns[SMTP:burnssal-at-pilot.msu.edu] } Sent: Thursday, September 07, 2000 9:39 AM } To: Microscopy-at-sparc5.microscopy.com } Subject: turf grass fixation for TEM } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } I am preparing turf grass samples for TEM. We will be examining the } crowns and lead blades for infectious agents. } Any tidbits on fixation and embedding of this tissue would be } appreciated! } } My starting point is this protocol, which has been used for wood } samples. If it is overkill, please let me know. } } Fix: 2% Glutaraldehyde in 50 mM NaCacodylate pH 7.2, with light vacuum } for 24 hours } Wash: 3X for 1 H each RT in buffer with mild agitation } PostFix: 1% OsO4 in water Overnight at 4C } Wash: 3X for 1H each at RT in water with mild agitation } en bloc stain: saturated UA for 30 min with mild agitation } Dehydration series: 25%, 50%, 75%, 100% Acetone, each 30 min with mild } agitation } 100% acetone + 1% DMP for 30 min with mild agitation } -- 100% acetone + 1% DMP overnight with mild agitation } 100% acetone + 1% DMP overnight with mild agitation } Infiltration: } 33% Spurr's in Acetone + 1% DMP for 2 H min with mild agitation } 66% Spurr's in Acetone + 1% DMP for 2 H min with mild agitation } 100 % Spurr's for 2H with mild agitation } 100 % Spurr's Overnight with mild agitation } 100 % Spurr's for 8-12 H with mild agitation } 100 % Spurr's for 8-12 H with mild agitation } Cast in fresh resin } } } Thanks, Sally } } Sally Burns } Center for Advanced Microscopy } B5 Center for Integrated Plant Studies } Michigan State University } East Lansing, MI 48823 } (517) 355-5004 } burnssal-at-msu.edu }
Further to my posting yesterday seeking a PCD for a JEOL 840, which, I am informed, is the same as that for a 6400, I am currently negotiating with a company to make me one.
Does anyone else out there want one?
It would probably ease my negotiations if there were to be more orders than just mine.
Please contact me direct.
rtch
Ritchie Sims Phone : 64 9 3737599 ext 7713 Department of Geology Fax : 64 9 3737435 The University of Auckland email : r.sims-at-auckland.ac.nz Private Bag 92019 Auckland New Zealand
Hello, Maybe you can help? I culture human preadipocytes in 35mm dishes } and then differentiate them to form lipid droplets. I have been staining } them with ORO for the past year, but have recently not been able to get the } ORO to penetrate the cells. I had been fixing the cells with formaldehyde } and calcium chloride (4g calcium chloride + 10ml of 37%formaldehyde into } ~90ml water to fix the cells for 1 hour. Then rinse with Hanks buffered } saline 2x and then stain with ORO for 15min. This has been working for 1 } year, then one day the ORO stain would not penetrate the cells any longer - } the lipid is clear, not red. What could be the problem? The formaldehyde } solution? Have you ever experienced something like this? Thank you for any } suggestions. Also, I have ordered all new fixative, CaCl2, ORO stain powder and isopropanol. The } lipid in my cells are not picking up the stain. Oh why, oh why???? I even } tried the stain on live cells without fixing and they won't stain either. Thanks for any word. } } Just for your information: I use .6g ORO in 100ml 100% isopropanol.
Georgia
Georgia Bachman NIH - Technical IRTA Gene Expression Unit 4212 N. 16th St. Phoenix, AZ 85016 Phone: 602-200-5310 or 5303.
I'm having (or trying to have) a go at doing serial ultrathin sections of Epon-embedded material. I'd like to see a long unbroken ribbon coming off the diamond knife, but after battling for a couple of hours nothing seemed to work - the sections floated off happily on their own little journeys and I wasn't going to chase them around. I tried cutting the pyramid sides with a new Weckprep blade to get them as smooth as possible, making them as parallel as railway tracks, lowering and raising the water level in the trough, and turning off vibrating things in the lab (unfortunately we're on the third floor), but nothing seemed to make any difference. I'm going home now (smiling) but would like to hear of any tips and experiences you've had that got you beautiful ribbons of sections (and any other tips for serial sectioning).
Looking forward to reading my mail tomorrow.
Mark
******************************************** Mark West, Unidad de Microscopia Electronica, (Electron Microscopy Unit) Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico, 04510 Mexico D.F.
I am looking for a knifeblock for a Reichert OMU2 to be able to bring one of these vintage machines back on-line. Should you have this part currently gathering dust or holding open a door in your lab, please contact me.
thanks in advance
Steve
Dr. Steven Barlow EM Facility/Biology Dept. San Diego State University 5500 Campanile Drive San Diego CA 92182-4614 phone: (619) 594-4523 fax: (619) 594-5676 email: sbarlow-at-sunstroke.sdsu.edu http://www.sci.sdsu.edu/emfacility
} Date: Tue, 12 Sep 2000 18:14:08 -0600 (=?ISO-8859-1?Q?Hora_est=E1ndar_de_M=E9xico?=) } From: Mark West {mwest-at-ifcsun1.ifisiol.unam.mx} } To: Microscopy-at-sparc5.microscopy.com } Subject: serial sections } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Hi, } } I'm having (or trying to have) a go at doing serial ultrathin sections of } Epon-embedded material. I'd like to see a long unbroken ribbon coming off } the diamond knife, but after battling for a couple of hours nothing seemed } to work - the sections floated off happily on their own little journeys } and I wasn't going to chase them around. I tried cutting the pyramid sides } with a new Weckprep blade to get them as smooth as possible, making them } as parallel as railway tracks, lowering and raising the water level in the } trough, and turning off vibrating things in the lab (unfortunately we're } on the third floor), but nothing seemed to make any difference. I'm going } home now (smiling) but would like to hear of any tips and experiences } you've had that got you beautiful ribbons of sections (and any other tips } for serial sectioning). } } Looking forward to reading my mail tomorrow. } } Mark } You should trim the top and bottom of your trapezoid **on the microtome with a glass knife.**
This means that the minute scores caused on the side facet by rough trimming run parallel to the top and bottom of your block face, not perpendicular as they do when you trim by hand pushing the razor blade down from the face.
Also, as you said, the top edge and bottom edge should be parallel. But the biggest thing is not to have any scores caused by a razor blade.
If you don't know how to rough-trim with the microtome, contact me, and I'll be happy to describe it.
} } } } } } ******************************************** } Mark West, } Unidad de Microscopia Electronica, } (Electron Microscopy Unit) } Instituto de Fisiologia Celular, } Universidad Nacional Autonoma de Mexico, } 04510 Mexico D.F. } } tel (unidad/lab) *(525) 622 5610* } (casa/home) (525) 619 3020 } Fax (525) 616 2282 } ******************************************** } } }
Sara E. Miller, Ph. D. P. O. Box 3712 Duke University Medical Center Durham, NC 27710 Ph: 919 684-3452 FAX: 919 684-3265
In my previous response I gave the method, though I've used Chloroform. HMDS may have a theoretical advantage, but I expect that the results would be identical. HMDS was introduced for rapid processing, particularly fast and complete dehydration. It chemically interacts with water and forms acetone. When at least the last dehydration is in HMDS, the user can be sure that dehydration is excellent even with doubtful ethanol or acetone.
During the air drying process residual water and solvent molecules exerts huge pressures onto membranes and these cause shrinkage and shriveling of any specimen. The drying mechanism: Using Peldri, drying only occurred at the Peldri/ air interface, but the specimen is held rigid in the solid Peldri material. As the Peldri slowly sublimes, more of the specimen is dried. During solvent drying the specimen is in a solvent saturated atmosphere and drying is very slow - over 48 hours. In this method there is little pressure exerted by molecules trying to pass through membranes and consequently shrinkage is minimized. Its a good alternative method and sometimes works very well. Given a choice I'll try CPD first. Cheers Jim Darley ProSciTech Microscopy PLUS PO Box 111, Thuringowa QLD 4817 Australia Ph +61 7 4774 0370 Fax:+61 7 4789 2313 service-at-proscitech.com Great microscopy catalogue, 500 Links, MSDS, User Notes ABN: 99 724 136 560 www.proscitech.com
On Wednesday, September 13, 2000 12:21 AM, Tobias Baskin [SMTP:BaskinT-at-missouri.edu] wrote: } } } Greetings, } Several replies have mentioned this mysterious solvent for } CPD, and that it works on some type of specimens. Could someone } outline how this is used (infiltrate and then evap to air, or what?) } and if there is any rhyme or reason about what kinds of specimens } that work??? } } THanks, } Tobias } } } } } } } We use HMDS (Hexamethyldisilazane) from Sigma. } } } } Dave } } } } } } On Tue, 12 Sep 2000 08:53:03 -0500 Cameron Hind } } {hindc-at-baxter.com} wrote: } } } } } ------------------------------------------------------------------------ } } } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } } } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } } } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } -----------------------------------------------------------------------. } } } } } } } } } } } } Hi Listservers, } } } } } } I have a memory lapse-can you remind me of the reagent used as an } } } alternative to CPD for SEM;or do you know of/recommend any safe (not too } } } toxic) alternative method to CPD?-any reply would be appreciated } } } } } } Cameron } } } } } } Mr. Cameron Hind } } } Research Scientist } } } Advanced Technologies group } } } Baxter R & D Europe S.C.R.L. } } } Rue du ProgrEs 7 } } } 1400 Nivelles } } } Belgium } } } } } } Tel:+32 67 882 511 } } } Fax:+32 67 217 191 } } } e-mail: hindc-at-baxter.com } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } ---------------------------------------- } } Patton, David } } Email: David.Patton-at-uwe.ac.uk } } "University of the West of England" } } -- } _ ____ __ ____ Tobias I. Baskin } / \ / / \ / \ \ University of Missouri } / | / / \ \ \ Biological } Sciences } /_ / __ /__ \ \ \__ 109 Tucker Hall } / / / \ \ \ } Columbia, MO 65211-7400 USA } / / / \ \ \ voice: } 573-882-0173 } / /____ / \ \__/ \____ fax: 573-882-0123 } }
My name is Sorin Lazar and I am scientist in Bucharest, Romania. I have received like donation a Hitachi S 500 #01-03 scanning electron microscope. Unfortunately I don't have the operating manual. I should be grateful if someone can help me to find an Operating Manual or a copy.
Hi, Try to trim the upper and the lower edges with a glass knife. Trimming with razor blade often results in non-perfectly parallel edges. Turn the block 90 degree (right or left) trim left and right sides with perfect glass knife. Use both sides of the glass knife. I used the angle of the knife towards the block around 60 degree. When you trim your block from both sides just turn it back 90 degree - you will get perfectly parallel edges.
Your problem could be caused also by irregular air flow around microtome or some grease on the blade you have used.
Hope it will help.
Sincerely, -- Alexander Mironov Jr. Division of Tumor Biology, H4 The Netherlands Cancer Institute Plesmanlaan 121 1066 CX Amsterdam
I have read the offerings with interest and would like to make a few comments.
Firstly - when a manufacturer designs an instrument they do not fit cold traps for fun, they believe that to obtain the best performance from their instrument you need the facilities that they fit. I have worked with design teams at two manufacturers and this is the attitude that they both took!
Secondly - I have in the past, in countries where LN2 was not available, used dry ice (Solid CO2) and acetone in LN2 traps. I believe this brings the temperature down to -80deg C and it works quite well much better than no cooling at all.
Thirdly there are imaging techniques that allow us to measure the contamination rate. Place a holey film in the TEM and without LN2 in use and take a high resolution picture of a hole. Leave the beam on the specimen and repeat the picture on the same hole 20 minutes later (that is if you still have a hole). Measure the two hole widths, take the smaller from the larger and divide by 2 (2 contaminated edges) divide by the time between the two pictures and divide by the magnification. You now have your contamination rate in nm/minute. Then try this with the LN2 systems in action and you will see just how well the LN2 systems work.
The same system can be used with a SEM but use a well coated latex sphere in place of the hole. In this case the sphere will grow with time as the contamination builds round the sphere.
Many years ago (1968) Hitachi produced a paper on LN2 systems. They gist was that if you used LN2 systems for a period of time and then stopped, it took an equal period of time before the vacuum level and contamination rate fell back to the original level. Of course you could not count the time if you let the column to air.
A few more things to think about and a way of proving just how clean your microscope is.
Steve Chapman Senior Consultant Protrain For consultancy and training by professional World Wide Tel +44 1280 814774 Fax +44 1280 814007 www.emcourses.com
} I've been out of touch with EM of membranes and tissues for many many years, } but as I recall acetone as a dehydration agent is rather harsh. After your } fixation and cross-linking steps it may be that a more gently dehydration } would help further preserve your plant tissue. I am definitely not up to } speed on these preps and I am sure some others will respond, but I think I } would try ethanol or another slightly less polar solvent for the early } dehydration steps. I only say this from my (ancient) experience with } acetone and its aggressive extraction behavior to plant tissue and } photosynthetic membranes. Otherwise, I'd stick to your recipe. } } Good Luck } Brad Huggins } BP Amoco
Brad, (and all)
My experience with Ethanol is that it makes the cell walls extremely brittle and hard to section, esp in concetrations above 70% ETOH.
Time is always the problem with Plant tissue. The cell walls really like to hold on to what ever it was in last. So dehydration must proceed at a snails pace. Grass leaf blades by being very thin make the process easier but I woud imagine that the crowns would be problematic if carried too fast through dehydration.
Sincerely, Geoff Williams
Electron Microscope Facility Supervisor Biology Department Brooks Hall Central Michigan University Mt. Pleasant, MI 48859
To glue serial sections together, make a gentle adhesive by dissolving the adhesive off of a piece of Scotch tape in chloroform. Paint the upper and lower block faces with your new adhesive, and, as you cut, the sections should stick together tenaciously.
Good luck!
Dotty Sorenson Microscopy and Image Analysis Laboratory Department of Cell and Developmental Biology University if Michigan Medical School Ann Arbor, Michigan dsoren-at-umich.edu
On Tue, 12 Sep 2000, Mark West wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Hi, } } I'm having (or trying to have) a go at doing serial ultrathin sections of } Epon-embedded material. I'd like to see a long unbroken ribbon coming off } the diamond knife, but after battling for a couple of hours nothing seemed } to work - the sections floated off happily on their own little journeys } and I wasn't going to chase them around. I tried cutting the pyramid sides } with a new Weckprep blade to get them as smooth as possible, making them } as parallel as railway tracks, lowering and raising the water level in the } trough, and turning off vibrating things in the lab (unfortunately we're } on the third floor), but nothing seemed to make any difference. I'm going } home now (smiling) but would like to hear of any tips and experiences } you've had that got you beautiful ribbons of sections (and any other tips } for serial sectioning). } } Looking forward to reading my mail tomorrow. } } Mark } } } } } } } ******************************************** } Mark West, } Unidad de Microscopia Electronica, } (Electron Microscopy Unit) } Instituto de Fisiologia Celular, } Universidad Nacional Autonoma de Mexico, } 04510 Mexico D.F. } } tel (unidad/lab) *(525) 622 5610* } (casa/home) (525) 619 3020 } Fax (525) 616 2282 } ******************************************** } }
For animal tissue: 10min. in 20%,30%,50%,70%,90% and 100% ethanol (or whatever times etc you use for CPD). 2x10min. in 100% ethanl 10min. in 1:1 ethanol HMDS (or 1:2, 1:1, and 2:1) 3x10min in HMDS I put tissue on filter paper in a petri dish with lid ajar and let it evaporate in fume cupboard.
Most papers say CPD is better eg for fine hairs on leaves. I believe the first report was on very satifactory use on insects. We use it mostly on bacteria which are pretty tough anyway. I have found it disappointing on cultured cells and am planning to dust down the CPD (cryoSEM and ESEM having relegated it to an historical artifact (sic!) for most purposes in this lab).
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Greetings, } Several replies have mentioned this mysterious solvent for } CPD, and that it works on some type of specimens. Could someone } outline how this is used (infiltrate and then evap to air, or what?) } and if there is any rhyme or reason about what kinds of specimens } that work??? } } THanks, } Tobias } } } } } } } We use HMDS (Hexamethyldisilazane) from Sigma. } } } } Dave } } } } } } On Tue, 12 Sep 2000 08:53:03 -0500 Cameron Hind } } {hindc-at-baxter.com} wrote: } } } } } ------------------------------------------------------------------------ } } } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } } } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } } } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } -----------------------------------------------------------------------. } } } } } } } } } } } } Hi Listservers, } } } } } } I have a memory lapse-can you remind me of the reagent used as an } } } alternative to CPD for SEM;or do you know of/recommend any safe (not too } } } toxic) alternative method to CPD?-any reply would be appreciated } } } } } } Cameron } } } } } } Mr. Cameron Hind } } } Research Scientist } } } Advanced Technologies group } } } Baxter R & D Europe S.C.R.L. } } } Rue du ProgrËs 7 } } } 1400 Nivelles } } } Belgium } } } } } } Tel:+32 67 882 511 } } } Fax:+32 67 217 191 } } } e-mail: hindc-at-baxter.com } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } ---------------------------------------- } } Patton, David } } Email: David.Patton-at-uwe.ac.uk } } "University of the West of England" } } -- } _ ____ __ ____ Tobias I. Baskin } / \ / / \ / \ \ University of Missouri } / | / / \ \ \ Biological Sciences } /_ / __ /__ \ \ \__ 109 Tucker Hall } / / / \ \ \ } Columbia, MO 65211-7400 USA } / / / \ \ \ voice: } 573-882-0173 } / /____ / \ \__/ \____ fax: 573-882-0123 }
---------------------------------------- Patton, David Email: David.Patton-at-uwe.ac.uk "University of the West of England"
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. Hello, You should trim the top and bottom sides of you face with a glass knife and try coating those sides with grid glue (soak double stick tape in chloroform, then remove the tape, coat with a filter paper wedge careful not to wet your face) Good Luck
Mike D.
} } } Hi, } } I'm having (or trying to have) a go at doing serial ultrathin sections of } Epon-embedded material. I'd like to see a long unbroken ribbon coming off } the diamond knife, but after battling for a couple of hours nothing seemed } to work - the sections floated off happily on their own little journeys } and I wasn't going to chase them around. I tried cutting the pyramid sides } with a new Weckprep blade to get them as smooth as possible, making them } as parallel as railway tracks, lowering and raising the water level in the } trough, and turning off vibrating things in the lab (unfortunately we're } on the third floor), but nothing seemed to make any difference. I'm going } home now (smiling) but would like to hear of any tips and experiences } you've had that got you beautiful ribbons of sections (and any other tips } for serial sectioning). } } Looking forward to reading my mail tomorrow. } } Mark } } } } } } } ******************************************** } Mark West, } Unidad de Microscopia Electronica, } (Electron Microscopy Unit) } Instituto de Fisiologia Celular, } Universidad Nacional Autonoma de Mexico, } 04510 Mexico D.F. } } tel (unidad/lab) *(525) 622 5610* } (casa/home) (525) 619 3020 } Fax (525) 616 2282 } ******************************************** } }
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. Hello, Fomvar thickness will effect stability, if you could make them slightly thicker than gray you should have no problem. If you need them really thin other than carbom coating you could try minimizing illumination/ intensity on the EM side, smaller spot size, smaller condenser aperture or lower beam current. good luck.
Mike D } } } Dear Microlisters, } } I'm having a bit of trouble with Formvar film stability under the TEM beam at 60 } kV. The films, attached to copper or nickel grids (cleaned with dilute HCl + } acetone in sonicator same day films picked up) develop holes which grow over a } few minutes time until eventually the entire film more or less disintegrates, } collapses. I also noticed that initially, the films are often pulled away from } the rim of the grid by about one square "hole" away. } } I'm using new Formvar 15/95 resin powder and new bottle of ethylene dichloride } solvent, mixing to 0.3% weight to volume. I use "Rite-On" glass slides, rinsed } with ethanol to clean of dust and particulates and air dried, to cast the films. } The films float off easily, and look very clean under the TEM. They were dried } overnight prior to inspection. I take pains to use very clean glassware for } mixing the Formvar solution. } } Of course, I could caarbon coat them to provide stability, but that's extra work } and I don't seem to have had this kind of problem before. } } I barely recall some discussion here some time ago about how Formvar has } "changed" due to different process of manufacturing, or whatever. } } Any clues? } } Thanks in advance. } } Gib } } Gib Ahlstrand } Electron Optical Facility, University of Minnesota, Dept. Plant Pathology } 495 Borlaug Hall, St. Paul, MN. USA. 55108 (612)625-8249 } 612-625-9728 FAX, giba-at-puccini.cdl.umn.edu } http://biosci.umn.edu/MIC/consortium.html } }
Rough trim the excess plastic from the specimen face with a razor blade, keeping all the tissue edges. Put the specimen chuck into the microtome arm, and put in a dry glass knife. Shave off enough plastic from the front (face) in very small increments (start with 1 um slices, decrease to 0.5 um, then 0.1 um until you get a mirror smooth face). If you must keep all the sample, then start with a very thin shaving just to get the face smooth. You should be able to tell when you are into the tissue by looking at the thin shavings. Mark the top edgeQthe one that will become the top of your trapezoid--with a magic marker on the chuck. Be sure to keep track of the plane of your sample because the clearance angle of the knife will necessitate that you return the specimen to the original position in which you trimmed the face. Rotate the chuck/specimen 90 degrees (e.g., clockwise). The edges that will become the bottom and top of your trapezoid are now vertical instead of parallel to the earth. Turn the knife holder about 30 degrees away from center (e.g., right) and approach the block carefully. Trim the left side of the block which will become the bottom of your trapezoid; this will make a smooth facet that will start with the face and go toward the microtome slanting outward at about 30 degrees. Leave the specimen in this position and turn the knife to the other side (e.g., left). Trim this side like the first; it will become the facet over the top of your trapezoid. The top and bottom should now be parallel. If they are not, you can make minute adjustments by slightly rotating the block. The purpose of this exercise it to make the scratches caused by rough trimming be parallel, not perpendicular as happens when trimming with a razor blade, to the top and bottom edges of the face.
If the face is too wide, you can trim it, either on the microtome or with a razor blade. I usually just chop the sides with a razor blade to save time; I make a trapezoid with the top edge slightly shorter than the bottom edge. However, folks with unsteady hands can make the same trapezoid by turning the block back to its original position with the magic marker at the top, then slightly rotating it right or left about 2 or 3 degrees. Trim this side with the knife still at 30 degrees away from center. Then rotate the block 2 or 3 degrees in the other direction and turn the knife 30 degrees to the opposite side. Trim the other side.
Before sectioning, turn the block back 2-3 degrees to be in the original position it was when you faced it with the mark at the top. Use a fresh knife, or even better if you have one, use a diamond. When aligning, be sure to have both the bottom edge of your block parallel to the edge of your knife as well as the plane of your block face parallel to the plane of the knife edge. To do the former, you may have to rotate your specimen very slightly. To do the latter, you may have to change the angle of your specimen in the microtome arm. These tiny adjustments may be necessary because the diamond may not be mounted in its holder exactly like the glass one you used for trimming.
Pick up the sections on slotted grids with a support film.
Good luck. Let me know if this doesn't make sense.
Sara E. Miller, Ph. D. P. O. Box 3712 Duke University Medical Center Durham, NC 27710 Ph: 919 684-3452 FAX: 919 684-3265
---------- Forwarded message ----------
Summary:
I am looking for a test image with control points with known positions in the warped and unwarped state and the known coefficients for a polynomial warp function to test a program I am writing. Can someone on the list help?
Details:
I am trying to write a program to correct center of gravity data for barrel distortion in the image. I am trying to implement the approach described by W. K. Pratt, Digital Image Processing, 2nd edition, pp.430-433 (1991). I imaged a rectangular lattice of squares [50 micron squares in a 100 micron square lattice] with a 5X objective. I fit the measured center of gravity coordinates to a square lattice using the points nearest the center. The difference between observed and computed values for each point shows the barrel distortion in the lens. I used the calculated value for each point as coordinate for the (unwarped) desired control point (x,y) and the observed coordinates as the coordinate for the warped control points (u,v). I'm trying to fit to the following orthogonal polynomials:
1 u1 v1 u1**2 u1v1 v1**2 1 u2 v2 u2**2 u2v2 v2**2 A = 1 u3 v3 u3**2 u3v2 v3**2 ..... 1 um vm um**2 umv2 vm**2
and the vectors u and v
u(transpose) = [u1 u2 u3 ... um] v(transpose) = [v1 u2 v3 ... vm]
I used Mathematica to compute the generalized inverse (Pseudoinverse[]) and generate the vector of coefficients, a and b by
a = PseudoInverse[A] u b = PseudoInverse[A] v
The non-unitary coefficients of a and b were much smaller than I expected and when I tried to use them to correct the center of gravity data, they did not remove the barrel distortion. I'd like a test image with known control points that produce known polynomial coefficients to test the code. Hoping that someone on the list can help....
Best Regards,
John Minter Eastman Kodak Company Analytical Technology Division Rochester, NY 14650-2152 Phone: (716) 722-3407 FAX: (716) 477-7781 email: john.minter-at-kodak.com
Hi all, Thank you all for your input regarding the typical salaries for Microscopists, it was very useful. I would now like to request that anyone who might be interested in an SEM operators position with a start-up company in the Atlanta, GA area of the USA please send me a copy of your resume and previous salary history. Although the type of position has not yet been finalized it will probably be at the entry level. Thank you to those of you who have already sent me their information, I have it on file for consideration.
Quesant Instrument Corp is looking for a customer support person whose primary duties will be: 1. Utilizing Quesant's QScopes (scanning probe microscopes) to scan samples and report back to sales prospects and customers. 2. Installation and taining of new customers. 3. Attending trade shows and demonstrating QScopes in the booth. 4. Work with marketing and R&D in testing and implementing new features.
Requirements: Advanced degree in Chemistry, Physics or Materials Science a plus but not mandatory. Must have good interpersonal skills and a profession al appearance. Must be willing to travel.
Contact Quesant for more information at 29397 Agoura Road, Suite 104 Agoura Hills, CA 91301 tel: 818-597-0311 fax: 818-991-5490 e-mail: qsales-at-quesant.com
We are closing down our EM facility. We have a Reichert Ultracut ultramicrotome available. Please make an offer, and be prepared to assume the cost of shipping to your facility.
Thanks, David Kerk
------------- David Kerk, Ph.D. Professor and Chair Department of Biology Point Loma Nazarene University 3900 Lomaland Dr San Diego, CA 92106 Ph: 619-849-2398 FAX: 619-849-2598 email: dkerk-at-ptloma.edu
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Steve, Thanks for this post. I try to get this through to our users and we faithfully use and require use of N2 on all scopes. However, there are other facilities on campus who do not use N2 or use it intermittantly. When this topic comes up again, I intend to hand them your post and ask them to determine contamination rates on their instruments to convince them of the necessity of using N2. One question that I have is that our main nitrogen trap on the SEM is above the diffusion pump to minimize backstreaming from the pump into the specimen chamber. (We also have a decontaminator close to the specimen that is cooled when we do cryo-SEM.) Now this is no doubt more of a problem with SEM than TEM due to the location of the diffusion pump. However, I wonder why most TEM manufacturers do not also use a nitrogen trap in this location, at least in the higher end scopes, in addition to the decontaminator in the specimen area. It would seem that, since contamination rate in the TEM is likely to be more critical to high resolution, it would also be used to help maintain the best possible vacuum in the column. I am aware that the majority of the contamination is produced in the TEM by interaction of the beam with the specimen thus the need for decontaminator close to the specimen. However, my understanding is that the better the vacuum, the fewer the interactions of the beam electrons with molecules in the column and the more coherent the beam....i.e. better potential resolution. Debby
Debby Sherman Phone: 765-494-6666 Microscopy Center in Agriculture FAX: 765-494-5896 Dept. of Botany & Plant Pathology E-mail: sherman-at-btny.purdue.edu Purdue University 1057 Whistler Building West Lafayette, IN 47907-1057
On Wednesday, September 13, 2000, Steve Chapman {PROTRAIN-at-CompuServe.COM} wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
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An elegant solution indeed, Dorothy (and others suggesting similar). Just keep in mind the type of TEM for which the sections are headed, however. For high-end TEM's in the physical sciences, where FEG sources, nanoprobes and EELS analysis are quite common, the TEM specialists running the instrument can be/should be very reluctant to allow specimens which might outgas even a bit and gunk up the specimen chamber area. Your glue 'rim' around the sections would do just that, I suspect.
Should anyone be doubtful of such effects, some years ago, Don Steele (Alcan Aluminum in Kingston, Canada) did some neat serial sectioning of an Al-SiC composite. (Really! Check the MSA '91 proceedings). I seem to recall that Don had to collect the sections on formvar-coated girds (maybe even a homemade formula?). In any event, when we tried to collect X-ray maps, the film breakdown and redeposition piled up the contamination so fast as to render mapping the Al and Si distribution very difficult. Any focused beam analysis resulted in 'cones' on the section surface, rendering good EDX analysis impossible. (People used to worry about column-borne contamination in the early days of AEM. With the recent generations of instruments, specimen-borne contamination is the issue). At least our case was of a very localized phenomenon, the glue rim would result in much more widespread 'fallout'.
Finally, I always remind workshop attendees contemplating 'materials science' ultramicrotomy, to be careful regarding C-analysis on embedded materials, especially particulate. The e-beam (at least at the 120 keV of our older scope) appears to instantly result in roughly a 20-30% mass loss from any embedding resin, even at low beam intensities. This breakdown produces a thin (~10 nm) film of C on the embedded bits which does not interfere with imaging, but can be clearly seen in an EEL spectrum.
Tom Malis Group Leader - Characterization Materials Technology Laboratory Natural Resources Canada (Govt. of Canada) 568 Booth St., Ottawa, Canada ph. 613-992-2310 FAX 613-992-8735 email: malis-at-nrcan.gc.ca
---------- From: Dorothy Roak Sorenson [SMTP:dsoren-at-umich.edu] Sent: Wednesday, September 13, 2000 8:52 AM To: Mark West Cc: Microscopy-at-sparc5.microscopy.com Subject: Re: serial sections
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To glue serial sections together, make a gentle adhesive by dissolving the adhesive off of a piece of Scotch tape in chloroform. Paint the upper and lower block faces with your new adhesive, and, as you cut, the sections should stick together tenaciously.
Good luck!
Dotty Sorenson Microscopy and Image Analysis Laboratory Department of Cell and Developmental Biology University if Michigan Medical School Ann Arbor, Michigan dsoren-at-umich.edu
On Tue, 12 Sep 2000, Mark West wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Hi, } } I'm having (or trying to have) a go at doing serial ultrathin sections of } Epon-embedded material. I'd like to see a long unbroken ribbon coming off } the diamond knife, but after battling for a couple of hours nothing seemed } to work - the sections floated off happily on their own little journeys } and I wasn't going to chase them around. I tried cutting the pyramid sides } with a new Weckprep blade to get them as smooth as possible, making them } as parallel as railway tracks, lowering and raising the water level in the } trough, and turning off vibrating things in the lab (unfortunately we're } on the third floor), but nothing seemed to make any difference. I'm going } home now (smiling) but would like to hear of any tips and experiences } you've had that got you beautiful ribbons of sections (and any other tips } for serial sectioning). } } Looking forward to reading my mail tomorrow. } } Mark } } } } } } } ******************************************** } Mark West, } Unidad de Microscopia Electronica, } (Electron Microscopy Unit) } Instituto de Fisiologia Celular, } Universidad Nacional Autonoma de Mexico, } 04510 Mexico D.F. } } tel (unidad/lab) *(525) 622 5610* } (casa/home) (525) 619 3020 } Fax (525) 616 2282 } ******************************************** } }
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I just wanted to say I am so sorry to hear that. Her e-mails were always excellent quides for me. She doesn't know me. However, I know her very well. During the M&M meeting in Portland last year, I saw her for a minute. I was willing to meet her. I just wanted to thank her and let her know that she was also a wonderful teacher for me and for my friends, young electron microscopists in Turkey. However, I could not get a chance to do that. I was so surprised when I heard that she had retired a few months ago. Now, I learn the reason.
Thank you very much all the way from Turkey, Hildy. I will really miss you.
Gulhan
Ranan Gulhan Aktas, M.D. Trakya University Faculty of Medicine Pathology Department Edirne 22030 TURKEY
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To the List:
I ran across this in an obscure book on electron microscopy technique.
"To obtain quality serial sections for electron microscopy the following procedure must be followed for consistent results. 1) Obtain one male goat. 2) Wait for new moon after a rain storm. 3) Obtain black robes which have been treated with a Z-stat gun. 4) Sacrifice goat in the light of the new moon. 5) Collect blood. 6) Spin down blood in centrifuge and collect goat plasma and store at 4 C 7) Take piece of plastic from sample block to be cut. Pulverize into dust. 8) Mix plastic dust with goat plasma. 9) Inject plastic/goat into rabbit. 10) Wait one pay period. 11) Wait for full moon. 12) Place black robes on which have been retreated with a Z-stat gun. 13) Sacrifice rabbit which has been injected with plastic/goat antigen under the full moon. 14) Collect antiplastic/goat antibody from rabbit. 15) Place one drop of antiplastic/goat antibody in the trough of diamond knife. 16) Cut serial sections. 17) The antiplastic antisera should cause all the plastic sections to arrange themselves in the proper order. 17) This should all make anti-sense."
Bob Blystone
-------------------------------- Robert V. Blystone, Ph.D. rblyston-at-trinity.edu
Department of Biology Trinity University 715 Stadium Drive San Antonio, Texas 78212 210.999-7243 FAX 210/999-7229
Wonderful! We have a similar protocol for FISHing particularly recalcitrant mRNAs, but we use a chicken instead of a goat (not much space here so we had to scale back to a smaller organism), and the blood goes in the hybridization buffer.......
Well, thanks for the flood of suggestions about getting a ribbon. I trimmed the block sides with a glass knife (and enjoyed Mike Delannoy's suggestion of 'trimming the top and bottom sides of your face with a glass knife' - didn't know microscopy was such a painful job) and quite a decent ribbon was the result. I tried the chloroform-glue idea and that seemed to make the sections stick together more, but sometimes they gummed up a bit on the knife. The next battle is picking up the ribbon on several slot grids - I suspect that it's more practice than anything else. The method I was shown was to go underneath the ribbon at an angle with the grid, raise it until the waterline is on the top of the exposed formvar of the slot, push the ribbon onto the waterline and draw the grid up at the same time, and touch the ribbon with the hair at the point where the slot finishes to break the ribbon, and remove the slot - all easier said than done! Any ideas would be much appreciated.
Thanks,
Mark
******************************************** Mark West, Unidad de Microscopia Electronica, (Electron Microscopy Unit) Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico, 04510 Mexico D.F.
A user of our facility needs to prepare specimens to determine if sub-micrometer pores are present in plant intervascular pit membranes. The user is very concerned that critical point drying or, especially, the dehydration steps prior to CPD, may introduce 'pores' that are really artifacts. He is similarly concerned that freezing or freeze-drying his specimens might also introduce 'pores' due to ice crystal formation. Work he has already done suggests that these are valid concerns.
Has anyone sufficient experience preparing pit membranes for SEM to suggest a procedure we might follow to minimize the possibility of creating small 'pores' where none may exist?
I am interested in knowing some details the Flourescence Microscopes, their principles and applications. Please e-mail me whatever details you have available on the subject or the internet address(s) of site(s) where I can obtain such information.
Thanks, Abdulrahman
__________________________________________________ Do You Yahoo!? Yahoo! Mail - Free email you can access from anywhere! http://mail.yahoo.com/
Dear Colleagues: I have made great contacts through this listserver in the past and hope that I can again this time. I am in search of a postdoctoral fellow or very independent technician to continue a program of research using modern optical imaging methods to follow membrane trafficking in vascular endothelial cells. It is a very exciting project that cuts across the fields of cell biology, immunology, and pathology, and uses techniques from standard transmission EM to tracking GFP-labeled proteins. If you have the right background and are interested, or know of a colleague who fits these criteria, please get in touch with me. The following is the text of an ad that appeared in the August 10 issue of Nature and the September issue of Nature Cell Biology:
Postdoctoral position in Membrane Trafficking Postdoctoral position open to study membrane trafficking in endothelial cells. The ideal candidate would have experience studying regulated membrane trafficking at the cellular and molecular levels using biochemical, microscopic, and molecular biology approaches. Experience with modern imaging systems is critical. Experience with endothelial cells is a definite "plus", but not necessary. The candidate should demonstrate excellent communication skills in English, be able to work independently, as well as to interact with a lively group of productive investigators studying leukocyte-endothelial cell interactions. We are based at the Weill Medical College of Cornell University in the nicest and safest area of New York City. We interact extensively with investigators Weill as well as at The Rockefeller University and Memorial Sloan-Kettering Cancer Center, which are both just across the street. The position is fully-funded and available immediately.
Submit CV and names (and contact numbers) of three references to:
William A. Muller, MD, PhD Department of Pathology and Program in Immunology Box 69 Weill Medical College of Cornell University 1300 York Avenue New York, NY 10021 e-mail: wamuller-at-med.cornell.edu
Thank you very much for your help.
Sincerely, Bill Muller
William A. Muller, MD, PhD Associate Professor of Pathology Weill Medical College of Cornell University 1300 York Avenue New York, NY 10021
We are attempting to put a ccd camera (high resolution, large area, sensitivity = SO163) to replace films on 1200EX JEOL at a cost below $100k. Would anyone be willing to share comments on recently acquired ccd camera for TEM? Manufacturers welcome. Used ccds considered.
Marek Malecki, M.D., Ph.D. Director and Principal Investigator
Molecular Imaging Laboratories University of California at San Diego address: 1500 Suite Bonner Hall, 9500 Gilman Drive, La Jolla, CA 92093-0368 phone - office: 8588223373 phone - lab: 8585342484 fax: 8588223715 pager: 8586161420 e.mail: mmm-at-ucsd.edu e.pager: 1620024619-at-alphapage.airtouch.com www site: http://mil.ucsd.edu/ ftp site: mil1.ucsd.edu
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At 11:42 AM 9/13/00 -0500, David Kerk wrote: } We are closing down our EM facility. We have a Reichert Ultracut } ultramicrotome available. Please make an offer, and be prepared to assume } the cost of shipping to your facility.
I've got a AO / Reichert Ultracut microtome, too. It's a model 701701. It's missing the 651102 control panel, though, as well as the knife holder and object holder. It looks just like the one at: http://www.labx.com/v2/adsearch/Detail3.CFM?adnumb=54745
If I were to sell what I have, what price might it fetch?
- John
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We currently have a Bench Top Turbo carbon evaporation unit made by Denton. We are very displeased with it. We consistently get non uniform coatings that peel off of our metallographic mounts. Any suggestions on a evaporation unit that works well or any information on 'sputtering" carbon? Any other experiences out there?
Dear Mark, With coated slot grids it's easier to come down on top of your ribbon of sections. Break up your ribbon into strips which will fit over your slot first. Then hold your coated slot grid over the sections (you can see the ribbon of sections through your coating and then slowly descend onto your stip of sections. The sections adhere immediately with the first touch. This method avoids any surface tension problems between the grid and water surface which can send the grids flying away from your grid.
Good Luck. Marilyn
Mark West wrote: } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Hi, } } Well, thanks for the flood of suggestions about getting a ribbon. I } trimmed the block sides with a glass knife (and enjoyed Mike Delannoy's } suggestion of 'trimming the top and bottom sides of your face with a glass } knife' - didn't know microscopy was such a painful job) and quite a decent } ribbon was the result. I tried the chloroform-glue idea and that seemed to } make the sections stick together more, but sometimes they gummed up a bit } on the knife. The next battle is picking up the ribbon on several slot } grids - I suspect that it's more practice than anything else. The method I } was shown was to go underneath the ribbon at an angle with the grid, raise } it until the waterline is on the top of the exposed formvar of the slot, } push the ribbon onto the waterline and draw the grid up at the same time, } and touch the ribbon with the hair at the point where the slot finishes to } break the ribbon, and remove the slot - all easier said than done! Any } ideas would be much appreciated. } } Thanks, } } Mark } } ******************************************** } Mark West, } Unidad de Microscopia Electronica, } (Electron Microscopy Unit) } Instituto de Fisiologia Celular, } Universidad Nacional Autonoma de Mexico, } 04510 Mexico D.F. } } tel (unidad/lab) *(525) 622 5610* } (casa/home) (525) 619 3020 } Fax (525) 616 2282 } ********************************************
} The next battle is picking up the ribbon on several slot } grids - I suspect that it's more practice than anything else.
Mark,
I got rather consistent results with Perfect Loop from EMS. They even describe how to pick up sections (http://www.emsdiasum.com/ems/grids/accessories.html). Nevertheless I used it in slightly different way. After step 3 I put the loop with sections in the holder (stand) in a way that I can see it looking in microtome oculars. Then I put the slot grid coated with formvar just above the loop in desired orientation and slowly lower it down until section attach to the formvar film. Then I moved the grid with attached sections to side (not up!) so that practically all water remains in the loop. Result: I got serial sections on the grid with desired orientation and without necessity to remove water (a lot of dust can go from filter papaer to the grid when you try to dry it).
I have no any interest in EMS.
Sincerely, -- Alexander Mironov Jr. Division of Tumor Biology, H4 The Netherlands Cancer Institute Plesmanlaan 121 1066 CX Amsterdam
Most modern TEMs are ion pumped (or turbo pumped) to prevent the diffision pump oil backstreaming problem. The diffusion pump is used when initially evacuating the column or gun, pumping the camera and viewing chamber and when warming up the specimen cold trap at the end of sessions. Whereas it would be good to have a N2 trap on the diffusion pump when it does pump the column the water vapour from the camera and viewing chamber (due to the films) would stall the backing pump when the N2 trap warmed up. Prior to ion pumped columns we did use a N2 trap on the TEM column diffusion pump (circa 1980). Ion pumped or turbo molecular pumped SEMs are also available.
Regards, Ron
On 13 Sep 2000 13:00:11 -0500 Debby Sherman {sherman-at-btny.purdue.edu} wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Steve, } Thanks for this post. I try to get this through to our users and we faithfully use and require use of N2 on all scopes. However, there are other facilities on campus who do not use N2 or use it intermittantly. When this topic comes up again, I intend to hand them your post and ask them to determine contamination rates on their instruments to convince them of the necessity of using N2. } One question that I have is that our main nitrogen trap on the SEM is above the diffusion pump to minimize backstreaming from the pump into the specimen chamber. (We also have a decontaminator close to the specimen that is cooled when we do cryo-SEM.) Now this is no doubt more of a problem with SEM than TEM due to the location of the diffusion pump. However, I wonder why most TEM manufacturers do not also use a nitrogen trap in this location, at least in the higher end scopes, in addition to the decontaminator in the specimen area. It would seem that, since contamination rate in the TEM is likely to be more critical to high resolution, it would also be used to help maintain the best possible vacuum in the column. } I am aware that the majority of the contamination is produced in the TEM by interaction of the beam with the specimen thus the need for decontaminator close to the specimen. However, my understanding is that the better the vacuum, the fewer the interactions of the beam electrons with molecules in the column and the more coherent the beam....i.e. better potential resolution. } Debby } } Debby Sherman Phone: 765-494-6666 } Microscopy Center in Agriculture FAX: 765-494-5896 } Dept. of Botany & Plant Pathology E-mail: sherman-at-btny.purdue.edu } Purdue University
} 1057 Whistler Building } West Lafayette, IN 47907-1057 } } On Wednesday, September 13, 2000, Steve Chapman {PROTRAIN-at-CompuServe.COM} wrote: } } ------------------------------------------------------------------------ } } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } -----------------------------------------------------------------------. } } } } } } Hi } } } } I have read the offerings with interest and would like to make a few } } comments. } } } } Firstly - when a manufacturer designs an instrument they do not fit cold } } traps for fun, they believe that to obtain the best performance from their } } instrument you need the facilities that they fit. I have worked with } } design teams at two manufacturers and this is the attitude that they both } } took! } } } } Secondly - I have in the past, in countries where LN2 was not available, } } used dry ice (Solid CO2) and acetone in LN2 traps. I believe this brings } } the temperature down to -80deg C and it works quite well much better than } } no cooling at all. } } } } Thirdly there are imaging techniques that allow us to measure the } } contamination rate. Place a holey film in the TEM and without LN2 in use } } and take a high resolution picture of a hole. Leave the beam on the } } specimen and repeat the picture on the same hole 20 minutes later (that is } } if you still have a hole). Measure the two hole widths, take the smaller } } from the larger and divide by 2 (2 contaminated edges) divide by the time } } between the two pictures and divide by the magnification. You now have } } your contamination rate in nm/minute. Then try this with the LN2 systems } } in action and you will see just how well the LN2 systems work. } } } } The same system can be used with a SEM but use a well coated latex sphere } } in place of the hole. In this case the sphere will grow with time as the } } contamination builds round the sphere. } } } } Many years ago (1968) Hitachi produced a paper on LN2 systems. They gist } } was that if you used LN2 systems for a period of time and then stopped, it } } took an equal period of time before the vacuum level and contamination rate } } fell back to the original level. Of course you could not count the time if } } you let the column to air. } } } } A few more things to think about and a way of proving just how clean your } } microscope is. } } } } Steve Chapman } } Senior Consultant Protrain } } For consultancy and training by professional World Wide } } Tel +44 1280 814774 Fax +44 1280 814007 } } www.emcourses.com } } } } } }
---------------------- Mr. R.C. Doole Department of Materials, University of Oxford. Parks Road, Oxford. OX1 3PH. UK. Phone +44 (0) 1865 273701 Fax +44 (0) 1865 283333 ron.doole-at-materials.ox.ac.uk
*Date sent: Wed, 13 Sep 2000 04:34:23 -0400 *From: Steve Chapman {PROTRAIN-at-CompuServe.COM} *Subject: LN2 and Contamination *To: American Soc {Microscopy-at-sparc5.microscopy.com}
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I understand the procedure regarding the TEM, however could you explain to what I should compare the contamination rate in the SEM.
Best regards,
Witold
* *Hi * *I have read the offerings with interest and would like to make a few *comments. * *Firstly - when a manufacturer designs an instrument they do not fit cold *traps for fun, they believe that to obtain the best performance from their *instrument you need the facilities that they fit. I have worked with *design teams at two manufacturers and this is the attitude that they both *took! * *Secondly - I have in the past, in countries where LN2 was not available, *used dry ice (Solid CO2) and acetone in LN2 traps. I believe this brings *the temperature down to -80deg C and it works quite well much better than *no cooling at all. * *Thirdly there are imaging techniques that allow us to measure the *contamination rate. Place a holey film in the TEM and without LN2 in use *and take a high resolution picture of a hole. Leave the beam on the *specimen and repeat the picture on the same hole 20 minutes later (that is *if you still have a hole). Measure the two hole widths, take the smaller *from the larger and divide by 2 (2 contaminated edges) divide by the time *between the two pictures and divide by the magnification. You now have *your contamination rate in nm/minute. Then try this with the LN2 systems *in action and you will see just how well the LN2 systems work. * *The same system can be used with a SEM but use a well coated latex sphere *in place of the hole. In this case the sphere will grow with time as the *contamination builds round the sphere. * *Many years ago (1968) Hitachi produced a paper on LN2 systems. They gist *was that if you used LN2 systems for a period of time and then stopped, it *took an equal period of time before the vacuum level and contamination rate *fell back to the original level. Of course you could not count the time if *you let the column to air. * *A few more things to think about and a way of proving just how clean your *microscope is. * *Steve Chapman *Senior Consultant Protrain *For consultancy and training by professional World Wide *Tel +44 1280 814774 Fax +44 1280 814007 *www.emcourses.com * * %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% Witold Zielinski, Ph.D. Warsaw University of Technology Department of Materials Science and Engineering 02-507 Warszawa, Woloska 141 POLAND
This may help? I have used a (no doubt frowned on) practice for many years for easy collection of sections:
Make up 0.2% sodium lauryl sulphate (a detergent) in distilled water. Grids are dipped in this just prior to collecting sections, then drained on filter paper before introducing under the water in the knife boat and bringing up under the sections. The sections can be manipulated along filmed slots with an eyelash before the residual fillm of water dries. You can play this game under the binocular for some minutes while watching the water evaporate. Its a bit like watching paint dry!
Sometimes, raising the water meniscus (i.e. to convex) is useful when trying to position sections prior to collection.
Too much carry-over of detergent can cause the sections to run together and bunch up (a surface tension effect of the detergent).
The main problem with this practice is that you can only do this after cutting a batch of sections. If you wish to continue cutting then it is adviseable to flush the boat with fresh water, otherwise the block face will probably "wet" if you resume cutting. I normally pipette out the water and replace it three times - that works.
Keith Ryan Marine Biological Association Plymouth, UK
Unfortunately our CPD is out of service and my bottle of PELDRI nearly empty, therefore I am forever looking for new, fast, inexpensive ways to prepare my samples.
Although I do not work with plant material I was very interested in a publication that describes the use of glycerol or triethylene glycol to infiltrate biological specimens (mostly plant material) so that they may be observed in the SEM.
("Liquid substitution: a versatile procedure for SEM specimen preparation of biological materials without drying or coating" Journal of Microscopy, Vol. 172, 3. 1993 pp195-203. Ensikat H.j. & Barthlott, W.)
.."The main objectives of these preparation methods are to stabilize the specimen, to prevent shrinkage and other artefacts during dehydration, and to render the sample electrically conductive....specimen are not dried, but their water is instead substituted for a liquid..."
If the researcher tries the method I would be very interested to hear how it worked.
Regards
Claudia
On 13 Sep 2000, at 15:50, Edward J. King wrote:
Date sent: Wed, 13 Sep 2000 15:50:14 -0600 } From: "Edward J. King" {king-at-biology.utah.edu} To: Microscopy Listserv {Microscopy-at-sparc5.microscopy.com}
I have no experience of looking at pits. Another possiblility to reduce artifacts would be to use an ESEM. I think it might be difficult to get the resolution on unfixed material with a tungsten instrument. A field emission ESEM would have the resolution at lower voltages but may still require a comparison of fixed and unfixed material. Unfixed material has to be handled carefully to avoid beam damage artifacts.
Dave
On Wed, 13 Sep 2000 15:50:14 -0600 "Edward J. King" {king-at-biology.utah.edu} wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } A user of our facility needs to prepare specimens to determine if } sub-micrometer pores are present in plant intervascular pit membranes. } The user is very concerned that critical point drying or, especially, } the dehydration steps prior to CPD, may introduce 'pores' that are } really artifacts. He is similarly concerned that freezing or } freeze-drying his specimens might also introduce 'pores' due to ice } crystal formation. Work he has already done suggests that these are } valid concerns. } } Has anyone sufficient experience preparing pit membranes for SEM to } suggest a procedure we might follow to minimize the possibility of } creating small 'pores' where none may exist? } } Thank you, } } Ed King }
---------------------------------------- Patton, David Email: David.Patton-at-uwe.ac.uk "University of the West of England"
From root Thu Sep 14 03:51:48 2000 Return-Path: {Microscopy-request-at-sparc5.microscopy.com} Received: (from daemon-at-localhost) by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) id DAA22959 for dist-Microscopy; Thu, 14 Sep 2000 03:36:26 -0500 (CDT) Received: from no_more_spam.com (sparc5 [206.69.208.10]) by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) with SMTP id DAA22955 for "MicroscopyFilteredEmail-at-msa.microscopy.com"; Thu, 14 Sep 2000 03:35:55 -0500 (CDT) Received: from haymarket.ed.ac.uk (haymarket.ed.ac.uk [129.215.128.53]) by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) with ESMTP id DAA22940 for {microscopy-at-sparc5.microscopy.com} ; Thu, 14 Sep 2000 03:35:44 -0500 (CDT) Received: from srv0.bio.ed.ac.uk (srv0.bio.ed.ac.uk [129.215.136.240]) by haymarket.ed.ac.uk (8.8.7/8.8.7) with ESMTP id JAA02352 for {microscopy-at-sparc5.microscopy.com} ; Thu, 14 Sep 2000 09:29:53 +0100 (BST) Received: from BIO-SRV0/SpoolDir by srv0.bio.ed.ac.uk (Mercury 1.44); 14 Sep 00 09:29:53 +0000 Received: from SpoolDir by BIO-SRV0 (Mercury 1.44); 14 Sep 00 09:29:25 +0000
Very satisfactory results have been reported for pollen (see ref. below) but I have found it worthless on plant cells in tissue culture, for which cryo-SEM must be the way to go. HMDS may work better on densely cytoplasmic material than on very vacuolate plant tissue.
I would be interested to know what the principles of operation of HMDS are thought to be. Does anyone have specific information about this?
Chris
Chissoe,W.F.; Vezey,E.L.; Skvarla,J.J. (1994) Hexamethyldisilazane as a drying agent for pollen scanning electron- microscopy. Biotechnic & Histochemistry69, 192-198 Abstract: Use of hexamethyldisilazane (HMDS) as a final dehydrating solution provides robust, undistorted secondary electron images of a variety of angiosperm and gymnosperm pollen grains, including those considered to be susceptible to collapse in the scanning electron microscope. Ease of handling, low cost, lack of specialized equipment, minimal expenditure of time, and high rate of success are factors that favor HMDS over other drying agents for preparing pollen grains for scanning electron microscopy
} From: "Patton, David" {David.Patton-at-uwe.ac.uk} To: Tobias Baskin {BaskinT-at-missouri.edu} Copies to: Microscopy-at-sparc5.microscopy.com
I confused myself with acronyms and broadcast the result! The HMDS may be used for drying specimens for SEM as noted. I also ventured that it can be used for dehydration - wrong. For rapid, complete dehydration dimethoxypropane (DMP) is used; it converts residual water to acetone. Thanks to Tobias for spotting that. No doubt a thousand others noted the error but were too polite (?) to write. I think DMP works only well to remove residual water after prior dehydration. Cheers Jim Darley ProSciTech
On Wednesday, September 13, 2000 11:43 PM, Tobias Baskin [SMTP:BaskinT-at-missouri.edu] wrote: } Jim, } Thanks for the info. How does HMDS compare with } dimethoxypropane as a water scavenger? I use a few % of latter in } freeze substitution brews as a "cemical sieve". I have heard to folks } using DMP as one shot dehydration before embedding. I even tried it. } Worthless for our stuff. Just curious about the HMDS. } } Tobias } } } In my previous response I gave the method, though I've used Chloroform. HMDS } } may have a theoretical advantage, but I expect that the results would be } } identical. } } HMDS was introduced for rapid processing, particularly fast and complete } } dehydration. It chemically interacts with water and forms acetone. When at } } least the last dehydration is in HMDS, the user can be sure that } } dehydration is } } excellent even with doubtful ethanol or acetone. } } } } During the air drying process residual water and solvent molecules exerts } } huge } } pressures onto membranes and these cause shrinkage and shriveling of any } } specimen. } } The drying mechanism: } } Using Peldri, drying only occurred at the Peldri/ air interface, but the } } specimen is held rigid in the solid Peldri material. As the Peldri slowly } } sublimes, more of the specimen is dried. } } During solvent drying the specimen is in a solvent saturated atmosphere and } } drying is very slow - over 48 hours. In this method there is little pressure } } exerted by molecules trying to pass through membranes and consequently } } shrinkage is minimized. } } Its a good alternative method and sometimes works very well. Given a choice } } I'll try CPD first. } } Cheers } } Jim Darley } } ProSciTech Microscopy PLUS } } PO Box 111, Thuringowa QLD 4817 Australia } } Ph +61 7 4774 0370 Fax:+61 7 4789 2313 service-at-proscitech.com } } Great microscopy catalogue, 500 Links, MSDS, User Notes } } ABN: 99 724 136 560 www.proscitech.com } } } } On Wednesday, September 13, 2000 12:21 AM, Tobias Baskin } } [SMTP:BaskinT-at-missouri.edu] wrote: } } } } } } } } } Greetings, } } } Several replies have mentioned this mysterious solvent for } } } CPD, and that it works on some type of specimens. Could someone } } } outline how this is used (infiltrate and then evap to air, or what?) } } } and if there is any rhyme or reason about what kinds of specimens } } } that work??? } } } } } } THanks, } } } Tobias } } } } } } } } } } } } } } } We use HMDS (Hexamethyldisilazane) from Sigma. } } } } } } } } Dave } } } } } } } } } } } } On Tue, 12 Sep 2000 08:53:03 -0500 Cameron Hind } } } } {hindc-at-baxter.com} wrote: } } } } } } } } } } } } } } ------------------------------------------------------------------------ } } } } } The Microscopy ListServer -- Sponsor: The Microscopy Society of } } } } } America } } } } } To Subscribe/Unsubscribe -- Send Email to } } } } } ListServer-at-MSA.Microscopy.Com } } } } } On-Line Help } } } } } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } } } } } } } } -----------------------------------------------------------------------. } } } } } } } } } } } } } } } } } } } } Hi Listservers, } } } } } } } } } } I have a memory lapse-can you remind me of the reagent used as an } } } } } alternative to CPD for SEM;or do you know of/recommend any } } } safe (not too } } } } } toxic) alternative method to CPD?-any reply would be appreciated } } } } } } } } } } Cameron } } } } } } } } } } Mr. Cameron Hind } } } } } Research Scientist } } } } } Advanced Technologies group } } } } } Baxter R & D Europe S.C.R.L. } } } } } Rue du ProgrEs 7 } } } } } 1400 Nivelles } } } } } Belgium } } } } } } } } } } Tel:+32 67 882 511 } } } } } Fax:+32 67 217 191 } } } } } e-mail: hindc-at-baxter.com } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } ---------------------------------------- } } } } Patton, David } } } } Email: David.Patton-at-uwe.ac.uk } } } } "University of the West of England" } } } } } } -- } } } _ ____ __ ____ Tobias I. Baskin } } } / \ / / \ / \ \ University } } } of Missouri } } } / | / / \ \ \ Biological } } } Sciences } } } /_ / __ /__ \ \ \__ 109 Tucker Hall } } } / / / \ \ \ } } } Columbia, MO 65211-7400 USA } } } / / / \ \ \ voice: } } } 573-882-0173 } } } / /____ / \ \__/ \____ fax: 573-882-0123 } } } } } } } } -- } _ ____ __ ____ Tobias I. Baskin } / \ / / \ / \ \ University of Missouri } / | / / \ \ \ Biological } Sciences } /_ / __ /__ \ \ \__ 109 Tucker Hall } / / / \ \ \ } Columbia, MO 65211-7400 USA } / / / \ \ \ voice: } 573-882-0173 } / /____ / \ \__/ \____ fax: 573-882-0123
Jim, I'm not sure if it is the same, but I have used 2,2-dimethoxypropene as an intermediate agent between 100% ethanol and air drying of the sample. It works very well and there is little distortion of the surface morphology.
Franklin Bailey Electron Microscopy Center University of Idaho Moscow, ID 83844-2204
} } We currently have a Bench Top Turbo carbon evaporation unit made by Denton. } We are very displeased with it. We consistently get non uniform coatings } that peel off of our metallographic mounts. Any suggestions on a } evaporation unit that works well or any information on 'sputtering" carbon? } Any other experiences out there? }
I would look very carefully at the cleanliness of the samples before coating. I use methanol then Freon then a dry Kimwipe, and the carbon coating is tenacious on brass.
cheers
rtch
Ritchie Sims Phone : 64 9 3737599 ext 7713 Department of Geology Fax : 64 9 3737435 The University of Auckland email : r.sims-at-auckland.ac.nz Private Bag 92019 Auckland New Zealand
Your first sentence is interesting, if I may stray a bit from the actual topic. I had formed the idea that the back-streamed oil is not diff pump fluid, but rotary pump oil. But if the phenomenon disappears when a turbo or ion pump replaces the diff pump, then that kindof exonerates the rotary pump, doesn't it? Or is it just that the turbo or ion pump is better than the diff pump at preventing rotary pump vapour from diffusing backwards through it? Anybody know the definitive answer to this?
} } Most modern TEMs are ion pumped (or turbo pumped) to } prevent the diffision pump oil backstreaming problem. The } diffusion pump is used when initially evacuating the column } or gun, pumping the camera and viewing chamber and when } warming up the specimen cold trap at the end of sessions. } Whereas it would be good to have a N2 trap on the diffusion } pump when it does pump the column the water vapour from the } camera and viewing chamber (due to the films) would stall } the backing pump when the N2 trap warmed up. } Prior to ion pumped columns we did use a N2 trap on the TEM } column diffusion pump (circa 1980). } Ion pumped or turbo molecular pumped SEMs are also } available. } } Regards, } Ron }
cheers
rtch
Ritchie Sims Phone : 64 9 3737599 ext 7713 Department of Geology Fax : 64 9 3737435 The University of Auckland email : r.sims-at-auckland.ac.nz Private Bag 92019 Auckland New Zealand
In response to Tamara Howard's predicament, here is an idea to consider. One can purchase bags of phase change material from Polar Tech Industries, Inc., in Genoa, IL (60135) that can be placed into a -20oC freezer and allowed to freeze. These can be placed into a styrofoam dry ice shipping box (commercially available from VWR with an external cardboard box for shipping) and should hold your samples between -20 and 0oC during shipment. This would be your ideal temperature range for shipment, and the temperature will hold for the duration of the air flight if your box size is reasonable. The foam refrigerant material stays in the bags, so there is no mess or hazard.
For more details, contact Polar Tech at 1-800-ICE-BRIX, or 815-784-9000, or at www.polar-tech.com.
I'd like to know how this works, if you haven't already opted for another approach.
Greg Fahy, Ph.D. 21st Century Medicine
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Does anyone have any information on Fourier Transform Infrared (FTIR) Spectroscopy used for organic contamination analysis? Specifically manufacturers and the appx. price for the equipment . Any information would be helpful.
Martin A. Izquierdo Analytical Laboratory Cerprobe, Inc.
Retch is right, but you could also have a poor cooling system on that pump, or just a badly designed system. In either case backstreaming of oil vapour would lead to poorly adhearing films. Cheers Jim Darley ProSciTech Microscopy PLUS PO Box 111, Thuringowa QLD 4817 Australia Ph +61 7 4774 0370 Fax:+61 7 4789 2313 service-at-proscitech.com Great microscopy catalogue, 500 Links, MSDS, User Notes ABN: 99 724 136 560 www.proscitech.com
On Friday, September 15, 2000 6:55 PM, Retch Sims [SMTP:r.sims-at-auckland.ac.nz] wrote: } } } } } } } We currently have a Bench Top Turbo carbon evaporation unit made by Denton. } } We are very displeased with it. We consistently get non uniform coatings } } that peel off of our metallographic mounts. Any suggestions on a } } evaporation unit that works well or any information on 'sputtering" carbon? } } Any other experiences out there? } } } } I would look very carefully at the cleanliness of the samples before } coating. } I use methanol then Freon then a dry Kimwipe, and the carbon } coating is tenacious on brass. } } cheers } } rtch } } } Ritchie Sims Phone : 64 9 3737599 ext 7713 } Department of Geology Fax : 64 9 3737435 } The University of Auckland email : r.sims-at-auckland.ac.nz } Private Bag 92019 } Auckland } New Zealand
I plan to buy CCD camera for TEM and want to remove wet dark room. So I need good printer. I think dye sublimation printer is the best choice. But I do not know what model is the best. Is there anybody who uses dye-sub printer in EM lab? Can you suggest what model is the best? My budget is about 5000 US$ or a little more. Thank you in advance.
Best regards,
Jondo Yun Kyungnam University Division of Advanced Materials Electron Microscopy Laboratory 449 Weolyeong-dong Masan, 631-701, Korea
Jondo Depends what the end-use of your prints is going to be. The most recent 6-colour + black 1440 dpi Epson Stylus printers produce absolutely stunning A4 size prints for a lot less capital and consumables cost. At arm's length you cannot really tell the difference between the Epson inkjet prints and a dye-sub print. With your budget you could buy a couple of these printers and still have enough left to buy a top-spec laptop! Epson also produce professional spec. inkjet printers, such as the Pro 5000 which are much more expensive, but may just be within your budget. see http://www.tssphoto.com/sp/dg/news/dot_comp.html for more details and links.
However, if you are determined to buy a dye-sub type printer, check out the Fuji Pictrography printers which combine laser exposed silver halide technology with dye transfer. The results are amazing. Chris
Send reply to: "Jondo Yun À±Á¸µµ" {jdyun-at-kyungnam.ac.kr} } From: "Jondo Yun À±Á¸µµ" {jdyun-at-kyungnam.ac.kr} To: "MicroscopyListserver" {Microscopy-at-sparc5.microscopy.com}
Hi Jondo,
I have no experience of dye sublimation printers but feel I must tell you how good our Epson Stylus Photo 1270 is. Using, admittedly expensive, photo quality paper we find it beats our very expensive colour laser printer for image quality. Unless you want dozens of copies done very quickly, I would certainly give the Epson a trial - and it costs a fraction of your budget, the remainder of which could buy a lot of consumables. BTW, I do not work for Epson
Kind regards,
Alan
********************************************* Alan Walker Dept of Electronic and Electrical Engineering University of Sheffield Mappin Street Sheffield S1 3JD United Kingdom
Glad I was able to help. I now number the "we do not use LN2" excuses 1 to 10 and simply ask which a client wishes to use as there is not such a thing as a "good excuse", I have heard them all!
Ron has given you a good explanation of DP traps, uses and problems but we should discuss pumping speed and TEM contamination a little further.
A vacuum in a TEM has fast and slow pumping areas. The electron gun is a fast pumping area but small pipes and pole piece apertures restrict gas flow and produce slow pumping areas, the specimen area in a TEM for example! No matter how good the vacuum may be at the DP it is only by improving the vacuum right by the TEM specimen that we see dramatic improvements, cold traps and cold fingers right by the specimen are the best route to lowering contamination.
Contamination in the TEM deposits on the top and the bottom of the specimen thickening the material and softening the image as the structures are gradually masked. Thicker specimens mean poorer resolution, often related as resolution is equal to one tenth of the specimen thickness. Improve the vacuum and you improve the contamination rate and an area that people do not relate to, you improve the gun vacuum and high voltage stability! So a better vacuum gives us a better contamination rate and better high voltage stability these two together will improve resolution.
I note you use a cold finger in the SEM when carrying out cryo experiments, I try to use such a device as much as possible if any high resolution study is taking place. Contamination rate tests would probably indicate as much as a five fold improvement when a trap is within inches of the specimen. I have designed several circular traps to fit round the pole piece of FEG SEMs, here contamination becomes the biggest barrier to very high resolution.
Good luck with those who have not taken to the LN2 gospel, keep at it!
Steve Chapman Senior Consultant Protrain For professional training and consultancy in EM world wide Tel +44 1280 814774 Fax +44 1280 814007 www.emcourses.com
I am trying to help a local high school set up an ISI SX-30 Super III SEM. It had been sitting for several years before being moved to the school. I am unfamiliar with this scope and we are trying to do some troubleshooting. We would greatly appreciate some help.
When the [Operate] button is depressed, it should pump down the chamber/column. However, it seems as though a valve is opened to atmosphere and loads the rotary pump. We hear hissing below the chamber which seems to be coming from a nozzle attached to a solenoid. We are wondering if there should be a hose attached to the nozzle and where it might go. Or is this an electrical problem?
Also, there is a loose snap wire connector in the back above the diffusion pump. It is marked JKA6 and contains a red and a yellow wire. I could not see the wires went to.
If anyone is familiar with this instrument, we would greatly appreciate some help in getting us started. The instrument was donated to the school and there are limited funds for bringing in service engineers.
Our FTIR expert here likes the complete system by Nicolet, but also said Perkin-Elmer and Biorad are a couple of other companies that make nice systems. I'm sure I'm leaving some out, as I'm not an FTIR expert, so forgive me if your company isn't on the list! As far as pricing, he mentioned a complete system goes for about $120,00US with only one lens. You can expect to add about $15,000US for each attachment, such as ATR and micro-ATR.
Again, I'm sure some vendors can give you more pertinent information, but this should put you in the ball park.
Hope this helps,
David Bell Scientist Electron Microscopy Lab Millipore Corporation 80 Ashby Road Bedford, MA 01730 (781) 533-2108
Martin Izquierdo To: "'Microscopy-at-MSA.Microscopy.Com'" {MartinI-at-ozte {Microscopy-at-sparc5.microscopy.com} k.com} cc: Subject: Fourier Transform Infrared Spectroscopy (FTIR) 09/14/00 09:13 PM
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Does anyone have any information on Fourier Transform Infrared (FTIR) Spectroscopy used for organic contamination analysis? Specifically manufacturers and the appx. price for the equipment . Any information would be helpful.
Martin A. Izquierdo Analytical Laboratory Cerprobe, Inc.
To the best of my knowledge, there is no such thing as "sputtering carbon". But I would suggest to look into your carbon source. Try different vendors. Also, make sure your vacuum is good.
Have a nice day!
Chao-Ying Ni Rodel Inc. 451 Bellevue Road Newark, DE 19713 (302) 366-0500 ext 2812
-----Original Message----- } From: jim [mailto:jim-at-proscitech.com.au] Sent: Thursday, September 14, 2000 9:53 PM To: 'Ritchie Sims'; Ekstrom, Harry; Microscopy-at-sparc5.microscopy.com
Retch is right, but you could also have a poor cooling system on that pump, or just a badly designed system. In either case backstreaming of oil vapour would lead to poorly adhearing films. Cheers Jim Darley ProSciTech Microscopy PLUS PO Box 111, Thuringowa QLD 4817 Australia Ph +61 7 4774 0370 Fax:+61 7 4789 2313 service-at-proscitech.com Great microscopy catalogue, 500 Links, MSDS, User Notes ABN: 99 724 136 560 www.proscitech.com
On Friday, September 15, 2000 6:55 PM, Retch Sims [SMTP:r.sims-at-auckland.ac.nz] wrote: } } } } } } } We currently have a Bench Top Turbo carbon evaporation unit made by Denton. } } We are very displeased with it. We consistently get non uniform coatings } } that peel off of our metallographic mounts. Any suggestions on a } } evaporation unit that works well or any information on 'sputtering" carbon? } } Any other experiences out there? } } } } I would look very carefully at the cleanliness of the samples before } coating. } I use methanol then Freon then a dry Kimwipe, and the carbon } coating is tenacious on brass. } } cheers } } rtch } } } Ritchie Sims Phone : 64 9 3737599 ext 7713 } Department of Geology Fax : 64 9 3737435 } The University of Auckland email : r.sims-at-auckland.ac.nz } Private Bag 92019 } Auckland } New Zealand
Much as I dislike cluttering up the list with complaints, I would really like to see everyone following Nestor's recommended guidelines which include posting the sender's full address, including geographical. Sometimes, obviously, we can tell from the email address but all too often we can't; and sometimes we cannot even tell the country of origin, much less the city and state. This information is especially when posting persons are seeking help with instrument problems.
I realize that full address is an option on many email programs, and I sometimes forget to choose it myself, but I urge everyone to give it more thought.
Thank you.
Don Marshall
Donald J. Marshall Relion Industries P.O. Box 12 Bedford, MA 01730 Ph: 781-275-4695 FAX: 781-271-0252 email dmrelion-at-world.std.com
Cathodoluminescence, mass spectroscopy, electron beam technology
"A weed is a flower out of place."
(Please note: Do not send email with attachments to this address. Instead, send it to dmrelion-at-aol.com. Thank you.)
The turbo pump is simply better than the diffusion pump at minimizing back streaming. Will Bigelow in his book on Vacuum Methods in Electron Microscopy talks about this and describes the care you should take in designing a pump-down protocol to minimize the backstreaming.
Tony.
} } Hi, Ron and all } } Your first sentence is interesting, if I may stray a bit from the } actual topic. } I had formed the idea that the back-streamed oil is not diff pump } fluid, but rotary pump oil. } But if the phenomenon disappears when a turbo or ion pump replaces } the diff pump, then that kindof exonerates the rotary pump, doesn't } it? } Or is it just that the turbo or ion pump is better than the diff pump } at preventing rotary pump vapour from diffusing backwards through it? } Anybody know the definitive answer to this? } }
** Anthony J. Garratt-Reed ** MIT Room # 13-1027 ** 77 Massachusetts Avenue ** Cambridge, MA 02139-4307 ** USA ** ** Phone: (+) 1-617-253-4622 ** Fax: (+) 1-617-258-6479 **
I use a Sony Mavigraph digital printer model UP-D1500 to print images from our SEM, its been reliable for the 4 years we have had it. I don't publish (industrial use- trade secrets, industrial hygiene, quality type stuff) so the resolution limit is OK for our use. The only problem that I have had is the print paper tends to curl sometimes and won't feed unless you straighten them. I also use a Sony Mavigraph color video printer model UP-3000 to print images from our light microscopes through a video camera and analog-digital converter and its been reliable and no problems at all. They both cost about $3000 when I bought them. Terry Ellis Of course I have no interests in that company, wish I did.
Can someone provide me with information on how to obtain microscope slides which can serve as standards for measuring optical density in transmission light microscopy? We would like to use these as a staining independent standard for the imaging and image analysis of histochemically stained control and experimental slides. Are there any commercially available ones? Alternative: can someone give me "recipes" for home made ones? We were thinking of eventually using small pieces of light exposed and developed film or neutral gray filters. Has anyone tried this already?
Thanks in advance.
**************************************************** Lauran C.J.M. Oomen oomen-at-nki.nl The Netherlands Cancer Institute tel. +31 20 5121889 Digital Microscopy Facility fax. +31 20 5121893 Plesmanlaan 121 1066CX Amsterdam The Netherlands ****************************************************
} Further to my posting yesterday seeking a PCD for a JEOL 840, which, } I am informed, is the same as that for a 6400, I am currently } negotiating with a company to make me one. } } Does anyone else out there want one?
While I am not looking for a PCD myself, I have customers who may be interested. If you can let me know the company you are dealing with I would appreciate it.
Joe _________________________________________ Joe Nabity, Ph.D. JC Nabity Lithography Systems E-Beam Lithography using Commercial SEMs & STEMs PO Box 5354, Bozeman, MT 59717 USA Voice: (406) 587-0848 FAX: (406) 586-9514 E-mail: info-at-jcnabity.com Web: www.jcnabity.com
I have just had a user approach me about looking at a water-saturated sample in our Hitachi S-2460N. While we have used VP-SEM for several years to look at insulating samples, this is the first time we have been asked to look at a moist sample. I have two questions as I plunge into this.
1-What instrumental concerns are there related to using water vapor as an atmosphere? I have heard that it may sorb onto the chamber surfaces and may be slow to desorb, so our ultimate vacuum may be poor for a while afterwards. There was also a concern over possible effects of the vapor on the precision leak valve. Are there any other components that might be affected, e.g., EDS window, BSE detector, etc.? Since the water will be present as vapor at less than 2 torr and will only be used for about an hour I don't envision problems, but I would rather not be surprised.
2-What are effective ways for introducing water vapor into the SEM? I am considering partially filling a vacuum desiccator with water and hooking our vent line to it and allowing the SEM vacuum to vaporize the water. Someone suggested a moistened rag in the vent line. What other means work well?
Thank you in advance. ---------------------- Warren E. Straszheim Materials Analysis and Research Lab Iowa State University 23 Town Engineering Ames IA, 50011-3232
I am a graduate student in the Materials Engineering Department at San Jose State University in California, and need some help with my thesis research. I was wondering if anyone out there has any experience with FIB imaging of recrystallized Cu films for the purposes of grain sizing. My research requires both plan view (at various tilt angles) and cross-sectional views of blanket films. I plan to work with an outside lab that is very experienced with SEM imaging, but relatively inexperienced with FIB imaging. They just got their first system online a couple of months ago. My questions are related to sample preparation from an 8" Si wafers and suggested FIB operating parameters, tips, and tricks to get quality images with good grain-to-grain contrast. If anyone has such experience or can point me in the right direction, it would be greatly appreciated. I am also seeking a good reference book or review article that covers FIB imaging theory (especially Ga ion channeling contrast). You can respond to the listserver, or contact me directly at attz-at-pacbell.net. Thanks in advance for your time and help.
LEE FILTERS Central Way Walworth industrial Estate Andover Hampshire SP10 5AN
http://www.leefilters.co.uk/
Lee make optical density filters in polyester squares 75mmx75mm which can be cut up into small squares or circles (paper punch) and mounted under a coverslip on a microsope slide. If you want to avoid contributions to the density from glass, mountant, etc drill an array of holes in a microscope slide sized piece of aluminium sheet, and mount samples of the filters over the apertures
Kodak also make ND Wratten filters in gelatin Chris
Date sent: Fri, 15 Sep 2000 15:56:33 +0200 } From: Lauran Oomen {Oomen-at-nki.nl} To: Microscopy-at-sparc5.microscopy.com
Good theory page: http://sis.bris.ac.uk/~sd9319/spec/IR.htm
Marc Helvey Sales Accounts Manager VLSI Standards, Inc. 3087 North First Street San Jose, CA 95134-2006 Phone: (408) 428-1800, ext. 108 FAX: (408) 428-9555 Mobile: (408) 307-3833 E-Mail: marc.helvey-at-vlsistd.com {mailto:marc.helvey-at-vlsistd.com} Internet: http://www.vlsistd.com
-----Original Message----- } From: Martin Izquierdo [mailto:MartinI-at-oztek.com] Sent: Thursday, September 14, 2000 6:14 PM To: 'Microscopy-at-MSA.Microscopy.Com'
Does anyone have any information on Fourier Transform Infrared (FTIR) Spectroscopy used for organic contamination analysis? Specifically manufacturers and the appx. price for the equipment . Any information would be helpful.
Martin A. Izquierdo Analytical Laboratory Cerprobe, Inc.
The answer is that rotary pump oil is able to pass through diffusion pumps easier than turbo pumps. Michael Postek of NIST wrote on article on this. "An Approach to the Reduction of Hydrocarbon Comtamination in the Scanning Electron Microscope" Scanning Vol. 18, 269-274 (1996). One of the techniques he used to reduce oil comtamination was to create a leak in the foreline of the Diff. Pump to create viscous flow conditions that stopped the backstreaming of rotary pump oil into the Diff. pump. This stopped condensation of pump oil on his EDS detector window.
I have a box of reprints of this article in my office, since it also discussed nitrogen purging which was used in my original SEM-CLEAN product. So if any one wants one, e-mail me back and ask for the "Postek" article. Please include your mailing address.
My web site SEMCLEAN.COM has full information on XEI's anti-contamination systems. No LN or turbopump required.
Ronald Vane XEI Scientific 3124 Wessex Way Redwood City, CA 94061 (650)369-0133
-----Original Message----- } From: Ritchie Sims {r.sims-at-auckland.ac.nz} To: message to: MSA list {microscopy-at-sparc5.microscopy.com}
I'm putting in my 2 cents on this thread because we have both in our lab. This is Tektronics Phaser 450 vs. the Epson 1270. The 1270 has cheaper supplies, more variety of papers, larger format and much much higher stability in it's favor. And the 450? I have to say I still prefer the dye-subs grey tones for images over the "grittiness" of the inkjet. The dye sub print still looks more like an RC print to me. When I requested materials from Epson demonstrating the capabilities of the 1270 and 870, we got this amazing test print from them where the gradation of tones really was spectacular. None of the prints from our own photos look nearly that good on the Epson, and in side by side comparisons with the dye sub, I think the dye sub prints always look better.
I guess it boils down to price considerations, is that extra $1.50 per print + the difference in cost between printers worth it?
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------------------------------------------------------------------------ The Microscopy ListServer -- Sponsor: The Microscopy Society of America Mark, I sent the method below to the list some time ago. It works great. Debby Sherman
A Fool-proof Method for Mounting Serial Sections on Single Hole Grids
I did serial sectioning for years on large single hole grids using a very simple technique that made the potential problems of film thickness, wrinkles and section loss very minor. I was not the original developer of the method and do not remember who originally gave it to me. It goes as follows:
1) Have your machine shop cut some thin pieces of Plexiglas into the size of glass slides. At one end, drill about a dozen holes, roughly 5mm in diameter, in an area about the size of a formvar film cast on glass slides. These slides will serve as your template for holding your films.
2) Cast the formvar films onto glass slides using your normal method. Usually a good silver film, not gray, will work fine. I routinely used 0.2% formvar in dichloroethane when casting by immersing the slide into the solution in a small jar, etc. We now use a film caster that lets us hold the slide in the dichloroethane vapors after lowering the formvar solution level. This method tends to give you thinner films consistently so the correct solution percentage and timing would have to be redetermined.
3) Float the film off the glass slide and pick it up with the Plexiglas slide so the film covers the holes. Then draw the water out of the holes by pressing the plastic slide down onto filter paper, or using small pieces of filter paper and capillary action to draw the water out of individual holes. The films should hold nicely over the holes in the slide. Store slides until needed.
4) Next, cut your sections using a block diameter that is fairly similar to the size of the slit in the grid. Pick up the sections on UNCOATED grids by gently lowering the grid to the surface of the knife boat. I put the dull side down on the premise that the rough surface would grab the film better during step 6. The surface tension of the water will hold the sections in the grid opening. Transfer the grid to a droplet of water until you have finished sectioning. Do not invert the grid. It is important that the top of the grid (shiny side) stay dry so that the grid will float on all subsequent solutions.
5) Transfer the grid + sections + water droplet to a drop of stain. A small amount of water will be transferred but this will not interfere with staining. If you are concerned about the dilution effect, increase your staining time slightly. Allow the section to stain, then wash by transferring through a series of droplets of clean water. Continue to post-stain if desired and wash the same way. Never let the grid dry. There is minimum problem with stain precipitation if you use very clean water and transfer the grid through a sufficient number of water droplets (6-12 recommended).
6) The final step is to transfer the grid to a film suspended over the hole in a Plexiglas slide and let it dry down. The sections will now be stuck to the film with NO wrinkles and minimum breakage. When ready to view, just punch out around the grid with the tip of your forceps, grab the grid and insert into the microscope.
Believe me....the sections will still be there at the end!
I found that as long as the sections cover a substantial portion of the open area of the grid, carbon coating was not essential. I used to do 50-100 grids worth of serial sections without loosing any. The films on the plastic slides would hold for months so I could make a lot and store until needed. The method really works...do give it a try.
Debby Sherman Microscopy Center in Agriculture Dept. of Botany & Plant Pathology Purdue University West Lafayette, IN 47907
Published: Sherman, D.M. (1998) A Full-proof Method for Mounting Serial Sections on Single Hole Grids. MSA Technologist’s Forum Newsletter 16:2
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Hi, } } Well, thanks for the flood of suggestions about getting a ribbon. I } trimmed the block sides with a glass knife (and enjoyed Mike Delannoy's } suggestion of 'trimming the top and bottom sides of your face with a glass } knife' - didn't know microscopy was such a painful job) and quite a decent } ribbon was the result. I tried the chloroform-glue idea and that seemed to } make the sections stick together more, but sometimes they gummed up a bit } on the knife. The next battle is picking up the ribbon on several slot } grids - I suspect that it's more practice than anything else. The method I } was shown was to go underneath the ribbon at an angle with the grid, raise } it until the waterline is on the top of the exposed formvar of the slot, } push the ribbon onto the waterline and draw the grid up at the same time, } and touch the ribbon with the hair at the point where the slot finishes to } break the ribbon, and remove the slot - all easier said than done! Any } ideas would be much appreciated. } } Thanks, } } Mark } } ******************************************** } Mark West, } Unidad de Microscopia Electronica, } (Electron Microscopy Unit) } Instituto de Fisiologia Celular, } Universidad Nacional Autonoma de Mexico, } 04510 Mexico D.F. } } tel (unidad/lab) *(525) 622 5610* } (casa/home) (525) 619 3020 } Fax (525) 616 2282 } ********************************************
by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) id MAA03852 for dist-Microscopy; Fri, 15 Sep 2000 12:23:25 -0500 (CDT) Received: from no_more_spam.com (sparc5 [206.69.208.10]) by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) with SMTP id MAA03837 for "MicroscopyFilteredEmail-at-msa.microscopy.com"; Fri, 15 Sep 2000 12:22:55 -0500 (CDT) Received: from corp.olin.com ([12.25.240.4]) by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) with ESMTP id MAA03828 for {Microscopy-at-sparc5.microscopy.com} ; Fri, 15 Sep 2000 12:22:41 -0500 (CDT) Received: from arch-exc-01.archchemicals.com ([172.20.132.14]) by igate.corp.olin.com with ESMTP id {115225} ; Fri, 15 Sep 2000 12:22:08 -0500 Received: by arch-exc-01.archchemicals.com with Internet Mail Service (5.5.2448.0) id {SRAZTQN5} ; Fri, 15 Sep 2000 12:18:35 -0500 Message-ID: {7531557A3D91D311B0320008C7E6708611A415-at-arch-exc-05.archchemicals.com}
I have a Kodak 8670 dye sub printer hooked up to my SEM, but it seems to me that it takes a long time to get it to print and also while it is in the printing process my computer (and hence my SEM) is locked up. Here are the details.
I have the printer (1 year old) hooked to a PC with a 333 Pentium II processor (128 MB RAM) using a parallel cable. I do get the same performance when printing from other computers so I doubt that it is anything peculiar to this one computer.
Now when I call for a printout it takes 5 minutes to get the 1MB file size print done, and almost all of the time is spent sending data to the printer - the actual printing takes a short time (30 sec). I have discussed this with Kodak Tech Support and they claim my performance is not unusual. HOWEVER, I also experienced much quicker printing times (I'm sure - I have notes, but it was a while ago) when I was first using the printer (1 minute / 1 MB of file size in the image) and this is confusing me.
My question is: how fast should this sort of printer print when using the parallel cable to feed data to it? Is 5 minutes / 1MB unusually slow? I get frustrated particularly as my computer is locked up while the data is being sent out and I cannot continue imaging until the data dump is done. I have the Win 95 spooler running of course and I don't have this problem with my HP 970 inkjet which I use normally.
I will welcome any good ideas or cold realities that apply.
The reason that you can back up a turbomolecular pump with an oil-sealed rotary vane pump and still have a system that is virtually free of oil contamination is that the compression ratio of turbo pumps varies exponentially with the square root of the molecular mass of the molecules being pumped. Thus, for nitrogen, which has M = 28, this value is about 200, whereas for a hydrocarbon molecule such as octane with M = 66 it is greater than 3000, and for a molecule of pump oil with M greater than 100 it is greater than a million. These values are for a single compression stage (rotor disk) of a turbo pump. Actual turbo pumps have from 15 to 20 such stages which act in series, so the overall value for nitrogen ends up being around 10^9, while for hydrocarbons it becomes astronimical (10^40 is often cited). Thus, if a turbo pump that is backed by a rotary vane pump is managed so that the rotor never drops below about 75% of full operating speed while the pressure in it is in the molecular flow range (below about 1 Torr is a safe figure), it is virtually impossible for oil molecules to backstream through it. These matters are all discussed in detail on pages 229 to 234 of my book 'Vacuum Methods in Electron Microscopy' (see http:/www.2spi.com/catalog/books/book48.html, and http:/pup.princeton.edu/titles/6484.html for a description) and appropriate operating protocols are are described on pp. 249 to 253 .
Wilbur C. Bigelow, Prof. Emeritus Materials Sci. & Engr., University of Michigan Ann Arbor, MI 48109-2136 e-mail: bigelow-at-umich.edu; Fx:734-763-4788; Ph:734-662-5237
Actually, any target material, including carbon and other high purity materials, that can be used in a vacuum evaporator or DC diode coater can be utilized in an Ion Beam Sputter Deposition System.
As for carbon, TEM electron diffraction images of ion beam sputtered carbon films are amorphous. The structure of a carbon grid before the carbon film was deposited cannot be distinguished from the carbon film!
TYPICAL SPUTTER RATES
Target Å/min
Cr 8-12 W 8-10 Ta 8-10 Pt 10-12 Ir 10-12 C 2-6
DISCLAIMER: South Bay Technology produces the IBS/e Ion Beam Sputter Deposition and Etching System for high resolution microscopy applications as described above and, therefore, has a vested interest in promoting its use.
David Henriks Vice President TEL: 800-728-2233 (toll free in the USA) South Bay Technology, Inc. +1-949-492-2600 1120 Via Callejon FAX: +1-949-492-1499 San Clemente, CA 92673 USA e-mail: henriks-at-southbaytech.com
} } } } } Please visit us at http://www.southbaytech.com { { { { {
Manufacturers of precision sample preparation equipment and supplies for metallography, crystallography and electron microscopy.
Message text written by "Ni, Chao-Ying" } ------------------------------------------------------------------------ The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Harry,
To the best of my knowledge, there is no such thing as "sputtering carbon". But I would suggest to look into your carbon source. Try different vendors. Also, make sure your vacuum is good.
Have a nice day!
Chao-Ying Ni Rodel Inc. 451 Bellevue Road Newark, DE 19713 (302) 366-0500 ext 2812 {
We currently use staff engineers to service instruments, assist in teaching classes, train users on operating equipment, and performing some minor administrative tasks (primarily in the physical sciences area). We have 12 pieces of analytical equipment and only 4 instruments on service contracts, so we rely heavily on our staff to keep our instruments running. I have performed some surveys from industry and service engineer pay scales vary approximately in the following manner:
$30's-40's: for starting engineers with little to no experience that require training mid $40's to mid $50's: for an experienced engineer mid $50's to mid $70's: for a very skilled engineer who can trouble shoot to the component level independently without any back-up help.
It would be of great help to me and my staff if you could take a few minutes and enlighten me as to your approximate salary ranges for your staff compared to their experience level, and compared to the numbers I have shown above.
Thanks very much for your time and help.
Regards, Lucille Giannuzzi
******************************************************************* Lucille A. Giannuzzi, Ph.D.
Associate Professor, Mechanical, Materials, and Aerospace Engineering Director, UCF/Cirent Materials Characterization Facility University of Central Florida 12443 Research Parkway, Suite 305 Orlando, FL 32826 email lag-at-mail.ucf.edu phone (407) 275-4354,5,6 fax (407) 275-4321 -------------------------------------------------------------------- "Good judgement comes from experience.
Experience comes from making bad judgement."
Mark Twain ********************************************************************
A colleague wishes to print "archival" quality grey scale images (micrographs) on an Epson 750 Photo printer. They have purchased CMY archival quality inks from a company called Lysonic. They are apparently unable to obtain a gray scale image but instead obtain one that is "off color". According to my colleague, the printer will not generate an image using the Black ink cartridge but only when CMY inks are used. Has anyone solved this problem (of color balance)?
Thank you,
#################################################################### John J. Bozzola, Ph.D., Director Micro-Imaging and Analysis Center 750 Communications Drive - MC 4402 Southern Illinois University Carbondale, IL 62901 U.S.A. Phone: 618-453-3730 Fax: 618-453-2665 Email: bozzola-at-siu.edu Web: http://www.siu.edu/departments/shops/cem.html ####################################################################
http://www.kodak.com/cgi-bin/searchKodak.cgi?searchText=neutral+density+fi lters+ will get you on a good start. I tried to send it direct and couldn't resolve your address.
Good luck Gordon Gordon Couger gcouger-at-couger.com Stillwater, OK www.couger.com/gcouger 405 624-2855 GMT -6:00
----- Original Message ----- } From: "Lauran Oomen" {Oomen-at-nki.nl} To: {Microscopy-at-sparc5.microscopy.com} Sent: Friday, September 15, 2000 8:56 AM
Edmund Scientific sell stepped neutral density filters with 11 discrete density steps in intervals of 0.1 OD which might be useful here, though the OD range may not be wide enough for all applications.
These filters are coated on glass 2" x 1" x 0.062" thick, cover the OD range 0.04 (92%) to 1.0 (10% transmittance). They are spectrally flat in the range 400 to 700nm. Tolerances are not stated, but they are recommended as "Ideal for applications requiring variable densities and known density values". £119 in UK, which probably translates into $119 in US.
} } Dear all, } } } } Can someone provide me with information on how to obtain microscope } } slides which can serve as standards for measuring optical density in } } transmission light microscopy? We would like to use these as a staining } } independent standard for the imaging and image analysis of } } histochemically stained control and experimental slides. Are there any } } commercially available ones? Alternative: can someone give me "recipes" } } for home made ones? We were thinking of eventually using small pieces of } } light exposed and developed film or neutral gray filters. Has anyone } } tried this already? } } } } Thanks in advance. } } } } **************************************************** } } Lauran C.J.M. Oomen oomen-at-nki.nl } } The Netherlands Cancer Institute tel. +31 20 5121889 } } Digital Microscopy Facility fax. +31 20 5121893 } } Plesmanlaan 121 } } 1066CX Amsterdam } } The Netherlands } } **************************************************** } } } } } } } } } } }
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Dr Chris Jeffree University of Edinburgh Biological Sciences EM Facility Daniel Rutherford Building King's Buildings EDINBURGH EH9 3JH Tel: +44 (0) 131 650 5345 FAX: +44 (0) 131 650 6563
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Dr. John Mansfield CPhys MInstP North Campus Electron Microbeam Analysis Laboratory 417 SRB, University of Michigan 2455 Hayward, Ann Arbor MI 48109-2143 Phone: (734) 936-3352 FAX (734) 763-2282 Cellular Phone: (734) 358-7555 (Leaving a phone message at 936-3352 is preferable to 358-7555) Email: jfmjfm-at-engin.umich.edu URL: http://emalwww.engin.umich.edu/people/jfmjfm/jfmjfm.html Location: Lat. 42° 16' 48" Long. 83° 43' 48"
We use a Hitachi 2250-N with a variety of gases. Attaching the inlet line to a desiccator or heavy conical flask of water works fine, but the vacuum gauge calibration may need checking if you need to know pressure accurately.
If you usually run with air or nitrogen in VP conditions, you will probably find the water gives you a resolution advantage (Helium is even better).
However your user should be aware that one or two torr of water does not do a lot to keep a specimen moist unless you also use a cold stage.
good luck,
Sally
Sally Stowe EM Unit Australian National University Canberra
} } } Warren E Straszheim {wesaia-at-iastate.edu} 09/16/00 02:01am } } } ------------------------------------------------------------------------ The Microscopy ListServer -- Sponsor: The Microscopy Society of America
I have just had a user approach me about looking at a water-saturated sample in our Hitachi S-2460N. While we have used VP-SEM for several years
to look at insulating samples, this is the first time we have been asked to
look at a moist sample. I have two questions as I plunge into this.
1-What instrumental concerns are there related to using water vapor as an atmosphere? I have heard that it may sorb onto the chamber surfaces and may
be slow to desorb, so our ultimate vacuum may be poor for a while afterwards. There was also a concern over possible effects of the vapor on
the precision leak valve. Are there any other components that might be affected, e.g., EDS window, BSE detector, etc.? Since the water will be present as vapor at less than 2 torr and will only be used for about an hour I don't envision problems, but I would rather not be surprised.
2-What are effective ways for introducing water vapor into the SEM? I am considering partially filling a vacuum desiccator with water and hooking our vent line to it and allowing the SEM vacuum to vaporize the water. Someone suggested a moistened rag in the vent line. What other means work well?
Thank you in advance. ---------------------- Warren E. Straszheim Materials Analysis and Research Lab Iowa State University 23 Town Engineering Ames IA, 50011-3232
FTIR is a very useful tool for analysis of organic contaminants. All FTIR systems consist of a spectrometer, or bench. The bench can be fitted with various accessories -- e.g., a beam condenser, in-compartment ATR, microscope, etc. -- that permit analysis of different states and amounts of materials. Analysis of contamination likely will require use of one or more of these sampling accessories. Manufacturers such as Nicolet, SensIR, Perkin-Elmer, Biorad, and other third-party vendors can provide specifications and application notes for each. The total system cost will depend largely on the sampling accessory and other add-ons such as cameras and databases. A standard bench and beam condenser or in-compartment ATR might cost $25K to $30K. Some benches and in-compartment accessories are sufficiently portable for field use (SensIR makes a portable diamond ATR system with integrated video that is quite nice). If your samples are routinely less than 100 microns in diameter or consist of multiple phases or layers, a microscope accessory likely will be required. A standard bench and microscope might cost $65K to $85K or more; add multiple detectors, objectives, and automated operation for QC work and the cost can exceed $125K.
I use a portable Nicolet bench coupled to a high-end Nicolet/Spectra-Tech microscope. The bench can be taken on-site for analysis of larger homogenous samples. The microscope is used in the lab for analysis of microscopic samples, including individual fibers or layer in composites. Dual detectors in the microscope cover a spectral range of 4000-450 wavenumbers for organic and many inorganic analyses, and imaging is enhanced by infinity-corrected optics, fluorescence and visible polarized illumination, and a digital/video imaging system.
Whatever you decide best suits your needs, I suggest you visit a manufacturer to test drive their equipment using your own samples. Most are only too pleased to assist.
Hope this helps, and good luck.
James Martin Principal/Research Scientist Orion Analytical, LLC PO Box 550 Williamstown, MA 01267 www.orionanalytical.com
----- Original Message ----- } From: Martin Izquierdo {MartinI-at-oztek.com} To: 'Microscopy-at-MSA.Microscopy.Com' {Microscopy-at-sparc5.microscopy.com} Sent: Thursday, September 14, 2000 9:13 PM
Hi,
I am interested in obtaining one or more Olympus POS (vertical tue monocular with rotating stage) microscopes in good condition. Anyone have one that is no longer being used?
Thanks.
Don Marshall
Donald J. Marshall Relion Industries P.O. Box 12 Bedford, MA 01730 Ph: 781-275-4695 FAX: 781-271-0252 email dmrelion-at-world.std.com
Cathodoluminescence, mass spectroscopy, electron beam technology
"A weed is a flower out of place."
(Please note: Do not send email with attachments to this address. Instead, send it to dmrelion-at-aol.com. Thank you.)
Speaking of such, check out this story at http://www.cnn.com/2000/TECH/computing/09/14/epson.image.fading.idg/ind ex.html
Ozone fades some Epson photos
Contrary to ads promising fade-resistant, long-lasting photos, color prints made with several Epson Stylus Photo ink jets turn orange in locations with heavy concentrations of ozone in the air, Epson officials confirm. The company offers to buy your printer if you're not satisfied with several recommended fixes, including using new types of paper Epson will introduce this fall. The problem came to light when some owners of Epson's Stylus Photo 870, 875DC, and 1270 reported photos they printed were turning orange.
} } John writes: } } When in doubt NEVER buy a hp printer. The HP1600 what a piece of the proverbial garbage! { {
John...you've gotta stop sugar-coating everything...just tell it like it is!
Larry ;-)
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Dr. Lawrence F. Allard Senior Research Staff Member High Temperature Materials Laboratory Oak Ridge National Laboratory 1 Bethel Valley Road Bldg. 4515, MS 6064 PO Box 2008 Oak Ridge, TN 37831-6064
As part of a gradulal upgrading of facilites, we're considering putting our booking sheets online to allow users to book themselves on the TEM, SEM, confocal or LM from the comfort of their own office.
Can anyone recommend Macintosh software capable of doing this? Ideally something which can be accessed by anyone with a web browser to avoid having to install new software on everyones computer.
Any feedback as to whether this sort of system really is better than a piece of paper nailed to the wall with a pen on a string would be welcomed too.
Thank you.
Andrew McNaughton -- ______________________________________________________________________________ Andrew McNaughton South Campus Electron Microscope Unit C/-Department of Anatomy and Structural Biology School of Medical Sciences University of Otago PO Box 913, Dunedin NEW ZEALAND
Is there a corresponding picture for a diff pump ie why do they let RP oil through? That's assuming, of course, that the oil that builds up on eds windows IS RP oil. It is, isn't it?
cheers
rtch
} } The reason that you can back up a turbomolecular pump with an oil-sealed } rotary vane pump and still have a system that is virtually free of oil } contamination is that the compression ratio of turbo pumps varies } exponentially with the square root of the molecular mass of the molecules } being pumped. Thus, for nitrogen, which has M = 28, this value is about } 200, whereas for a hydrocarbon molecule such as octane with M = 66 it is } greater than 3000, and for a molecule of pump oil with M greater than 100 } it is greater than a million. These values are for a single compression } stage (rotor disk) of a turbo pump. Actual turbo pumps have from 15 to 20 } such stages which act in series, so the overall value for nitrogen ends up } being around 10^9, while for hydrocarbons it becomes astronimical (10^40 is } often cited). Thus, if a turbo pump that is backed by a rotary vane pump is } managed so that the rotor never drops below about 75% of full operating } speed while the pressure in it is in the molecular flow range (below about } 1 Torr is a safe figure), it is virtually impossible for oil molecules to } backstream through it. These matters are all discussed in detail on pages } 229 to 234 of my book 'Vacuum Methods in Electron Microscopy' (see } http:/www.2spi.com/catalog/books/book48.html, and } http:/pup.princeton.edu/titles/6484.html for a description) and } appropriate operating protocols are are described on pp. 249 to 253 . } } Wilbur C. Bigelow, Prof. Emeritus } Materials Sci. & Engr., University of Michigan } Ann Arbor, MI 48109-2136 e-mail: bigelow-at-umich.edu; } Fx:734-763-4788; Ph:734-662-5237 } } } } Ritchie Sims Phone : 64 9 3737599 ext 7713 Department of Geology Fax : 64 9 3737435 The University of Auckland email : r.sims-at-auckland.ac.nz Private Bag 92019 Auckland New Zealand
} Hello } } As part of a gradual upgrading of facilities, we're considering } putting our booking sheets online to allow users to book themselves } on the TEM, SEM, confocal or LM from the comfort of their own office. } } Can anyone recommend Macintosh software capable of doing this? } Ideally something which can be accessed by anyone with a web browser } to avoid having to install new software on everyones computer. } } Any feedback as to whether this sort of system really is better than } a piece of paper nailed to the wall with a pen on a string would be } welcomed too. } Andrew,
A web page that emulated the sheet nailed to the wall should work fine and the use won't have to walk to the machine to sign up. I think it is a pretty simple job to write there should be dozens of kids around that can do it. Just don't let them try to improve on the sheet of paper on the wall concept or the users will get upset.
Gordon Gordon Couger gcouger-at-couger.com Stillwater, OK www.couger.com/gcouger 405 624-2855 GMT -6:00
Dear All has anyone any experience with Agar's melinex film for growing cells on before fixation and embedding for the TEM? I would like to find out why it is necessary to fix the film with warm fixative - doesn't this introduce some artefacts due to membrane mobility at 37 degrees? I am looking at membrane antigens! Carole Elleman
} A colleague wishes to print "archival" quality grey scale images } (micrographs) on an Epson 750 Photo printer. They have purchased CMY } archival quality inks from a company called Lysonic. They are } apparently unable to obtain a gray scale image but instead obtain one } that is "off color". According to my colleague, the printer will not } generate an image using the Black ink cartridge but only when CMY } inks are used. Has anyone solved this problem (of color balance)? }
R. Howard Berg, Ph.D. Director, Integrated Microscopy Facility, Associate Member Donald Danforth Plant Science Center/Nidus Center 893 North Warson St. Louis, MO 63141
1 MB/minute is just under 20K/second which is what we typically see with our printers. It doesn't seem to matter what printers or computers we use. We have most of our printers hooked up via ethernet connections, but even those do not seem to be much faster than those using the parallel cable. That surprises me since ethernet (10 Mbit/sec) should be able to handle almost 1000K/second (if we had the whole cable to ourselves).
It seems that there must be something about Windows that restricts throughput to 20K//sec. Perhaps someone out there knows how to speed things up. Maybe there is some Windows setting to tweak.
The fact that your speed has dropped to about 4K/second indicates that something must have changed - I don't know what. I am also surprised that your computer is tied up while printing. You might want to check out your printer properties. On the second tab (details) there is a button for spool settings. I am used to the top selection being clicked which is "Spool print jobs so program finishes printing faster", rather than the alternate choice of "print directly to printer". Our programs are ready for action in just a couple of seconds (which depends on the computer and the printing task), and other programs continue to run even while the job is spooled up. If the other box is checked, the computer will be tied up until the job is fully transmitted.
I hope some of this help.
Warren
At 12:17 PM 9/15/2000 -0500, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Hi All, I beg your pardon for bringing up this issue again, but when I did not need it, I did not pay attention to postings...
Now I have to include in a construction grant a price for antivibration table for FEG-ESEM XL30 and, possibly, for STEM CM12. In XL30 I have problems with vibrations at magnifications 100-200k (and sometimes starting at 50k).
Who got good results with antivibrition platforms? What type? Should only a column be isolated or a whole instrument? What is a probable price range (including installation)?
Thanks,
Vladimir
Vladimir M. Dusevich, Ph.D. Electron Microscope Lab Manager 3127 School of Dentistry 650 E. 25th Street Kansas City, MO 64108-2784
} -----Original Message----- } From: Ni, Chao-Ying [mailto:CYNi-at-rodel.com] } Sent: Friday, September 15, 2000 7:56 AM } To: Ekstrom, Harry; Microscopy-at-sparc5.microscopy.com } Cc: 'Ritchie Sims'; 'jim-at-proscitech.com.au' } Subject: RE: Carbon Evaporation Units } } } -------------------------------------------------------------- } ---------- } The Microscopy ListServer -- Sponsor: The Microscopy Society } of America } To Subscribe/Unsubscribe -- Send Email to } ListServer-at-MSA.Microscopy.Com } On-Line Help } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -------------------------------------------------------------- } ---------. } } } Harry, } } To the best of my knowledge, there is no such thing as } "sputtering carbon". } But I would suggest to look into your carbon source. Try } different vendors. } Also, make sure your vacuum is good. } } Have a nice day! } } Chao-Ying Ni } Rodel Inc. } 451 Bellevue Road } Newark, DE 19713 } (302) 366-0500 ext 2812 } } } -----Original Message----- } } From: jim [mailto:jim-at-proscitech.com.au] } Sent: Thursday, September 14, 2000 9:53 PM } To: 'Ritchie Sims'; Ekstrom, Harry; Microscopy-at-sparc5.microscopy.com } Subject: RE: Carbon Evaporation Units } } } -------------------------------------------------------------- } ---------- } The Microscopy ListServer -- Sponsor: The Microscopy Society } of America } To Subscribe/Unsubscribe -- Send Email to } ListServer-at-MSA.Microscopy.Com } On-Line Help } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } -------------------------------------------------------------- } ---------. } } } Retch is right, but you could also have a poor cooling system } on that pump, } or } just a badly designed system. In either case backstreaming of } oil vapour } would } lead to poorly adhearing films. } Cheers } Jim Darley } ProSciTech Microscopy PLUS } PO Box 111, Thuringowa QLD 4817 Australia } Ph +61 7 4774 0370 Fax:+61 7 4789 2313 service-at-proscitech.com } Great microscopy catalogue, 500 Links, MSDS, User Notes } ABN: 99 724 136 560 www.proscitech.com } } On Friday, September 15, 2000 6:55 PM, Retch Sims } [SMTP:r.sims-at-auckland.ac.nz] } wrote: } } } } } } } } } } } } We currently have a Bench Top Turbo carbon evaporation } unit made by } Denton. } } } We are very displeased with it. We consistently get non uniform } coatings } } } that peel off of our metallographic mounts. Any suggestions on a } } } evaporation unit that works well or any information on } 'sputtering" } carbon? } } } Any other experiences out there? } } } } } } } I would look very carefully at the cleanliness of the samples before } } coating. } } I use methanol then Freon then a dry Kimwipe, and the carbon } } coating is tenacious on brass. } } } } cheers } } } } rtch } } } } } } Ritchie Sims Phone : 64 9 3737599 ext 7713 } } Department of Geology Fax : 64 9 3737435 } } The University of Auckland email : r.sims-at-auckland.ac.nz } } Private Bag 92019 } } Auckland } } New Zealand } }
I am trying to locate a Zeiss (Oberkochen)phase-contrast condenser for their 160mm tubelength classic grey series of microscopes.
The phase-contrast condenser needs to have achromatic-aplanatic top lenses, for the phase ring inserts that I have. I am prepared to buy any condenser that i can locate. Can anyone out there help me?
(I hope that this is the right place to post this request, Nestor)
Please reply privately to me on jb_sanderson-at-yahoo.com
Regards, Jeremy Sanderson
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Basically a distillation process allows roughing pump oil to leak through the diffusion pump. The rough pump oil boils up through the top jet of the diff. pump where as a lighter weight component it may collide with another molecule and bounce up into the chamber and backstream. The heavier diffusion pump oil is less likely to lose its downward and forward momentum during a collision.
Ron Vane XEI Scientific Redwood City, CA 94061 650-369-0133
Is there a corresponding picture for a diff pump ie why do they let RP oil through? That's assuming, of course, that the oil that builds up on eds windows IS RP oil. It is, isn't it?
cheers
rtch
} } The reason that you can back up a turbomolecular pump with an oil-sealed } rotary vane pump and still have a system that is virtually free of oil } contamination is that the compression ratio of turbo pumps varies } exponentially with the square root of the molecular mass of the molecules } being pumped. Thus, for nitrogen, which has M = 28, this value is about } 200, whereas for a hydrocarbon molecule such as octane with M = 66 it is } greater than 3000, and for a molecule of pump oil with M greater than 100 } it is greater than a million. These values are for a single compression } stage (rotor disk) of a turbo pump. Actual turbo pumps have from 15 to 20 } such stages which act in series, so the overall value for nitrogen ends up } being around 10^9, while for hydrocarbons it becomes astronimical (10^40 is } often cited). Thus, if a turbo pump that is backed by a rotary vane pump is } managed so that the rotor never drops below about 75% of full operating } speed while the pressure in it is in the molecular flow range (below about } 1 Torr is a safe figure), it is virtually impossible for oil molecules to } backstream through it. These matters are all discussed in detail on pages } 229 to 234 of my book 'Vacuum Methods in Electron Microscopy' (see } http:/www.2spi.com/catalog/books/book48.html, and } http:/pup.princeton.edu/titles/6484.html for a description) and } appropriate operating protocols are are described on pp. 249 to 253 . } } Wilbur C. Bigelow, Prof. Emeritus } Materials Sci. & Engr., University of Michigan } Ann Arbor, MI 48109-2136 e-mail: bigelow-at-umich.edu; } Fx:734-763-4788; Ph:734-662-5237 } } } } Ritchie Sims Phone : 64 9 3737599 ext 7713 Department of Geology Fax : 64 9 3737435 The University of Auckland email : r.sims-at-auckland.ac.nz Private Bag 92019 Auckland New Zealand
} } My question is: how fast should this sort of printer print when using the } parallel cable to feed data to it? Is 5 minutes / 1MB unusually slow? I } get frustrated particularly as my computer is locked up while the data is } being sent out and I cannot continue imaging until the data dump is done. I } have the Win 95 spooler running of course and I don't have this problem with } my HP 970 inkjet which I use normally.
Richard,
I have the immediate predecessor to the 8670, the 8650. We have 64 meg ram in the printer. We have the ethercard connector because we were told it printed faster. There is a user sitting at the printer workstation right now printing a 14 meg image. It is taking about 35 seconds for Photoshop to process the file and export to our print server. The print server sends the image to the printer and it is taking 90 seconds to print. The NT Workstation with Photoshop is a 3 year old dual Pentium Pro with 512 meg ram. The Photoshop preferences are set to 3 scratch disks on an unused 9 gig HDD on the same machine. The NT print server is a Pentium II 400 with 256K ram. Postscript takes longer. Very large files take longer (sizes 30 meg and up). The delay occurs in Photoshop processing and between color panels once printing has started and for very large files the time can be as long as several minutes. B&W 1 meg files will print in 70 seconds.
Rick A. Harris, Director Microscopy and Imaging Facility Section of Molecular and Cellular Biology 1241 Life Sciences Addition University of California Davis, CA 530 752 2914 530 754 7536 fax http://katie.ucdavis.edu raharris-at-ucdavis.edu
Lots of antibodies don't work at LM immunocytochemistry and even more don't work at the EM level. I would like to make an informal survey. If you respond to me or the list, I will post a summary to the list.
Here are the questions:
Have you generated antibodies that work in some method (ELISA, Westerns, immunoprecipitation) but not LM immunocytochemistry?
Were they monoclonals or polyclonals?
What percentage of the antibodies you have made work at the LM level but not for EM immunocytochemistry?
If you have made antibodies against synthetic peptides, what percentage work in either LM or EM?
Thanks, Tom
-- Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility
3 Tucker Hall Division of Biological Sciences University of Missouri Columbia, MO 65211-7400 (573)-882-4712 (voice) (573)-882-0123 (fax)
This is going to be a *very* depressing survey.......Maybe you should include a salary survey and get all the glum stuff out of the way at one time?
Tamara Howard CSHL
On Mon, 18 Sep 2000, Tom Phillips wrote:
} Lots of antibodies don't work at LM immunocytochemistry and even more } don't work at the EM level. I would like to make an informal survey. } If you respond to me or the list, I will post a summary to the list. } } Here are the questions: } } Have you generated antibodies that work in some method (ELISA, } Westerns, immunoprecipitation) but not LM immunocytochemistry? } } Were they monoclonals or polyclonals? } } What percentage of the antibodies you have made work at the LM level } but not for EM immunocytochemistry? } } If you have made antibodies against synthetic peptides, what } percentage work in either LM or EM? } } Thanks, Tom } } -- } Thomas E. Phillips, Ph.D. } Associate Professor of Biological Sciences } Director, Molecular Cytology Core Facility } } 3 Tucker Hall } Division of Biological Sciences } University of Missouri } Columbia, MO 65211-7400 } (573)-882-4712 (voice) } (573)-882-0123 (fax) }
One more note on the oil found on EDS detector windows. It isn't always roughing pump oil. Rick Felten found through IR analysis that the plasticizer from the vacuum hose connecting the roughing pump to the diff pump was at fault. Black vacuum hose from Japan is a bad actor in this way. The Red Vacuum Hose made in the USA has less problems.
One more note on the oil found on EDS detector windows. It isn't always roughing pump oil. Rick Felten found through IR analysis that the plasticizer from the vacuum hose connecting the roughing pump to the diff pump was at fault. Black vacuum hose from Japan is a bad actor in this way. The Red Vacuum Hose made in the USA has less problems.
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Reply to: RE: % of antibodies that work in immuno Dear Tom,
Why bother? It is obvous that some antibodies will only work with a specific technique. Why this happens is still open to speculation, but it is most likely due to masking of antigenic sites. If an antibody is made that recognises a protein only when it is configured in its natural state then the antibody will not work by Western blot. It may work really well after the sample has been fixed in high aldehyde concentration (or after osmium fixation!) as long as it has been fixed in its "natural" configuration. I wonder how many really good antibodies were discarded because they did not label denatured protein on a Western?
If the fixation process is so efficient that other proteins are retained around the antigenic site, then the antibody will not work either. Maybe the antibody has been diluted in inappropriate blocking agent and does not work for EM when it worked for LM. One example I know of occurred when the researcher was using rabbit serum as a blocking agent. Everything worked well by LM but at the EM level, when he used protein A-gold, there was no signal. He diluted the gold probe in the rabbit serum which bound very efficiently to the gold and stopped it from binding to the antibody on the section.
Monoclonal antibodies are more specific than polyclonals, and anti-peptide antibodies are usually even more specific than many monoclonals. If the antigenic sequence is contained inside a molecule then is may become inaccessible after good fixation. If the antigen is a small peptide chain that can be easily dislodged, then it may be washed away duing the fixation or labeling process. Labeling success will depend on the preparation protocols used and each will be usiue for each antibody-antigen system.
All of these problems have solutions available to fix them. Saying an antibody works for one method but not another only serves to re-enforce the "black magic" aspect of our VERY sceintific discipline. All these statements really mean is that we either do not fully understand our preparation and labeling protocols, or that we have no idea what our antibodies are binding to. Regards,
Paul Webster
Paul Webster, Ph.D. Scientist II & Director Ahmanson Advanced Electron Microscopy & Imaging Center House Ear Institute 2100 West Third St. Los Angeles, CA 90057
First of all I would like to thank all of you who responded to my question on standards for measuring optical density in transmission light microscopy. As a service to the "list" (at least I hope it is) I post a summary of the responses I have got. Most of them were 'off line'. Different types of ND filters can be obtained from: 1. Edmund Industrial optics (http://www.edmundoptics.com/IOD/DisplayProduct.cfm?Productid=1945) A range of mounted or unmounted glass filters (ND 0.15-2.5) and a stepped one with 11 discrete density steps (0.1 steps from ND 0.04 to 1.0) 2. Lee Filters (http://www.leefilters.co.uk/) A range of unmounted filters (ND 0.1-0.9) and as a set of three (ND 0.3, 0.6 and 0.9) (material not specified, but might be available as resin or gelatin filters, according to the representative in the Netherlands) 3. Kodak (http://www.kodak.com/country/US/en/motion/support/h1/filtration.shtml#camfilm)
A range of unmounted filters (gelatin WRATTEN; ND 0.1-4.0) 4. Cargill Industries in NJ Not been able to find info (yet) 5. Densichron Not been able to find info (yet)
-- **************************************************** Lauran C.J.M. Oomen oomen-at-nki.nl The Netherlands Cancer Institute tel. +31 20 5121889 Digital Microscopy Facility fax. +31 20 5121893 Plesmanlaan 121 1066CX Amsterdam The Netherlands ****************************************************
Our ESEM has developed a problem with its beam. I don't know if many other instruments have this, but there is a "source" mode button which , when hit, provides a sort of view "back up the column towards the gun". It's used for saturating the filament - when properly saturated, the beam should look like a nice round to oval bright spot. Our instrument has a LaB6 source, which, because of the shape of the tip of the crystal, may produce four weaker "satellite" beams around the "main" beam - which is OK, you just want to be sure you're using the main beam. Just yesterday, and quite suddenly, this view changed somewhat - we no longer seem to have a nice oval spot, but instead a sort of cross shape, with one side smeared out into a separate, smaller beam. Signal has been dramatically reduced - I have to open up the condensor 'way more than I should, to get any kind of reasonable signal, and then the resolution is pretty degraded. I'm thinking the LaB6 has failed, perhaps with some small bit falling off one side. Previously, when one of these has died on us, it's pretty sudden and dramatic - suddenly no signal, period. Our gun vacuum is generally pretty good, but we have at least a thousand hours on this filament, so I suppose it may be on its last legs. Assuming I'm right in blaming it on the LaB6 - I can't think of any other reason for this problem - has anyone else had a filament fail in this way? I'd just like to hear of any other possible causes before I open up the gun. I suppose it's possible some little bit of crud found its way partially up the column and is causing severe astigmatism, but that sounds like a major disassembly effort, too.
F.C. Thomas MicroAnalysis Facility Bedford Institute of Oceanography Dartmouth, Nova Scotia Canada Geological Survey of Canada (Atlantic) Bedford Institute of Oceanography Dartmouth, Nova Scotia Canada B2Y 4A2
Thanks to all who responded with my question about slow printing from my Kodak dye sub printer. I have tried some things and have tracked the problem down some.
I found I can print via the parallel port to the printer just fine from a second computer (Dell 450) in the lab, using freshly reinstalled postscript drivers. A 1.2MB file prints in about 1 minute and the transfer to the printer (Progress in the Systray Printer icon box) goes in a few seconds. In turn the same file, same cable, same software, both win 95, but different computer (Compaq 333 attached to my Hitachi SEM) takes 4.5 minutes to get over the printer, before printing starts, transferring in blocks of about 60kB, 1 every 8-10 seconds. I am not logged onto the network, and have disabled my Norton anti-virus software.
This experiment rules out a lot of issues and now I'm focusing on the setup of my SEM computer to see why the Progress goes so slowly on it while it goes so much faster on the Dell. If anyone has ideas about THIS issue I'll gladly try them out. Maybe Hitachi's computer guy has some ideas.
Richard Shalvoy --------------------- original note
I have a Kodak 8670 dye sub printer hooked up to my SEM, but it seems to me that it takes a long time to get it to print and also while it is in the printing process my computer (and hence my SEM) is locked up. Here are the details.
I have the printer (1 year old) hooked to a PC with a 333 Pentium II processor (128 MB RAM) using a parallel cable. I do get the same performance when printing from other computers so I doubt that it is anything peculiar to this one computer.
Now when I call for a printout it takes 5 minutes to get the 1MB file size print done, and almost all of the time is spent sending data to the printer - the actual printing takes a short time (30 sec). I have discussed this with Kodak Tech Support and they claim my performance is not unusual. HOWEVER, I also experienced much quicker printing times (I'm sure - I have notes, but it was a while ago) when I was first using the printer (1 minute / 1 MB of file size in the image) and this is confusing me.
My question is: how fast should this sort of printer print when using the parallel cable to feed data to it? Is 5 minutes / 1MB unusually slow? I get frustrated particularly as my computer is locked up while the data is being sent out and I cannot continue imaging until the data dump is done. I have the Win 95 spooler running of course and I don't have this problem with my HP 970 inkjet which I use normally.
I will welcome any good ideas or cold realities that apply.
The XL30 FEG-ESEM is already mounted on a built-in air-supported anti-vibration system. Before you go ahead and specify (or buy) an air table, I would suggest you look very carefully at the resonances, and how they would be affected by the additional isolation. Things don't always get better - in fact, an air table could make your situation worse if the resonances are not carefully thought out.
At the very least, have a professional vibration analysis performed to show what vibrations are present at your site and how the built-in system is responding.
I don't know about the CM12 and how it is supported.
Tony Garratt-Reed.
} } Hi All, } I beg your pardon for bringing up this issue again, but } when I did not need it, I did not pay attention to postings... } } Now I have to include in a construction grant a price for } antivibration table for FEG-ESEM XL30 and, possibly, } for STEM CM12. In XL30 I have problems with vibrations } at magnifications 100-200k (and sometimes starting at 50k). } } Who got good results with antivibrition platforms? What } type? Should only a column be isolated or a whole instrument? } What is a probable price range (including installation)? } } Thanks, } } Vladimir }
** Anthony J. Garratt-Reed ** MIT Room # 13-1027 ** 77 Massachusetts Avenue ** Cambridge, MA 02139-4307 ** USA ** ** Phone: (+) 1-617-253-4622 ** Fax: (+) 1-617-258-6479 **
My use of Alar was a veerrryyy looong time ago, but, my recall is that the use of the warm fixative was more to fix the cells at the same temperature as the culture medium. This is due to the fact that many of the cells contract/round up/do nasty things when exposed to cold fixative. If you are concerned about membrane mobility artifacts, I would have thought that using a fixative that is the same temperature as the culture medium would be most appropriate. However, it might be useful to compare cells fixed at 37 and 4 C for your experiment. I know that this adds another layer of complexity onto the experimental design, but if this is a major issue, then I think it should be done. Let us know how it turns out.
Roger Moretz, Ph.D. Dept of Toxicology Boehringer Ingelheim Pharmaceuticals On Mon, 18 Sep 2000 11:44:12 +0100, Carole Elleman wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Dear All has anyone any experience with Agar's melinex film for growing } cells on before fixation and embedding for the TEM? I would like to find out } why it is necessary to fix the film with warm fixative - doesn't this } introduce some artefacts due to membrane mobility at 37 degrees? I am } looking at membrane antigens! } Carole Elleman } }
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As a coincidence, one of my technologists pulled your request for survey response off the web to tell me that I was under paying him. I presume that your classification of "engineer" refers to technical degree level people. I manage a group of 6 technical people with about ten state of the art level SEM's, TEM, EMP's and XRD equipment for a very long term AFTAC contract. AFTAC HQ is at Patrick AFB. Because we reside near Silicon Valley (just West of LLNL) we must reach deeper for starting rates here where entry level housing is about $300K. We cannot compete with the high tech firms that offer stock options, signing bonuses, etc. I just lost a materials scientist who about doubled her salary at a firm in Fremont. Obviously, our starting rates are a little higher than you indicate for engineers/scientists as well as technicians with little difference for degree status. I don't think that we have started anyone in our organization lower than the mid $40's in the past couple of years. Otherwise, we work with a similar salary range for non senior level professionals without significant supervisory responsibility.
Since no one has responded to this question in the past month I will provide my perspective. I have not seen any epoxy that will adhere to chitinous structures during sectioning. If someone else has please post the information. What I typically do when sectioning the eyes is to cut ten two micrometer sections on a dry glass knife then pick up the tissue (discard the piece of surrounding epoxy) with a fine needle probe and transfer it to a drop of water on a glass microscope slide. When I have enough sections the slide is moved to a hot plate for drying and later staining with toluidine blue. This works for the eye because epoxy infiltrates the underlying tissue from the backside. The proboscis is cut away and sometimes the head is cut in half during fixation. For fixing and infiltrating legs I would expect that this technique would work if they are cut in order to provide an opening at each end for fluid transfer. Best wishes.
Larry D. Ackerman Lily & Yuh Nung Jan Laboratories Howard Hughes Medical Institute UCSF, Box 0725, Rm U226 533 Parnassus Ave. San Francisco, CA 94143
When you solve this problem, I'd be really interested to hear what it was. Printers drive me nuts, too.
rtch
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Thanks to all who responded with my question about slow printing from my } Kodak dye sub printer. I have tried some things and have tracked the } problem down some. } } I found I can print via the parallel port to the printer just fine from a } second computer (Dell 450) in the lab, using freshly reinstalled postscript } drivers. A 1.2MB file prints in about 1 minute and the transfer to the } printer (Progress in the Systray Printer icon box) goes in a few seconds. } In turn the same file, same cable, same software, both win 95, but } different computer (Compaq 333 attached to my Hitachi SEM) takes 4.5 minutes } to get over the printer, before printing starts, transferring in blocks of } about 60kB, 1 every 8-10 seconds. I am not logged onto the network, and } have disabled my Norton anti-virus software. } } This experiment rules out a lot of issues and now I'm focusing on the setup } of my SEM computer to see why the Progress goes so slowly on it while it } goes so much faster on the Dell. If anyone has ideas about THIS issue I'll } gladly try them out. Maybe Hitachi's computer guy has some ideas. } } Richard Shalvoy } --------------------- } original note } } I have a Kodak 8670 dye sub printer hooked up to my SEM, but it seems to me } that it takes a long time to get it to print and also while it is in the } printing process my computer (and hence my SEM) is locked up. Here are the } details. } } I have the printer (1 year old) hooked to a PC with a 333 Pentium II } processor (128 MB RAM) using a parallel cable. I do get the same } performance when printing from other computers so I doubt that it is } anything peculiar to this one computer. } } Now when I call for a printout it takes 5 minutes to get the 1MB file size } print done, and almost all of the time is spent sending data to the printer } - the actual printing takes a short time (30 sec). I have discussed this } with Kodak Tech Support and they claim my performance is not unusual. } HOWEVER, I also experienced much quicker printing times (I'm sure - I have } notes, but it was a while ago) when I was first using the printer (1 minute } / 1 MB of file size in the image) and this is confusing me. } } My question is: how fast should this sort of printer print when using the } parallel cable to feed data to it? Is 5 minutes / 1MB unusually slow? I } get frustrated particularly as my computer is locked up while the data is } being sent out and I cannot continue imaging until the data dump is done. I } have the Win 95 spooler running of course and I don't have this problem with } my HP 970 inkjet which I use normally. } } I will welcome any good ideas or cold realities that apply. } } Richard Shalvoy } Arch Chemicals } 350 Knotter Drive } Cheshire, CT 06410 } 203-271-4394 } } Ritchie Sims Phone : 64 9 3737599 ext 7713 Department of Geology Fax : 64 9 3737435 The University of Auckland email : r.sims-at-auckland.ac.nz Private Bag 92019 Auckland New Zealand
I work in a lab that has great success with immuno labeling on a fluoresce laser confocual level, but taking the same antibodies to TEM thin sections is generally a lost cause. My success rate is probably around 5 to 10% (and that's a lot higher then my annual raises). Tim
Timothy G. Schneider Director of Electron Microscopy Department of Pathology Room 229 Jefferson Hall Thomas Jefferson University 1020 Locust St. Philadelphia Pa. 19107 215-503-4798 work 610-613-8170 cellular timothy.schneider-at-mail.tju.edu
I am having trouble obtaining good SEM preparations of cultured endothelial cell monolayers. The monolayers show evidence of artefactual cracking along cell borders. The cracks appear whether the cells are grown on glass coverslips or transwell filters. I have tried various fixatives (buffered glutaraldehyde alone, glutaraldehyde followed by osmium and uranyl acetate), different dehydrating agents (ethanol or acetone), followed by critical point drying (CPD). I have even tried tetramethylsilane (Ted Pella) in place of CPD. The only time I get good images (i.e., no cracks) is when a small portion of the monolayer has inadvertently detached from its substrate during tissue processing. The cells on this small flap look marvelous (no cracks). I believe, in the absence of substrate adhesion, the cells on the flap shrink uniformly during dehydration when surface tension forces exert their effects. Does anyone know how to prepare cell monolayers for SEM that leaves them intact and free from artefactual cracking?
Thanks.
Alan Burns, Ph.D. Assistant Professor Cardiovascular Sciences Department of Medicine Baylor College of Medicine
This relates to the question of printing from the computer that runs the SEM.
It is my policy in our facility that computers that operate microscopes such as our Hitachi S3500N and our confocals are not used for any other purpose. Images acquired are not saved on the computer that operates the device but rather are saved to a network drive (on a server) that is mapped to the individual user. All images from all devices go to the same server. The function of the server is to distribute these images, via direct connection, http, ftp to the individual users. The computers that run the devices are thus never clogged with images and are free of any programs other than those needed to run the device. The computers run faster and have fewer failures. They are setup the same as the day they were installed except for the network connection. I have often seen computer support personnel make the mistake of assuming that they can troubleshoot a computer than runs an SEM or confocal or other high end device only to discover that these computers are integral parts of the imaging device and should only be repaired by qualified technicians familiar with the device, not the computer.
Whenever possible, I suggest isolating the computer that drives your microscope from any other function.
Rick A. Harris, Director Microscopy and Imaging Facility Section of Molecular and Cellular Biology 1241 Life Sciences Addition University of California Davis, CA 530 752 2914 530 754 7536 fax http://katie.ucdavis.edu raharris-at-ucdavis.edu
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Listers- } } Our ESEM has developed a problem with its beam. I don't know if many other } instruments have this, but there is a "source" mode button which , when } hit, provides a sort of view "back up the column towards the gun". It's } used for saturating the filament - when properly saturated, the beam should } look like a nice round to oval bright spot. Our instrument has a LaB6 } source, which, because of the shape of the tip of the crystal, may produce } four weaker "satellite" beams around the "main" beam - which is OK, you } just want to be sure you're using the main beam. } Just yesterday, and quite suddenly, this view changed somewhat - we no } longer seem to have a nice oval spot, but instead a sort of cross shape, } with one side smeared out into a separate, smaller beam. Signal has been } dramatically reduced - I have to open up the condensor 'way more than I } should, to get any kind of reasonable signal, and then the resolution is } pretty degraded. } I'm thinking the LaB6 has failed, perhaps with some small bit falling off } one side. Previously, when one of these has died on us, it's pretty sudden } and dramatic - suddenly no signal, period. Our gun vacuum is generally } pretty good, but we have at least a thousand hours on this filament, so I } suppose it may be on its last legs. } Assuming I'm right in blaming it on the LaB6 - I can't think of any other } reason for this problem - has anyone else had a filament fail in this way? } I'd just like to hear of any other possible causes before I open up the } gun. I suppose it's possible some little bit of crud found its way } partially up the column and is causing severe astigmatism, but that sounds } like a major disassembly effort, too. } } F.C. Thomas } MicroAnalysis Facility } Bedford Institute of Oceanography } Dartmouth, Nova Scotia } Canada } Geological Survey of Canada (Atlantic) } Bedford Institute of Oceanography } Dartmouth, Nova Scotia } Canada B2Y 4A2
I am trying to create a positive control for a immunohistochemistry protocal. I have lyophilized protein which I have reconstituted in PBS. Does anyone have any ideas how I can adhere this protein to a slide so that it can be stained with the normal histo protocal.
I have tried drying the solution in the oven at 37C for different lengths of time. Should I use a gelatin solution.
Frank, Sounds like on of two things - either it failed that way because of the amount of hours(as you suggested), or, the tip was brought up to "saturation" way to fast.
Gary M. Easton Scanners Corporation Third Party EM Service ----- Original Message ----- } From: Frank Thomas {thomasf-at-AGC.BIO.NS.CA} To: {Microscopy-at-sparc5.microscopy.com} Sent: Tuesday, September 19, 2000 7:25 AM
Hi Alan We also had this problem but solved it by using tannic acid and making sure the cells were never fully uncovered.
I use two plastic pipettes - as I remove the liquid from the bottom of the culture dish, I add the new liquid to the top always making sure there is a thin layer of liquid on the cells. Three fillings of the chambers at each change ensures a good exchange.
Wash: 0.1M cacodylate buffer 3x5minutes
Postfix: 1%osmium tetroxide in 0.1M cacodylate buffer 30 minutes 1% tannic acid in 0.1M cacodylate buffer 30-60 minutes 1%osmium tetroxide in 0.1M cacodylate buffer 30 minutes
Rinse: ddw
Dehydrate: 5 minutes each in 30%, 50%, 70%, 85%, 95%, 100%, 100%, 100%
CPD.
If you remove all the liquid at a change of solutions, there can be damage to cellular processes such as filopodia. The tannic acid acts as a mordant and makes a real difference to the surface of the cells. I think this method is called the OTO method (Osmium, Tannic Acid, Osmium).
I have also used Tannic Acid in the buffer wash and then just used Osmium as normal. That worked too and was less expensive on Osmium. If you have empty wells when you use the OTO method, you can take off the first osmium and put it into an adjacent well. Then after the tannic acid, put the same osmium back.
Elaine
} } I am having trouble obtaining good SEM preparations of cultured endothelial } cell monolayers. The monolayers show evidence } of artefactual cracking along cell borders. The cracks appear whether the } cells are grown on glass coverslips or transwell filters. I have tried } various } fixatives (buffered glutaraldehyde alone, glutaraldehyde followed by osmium } and uranyl acetate), different dehydrating agents (ethanol or acetone), } followed by critical point drying (CPD). I have even tried } tetramethylsilane (Ted Pella) in place of CPD. The only time I get good } images (i.e., } no cracks) is when a small portion of the monolayer has inadvertently } detached from its substrate during tissue processing. The cells on this } small flap look marvelous (no cracks). I believe, in the absence of } substrate adhesion, the cells on the flap shrink uniformly during dehydration } when surface tension forces exert their effects. Does anyone know how to } prepare cell monolayers for SEM that leaves them intact and free } from artefactual cracking? } } Thanks. } } Alan Burns, Ph.D. } Assistant Professor } Cardiovascular Sciences } Department of Medicine } Baylor College of Medicine
-- Dr. Elaine Humphrey Biosciences Electron Microscopy Facility University of British Columbia 6270 University Blvd, mail-stop Botany Vancouver, BC CANADA, V6T 1Z4 Phone: 604-822-3354 FAX: 604-822-6089 e-mail: ech-at-unixg.ubc.ca
It could be that the printer port on the "slow computer" could be set up in bios "wrong", i.e. as a standard printer port, with no bi-directional capability. When booting up the Compaq, hit the appropriate key to enter bios setup. Look for an "integrated peripherals" section. Then, check your printer port selection. It should be set at least to the EPP mode or the EPP+ECP(this mode gives the port DMA capabilities which further enhance high speed transfers) mode. Save, and re-boot. Win95 should automatically recognize the change and load the appropriate drivers. If that doesn't work, upgrade the Compaq's memory by 64Mb's. Win95 spools print jobs to memory(if available - if not, the hard drive). Also, check the printers settings. Make sure it is set to "Spool" rather than "Print directly to printer". Good luck!
Gary M. Easton Scanners Corporation Third Party SEM Service ----- Original Message ----- } From: Shalvoy, Richard B **CHES {RBShalvoy-at-archchemicals.com} To: {Microscopy-at-sparc5.microscopy.com} Sent: Tuesday, September 19, 2000 8:50 AM
How is this really different from what others are talking about? If the computer has to talk to a printer, or in your case, to a network, the path is still external. A pristine example is when the computer saves to a local disk. Then, the disk is physically carried to some other computer and accessed at that point. A network interface requires system resources just as any other interface does. Arguably, the network can take more resources than other interfaces. It is not benign.
The method which SEM makers utilize a controlling computer is a big variable. Some use programmed data transfers while others use IRQs. The days of PIO are waning as PC OSs move to NT-type systems. PIO is not supported. Furthermore, this type of interface eats CPU bandwidth...in stark contrast to IRQ approaches. A big problem with Wintel systems is the lack of interrupts. It is an old and still aging architecture. Watch for Win to change in many ways. A DOS-less system is one of the key changes.
System management is a core function. This applies very well to computers and computer systems. A single computer shackled to a single unit is not a very useful application of resources. If the computer is so inextricably tied to the controlled system, I'd be very shy of such a system and architecture.
If a computer is "clogged" in any respect, then I would submit that these systems are poorly designed. The sore sticking point is whether these systems will be or are supported in the future. Too many computer-based products are here today, gone tomorrow. How many folks are suffering with Win3.1 or Win3.11 systems? Not much fun.
At 12:30 PM 9/19/00, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Reply to: Re: Immuno TEM Tim Schneider writes: I work in a lab that has great success with immuno labeling on a fluoresce laser confocual level, but taking the same antibodies to TEM thin sections is generally a lost cause. My success rate is probably around 5 to 10% (and that's a lot higher then my annual raises). Tim
Hi Tim,
I look forward to seeing more details about your preparation protocols before commenting on your specific case. However, in all instances it is important that general statements regarding antibody reactivity be accompanied by specific examples of protocols. Only then can we compare antibody performance.
Here is a more general reply that I hope stimulates some thought:
As an addendum to my last posting, I will make the very bold statment that if an immuno-signal is seen by light microscopy the signal will be seen by EM. How so? First look at the preparation protocol used for the LM preparation. Was it on unfixed, air dried, methanol-fixed cryostat sections? Maybe. It is a routine protocol for LM immunocytochemistry. Now look at the typical EM protocol. Good fixation to preserve morphology (maybe 4% formaldehyde and 0.1% gutaraldehyde), rapid dehydration because we do not want to wash away our antigens, finally embedding in LR White or maybe even freeze substitution into Lowicryl after rapid freezing and no chemical fixative. The morphology in the sections looks good, but where is the signal?
One possibility is that the signal cannot be generated because the sample has been so well fixed and embedded that the antigen, or the gold probe, or both cannot gain access. I bet if the same protocol used for LM was also used for the EM preparation, the signal would be there. If it could not, I would start looking for the smelly rat. Maybe the morphology would suffer a little, but as this isn't a problem for LM, why should this trouble us at the EM level? We only require the morphology to enable us to identify labeled structures.
Another possibility is that the signal really is there, but because it is in such low amount we discard it as background labeling. However, this may be the total amount of signal we will ever get on thin sections. A cryostat section is at least 10 microns thick. Dry this down to a thin smudge on the slide, make it totally accessible to antibodies by exposure to methanol and you have an 2-D sample with antigen concentrated down and totally accessible to antibody. Apply antibody, add a secondary fluorescent antibody (and maybe a bridging antibody to amplify the signal) and a very obvious result is obtained.
Take this same antibody and apply it to thin resin sections (the thinner the better to get good resolution) and there will be much less antigen present in the section (60nm v 10,000nm). Also the antibody or gold markers usually do not penetrate the section. This means that the only antigen available for binding will be the small fraction that is exposed on the surface of the section. If the antigen is present in small numbers anyway, then the specific signal will be low. If protein A-gold is used, this usually binds with antibody at a 1:1 ratio so it is possible that the specific signal for any section may be one gold particle over one structure on the whole section. This might be the result for this antibody. This is easy to check. First find out how many antigens are present in the sample and work out how many should be exposed on the section surface. Labeling efficiency (the relationship between antigen number to observed signal) is usually between 5-20% (ie. if 100 antigens are available for binding only between 5 and 20 will label). From this it is possible to get an estimate of how many antibodies will bind to the section. It is often surprising that signal is detected at all.
In conclusion: I repeat - if an antibody doesn't work, then we probably do not understand enough about the antigen to design a good labeling protocol.
Also: If the antibody works for LM it WILL work for EM. Keep preparation and labeling protocols the same for both and you will enjoy much success. I don't think that any one of my collection of 100's of antibodies works only by LM. They all also work by EM. Some do not work on resin sections and some do not work if I fix cells with glutaraldehyde, but they all label at the EM level. Some only give me one gold particle for every 20 vesicles they are supposed to label, but the particle is always over one of these vesicles. I look forward to a good discussion about this (I just finished the official writing!), but don't ask me about salaries.
Regards,
Paul Webster, Ph.D. Scientist II & Director Ahmanson Advanced Electron Microscopy & Imaging Center House Ear Institute 2100 West Third St. Los Angeles, CA 90057
Alan, problems with cells cracking when cultured as monolayers are mostly depending on artifacts induced when dehydrating or drying the samples. The most probable reason for your problem is shrinkage of the cells, which gives severe tensions between your rigid support and the fragile cells. It's very typical that you get good preservation with no shrinkage artifacts when the monolayer has lost contact with the support, as they are free to alter their volume without any hindrance from the support. In my experience, fixation with 2.5 % buffered glutar aldehyde is normally sufficient as a fixing agent. Acetone should be avoided as the rate of dehydrating is too rapid and will in may tissues induce uncontrolled shrinkage. As I don't know your exact protocol for dehydration and CP-drying, I can only suggest some hints, so what I would try is dehydrating with ethanol followed by drying from liquid CO2. I would start dehydration in a medium-low percentage, 60 - 65 % ethanol in water, and work through a series of steps with 5% increase in concentration up to 100% ethanol. Time in every step would be at least 10 - 15 minutes. Substituting the ethanol with liquid CO2 in the CPD is critical, so if you can flush the chamber with liquid gas with the chamber closed, give it at least 10 minutes before closing the unit completely. Wait at least 2 hours before another 10 minutes flush before starting the drying procedure. The temperature has to be very slowly increased, normally it takes us 20 minutes to reach the state when the pressure can be released, and this last step has also to be done very slowly, at least in terms of 30 minutes to reach normal pressure.
This would be the way I'd go for any type of a cultured monolayer, and normally works OK. Good luck! Yours sincerely
Per Hörstedt Department of Medical Biosciences Pathology Unit for Electron Microscopy University of Umeå S-90187 Umeå Sweden
Late comer to this thread, but... A few things to check: Do the 2 computers (other than the Dell's slightly faster CPU) have similar resources? That includes system memory & bus speed, open hard drive space for virtual memory, similar setup size constraints on swap file size (if any), similar background programs running, similar hard drive access speeds... Humm, can't think of others to check presently.
Also, are both parallel ports set to the same mode in the system BIOS?
If the Hitachi system is like mine, it is using a SCSI I/O to the SEM, but I don't believe that will seriously affect resources unless in use (not positive).
Given a choice I would dump Dell and/or Compaq and go for a custom, generic clone PC. I (we) had no choice on the Hitachi (Compaq), but I managed to do so for my IXRF EDS because of special circumstances - and have been very happy with the performance. I am somewhat biased (could you tell) about the proprietary stuff the bigger manufacturers add to their PCs. good luck... BTW, I am using an Epson 900, parallel I/O.
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Thanks to all who responded with my question about slow printing from my Kodak dye sub printer. I have tried some things and have tracked the problem down some.
I found I can print via the parallel port to the printer just fine from a second computer (Dell 450) in the lab, using freshly reinstalled postscript drivers. A 1.2MB file prints in about 1 minute and the transfer to the printer (Progress in the Systray Printer icon box) goes in a few seconds. In turn the same file, same cable, same software, both win 95, but different computer (Compaq 333 attached to my Hitachi SEM) takes 4.5 minutes to get over the printer, before printing starts, transferring in blocks of about 60kB, 1 every 8-10 seconds. I am not logged onto the network, and have disabled my Norton anti-virus software.
This experiment rules out a lot of issues and now I'm focusing on the setup of my SEM computer to see why the Progress goes so slowly on it while it goes so much faster on the Dell. If anyone has ideas about THIS issue I'll gladly try them out. Maybe Hitachi's computer guy has some ideas.
Richard Shalvoy --------------------- original note
I have a Kodak 8670 dye sub printer hooked up to my SEM, but it seems to me that it takes a long time to get it to print and also while it is in the printing process my computer (and hence my SEM) is locked up. Here are the details.
I have the printer (1 year old) hooked to a PC with a 333 Pentium II processor (128 MB RAM) using a parallel cable. I do get the same performance when printing from other computers so I doubt that it is anything peculiar to this one computer.
Now when I call for a printout it takes 5 minutes to get the 1MB file size print done, and almost all of the time is spent sending data to the printer - the actual printing takes a short time (30 sec). I have discussed this with Kodak Tech Support and they claim my performance is not unusual. HOWEVER, I also experienced much quicker printing times (I'm sure - I have notes, but it was a while ago) when I was first using the printer (1 minute / 1 MB of file size in the image) and this is confusing me.
My question is: how fast should this sort of printer print when using the parallel cable to feed data to it? Is 5 minutes / 1MB unusually slow? I get frustrated particularly as my computer is locked up while the data is being sent out and I cannot continue imaging until the data dump is done. I have the Win 95 spooler running of course and I don't have this problem with my HP 970 inkjet which I use normally.
I will welcome any good ideas or cold realities that apply.
Interesting post, but I have totake issue with one statement - I've read it over and I hopeI'm not taking this out of context; please excuse me if I have:
On 19 Sep 2000, Paul Webster wrote:
Lots of snipping.....} -----
} Maybe the morphology would suffer a little, but as this isn't a } problem for LM, why should this trouble us at the EM level? We only } require the morphology to enable us to identify labeled structures. } I depend on decent morphology to tell me that I haven't cause redistribution of the antigen/mRNA/gene at some time during the processing. If the sample looks like mush but has nice label, how am I to know that the label is where it was in vivo? I realize this may be extreme, to assume that everything is going to move if not properly fixed, but I think that it critical to keep this possibility in mind when localizing anything, at any level of detection.
My $0.02 this morning;hope I haven't ticked anyone off!
Tamara Howard CSHL (NY, USA for those of you who wonder!)
We have an old LKB III microtome that needs service. I called Leica but have not heard back -- and maybe never will-- they said they weren't sure they had anyone in the Washington DC area who could work on them.
Does anyone know of someone in this area who might be able to help? THere used to be a service engineer named Patricia Cappagrossi (I think) but I can't find any numbers for her...
TIA
Margaret -- Margaret Dienelt Plant Pathologist
Electron Microscopy Lab FNPRU, National Arboretum ARS, USDA
B. 010A R. 238 BARC-W 10300 Baltimore Avenue Beltsville, MD. 20705
Paul Webster said "I don't think that any one of my collection of 100's of antibodies works only by LM. They all also work by EM. "
Wow, that is amazing. I have never heard anyone having any where near that level of success. What type of epitopes are you looking at? What tissues are they located in? I could understand it if you were routinely using polyclonal antisera against carbohydrate epitopes - my own work with mucin molecules has shown me that carbohydrates are relatively easy to localize. How many of your antisera were made against synthetic peptides?
Can you tell me what you do when you get an IgM monoclonal to make it work at the EM level? I have never succeeded with one of them at the EM level.
You don't give your success rate for antibodies at the LM level? Have you succeeded with every antibody you have tried at the LM level? I have tried numerous ones that work for westerns or immunoppt that don't work at all at the light level (chemical fixation, precipitant fixes, quick-freeze) on cryo, paraffin, resin embedded protocols with or without a host of antigen retrival. Can you give me some references to your publications that outline your EM protocols for the "hard ones" or post your protocols on the list?
I routinely use 2% paraformaldehyde as a fix but vary this when I run into failure. I routinely screen in LR White, LR Gold, and Lowicryl K4M but in some (admittedly only a small percentage) have tried other plastics and heroic procedures. I have used ultra-thin cryo sections but, that it not really practical for some tissues (e.g., seed tissues with their hard coat and softer insides). Likewise, not every tissue is ammenable to nanogold permeabilization pre-embedding approaches (e.g., seed tissue again) . What do you do when you have tried 2% paraformaldehyde fixed tissues in LR White, LR Gold and Lowicryl and it still doesn't work? I routinely use labeled secondary antibodies to avoid the limitations with protein A. In most of my cases, the antigen level should be sufficient to see if the antibody worked.
How long does it take you to determine a successful protocol for each antibody?
You say "Keep preparation and labeling protocols the same for both and you will enjoy much success." I am not sure how to translate a positive result at the LM level with an acetone fixed whole mount of tissue culture cells grown on coverslips to the EM level. Do you use acetone or other precipitant fixes at the EM level? I have actually tried that but, not surprisingly, the quality of tissue preservation was not good enough for publication?
I know this is a lot of questions but your success rate is so much higher than mine, I am quite interested in hearing the details of your protocols.
Thanks, Tom
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-- Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility
3 Tucker Hall Division of Biological Sciences University of Missouri Columbia, MO 65211-7400 (573)-882-4712 (voice) (573)-882-0123 (fax)
Date sent: Tue, 19 Sep 2000 17:31:45 -0400 } From: "Spaulding, Robert F" {SpauldinRF-at-corning.com}
Hi,
A question about SEM interference. We have high electromagnetic field levels (about 5 times the Jeol recommended limits) and our SEM isn't much use above 10 000x mag (a Jeol 5410 low vacuum) - the focus is fuzzy and astigmatism hard to correct. I've had water and air pipes removed from the room, and we've been everywhere measuring fields but the fields and the poor performance don't change. Changing the room is not an option (to the people in charge of the institute!). Jeol recommended, ruling out a room change, that we fit a mu-metal shield around the column. I have contacted the company that makes the mu-metal and the thing would cost about US$6000.00 and a hassle to ship to Mexico, so I don't want to go ahead and find that we're in the same place with our performance.
Does anyone have experience with fitting a mu-metal shield to their microscope (especially a scanning EM) and resolving (or not resolving!) interference problems. I'd be very interested in hearing about your experiences with this stuff.
Thanks,
Mark
PS - the serial sections are coming along nicely (not perfect,but heading that way!), but I've still got a lot of ideas to try yet. Many thanks.
******************************************** Mark West, Unidad de Microscopia Electronica, (Electron Microscopy Unit) Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico, 04510 Mexico D.F.
At 08:11 PM 9/19/00 -0700, Gary Gaugler wrote: } How is this really different from what others are talking about? If the } computer has to talk to a printer, or in your case, to a network, } the path is still external. A pristine example is when the computer } saves to a local disk. Then, the disk is physically carried to } some other computer and accessed at that point. A network } interface requires system resources just as any other interface } does. Arguably, the network can take more resources than } other interfaces. It is not benign.
I disagree. Very few system chores are as demanding as printing while networking is inconsequential. Writing to a mapped drive on a 10/100 mbit card is virtually transparent. If you have a local switch then it would be entirely transparent. However, printing a 1 meg file, especially to a postscript printer, can bring your system to it's knees (as it does in this case). PS translation can easily increase a 1 meg file to many times it's original size with a corresponding hit on performance while the information is generated then spooled then sent to the printer. But that is not really the point I was trying to make. My point is that it is well to consider isolating your dedicated computers from everyday tasks such as printing and allow them to manage the device for which they were designed.
Also, we do not use removable media to transfer images from devices and prefer to save from the image acquisition device to a RAID 5 server. Removable media have too many failure points and generally require more system resources. If you start the Task Manager on your NT workstation and watch the performance hit while writing to a ZIP you can see it is considerable. Writing to a HDD or a NIC are about equivalent and much less than the ZIP. Printing locally takes a big chunk of CPU time and sending the print to a print server takes the least of all. You should also consider the printer setup. If you are sending 600 dpi files to a 300 dpi printer you are wasting system resources and gaining nothing in return so it is well to check the printer setup on the two machines our original letter writer was referring to. In my opinion, I think you should let the server do the grunt work and let the dedicated computer manage the device.
Rick A. Harris, Director Microscopy and Imaging Facility Section of Molecular and Cellular Biology 1241 Life Sciences Addition University of California Davis, CA 530 752 2914 530 754 7536 fax http://katie.ucdavis.edu raharris-at-ucdavis.edu
Frank, The other thing that can happen with a LaB6 is a rather severe accumulation of "crud" on and around the Wehnelt aperture. With the very long lifetime of the LaB6 crystal, you can accumulate significant deposit in this area. I have seen similar instances where the crystal is still OK, but the contaminants on the Wehnelt get very thick and begin to pull away from the wall of the aperture and create distortions in the emission through the Wehnelt assembly. In order avoid this I try to clean the Wehnelt cap approximately every six months. Good Luck, Brad Huggins BP Amoco, Naperville, IL
} ---------- } From: Frank Thomas[SMTP:thomasf-at-AGC.BIO.NS.CA] } Sent: Tuesday, September 19, 2000 6:25 AM } To: Microscopy-at-sparc5.microscopy.com } Subject: ESEM beam problem } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Listers- } } Our ESEM has developed a problem with its beam. I don't know if many } other } instruments have this, but there is a "source" mode button which , when } hit, provides a sort of view "back up the column towards the gun". It's } used for saturating the filament - when properly saturated, the beam } should } look like a nice round to oval bright spot. Our instrument has a LaB6 } source, which, because of the shape of the tip of the crystal, may produce } } four weaker "satellite" beams around the "main" beam - which is OK, you } just want to be sure you're using the main beam. } Just yesterday, and quite suddenly, this view changed somewhat - we } no } longer seem to have a nice oval spot, but instead a sort of cross shape, } with one side smeared out into a separate, smaller beam. Signal has been } dramatically reduced - I have to open up the condensor 'way more than I } should, to get any kind of reasonable signal, and then the resolution is } pretty degraded. } I'm thinking the LaB6 has failed, perhaps with some small bit } falling off } one side. Previously, when one of these has died on us, it's pretty sudden } and dramatic - suddenly no signal, period. Our gun vacuum is generally } pretty good, but we have at least a thousand hours on this filament, so I } suppose it may be on its last legs. } Assuming I'm right in blaming it on the LaB6 - I can't think of any } other } reason for this problem - has anyone else had a filament fail in this way? } I'd just like to hear of any other possible causes before I open up the } gun. I suppose it's possible some little bit of crud found its way } partially up the column and is causing severe astigmatism, but that sounds } like a major disassembly effort, too. } } F.C. Thomas } MicroAnalysis Facility } Bedford Institute of Oceanography } Dartmouth, Nova Scotia } Canada } Geological Survey of Canada (Atlantic) } Bedford Institute of Oceanography } Dartmouth, Nova Scotia } Canada B2Y 4A2 }
I am awaiting delivery of an "engineering kit" of muMetal from Advance (about $100, online order). We have some EMI from the SEM CRT which doesn't sound as bad as your situation, but does limit resolution. Since it is not practical to move the monitor far enough to mitigate the situation, I decided to try shielding. I am not yet sure how I will fit in/around the chamber , but am going to see if it will make a difference. Will let you know if it successfully eliminates the "vibes".
Woody White McDermott Technology, Inc.
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Hi,
A question about SEM interference. We have high electromagnetic field levels (about 5 times the Jeol recommended limits) and our SEM isn't much use above 10 000x mag {SNIP} ******************************************** Mark West, Unidad de Microscopia Electronica, (Electron Microscopy Unit) Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico, 04510 Mexico D.F.
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Reply to: Re: Immuno TEM Hi Tom,
Thanks for joining in. My posting was a good excuse to get people thinking about stuff they usually take for granted. I am surprized when people tell me about their lack of success with EM immunocytochemistry when often the problem can be solved by minor protocol modifications.
My antibodies really do all work by EM. I guess I have never really thought about why this is so, but one reason may be that I get to know the people who make them, find out as much as I can about what the antibodies label, and try to understand as much as I can about the biology of the system I work with. Some antibodies will only work on tissue culture cells that have been cultured for more than 15 days. Before this time, the antigen has not been produced in sufficent number and has not been inserted into it's functional site. I can go on with examples, but this really does not help you.
As for making IgM's work by EM, how do you store your antibodies? Do you make sure that the IgM's are not subject to freeze-thaw conditions? One round of this will break them up sufficiently to make them unuseable. Sometimes one freezing is enough if there is no cryoprotectant in the solution. Ask whoever made the antibodies if they had been frozen at least once. The surprizing thing is that for LM, even low affinity antibodies can be detected. Imagine that only about 10% of the IgM is in good shape and all the rest is totally fragmented. This will have two effects. First, the antibody dilution will have to be increased for EM detection. Second, the fragments may still bind to antigens on your section, but because they are fragments, the secondary antibody will not recognise them (know your reagents). They have competed for the specific binding sites, have won, but cannot be detected by your visualization antibody. At the EM level, this is a disaster. For LM, where there will be many more antigens present, and where amplification techniques can be used, the signal will still be detected. Know how the IgM fragments, and know what the secondary antibody recognises, and you may be on the way to understanding why the antibody does not work.
Have you ever purchased streptavidin-gold to localize biotin at the EM level? This is another tricky reagent to work with because it always seems to detect biotin at the LM level using silver enhancer. At the EM level there is never a signal. Usually, the first question I ask is what blocking agent is being used. If serum is used, then the gold probe will bind to biotin-like molecules in the serum, thus eliminating the specific label. More importantly, it seems that this probe is very unstable with the gold seperating from the protein after only short storage times. The presence of free streptavidin in the solution is not sufficient to inhibit labeling at the LM level, but it does remove almost all the EM label.
ANother example I have concerns an antibody that worked by LM but not by EM. This was an antibody that was made to a protein synthesized by endothelial cells. The solution took me a week to work out. My colleagues could see immunofluorescence labeling if they followed the protocols in published papers using the same antibody, and could even repeat the published result. Whenever they tried the antibody by EM, there was literally no signal. Now usually, it is possible to see one or two gold aprticiles on a grid that has been exposed to gold probe. After lots of thinking, and a few experiments, we found out that the specific antibody was being removed from our labeling solution. The blocking agent initially used to dilute the antibody was 10% serum. As the protein being studied was also a serum protein, the antibody was reacting to this molecule and we were getting a very good negative result (adsorption control). The authors of the published papers had never bothered to do obvious controls and were happy when they saw signal at the LM level. What were they looking at? It turns out that the cells they (and us) were using contained lots of autofluorescent organelles that were being interpreted as specific signal.
As for antibodies not working by LM. If I do not get a signal by LM, I give up the antibody as being no use to me. There is no way I would expect a signal by EM if I couldn't see it first by LM. I will usually screen antibodies first on the system I expect to use for EM labeling. Thin sections of Lowicryl-embedded material make really good LM preparations. It is possible to also test the EM immunoreagents by LM too. The silver enhancers are great for visualizing protein A-gold.
Limitations of protein A-gold, what are they? I think there are more disadvantages to using gold conjugated secondary antibodies. First, the resolution is not as good as with PAG. Second, the antibodies will deteriorate when stored. Antibody without gold will compete for binding sites and reduce the label. With PAG, one reagent, used by everyone in the lab will be less expensive and can also be easily monitored for quality. If everyone is using it successfully and one researcher has a problem, it is easy to identify where the problem is. If someone is using a secondary antibody that has not been used for 6 months, the troubleshooting procedure will be boring and time consuming. My work is mostly with mammalian cells and pathogens and I do not have many antibodies to carbohydrates. Mostly they are to membrane proteins or to other cellular components. I think I have about 6 anti-peptide antibodies that work for me.
The mention of pathogens and antibodies brings up another point. Many antibodies are made using Freund's adjuvant, a common protocol for antibody preparation. This is a homogenate of ground-up bacterial proteins. If the study involves looking at bacteria it is inevitable that some background labeling on bacteria will be observed. If we know how the antibody was prepared, then we will not be surprized by this result. If we do not know the preparation protocols, then we will spend lots of time trying out different fixation and blocking solutions, eventually giving up saying that the antibody is no good.
I hope these posts help answer some of your questions. Contact me again if there are issues I didn't address. Regards,
Paul.
Paul Webster, Ph.D House Ear Institute 2100 West Third Street Los Angeles, CA 90057 phone:213 273 8026 fax: 213 413 6739 e-mail: pwebster-at-hei.org http://www.hei.org/htm/aemi.htm
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Mark-
Are you sure that your problem is external EM interference? If it were 60Hz, then I would expect to see (at some scan rates and magnifications, anyway) a "jagginess" in the images, rather than "fuzziness". Also, if it is possible to lock the scan to the local power frequency, you could get some other recognizable effects in the image. If it is not 60Hz, but a much higher frequency, it may be getting into the 'scope circuitry and affecting the scan drives or other parts of the optics, rather than the magnetic field directly affecting the beam. A mumetal enclosure would not help this. Even if it is 60Hz, it can get in via ground loops (often accidentally set up) and would not be affected by mumetal.
Another totally different possibility is that you are blaming the wrong thing, and in fact you have a problem elsewhere, but because of the known high fields the service people have a "get out".
You say that your fields are 5 times the recommended, and that you cant use the scope above 10,000. This would imply that with the recommended fields the scope could be used up to 50,000. Is this realistic? (I don't know the specs of the 5410). If not, the implication is that there is another fault.
These are just thoughts, I know, not solutions, but hope they help.
Tony G-R
} } Hi, } } A question about SEM interference. We have high electromagnetic field } levels (about 5 times the Jeol recommended limits) and our SEM isn't much } use above 10 000x mag (a Jeol 5410 low vacuum) - the focus is fuzzy and } astigmatism hard to correct. I've had water and air pipes removed from the } room, and we've been everywhere measuring fields but the fields and the } poor performance don't change. Changing the room is not an option (to the } people in charge of the institute!). Jeol recommended, ruling out a room } change, that we fit a mu-metal shield around the column. I have contacted } the company that makes the mu-metal and the thing would cost about } US$6000.00 and a hassle to ship to Mexico, so I don't want to go ahead and } find that we're in the same place with our performance. } } Does anyone have experience with fitting a mu-metal shield to their } microscope (especially a scanning EM) and resolving (or not resolving!) } interference problems. I'd be very interested in hearing about your } experiences with this stuff. } } Thanks,
** Anthony J. Garratt-Reed ** MIT Room # 13-1027 ** 77 Massachusetts Avenue ** Cambridge, MA 02139-4307 ** USA ** ** Phone: (+) 1-617-253-4622 ** Fax: (+) 1-617-258-6478 **
Accurel Systems International Corp. has an immediate opening for a Dual Beam FIB Engineer.
Responsibilities include operating an FEI 820 and/or 835 Dual Beam FIB system to prepare SEM and TEM cross-sections for external customers on a commercial basis.
Qualified candidates must possess a B.S. or M.S. degree in the physical sciences or engineering or equivalent expertise. Although prior experience with FIB is not required, it is advantageous. Familiarity with IC processing and magnetic materials would also be advantageous.
Accurel Systems is an independent commercial laboratory located in the heart of Silicon Valley, 40 miles south of San Francisco. The laboratory is also well equipped for SIMS, Auger, ESCA, TEM, SEM AFM, FIB, XRD, TXRF, Failure Analysis Services etc.
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Does anyone have an old Edwards E306A (manual) vacuum evaporator mouldering in their lab? I need either a rototilt 3 electrical feedthrough or a blank plug for a size "B" hole. The same parts for the *A*306 (automatic) won't work, as Edwards changed the hole size when they changed models.
Thanks!
Phil -- }}}}}}}}}}}}}}}}}{{{{{{{{{{{{{{{{{ Philip Oshel AMFSC and BBPIC Dept. of Animal Health and Biomedical Sciences University of Wisconsin 1656 Linden Drive Madison, WI 53706-1581 voice: (608) 263-4162 fax: (608) 262-7420 (dept. fax)
Mark, Are you sure you have a em field problem? They usually show up as a large(assuming the frequency is low, 50-60hz) sawtooth on the displayed image. This sawtooth will change in appearance with different sem scan rates, i.e., at TV rate, the images appears to "shake" back & forth, and at the slowest visual rate, will exhibit a very well defined sawtooth. If you do have a photo CRT(probably not), the photo scan is usually synchronized to the AC line frequency, hence negating the effect of any EM interference, again, assuming your "external fields" are the same frequency. Did you shut down the SEM completely, and disconnect it from its power source when making your field checks? This is very important. If the symptoms are a fuzzy image and high astigmatism, I would check my SEM parameters to ensure that the controls are properly set for high resolution - WD, spot size, aperture size, tilt, etc.. If these are okay, look for dirt somewhere in the column, column mis-alignment, poor vacuum levels. Also make sure that the beam crossover pole pieces are inserted correctly and that the column liner tube apertures are in the correct spot. Has this problem always been there since the instrument was initially installed, or did it develop afterwards. I really don't think mu-metal will fix your problem. Good Luck!
Gary M. Easton Scanners Corporation Third Party SEM Service
----- Original Message ----- } From: "Mark West" {mwest-at-ifcsun1.ifisiol.unam.mx} To: {microscopy-at-sparc5.microscopy.com} Sent: Wednesday, September 20, 2000 12:13 PM
We have a very low success rate as well, but I am convinced that it is because we are a fee for service lab and the PI's do not want to take the time or spend the money on the numerous trials that might be required to get acceptable results. I think, that if given the opportunity, we could have a much better success rate. But we are treated as a commodity and not a research endeavor. Greg Erdos Gregory W. Erdos, Ph.D. Assistant Director, Biotechnology Program PO Box 110580 University of Florida Gainesville, FL 32611
I would be interested in heaing from anyone who has done a market survey on JEOL 8200 versus CAMECA SX100 microprobes. Which ( in the responders opinion ) has the better system for geological quantitative WDS analysis, or performs better in the SEM mode. Please give the reasons behind your preference, if at all possible. PLEASE, NO VENDOR RESPONSE.
Gary L. Lovell Phillips Petroleum Company 245a GB Bartlesville, Ok. 74004 Phone: (918)661-9691 Fax : (918)662-2047 Email: gllovel-at-ppco.com
One of our students needs a receipe for citrated saline and cannot find it in the very limited references we have available here (a materials lab where one group is doing some tissue culture). She has seen it mentioned in articles but it must be so basic (or maybe acidic) that no one tells how it is made. I hope someone can enlighten us.
Microscopy and Digital Imaging: Advances and Applications
University of Washington Seattle, WA
October 5, 2000 Room CD150, CHDD Early Education Unit 8:30 AM to 4:30 PM
A day of presentations, demonstrations and refreshments. The first in a series of opportunities to learn and share knowledge about all aspects of microscopy and digital imaging for biomedical research.
Confocal Microscope Demonstrations October 4-6th: Bio-Rad - Radiance laser scanning confocal Solamere Technologies - Spinning disk confocal
Sponsored by: The UW Center on Human Development and Disability The W.M. Keck Center for Advanced Studies in Neural Signaling The Virginia Merrill Bloedel Hearing Research Center Supported by the Bio-Rad Corporation.
Please RSVP to Glen MacDonald, glenmac-at-u.washington.edu, 616-4156, to register for attendance to the workshop, confocal demonstrations or to meet with Dr. Johnson and Dr. Danilchik. Lunch will be provided for all who pre-register by October 2nd.
Schedule: 8:30 Welcome 8:45 Glen MacDonald, UW CHDD/VMBHRC - “What is in a Digital Image, What Happens When You Enhance It?” 9:45 Coffee and snacks 10:00 Iain Johnson, Molecular Probes - “Introduction to Fluorescence and Fluorescent Probes” 11:00 Mike Danilchik, University of Oregon Health Sciences Center - “Using Confocal Microscopy to Study Early Events in Xenopus laevi Development” 12:00 Lunch 1:00 Peter Rabinovitch, UW Dept. Pathology - “ Telomere Length Measurements in Tissue Sections by Quantitative FISH Confocal Microscopy” 2:00 John Jordan, Bio-Rad - “Recent Developments in Confocal Microscopy at Bio-Rad” 2:40 George Peeters, Solamere Technology - “Introducing the Spinning Disk Confocal Microscope and its Applications ” 3:00 Coffee and snacks 3:15 Iain Johnson - “A Demonstration of Web-based Resources for Fluorescent Microscopy, Where Does the Molecular Probes’ Technical Support Staff Get Their Information?”
--
Glen MacDonald Virginia Merrill Bloedel Hearing Research Center Box 357923 University of Washington Seattle, WA 98195-7923 glenmac-at-u.washington.edu (206) 616-4156 (206) 616-1828 fax ************************************************************************ C:} The box said "Requires Windows95 or better". So I bought a Macintosh. ************************************************************************
We have not done this for a SEM, but have mu-metal around our TEM, and it works very well. Before you go to the expense, you may want to try some soft iron as an experimental shield. It is not the best (and certainly not pretty) but it works and will tell you if the problem really is fields and not something else.
++++++++++++++++++++++++++++++++++++++++++++++++ Laurence Marks Department of Materials Science and Engineering Northwestern University Tel: (847) 491-3996 Fax: (847) 491-7820 mailto:ldm-at-risc4.numis.nwu.edu http://www.numis.nwu.edu ++++++++++++++++++++++++++++++++++++++++++++++++
Are you running the scope in high vacuum mode when you have these problems? What are the operating conditions and sample characteristics when you have this trouble?
I have trouble on our Hitachi 2460N getting decent pictures above 5kx using 40Pa of He. I also re-proved the point that gold coatings are beneficial. I was looking at some silicon samples in our JEOL 840A at 10kx. I could get images without charging, but the quality was poor. The pictures looked much better with gold since the secondary signal was stronger and generally coming from an area closer to the beam.
At 10:13 AM 9/20/2000 -0600, you wrote: } Hi, } } A question about SEM interference. We have high electromagnetic field } levels (about 5 times the Jeol recommended limits) and our SEM isn't much } use above 10 000x mag (a Jeol 5410 low vacuum) - the focus is fuzzy and } astigmatism hard to correct. I've had water and air pipes removed from the } room, and we've been everywhere measuring fields but the fields and the } poor performance don't change. Changing the room is not an option (to the } people in charge of the institute!). Jeol recommended, ruling out a room } change, that we fit a mu-metal shield around the column. I have contacted } the company that makes the mu-metal and the thing would cost about } US$6000.00 and a hassle to ship to Mexico, so I don't want to go ahead and } find that we're in the same place with our performance. } } Does anyone have experience with fitting a mu-metal shield to their } microscope (especially a scanning EM) and resolving (or not resolving!) } interference problems. I'd be very interested in hearing about your } experiences with this stuff. } } Thanks, } } Mark } } PS - the serial sections are coming along nicely (not perfect,but heading } that way!), but I've still got a lot of ideas to try yet. Many thanks.
---------------------- Warren E. Straszheim Materials Analysis and Research Lab Iowa State University 23 Town Engineering Ames IA, 50011-3232
Scanning electron microscopy, x-ray analysis, and image analysis of materials Computer applications and networking
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Hi
Any one still awake who can tell me which ports to mount 3 x wds spectros on an 840 which has the OM?
The three spectros are
STE/RAP PET/LIF PET/LIF
The three ports on the head of the OM aren't the same, which leads me to suspect that the positioning may not be arbitrary.
Please express your answer numbering the ports clockwise from the operator, bird's-eye view, 1, 2, 3.
rtch
Ritchie Sims Phone : 64 9 3737599 ext 7713 Department of Geology Fax : 64 9 3737435 The University of Auckland email : r.sims-at-auckland.ac.nz Private Bag 92019 Auckland New Zealand
Does anyone out there know of a SEM/TEM facility in the DFW area that is willing to rent time on their machine? .
I'm posting this for a colleague who has been using both SEM and TEM facilities on a pay for use basis for forever. Unfortunately those facilities are under new management who are reluctant to give outside users access to their equipment. So he is looking for alternatives. Thanks in advance for your input.
Email: laniven-at-julian.uwo.ca Name: Leigh Ann School: UWO Question: What is GFP? How do you convert a microscope for birefringence and what type of microscopy is this?
Listers, Anyone out there know of a good(hopefully easy!) way to remove carbon tracking deposits on a ceramic electon gun? Thanks in advance. Gary M. Easton Scanners Corporation
Date sent: Wed, 20 Sep 2000 10:13:03 -0600 (Hora estándar de México) } From: Mark West {mwest-at-ifcsun1.ifisiol.unam.mx} To: microscopy-at-sparc5.microscopy.com
} I found I can print via the parallel port to the printer just fine from a } second computer (Dell 450) in the lab, using freshly reinstalled postscript } drivers. A 1.2MB file prints in about 1 minute and the transfer to the } printer (Progress in the Systray Printer icon box) goes in a few seconds. } In turn the same file, same cable, same software, both win 95, but } different computer (Compaq 333 attached to my Hitachi SEM) takes 4.5 minutes } to get over the printer, before printing starts, transferring in blocks of } about 60kB, 1 every 8-10 seconds. I am not logged onto the network, and } have disabled my Norton anti-virus software. } } This experiment rules out a lot of issues and now I'm focusing on the setup } of my SEM computer to see why the Progress goes so slowly on it while it } goes so much faster on the Dell. If anyone has ideas about THIS issue I'll } gladly try them out. Maybe Hitachi's computer guy has some ideas.
Basically, you are using the wrong kind of OS and hardware for trying to do two things at once. "Go unix :-("
It seems that the key feature here is that the computer is also trying to control the SEM at the same time. Presumambly there is a resources bottlenck because the computer is desperately trying to do too many things in too many little chunks too often? If you've checked out the printing side and that's OK then maybe take a look at how the computer is talking to the microscope. At what rate does the computer normally try to "converse" with the microscope for example. If you used to be able to run the microscope and simultaneously print in reasonable time when the system was first installed, then perhaps this microscope polling rate (or whatever) has been subsequently increased excessively. Too fast a rate could cause an excessive slowdown when you try to get the computer to do something else intensive such as printing. Will your microscope software allow you to find out how often the computer talks to the microscope and allow you to change it? You might only need to slow down the rate of conversation with the microscope slightly to get a big improvement in both operations. Some experimentation might allow you to find an acceptable balance between printing time and microscope responsiveness.
Just a thought.
} } Richard Shalvoy } --------------------- } original note } } I have a Kodak 8670 dye sub printer hooked up to my SEM, but it seems to me } that it takes a long time to get it to print and also while it is in the } printing process my computer (and hence my SEM) is locked up. Here are the } details. } } I have the printer (1 year old) hooked to a PC with a 333 Pentium II } processor (128 MB RAM) using a parallel cable. I do get the same } performance when printing from other computers so I doubt that it is } anything peculiar to this one computer. } } Now when I call for a printout it takes 5 minutes to get the 1MB file size } print done, and almost all of the time is spent sending data to the printer } - the actual printing takes a short time (30 sec). I have discussed this } with Kodak Tech Support and they claim my performance is not unusual. } HOWEVER, I also experienced much quicker printing times (I'm sure - I have } notes, but it was a while ago) when I was first using the printer (1 minute } / 1 MB of file size in the image) and this is confusing me. } } My question is: how fast should this sort of printer print when using the } parallel cable to feed data to it? Is 5 minutes / 1MB unusually slow? I } get frustrated particularly as my computer is locked up while the data is } being sent out and I cannot continue imaging until the data dump is done. I } have the Win 95 spooler running of course and I don't have this problem with } my HP 970 inkjet which I use normally. } } I will welcome any good ideas or cold realities that apply. } } Richard Shalvoy } Arch Chemicals } 350 Knotter Drive } Cheshire, CT 06410 } 203-271-4394
Arthur Day, Electron Microscope Unit Phone: 61-2-9717-3457 Ansto Materials Division Fax: 61-2-9543-7179 PMB 1, Menai (Sydney), NSW, 2234 Email: ard-at-ansto.gov.au Australia www: http://www.ansto.gov.au/
Before heading down an expensive path with the mu-metal, check out a few basic things first. The power source and step-down transformer would be easy to verify if they are correct.
Make sure the power is wired with a ground that runs all the way back to the distribution panel and is not grounded to the local disconnect box which would be a "conduit" ground. This type of ground is a good source
for noise to the microscope. Also, have a look at the step-down transformer. This could be incorrectly wired too.
I would imagine with a 5410 you could roll it down the hall, plug it in and see if it changes anything.
Bob Roberts EM Lab Services, Inc. 2409 S. Rural Rd Suite C Tempe, Arizona 85282 (480) 967-3946
I am after a supplier (preferably in Australia) for some doncaster dishes. These are glass petri dishes / watchglass dishes containing raised, concentric circles. Thanks Garry
Dr. Garry Rosewarne Plant Industry CSIRO, Black Mountain
On Wed, 20 Sep 2000 20:26:39 -0500, laniven-at-julian.uwo.ca wrote:
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| | Email: laniven-at-julian.uwo.ca | Name: Leigh Ann | School: UWO | Question: What is GFP? How do you convert a microscope for birefringence | and what type of microscopy is this? |
Hi Leigh Ann - I can answer the first part of your question (to some extent), but I'm not very sure about the second part. GFP = Green Fluorescent Protein ; it's a protein that naturally fluoresces, and I believe that it was first discovered as a natural product from some marine animal (name ?). What I know of GFP is that it's gaining usage as a fluorescent marker. I believe it's been used as a marker for determining the location of X compound in a cell when viewed by fluorescent microscopy, but I'm sure that other usages have been cited in other research papers. As for the second part ... someone in this list may correct me, but I believe that birefringence is correlated with polarized light. You'll definitely need to consult with someone else in this list to get more information. Good luck getting more answers. Nelson Conti
_______________________________________________________ Say Bye to Slow Internet! http://www.home.com/xinbox/signup.html
I assume that the tracks are on a ceramic insulator which is not glazed, I am not sure what might happen if a glazed surface is abraded.
Alumina powder made up to a paste in Alcohol is good for polishing out tracks on high voltage insulators (guns and accelerators). If it is really bad then grit blasting with Alumina powder may be needed. Make sure you don't damage seals or other surfaces and that you wash thoroughly to remove all traces of the powder. (That's wash the gun of course, you should not get it all over yourself.) The insulator should be gently baked out in a (clean) vacuum oven to remove the final traces of solvent, but you may get away with baking out gently in air and pumping out for a long time before applying the voltage.
Beware of the limitations imposed by other materials used in the constuction of the component, epoxy resins etc.
Good luck, Ron
On Wed, 20 Sep 2000 20:15:50 -0500 "Gary M. Easton" {gary.easton-at-scannerscorp.com} wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Listers, Anyone out there know of a good(hopefully easy!) way to } remove carbon tracking deposits on a ceramic electon gun? Thanks in } advance. Gary M. Easton Scanners Corporation } } }
---------------------- Mr. R.C. Doole Department of Materials, University of Oxford. Parks Road, Oxford. OX1 3PH. UK. Phone +44 (0) 1865 273701 Fax +44 (0) 1865 283333 ron.doole-at-materials.ox.ac.uk
the dryers we're using are a couple of Polaron E-3000 CPD's, and they are working perfectly. The main advantage is that you manually control inlet- outlet- and evacuation valves, which means you can totally control the substitution of dehydration agent when the chamber is closed. Temperature is controlled with ordinary tap water, controlled by an ordinary thermostat-mixer. They also have a large viewing window that makes it very easy to visualize everything that goes on inside the chamber, i.e. for example the level of liquid CO2 which is very important when flushing the chamber. The chamber has a large volume that can contain many and/or large samples. I've had a workshop making some sets of specimen carriers for different types of specimen, so for example when working with biopsies, we can prepare 24 at the same time. We've used them for many years now and I find them highly recommendable. Yours sincerely
Per Hörstedt Department of Medical Biosciences Pathology Unit for Electron Microscopy University of Umeå S-90187 Umeå Sweden
Building test shields from 0.30 cent per pound iron is a good test for how well MU metal will work.. If you have an Ag Engineering department you might be able to borrow it from them at no cost except on favor to be announced at a later date. .30 cent iron my be less expensive.
Gordon Gordon Couger gcouger-at-couger.com Stillwater, OK www.couger.com/gcouger 405 624-2855 GMT -6:00 ----- Original Message ----- } From: {"bobrob-at-uswest.net"-at-sparc5.microscopy.com} To: {Microscopy-at-sparc5.microscopy.com} Sent: Thursday, September 21, 2000 12:10 AM
HCL works great as long as you protect the other (metal) elements.
Earl
"Gary M. Easton" wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Listers, Anyone out there know of a good(hopefully easy!) way to } remove carbon tracking deposits on a ceramic electon gun? Thanks in } advance. Gary M. Easton Scanners Corporation
-----Original Message----- } From: Granville Sent: Thursday, September 21, 2000 1:51 PM To: Luc
Hi Ann, GFP is the Green Fluorescence Protein. Jellyfish Aequorea produces bioluminescence from two proteins, aequorin, which is a Ca++ binding protein that produces blue light. GFP emits green light by absorption of the aequorin. GFP was discovered in1962 by O. Shimomura. In 1992 D.Prasher and colleagues sequenced it. Now there are variants of GFP. GFP is a 238 amino acid protein. Absorption at 395 nm and 475 nm, emission at 508 nm. It can expressed in a cell/tissue /animal with along another protein of interest. It is, therefore, a reporter or label. It can be used to report gene expression or gene transfer, for a highly specific label for protein localization, quantification or dynamics. greetings Werner
Margaret, You may call Norman J. Woodside, an ex-LKB employee, and he may help you. His address and phone and fax numbers: NJW Supplies 7 Tanglewood Rd. Catonsville, MD 21228 Phone: 410-744-9574; Fax 410-744-1580 Thanks! Pat
Birefringence is observed under a light microscope by putting a polarizer before the specimen (and generally before the condenser) and a similar device called the analyser between the specimen and the eyepiece: the analyser is usually set at 90^ to the polarizer. If the crystal (or even a piece of stretched plastic or a blob of liquid crystal) has different refractive indices in different directions, the polarized light passing through the specimen gets divided into two out-of-phase components which are recombined in the analyzer to give pretty interference colours, from which one can also make quantitative deductions. The people that use this technique most are geologists and mineralogists looking at thin polished rock sections, but we plastics people also look at polymers in this way. I'm not sure if it's used much in bio/med applications.
A good book to explain the physics and applications of the technique is:
Wood, Elizabeth Armstrong Crystals and light.
while there are many books which concentrate more on the setup of the microscope. Here's a selection out of our library catalogue:
Hartshorne, N.H. Crystals and the polarising microscope.
Polarized light microscopy by Walter C. McCrone, Lucy B.McCrone and John Gustav Delly
The polarizing microscope by A. F. Hallimond Hallimond A.F.
+------------------------------------------------------------------------+ | Robert H.Olley Phone: | | J.J.Thomson Physical Laboratory {direct line +44 (0) 118 9318572 | | University of Reading {University internal extension 7867 | | Whiteknights Fax +44 (0) 118 9750203 | | Reading RG6 6AF Email: R.H.Olley-at-reading.ac.uk | | England URL: http://www.reading.ac.uk/~spsolley | +------------------------------------------------------------------------+
} Email: laniven-at-julian.uwo.ca } Name: Leigh Ann } School: UWO } Question: What is GFP? How do you convert a microscope for birefringence } and what type of microscopy is this?
Tamara Howard writes: } Maybe the morphology would suffer a little, but as this isn't a } problem for LM, why should this trouble us at the EM level? We only } require the morphology to enable us to identify labeled structures. } I depend on decent morphology to tell me that I haven't cause redistribution of the antigen/mRNA/gene at some time during the processing. If the sample looks like mush but has nice label, how am I to know that the label is where it was in vivo? I realize this may be extreme, to assume that everything is going to move if not properly fixed, but I think that it critical to keep this possibility in mind when localizing anything, at any level of detection.
I seem to have caught someone's attention with my very controvertial post. First let me address Tamara's question, which also covers part of an issue brought up by Tom Phillips. You take exception that I should advocate the use of poorly fixed material for EM localization. Yet, at the LM level this same poor fixation is considered a routine part of the protocol. How many people who perform LM immunolocalizations routinely freeze the tissue without aldehyde fixation or cryoprotection and then fix the sections with acetone or methanol, air-drying them before or after this treatment? Who would conside that they are working with a) badly fixed material?, b) material where the target antigens have either been removed or relocated? My guess is that very few people have even given this a thought. [If you think that performing LM immunochtochemistry using aldehyde fixed material followed by detergent permiabilization (without drying the sample) may I direct you to Hannah, Weiss & Huttner 1998 Differential extraction of proteins from paraformaldehyde-fixed cells: Lessons from synaptophysin and other membrane proteins Methods (a companion to Meth. Enzymol.) vol16 pp170-181)].
These issues about morphology are never taken into consideration at the LM level. Why then should we decide it is important to have good morphology at the EM level? One reason is that we are attempting perfection and feel that good morphology is aesthetically pleasing. More importantly we need to have sufficient contrast information within the sections to obtain a colocalization with recognizable subcellular structures (and hopefully see the antigen location at sites where it exists in vivo).
If we are able to perform multiple labeling experiments, where we can use one antibody to identify specific organelles, then absolute morphology is not important. We can disrupt our cells so that they become bags of loosely packed organelles and our antibodies have total accessibility to antigens. We can even partially purify these organelles and work with them as a pellet or adsorbed onto specimen grids. We will loose spatial information if we do this, but we will get a signal.
Similarly, if the only way we can get a signal by LM is by air drying and acetone fixing, then use the same protocol for EM. The protocol will be easy (pre-embedding label) but the morphology will be awful. Remember, this is what light microscopists accept as good morphology! Once the label system has been established to work by EM (ie all the reagents work to give a result), then it will be possible to start modifying the protocol to improve morphology. Perhaps fixing the sample in 2% formaldehyde for 30 seconds, followed by a short homogenation (improve membrane preservation and control cellular disruption) is the answer. Maybe a fixation in 5% glutaraldehyde in a low osmolality buffer, so that the cells swell, is the answer. The exact recipe will depend on what is known about the antigen. If it is a membrane protein, is the epitope deeply embedded inside the membrane? If so, will this require membrane disruption (freeze-thaw, detergent?) or will it be sufficient to just remove the cytoplasm? Each system has to be approached as a completely new project and each will have an answer. Knowing as much as we can about the antigen is our first step to success, allowing ourselves to work with samples that do not have "text-book" quality morphology is the next step. Being totally open about how we are willing to prepare samples and look at our results, completes the process. My protocols will not help many people with their own particular immunolabeling problems but my experience might. So will the experience of all the other researchers who have faced similar problems. Many different immunolabeling protocols are published but, not surprizingly, are not broadcast in technical journals. The best protocols are hidden in scientific papers where the approach was unique and led to an interesting discovery about a biological system. As I said, the methods are unique to each system. There is little point in publishing lists for someone to follow without thinking.
We have to face the fact that whatever method we use to prepare our sample, each will have limitations. Our role is to try to make these limitations work in our favor. Knowing what the limitations are for each preparation protocol makes our job a little easier.
Do you want my replies to Tom Phillips posted here, or should I reply to him off-line? I am sure there are many people already bored by this discussion.
Regards,
Paul Webster.
Paul Webster, Ph.D House Ear Institute 2100 West Third Street Los Angeles, CA 90057 phone:213 273 8026 fax: 213 413 6739 e-mail: pwebster-at-hei.org http://www.hei.org/htm/aemi.htm
At 3:04 PM -0400 9/20/00, Greg Erdos wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Lee
Leona Cohen-Gould, M.S. Sr. Staff Associate Director, Electron Microscopy Core Facility Manager, Optical Microscopy Core Facility Joan & Sanford I. Weill Medical College of Cornell University voice (212)746-6146 fax (212)746-8175
At 8:26 PM -0500 9/20/00, laniven-at-julian.uwo.ca () wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Dear Leigh GFP stand for green fluorescent protein. It is a naturally fluoescent protein, originall isolated from a jelly fish and nowavailable as a construct that you can insert into your protein of choice using routine molecular biology techniques. It obviates the need for immuno-labelling. In order to see it, you need a UV light source that emits at/around 488nm and the appropriate fluorescence filter sets. The ones designed for FITC work just fine. YOu'd need to add a fair number of components to your 'scope...if you can convert it. Your local microscope sales/service person will be able to gie you the details.
Lee
Leona Cohen-Gould, M.S. Sr. Staff Associate Director, Electron Microscopy Core Facility Manager, Optical Microscopy Core Facility Joan & Sanford I. Weill Medical College of Cornell University voice (212)746-6146 fax (212)746-8175
I'm sorry, but I think questions like yours aren't fair. You may get a number of very public gripes from people who may have a good point OR are getting bad results because of their poor skills. Respectable vendors are defenceless against these things because if they rebutted all complaints it could look like they were petty or argumentative or had something to hide. Consider the Golden Rule.
I can remember seeing a respected vendor raked over the coals by a TEM operator who claimed that the TEM in question was unstable. It turns out that he liked bright screens (easier to focus) and recorded all of his pictures of biological tissue specimens with the largest beam size and the beam at crossover.
It's best that you conduct your own comparisons, using your own bread-and-butter specimens. If you are just at the info gathering stage, it would have been MUCH better, in my opinion, to REQUIRE private responses to you instead of having them all go the listserver. You've already required vendors to remain silent!
Ron
Ron Anderson, IBM, Hopewell Jct., New York, USA. anderron-at-us.ibm.com
IBM Analytical Services; http://www.chips.ibm.com/services/asg
"Gary Lovell" {gllovel-at-ppco.com} on 09/20/2000 04:22:52 PM
To: Microscopy-at-sparc5.microscopy.com cc:
I would be interested in heaing from anyone who has done a market survey on JEOL 8200 versus CAMECA SX100 microprobes. Which ( in the responders opinion ) has the better system for geological quantitative WDS analysis, or performs better in the SEM mode. Please give the reasons behind your preference, if at all possible. PLEASE, NO VENDOR RESPONSE.
Gary L. Lovell Phillips Petroleum Company 245a GB Bartlesville, Ok. 74004 Phone: (918)661-9691 Fax : (918)662-2047 Email: gllovel-at-ppco.com
Greetings, Paul Webster asks: Why then should we decide it is important to have good morphology at the EM level?
One answer is that the questions at light and electron levels are distinct. At the light level, we might interested in finding out what tissue expresses a particular antigen, whereas at the electron level, we might be wondering whether it is in the outer or the inner membrane of the mitochondrion. In the latter case, if we can't distinguish outer from inner membrane, let alone a mitochondrion from a lysosome, we are in trouble.
Greetings, Paul Webster asks: Why then should we decide it is important to have good morphology at the EM level? snip snip
One answer is that the questions at light and electron levels are distinct. At the light level, we might interested in finding out what tissue expresses a particular antigen, whereas at the electron level, we might be wondering whether it is in the outer or the inner membrane of the mitochondrion. In the latter case, if we can't distinguish outer from inner membrane, let alone a mitochondrion from a lysosome, we are in trouble.
Years ago while doing lithography using a JEOL 840A, we observed definite* line frequency interference in some of the patterns. The source of the noise was traced to a power bus running in the ceiling to another lab, which could not easily be changed.
JEOL provided a mu-metal shield on a trial basis that was specifically made for the 840. As I recall, their price (~10 years ago) was $6k US. We could quantitatively measure the change in the patterns and found that the interference was reduced by about a factor of 3. Note that this shield surrounded just the lower part of the column, i.e., it extended just above the sample chamber.
With that said, I will now add that I agree with the other responses that suggest the external noise may not be your primary problem. In my experience, I have not seen that line frequency interference causes "fuzzy" focus and astigmatism problems. Since the SEM scan is (or should be) in sync with the line frequency, line frequency interference problems can show up as localized distortions in the image position, depending on the scan frequency.
There are so many possible reasons for fuzzy focus and astigmatism problems (as other responses have pointed out), that it is hard to suggest where you should start. I think you could get better suggestions if you could describe the recent history of the SEM related to its performance. For example, has the SEM always been in its present location and has it always had this problem? If it was moved to the room with the interference, did it work properly before this? What is the best performance that has been seen on your 5410? Have you contacted other 5410 users to check on their experiences? (JEOL should have a user list. If not, I have two customers with 5400's that may be able to offer some guidance.) Do you have an experienced SEM operator who can work with your SEM and diagnose the problems? (I know you have JEOL working on it, but while many SEM technicians - from all SEM vendors - are very good, there are always those who are just learning or just don't really care. Also, as pointed out previously, since your room is out of spec, they have an easy excuse and may not really be trying to solve the problem.)
Joe _________________________________________ Joe Nabity, Ph.D. JC Nabity Lithography Systems E-Beam Lithography using Commercial SEMs & STEMs PO Box 5354, Bozeman, MT 59717 USA Voice: (406) 587-0848 FAX: (406) 586-9514 E-mail: info-at-jcnabity.com Web: www.jcnabity.com
(*When doing lithography, you can often measure the frequency of the interference in the patterns. Not that it really helps you, but if you are interested, example lithography patterns with line frequency noise problems can be seen at "www.jcnabity.com/pictures.htm" under the Diagnostic Images heading.)
To 1 litre with deionised water, pH to 7.8 with hydrochloric acid.
Hope this helps.
Lesley Weston.
On Wed, 20 Sep 2000, Ronald LHerault wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } One of our students needs a receipe for citrated saline and cannot find it } in the very limited references we have available here (a materials lab } where one group is doing some tissue culture). She has seen it mentioned } in articles but it must be so basic (or maybe acidic) that no one tells } how it is made. I hope someone can enlighten us. } } Thanks. } } Ron } lherault-at-bu.edu } } } }
I must have missed the original question, but Birefringence is the difference between the two refractive indices displayed by an anisotropic substance under polarized light observation.
Phil Aviles Forensic Microanalyst
} -----Original Message----- } From: Nelson Conti [SMTP:NelsonC51-at-excite.com] } Sent: Thursday, September 21, 2000 12:50 AM } To: Leigh Ann } Cc: Microscopy-at-sparc5.microscopy.com } Subject: Re: What is GFP? } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } } On Wed, 20 Sep 2000 20:26:39 -0500, laniven-at-julian.uwo.ca wrote: } } | } ------------------------------------------------------------------------ } | The Microscopy ListServer -- Sponsor: The Microscopy Society of America } } | To Subscribe/Unsubscribe -- Send Email to } ListServer-at-MSA.Microscopy.Com } | On-Line Help } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } | } -----------------------------------------------------------------------. } } | } | Email: laniven-at-julian.uwo.ca } | Name: Leigh Ann } | School: UWO } | Question: What is GFP? How do you convert a microscope for } birefringence } | and what type of microscopy is this? } | } } } Hi Leigh Ann - } I can answer the first part of your question (to some } extent), but I'm not } very sure about the second part. } GFP = Green Fluorescent Protein ; it's a protein that } naturally } fluoresces, and I believe that it was first discovered as a natural } product } from some marine animal (name ?). What I know of GFP is that it's gaining } usage as a fluorescent marker. I believe it's been used as a marker for } determining the location of X compound in a cell when viewed by } fluorescent } microscopy, but I'm sure that other usages have been cited in other } research } papers. } As for the second part ... someone in this list may correct } me, but I } believe that birefringence is correlated with polarized light. You'll } definitely need to consult with someone else in this list to get more } information. } Good luck getting more answers. } Nelson Conti } } } } } } _______________________________________________________ } Say Bye to Slow Internet! } http://www.home.com/xinbox/signup.html }
Dear Listers, I am seeking input from those of you who use an automated critical point dryer. The chamber on all of those I've considered is much smaller than that of the Polaron E3000 which I have used for many years. What are the advantages of automated / semi-automated critical point dryers? Rosemary
Rosemary Walsh, Manager The Electron Microscope Facility for the Life Sciences, A Shared Technology Facility, The Life Sciences Consortium 1 South FrearLab Penn State University University Park, PA 16802 (814) 865-0212 rw9-at-psu.edu http://www.lsc.psu.edu/stf/em/home.html
Hello, I am looking for a supplier of Mitsubishi thermal dye-sub paper (with ribbon) catalog CK 200sa for printer. I was supplied by Kipp and Sons, apparently now out of business. Preferable that supplier be close to DC Baltimore region. Thank You.
Hi All, I have a picky question that I think would be good for all of us to be clear on, so I'll ask. Can someone define the difference between formaldehyde and formalin? My impression has always been that formalin is to formaldehyde as Kleenex is to tissues (aka a type of brand). I have just come across a protocol that specifically says "formalin (not formaldehyde)". Thanks for your help, Kristen
Kristen A. Lennon Department of Plant Pathology 351 Bessey Hall Iowa State University Ames, IA 50011 email: kalen-at-iastate.edu
Observing birefringence is a very useful means for identifying and locating optically anisotropic materials, i.e. crystalline and polymeric materials. For example, oxalates in tissues would display obvious birefringence, whereas the rest of the tissue would not and would appear black with the polarizer and analyzer inserted (termed "crossed nicols"). See the response below from Olley for setup, but I would recommend the purchase of a polarizing (transmitted light) microscope rather than converting something. The "pretty interference colors" are termed birefringence and should be viewed without the use of the condenser lens. Conoscopic light can be used, however, for such things as the determination of the orientations of the maximum and minimum refractive indices, the magnitude of the anisotropy, and the orientation of the optic axes. These are best determined by observing the interference figure that forms above the objective lens with the nicols crossed and a Bertrand lens. A retardation plate (gypsum, mica, or quartz wedge) is often inserted between the sample and the analyzer. If you don't have a Bertrand lens, simply remove the ocular to view the figure (be sure that the condenser inserted).
All this seems totally unrelated to GFP. You can put a UV source on a polarizing light microscope if necessary.
Donald Miser
} -----Original Message----- } From: Robert H. Olley [SMTP:r.h.olley-at-reading.ac.uk] } Sent: Thursday, September 21, 2000 9:14 AM } To: laniven-at-julian.uwo.ca } Cc: Microscopy-at-sparc5.microscopy.com } Subject: Re: What is GFP? } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } } Leigh Ann, } } Birefringence is observed under a light microscope by putting a polarizer } before the specimen (and generally before the condenser) and a similar } device called the analyser between the specimen and the eyepiece: the } analyser is usually set at 90^ to the polarizer. If the crystal (or even } a piece of stretched plastic or a blob of liquid crystal) has different } refractive indices in different directions, the polarized light passing } through the specimen gets divided into two out-of-phase components which } are recombined in the analyzer to give pretty interference colours, from } which one can also make quantitative deductions. The people that use this } technique most are geologists and mineralogists looking at thin polished } rock sections, but we plastics people also look at polymers in this way. } I'm not sure if it's used much in bio/med applications. } } A good book to explain the physics and applications of the technique is: } } Wood, Elizabeth Armstrong } Crystals and light. } } while there are many books which concentrate more on the setup of the } microscope. Here's a selection out of our library catalogue: } } Hartshorne, N.H. } Crystals and the polarising microscope. } } Polarized light microscopy by Walter C. McCrone, Lucy B.McCrone } and John Gustav Delly } } The polarizing microscope by A. F. Hallimond } Hallimond A.F. } } } +------------------------------------------------------------------------+ } | Robert H.Olley Phone: } | } | J.J.Thomson Physical Laboratory {direct line +44 (0) 118 9318572 } | } | University of Reading {University internal extension 7867 } | } | Whiteknights Fax +44 (0) 118 9750203 } | } | Reading RG6 6AF Email: R.H.Olley-at-reading.ac.uk } | } | England URL: http://www.reading.ac.uk/~spsolley } | } } +------------------------------------------------------------------------+ } } } Email: laniven-at-julian.uwo.ca } } Name: Leigh Ann } } School: UWO } } Question: What is GFP? How do you convert a microscope for birefringence } } and what type of microscopy is this?
Hello everyone on the list who is doing Immuno-EM! Dear Greg, Paul, Tom and Tim
I have read your contributions with great interest and smiled everytime. Ok, since my bible in Immunoelectronmicroscopy is the outstanding book of G.Griffiths ("Fine structure immunocytochemistry", 1993), and as I am trying to do good IEM for over three years, I do agree with all of you.
First, let me state that it is particulary difficult to enable monoclonal antibodies for EM immunoprocedures. In fact, I was not able to run a good EM-Immuno with a monoclonal one (but the polyclonals do work.). And I agree with you, most of the (monoclonal) antibodies provided today are very well suited for Western blotting and sometimes even for LM because they were fitted for degenerative conditions applied in these techniques. I am always aware of that approximately 90% of the epitopes are gone, due to embedding and sectioning.
Second, many collegues who do have quite satisfactorily results at LM ask me why, the signal at EM is not worth looking at, to poor. Many of them are not aware of the dimensions we are talking about, a few milimetres in square and a section thickness of 50-70 nms is little, and fixation and post-processing leads to a loss of antigenicity.
Third, if you do have an antibody running well in IEM, use it. There are, in fact too few AB suitable for IEM. I am wondering why there isn´t any company already which is offering AB´s for IEM which recognize their epitope after aldehyde degeneration/fixation and acrylic plastic embedding.
At least, counting your signal should help. Sometimes you don´t recognize specific signals because you only see almost nothing at a magnification of x20,000 or more. If you start counting more fields, you are often surprised by the high specifity, althoug it´s very weak.
So, my two cents to this topic are: Try different antibodies from different Labs made against the same epitope! If it works, you should always use it as reference marker. And don´t forget: IEM does take so much more time and spirit as LM, this business is more tricky.
The best wishes for the IEM´s on this list.
Michael Reiner
PS: In memoriam Hildy Crowley, I already do miss her reply to this topic. _______________________________________________________________________ 1.000.000 DM gewinnen - kostenlos tippen - http://millionenklick.web.de IhrName-at-web.de, 8MB Speicher, Verschluesselung - http://freemail.web.de
Formaldehyde is a gas which may be dissolved in water.
A 40% solution of formaldehyde dissolved in water is called Formalin. Normally, various additives are put into the 40% solution of formaldehyde in order to stabilize it (I believe that traces of methanol may be added). Because of impurities, Formalin is usually considered unsuitable for electron microscopy studies. Confusion often arises when people refer to percentages of these solutions. For example, a 10% solution of formaldehyde is quite different than a 10% solution of Formalin ((10% versus 4%, respectively).
Hope this answers your question.
} I have a picky question that I think would be good for all of us to } be clear on, } so I'll ask. Can someone define the difference between formaldehyde and } formalin? My impression has always been that formalin is to formaldehyde as } Kleenex is to tissues (aka a type of brand). I have just come across } a protocol } that specifically says "formalin (not formaldehyde)".
#################################################################### John J. Bozzola, Ph.D., Director Micro-Imaging and Analysis Center 750 Communications Drive - MC 4402 Southern Illinois University Carbondale, IL 62901 U.S.A. Phone: 618-453-3730 Fax: 618-453-2665 Email: bozzola-at-siu.edu Web: http://www.siu.edu/departments/shops/cem.html ####################################################################
} Hi All, } I have a picky question that I think would be good for all of us to be clear on, } so I'll ask. Can someone define the difference between formaldehyde and } formalin? My impression has always been that formalin is to formaldehyde as } Kleenex is to tissues (aka a type of brand). I have just come across a protocol } that specifically says "formalin (not formaldehyde)". } Thanks for your help, } Kristen } } Kristen A. Lennon } Department of Plant Pathology } 351 Bessey Hall } Iowa State University } Ames, IA 50011 } email: } kalen-at-iastate.edu
Hi Kristen:
Formaldehyde is a gas, when bubbled through water the solution becomes saturated to about 37-40%. Methanol is usually added as a stabilizer. Formalin is the name often given to a 1:9 dilution of the "formaldehyde" solution, it is about 3.7-4% formaldehyde. The nomenclature is not exact, to put it mildly. To know exactly the person who wrote the protocol means, you will probably have to ask him/her. I don't know how you could have formalin without formaldehyde!
Geoff -- ********************************************** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854 voice: (732)-235-4583; fax: -4029 mcauliff-at-umdnj.edu **********************************************
I concur with everything John said and point out that one also commonly sees 3.7% formalin in protocols. This is the "same" as 4% usually since it depends on whether you mean % w/w or % w/v (i can't remember which is which).
Also, besides the impurities, formalin is often bought in gallon or barrel sizes so it can sit around a long time and form polymers not present in freshly depolymerized paraformaldehyde.
So the words should never be taken as synonomous.
Tom
} } } Formaldehyde is a gas which may be dissolved in water. } } A 40% solution of formaldehyde dissolved in water is called } Formalin. Normally, various additives are put into the 40% solution } of formaldehyde in order to stabilize it (I believe that traces of } methanol may be added). Because of impurities, Formalin is usually } considered unsuitable for electron microscopy studies. Confusion } often arises when people refer to percentages of these solutions. } For example, a 10% solution of formaldehyde is quite different than } a 10% solution of Formalin ((10% versus 4%, respectively). } } Hope this answers your question. } } } I have a picky question that I think would be good for all of us to } } be clear on, } } so I'll ask. Can someone define the difference between formaldehyde and } } formalin? My impression has always been that formalin is to formaldehyde as } } Kleenex is to tissues (aka a type of brand). I have just come } } across a protocol } } that specifically says "formalin (not formaldehyde)". } } #################################################################### } John J. Bozzola, Ph.D., Director } Micro-Imaging and Analysis Center } 750 Communications Drive - MC 4402 } Southern Illinois University } Carbondale, IL 62901 U.S.A. } Phone: 618-453-3730 } Fax: 618-453-2665 } Email: bozzola-at-siu.edu } Web: http://www.siu.edu/departments/shops/cem.html } ####################################################################
-- Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility
3 Tucker Hall Division of Biological Sciences University of Missouri Columbia, MO 65211-7400 (573)-882-4712 (voice) (573)-882-0123 (fax)
Formaldehyde is a gas. An aqueous solution saturated with the gas is called formalin. This saturated solution is called 100% formalin, which contains approximately 37% formaldehyde. Thus, a solution of 10% formalin is very different in strength from a solution that is 10% formaldehyde. Thus, an instruction may say to use "10% formalin (not formaldehyde)" to make sure that you indeed are using the correct strength.
DL
Kristen A. Lennon wrote: } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Hi All, } I have a picky question that I think would be good for all of us to be clear on, } so I'll ask. Can someone define the difference between formaldehyde and } formalin? My impression has always been that formalin is to formaldehyde as } Kleenex is to tissues (aka a type of brand). I have just come across a protocol } that specifically says "formalin (not formaldehyde)". } Thanks for your help, } Kristen } } Kristen A. Lennon } Department of Plant Pathology } 351 Bessey Hall } Iowa State University } Ames, IA 50011 } email: } kalen-at-iastate.edu
-- ____________________________________________________________________ Donald L. Lovett voice: 609-771-2876 Assoc. Professor fax: 609-637-5118 Dept. of Biology e-mail: lovett -at- tcnj.edu The College of New Jersey P.O. Box 7718 www.tcnj.edu/~scholars/lovett.html Ewing, NJ 08628-0718
Kristen: Formalin is commonly used to describe a mixture of37% to 38% formaldehyde in water (this reagent grade chemical is also called formaldehyde solution). This solution of dissolved formaldehyde gas generally contains some small amount of methanol in it also. Now for the good part! Traditionally this reagent grade mixture of the 37% or so formaldehyde in water is considered a 100%formalin concentration (the maximum concentration range you can buy commercially). A traditional 10% formalin solution would then contain about 3.7% to 3.8% aldehyde concentration. Many E-M folks prefer to make their formaldehyde solutions from the polymer paraformaldehyde--in part I think believing that there is less methanol in the final product yet folks have had success using both kinds in the right application. I hope this helps to clarify the common usage of these terms.
Dear Listers, For preembedment EM-ISH, is a two-step detection method more sensitive than a single-step method? Specifically, I am planning to detect digoxigenin-labeled RNA probes with mouse anti-dig antibody, followed by anti-mouse gold-conjugated secondary antibody (then enhancing ultrasmall gold particles). Or would direct detection of the dig probes with an anti-dig gold-conjugated antibody lend any advantage? One review attributes higher sensitivity for two-steps, but I think this might be restricted to postembedment methods only (J. Histochem. Cytochem. 45:481-92,1997).
If a two-step method is used, would gold-conjugated antibodies (against biotin probes) have advantages over streptavidin-gold? As recently discussed on this list, unconjugated streptavidin will compete with the gold-labeled streptavidin, thus reducing signal strength. Nanoprobes makes covalently bound gold antibodies, but I have seen a higher background with these than with noncovalently bound antibodies, at least in cultured rat neurons.
Finally, will glutaraldehyde levels greater than 0.05% tend to inhibit hybridization? Rat glioma monolayers have been labeled with EM-ISH by Punnonen, et al., using 4% paraformaldehyde and 0.05% glut for 30 min. (J. Histochem. Cytochem. 47:99-112, 1999). However, this protocol included 0.1% saponin permeabilization for 30 min. Their probes were up to 850 bp and "partially hydrolyzed" while my probes are only 50 bp long. I have immunolabeled neurons fixed in 2% glut with no permeabilization; so if the antibodies can make it into the cells, then the smaller RNA probes should too, right?
I know that target abundance is an issue here. I plan to include positive controls for abundant RNAs (e.g., beta-actin), and I will consider Tyrimide Signal Amplification, if necessary.
Thank you for your helpful comments on any of these questions. I will be happy to elaborate if this inquiry is too brief.
Michael Plociniak Research Technician Albert Einstein College of Medicine Neuroscience Dept. Rose Kennedy Center, 529 Bronx, NY 10461 (718) 430-3509 plocinia-at-aecom.yu.edu
Win95 has a utility named system monitor (Start | Programs | Accessories | System Tools |). I don't believe it's installed by default. You may have to add it through the custom install section. It could help you rule out the physical disk, CPU, and RAM as bottlenecks. You might compare the charts using your different printers. It's a "watch it in real time" affair though you might be able to use {Alt-Print Screen} to get a snapshot for comparison or baselining (it does work, I tried it : ) ).
It contains counters to monitor your CPU, physical disk, virtual memory, and network connections. A knowledgeable person in your computer department should be able to help you sort which counters might be useful and what they mean. Or you could play with different counters. Unfortunately the help portion of the utility is the stuff of legends.
I'd probably start with Kernel Processor Usage %, File System Bytes Written/Second & Dirty Data (This sounds like the amount of queued data), and Memory Manager Disk Cache Size & Page Faults.
Hope it helps,
Chuck ------------------------------------- Name: Charles Gilbert VOC: (704) 355-5261 Carolinas Medical Center FAX: (704) 355-8424 Dept of Pediatric Research digPager: (704) 355-4088 : 2058 PO Box 32861 Charlotte, NC 28232-2861
Please keep the discussion coming! I have found it extremely informative and helpful, as I am attempting pre-embedding anti-GFP immunolabelling with nanogold and silver enhancement. ImmunoEm is so important now that many people have localized their GFP transfected proteins by confocal. They want to know what are the fluorescent blobs they cannot quite resolve. Before I attempt any immunoEm with anyone, they must do a fixation series of formaldehyde and glutaraldehyde dilutions to see where they lose their fluorescent signal. We attempt to get the best fixation possible, yet penetration of the ab is an issue too. It is always a compromise. There are many ways to skin a cat, and only time and effort will hopefully give you an answer. JoAnn Buchanan Stanford University School of Medicine Stanford, CA
} I have a picky question that I think would be good for all of us to be clear on, } so I'll ask. Can someone define the difference between formaldehyde and } formalin? My impression has always been that formalin is to formaldehyde as } Kleenex is to tissues (aka a type of brand). I have just come across a protocol } that specifically says "formalin (not formaldehyde)". } Thanks for your help, } Kristen
As I have understood it, formalin is the (fairly concentrated) aqueous solution, formaldehyde is the actual pure compound HCHO.
Not that I've ever used, the stuff, though.
cheers
rtch
Ritchie Sims Phone : 64 9 3737599 ext 7713 Department of Geology Fax : 64 9 3737435 The University of Auckland email : r.sims-at-auckland.ac.nz Private Bag 92019 Auckland New Zealand
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Reply to: Re: Immuno TEM Tobias Baskin writes: One answer is that the questions at light and electron levels are distinct. At the light level, we might interested in finding out what tissue expresses a particular antigen, whereas at the electron level, we might be wondering whether it is in the outer or the inner membrane of the mitochondrion. In the latter case, if we can't distinguish outer from inner membrane, let alone a mitochondrion from a lysosome, we are in trouble.
Hi Tobias,
I agree totally. The level of acceptance for poor morphology will depend on the result expected. Again, each system will have its own protocol for getting that particular result. I am not encouraging everyone to go out and prepare samples with such poor morphology that any result they get will be meaningless. This would be irresponsible for me to advocate and a waste of time for anyone who was foolish enough to follow this advice.
Rather, I suggest that if it is possible to label purified (or semi-purified) sub-cellular particles, then do that. If you are looking for antigens on the outside of mitochondria, perform a pre-embedding labeling on purified fractions. As the mitochondria are so recognizable, it is not even necessary to get very pure fractions. If the antigen is inside the mitochondria, then sectioning is probably the best way. But even then, if the profile that is labeled is so obviously a mitichondion, then it doesn't matter if the cristae are not perfectly preserved (unless there is extraction or redistribution of antigen). If specific membrane markers are available, structural morpholgy is even less important. Co-localizing the marker with the test antibody on a membrane or organelle fragment is often very acceptable. Sometimes semi-purified fractions can be obtained just by breaking open cells to let out the antigens we are not interested in (unless the antigens are cytoplasmic of course). Gently lysed cells have wonderful contrast and it is possible to recognize a great number of the intracellular organelles if they have been fixed correctly. The added accessibility obtained by removing the cytoplasm makes it possible to reach antigens that are usually covered. I know an investigator who labeled the lumen of the RER with a very specific antibody. He couldn't do this on well fixed, heathly cells as the antibody could not gain access to the luminal antigens. He solved his problem by noticing that some of the cells in the pellet had obviously died before being fixed, and had really poor morphology. However, the RER was easily recognizable and had lots of gold particles in the swollen lumen. Is this unacceptable because the cells were not perfectly preserved? The journal reveiwers didn't think so.
I think that immunocytochemistry is a very special branch of EM that is almost impossible to provide as a service. I know that this is what is being asked of EM labs all over the world and I know it will be impossible to change this. However, it is our responsibility to educate our colleagues and to allow them to become involved in the discovery process of how their antibodies work. I have no good solution to this, but do know that if someone is involved in their own specimen preparation and data collection, their work will progress much faster. If new EM users are taught why particular approaches should be applied instead of being given one protocol to apply exactly as written, pretty neat ideas originate from their work.
We must stop being the "black box" of science. Regards,
Paul Webster.
Reccommended reading: G. Griffiths 1993 Fine Structure Immunocytochemistry. Springer Verlag, Heidelberg & Berlin {http://www.amazon.com/exec/obidos/ASIN/038754805X/qid%3D969583271/102-2138449-1518557}
Discalimer: I have no financial interest in Amazon or Springer. I have co-authored papers with Griffiths and we teach immunocytochemical methods together.
Paul Webster, Ph.D House Ear Institute 2100 West Third Street Los Angeles, CA 90057 phone:213 273 8026 fax: 213 413 6739 e-mail: pwebster-at-hei.org http://www.hei.org/htm/aemi.htm
Tobias Baskin wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Greetings, } Paul Webster asks: Why then should we decide it is } important to have good morphology } at the EM level? snip snip } } One answer is that the questions at light and electron levels } are distinct. At the light level, we might interested in finding out } what tissue expresses a particular antigen, whereas at the electron } level, we might be wondering whether it is in the outer or the inner } membrane of the mitochondrion. In the latter case, if we can't } distinguish outer from inner membrane, let alone a mitochondrion from } a lysosome, we are in trouble. } } Tobias } -- } _ ____ __ ____ Tobias I. Baskin } / \ / / \ / \ \ University of Missouri } / | / / \ \ \ Biological Sciences } /_ / __ /__ \ \ \__ 109 Tucker Hall } / / / \ \ \ } Columbia, MO 65211-7400 USA } / / / \ \ \ voice: } 573-882-0173 } / /____ / \ \__/ \____ fax: 573-882-0123 } } } } RFC822 header } ----------------------------------- } } Received: from ultra5.microscopy.com [206.69.208.10] by mailhouse.hei.org } with ESMTP } (SMTPD32-6.00) id A14011010214; Thu, 21 Sep 2000 17:01:04 -0700 } Received: (from daemon-at-localhost) } by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) id LAA02943 } for dist-Microscopy; Thu, 21 Sep 2000 11:06:33 -0500 (CDT) } Received: from no_more_spam.com (sparc5 [206.69.208.10]) } by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) with SMTP id LAA02940 } for "MicroscopyFilteredEmail-at-msa.microscopy.com"; Thu, 21 Sep 2000 } 11:06:03 -0500 (CDT) } Received: from umc-mail01.missouri.edu (umc-mail01.missouri.edu } [128.206.10.216]) } by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) with ESMTP id LAA02932 } for {Microscopy-at-sparc5.microscopy.com} ; Thu, 21 Sep 2000 11:05:52 -0500 (CDT) } Received: from [128.206.81.167] (houndbyte.biosci.missouri.edu } [128.206.81.167]) by umc-mail01.missouri.edu with SMTP (Microsoft Exchange Internet Mail } Service Version 5.5.2650.21) } id RL53XF67; Thu, 21 Sep 2000 10:59:59 -0500 } Mime-Version: 1.0 } X-Sender: baskint-at-pop.email.missouri.edu } Message-Id: {p04320412b5efe08d6ca7-at-[128.206.81.167]} } Date: Thu, 21 Sep 2000 11:00:03 -0500 } To: Microscopy-at-sparc5.microscopy.com } From: Tobias Baskin {BaskinT-at-missouri.edu} } Subject: Re: Immuno TEM } Content-Type: text/plain; charset="us-ascii" ; format="flowed" } Errors-to: Microscopy-request-at-sparc5.microscopy.com } X-RCPT-TO: {pwebster-at-hei.org} } X-UIDL: 257555211 } Status: U }
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Another thing that can be critical is service from the local reps in your area. This varies enormously around the globe and can also vary between different regions of a country. Ask others in your area what the service has been like over the last few years. If you've got a complex piece of machinery you'll appreciate good service, especially as it ages.
Rosemary
Rosemary White Microscopy Centre CSIRO Plant Industry GPO Box 1600 Canberra, ACT 2601 Australia
Dear all, we have received a SEM Topcon SM510 and we would like to upgrade this system with LaB6 filament. It is clear that the system is almost ready for this upgrade but we have a lot of problems to obtain information from Topcon in Europe (our SEM is in Italy). We need help to know the ion pump necessary to upgrade our system, how to install it and the procedures to convert the SEM for LaB6 filament use. We need also one complete copy of Topcon SM510 manual in order to clearly understand how to correctly work with LaB6 filament.
Hi All, Thanks to all of you who responded to my query. It was a question that had been sitting in the back of my mind for several years (since the time a mentor of mine told me that formalin was a "safer" alternative to formaldehyde as my fellow students and I proceeded to be pretty much covered with it while doing some mass field fixes). When that protocol hit, it was a good excuse to settle it once and for all! Thanks, Kristen
Kristen A. Lennon Department of Plant Pathology 351 Bessey Hall Iowa State University Ames, IA 50011 email: kalen-at-iastate.edu
I am writing in response to Paul Webster's recent description of gold labeling the lumen of RER by way of lyseing cells and isolating the resulting membranes and vesicles in a fraction. The basic question I have is, at what point was the fraction exposed to the primary antibody and the secondary antibodie that would have been conjugated to colloidal gold? Did these steps occur before any fixation? Was the fraction fixed, dehydrated, embedded, thin sectioned and then exposed to the primary and secondary antibodies? Or was a entirely different procedure used.
For the past 9 years I have been working with Plasmodium falciparum during its 48 hour asexual life cycle in human erythrocytes. I have been able to isolate various membranous structures which we believe contain proteins that are of interest to us. We have had only marginal success in labeling said structures in lightly fixed, L. R. White embedded sections. I might be able to adept your mentioned RER fractionation technique to meet my needs. Thanks in advance for any help, Tim
Timothy G. Schneider Director of Electron Microscopy Department of Pathology Room 229 Jefferson Hall Thomas Jefferson University 1020 Locust St. Philadelphia Pa. 19107 215-503-4798 work 610-613-8170 cellular timothy.schneider-at-mail.tju.edu
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Reply to: RE: TEM in-situ hybridization
Michael Plociniak wrote: } } Dear Listers, } For preembedment EM-ISH, is a two-step detection method more sensitive than } a single-step method? Specifically, I am planning to detect } digoxigenin-labeled RNA probes with mouse anti-dig antibody, followed by } anti-mouse gold-conjugated secondary antibody (then enhancing ultrasmall } gold particles). Or would direct detection of the dig probes with an } anti-dig gold-conjugated antibody lend any advantage? One review } attributes higher sensitivity for two-steps, but I think this might be } restricted to postembedment methods only (J. Histochem. Cytochem. } 45:481-92,1997).
Hi Michael,
As I have been getting lots of off-line e-mail encouraging me to continue writing, I will attempt to offer my thoughts on your question for all to see. As you know, in situ hybridization is just another form of affinity localization labeling protocol. In this instance the probe is a nucleic acid sequence instead of an antibody. Much the same rules apply. I certainly don't know all the answers but can share some of what I have learned the hard way. I have embedded my comments in your text. :
Two step, sequential detection methods (primary label, followed by secondary with attached visualization probe) are usually more sensitive. This was first observed by Coons in the 1950's, and he is generally considered to be the father of immunoctyochemistry. This sequential labeling can be applied to any labeling system you want to use. You must be careful to differentiate between signal amplification and sensitivity. What you (and all of use) are looking for is to maximize the amount of target that reacts with affinity marker (increasing sensitivity). Signal amplification refers to the process of making the reacted agents more visible. } } If a two-step method is used, would gold-conjugated antibodies (against } biotin probes) have advantages over streptavidin-gold? As recently } discussed on this list, unconjugated streptavidin will compete with the } gold-labeled streptavidin, thus reducing signal strength. Nanoprobes makes } covalently bound gold antibodies, but I have seen a higher background with } these than with noncovalently bound antibodies, at least in cultured rat } neurons.
If you are using antibodies to digoxigenin, then a secondary antibody-gold probe should be sufficient. In your system this would be mouse anti-digoxigenin followed by anti-mouse-gold. Remeber that if you choose to use nanoprobes, it might be difficult to later perform multiple labeling experiments. If you want to look for biotin labeled nucleic acid probes try using anti-biotin antibodies and cold water fish skin gelatin as a blocking agent - not serum. } } Finally, will glutaraldehyde levels greater than 0.05% tend to inhibit } hybridization? Rat glioma monolayers have been labeled with EM-ISH by } Punnonen, et al., using 4% paraformaldehyde and 0.05% glut for 30 min. (J. } Histochem. Cytochem. 47:99-112, 1999). However, this protocol included } 0.1% saponin permeabilization for 30 min. Their probes were up to 850 bp } and "partially hydrolyzed" while my probes are only 50 bp long. I have } immunolabeled neurons fixed in 2% glut with no permeabilization; so if the } antibodies can make it into the cells, then the smaller RNA probes should } too, right? } The only answer to this question is what you will tell us when you have performed the experiment. Each system has its own unique qualitites and sensitivity to fixatives will be one of these variables. If it bothers you to include glutaraldehyde then leave it out. Fix with a stong glutaraldehyde solution after you have performed your hybridization and silver enhancing steps.
Watch out for the mistake that many people make in assuming that immunoreagents are inert objects and that their size is the only property to be taken into account. As someone who mailed me said: immunoreagents are biological molecles and not chemicals. They are charged molecules and have many complex, variable interactive and binding properties that we cannot begin to know. Try out your protocol and see if it works. If is does then the question about your RNA probe is answered.
} I know that target abundance is an issue here. I plan to include positive } controls for abundant RNAs (e.g., beta-actin), and I will consider Tyrimide } Signal Amplification, if necessary.
One possible step you could put in is to first try out your whole preparation and immunolabeling protocol at the LM level. Use the same reagents and same preparation protocols as you plan to use for EM and expose to silver enhancment for a longer time so that you can see the signal by LM. If you see a signal then you will be confident that you will see a signal when you go to examine the EM experiment. You might even try to find a way of even preparing the LM sample that incorporates the resin embedding so that you can control for possible digestion of the silver signal by osmium tetroxide.
Regards,
Paul Webster
} } Thank you for your helpful comments on any of these questions. I will be } happy to elaborate if this inquiry is too brief. } } } Michael Plociniak } Research Technician } Albert Einstein College of Medicine } Neuroscience Dept. } Rose Kennedy Center, 529 } Bronx, NY 10461 } (718) 430-3509 } plocinia-at-aecom.yu.edu } } transmission and scanning electron microscopy } Paul Webster, Ph.D House Ear Institute 2100 West Third Street Los Angeles, CA 90057 phone:213 273 8026 fax: 213 413 6739 e-mail: pwebster-at-hei.org http://www.hei.org/htm/aemi.htm } } } } RFC822 header } ----------------------------------- } } Received: from ultra5.microscopy.com [206.69.208.10] by mailhouse.hei.org } with ESMTP } (SMTPD32-6.00) id AA6F3AA023A; Thu, 21 Sep 2000 23:21:35 -0700 } Received: (from daemon-at-localhost) } by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) id SAA04892 } for dist-Microscopy; Thu, 21 Sep 2000 18:10:29 -0500 (CDT) } Received: from no_more_spam.com (sparc5 [206.69.208.10]) } by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) with SMTP id SAA04889 } for "MicroscopyFilteredEmail-at-msa.microscopy.com"; Thu, 21 Sep 2000 } 18:09:59 -0500 (CDT) } Received: from post.aecom.yu.edu (post.aecom.yu.edu [129.98.1.4]) } by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) with ESMTP id SAA04882 } for {Microscopy-at-sparc5.microscopy.com} ; Thu, 21 Sep 2000 18:09:47 -0500 (CDT) } Received: from amca (amca.aecom.yu.edu [129.98.90.216]) } by post.aecom.yu.edu (8.9.3/8.9.3) with SMTP id TAA05342 } for {Microscopy-at-MSA.Microscopy.com} ; Thu, 21 Sep 2000 19:03:54 -0400 (EDT) } Message-Id: {3.0.6.32.20000921185842.0079b8e0-at-mailserver.aecom.yu.edu} } X-Sender: plocinia-at-mailserver.aecom.yu.edu (Unverified) } X-Mailer: QUALCOMM Windows Eudora Light Version 3.0.6 (32) } Date: Thu, 21 Sep 2000 18:58:42 -0400 } To: Microscopy-at-sparc5.microscopy.com } From: Michael Plociniak {plocinia-at-aecom.yu.edu} } Subject: TEM in-situ hybridization } Mime-Version: 1.0 } Content-Type: text/plain; charset="us-ascii" } Errors-to: Microscopy-request-at-sparc5.microscopy.com } X-RCPT-TO: {pwebster-at-hei.org} } X-UIDL: 257555229 } Status: U }
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Daniela: I have a TOPCON 500/510 wet SEM and I have the manual although I don't know how to get a copy to you. I use the tungsten filament, don't have theLaB6 filament. You might ask the people I have repair our SEM, they all used to work for TOPCON and they have parts for them.
Chuck Humphreys Image Control Inc. P.O. Box 720596 Orlando, FL 32876-0596 U.S.A. email : cchumph-at-ibm.net
Terry Ellis Hallmark Cards Inc. 2501 McGee Kansas City, MO 64141 U.S.A. email: tellis2-at-hallmark.com
THE NORTH CAROLINA SOCIETY FOR MICROSCOPY AND MICROBEAM ANALYSIS
present the
NINETEENTH ANNUAL SYMPOSIUM ON ADVANCES IN MICROSCOPY
"Molecules, Cells & Microchips"
Coastline Convention Center, Wilmington, North Carolina
October 13 - 15, 2000
FINAL ANNOUNCEMENT!
The Nineteenth Annual Symposium, sponsored by the North Carolina Society for Microscopy and Microbeam Analysis (NCSMMA), has been planned with a theme of "Molecules, Cells & Microchips" Continuing with the tradition of the symposium, the guest lecturers are composed of both nationally and internationally distinguished scientists.
Speakers who have agreed to participate (so far) this year include:
* Ian Anderson (ORNL, Oak Ridge, TN)Spectrum Imaging: Microanalysis for a New Millennium * Ken Downing (University of California, Berkeley, CA) High Resolution Protein Structure * Jan Ho (Johns Hopkins University, Baltimore, MD) Scanning Probe Microscopy in Biology * Stefan Jeglinski (4pi Analysis Inc.) Open Source software as a Business Model for a Microscopy Company? * Dan Kiehart (Duke University Medical Center, Durham NC) Flies 'R Us * Richard Palmer (Duke Univ. NC) Recent Developments in Infrared Imaging and Microscopy * Peter So (MIT Cambridge, MA) New Microscopy Instrumentation for Biomedical Research * Rich Superfine (UNC, Chapel Hill, NC)Remote Nanomanipulation: from Viruses to Nanotubes
* The meeting has several purposes, not the least of which is to draw attention of the scientific community to emerging developments in the practical and basic research aspects of exciting new fields, and to bring people together from diverse disciplines to discuss how innovative techniques will be relevant to the future direction of microscopy and microprobe analysis. In particular, this year, special emphasis will be placed on how recent advances in electron, optical and probe microscopies have resulted in new knowledge that has benefited microscopy in general and are having a significant impact in the biological and physical sciences. The symposium also offers an opportunity for interested participants including students to submit abstracts of related studies for poster display.
* 3 special WORKSHOPS/TUTORIALS will be offered at NO ADDITIONAL CHARGE to participants in the Symposium: (a) Immuno/Microwave EM Techniques, (Cindy Hastings, VA Little Rock AR) (b) Atomic Force Microscopy (Jan Ho) (c) Digital Imaging Methods (John Mackenzie, North Carolina State Univ). These are practical, introductory sessions and no previous experience or knowledge is necessary.
Registration Fees, Hotel rates
The $90 ($100 on site) per person and $50 for students ($60 on site). Registration fee includes: symposium attendance and materials, Saturday lunch, breaks, and Friday and Saturday evening meals. Additional Friday evening tickets are available for Adults - $20; Children 10 years of age and under - $10. Additional Saturday evening tickets are available for Adults - $20; Children 10 years of age and under - $10. There is a $15 fee for all cancellations.
COAST LINE INN special rates $80/room/night (single or double). Tel: 1-800 617-7732
For questions or further information on Registration, please telephone Betty Gooch, Duke University Medical Center: (919) 286-0411 x 6508 or email: b.gooch-at-cellbio.duke edu
Website: http://152.3.167.174/NCAnnSymp2000.html
Or call Peter Ingram/Ann LeFurgey (919) 660-2671 email: p.ingram-at-cellbio.duke.edu
Today's question is, how will PMMA react in the PIPS? I have a thin film of Ti/Cu/Ti grown on a substrate of (1 1/2um thick) PMMA . My plan is to do a regular film to film x-section, dimple down to about 10um and finish it in the PIPS...I'm just not sure how PMMA mills?
If anyone has done this sort of sampIe I would love to hear about it. As always any suggestions are gladly received.
Looking for a TEM sample Prep technician at SEMATECH in Austin TX
SEMATECH is a research and development consortium working on advanced materials and processes for semiconductor manufacturing.
JOB RESPONSIBILITIES: Prepare samples, both cross sectional and plan view, of semiconductor material and devices for TEM analysis. Using flat-polishing, wedge-polishing, mechanical dimpling, ion milling, Focused Ion Beam milling and additional techniques as needed. Maintain lab equipment in working order, maintain detailed records, lab safety, housekeeping, order supplier and perform additional tasks as needed. QUALIFICATIONS: Two year technical degree, Superior fine motor skills and near vision, Ability to work with limited supervision and direction, Ability to multitask effectively, Strong organizational skills Ability to work with limited supervision and direction, Experience with sample polishing and the semiconductor industry is preferred Experience with FIB and TEM prep greatly preferred
Interested parties should send resumes to:
Carolyn Gondran 2706 Montopolis Dr. Austin TX 78741
} we have received a SEM Topcon SM510 and we would like to } upgrade this system with LaB6 filament. } It is clear that the system is almost ready for this } upgrade but we have a lot of problems to obtain } information from Topcon in Europe (our SEM is in Italy). } We need help to know the ion pump necessary to upgrade our } system, ...
I believe an ion pump is definitely necessary ... LaB6 emitters are extremely susceptable and unstable with respect to contamination. For adding an ion pump, you will need (1) the ion pump itself and its power supply ... (2) and you'll need add a port near the gun for the ion pump, and an isolation valve for isolating the gun from the rest of the vacuum system while the ion pump is working. For LaB6 emitters, (1) you'll likely need modify your power supply for the voltage/current requirements for heating the emitter, and (2) you'll most likely need a different Wehnelt. (...hmmmmm? ... have I forgot anything? ....)
cheerios, =shAf= :o)
{} /\ {\/} /\ {\/} /\ {\/} /\ cogito, ergo zZOooOM /\ {\/} /\ {\/} /\ {\/} /\ {} Michael Shaffer, R.A. - mshaf-at-darkwing.uoregon.edu Geological Science's Electron Probe Facility - University of Oregon http://epmalab.uoregon.edu/
I am interested in finding out more about the Oklahoma City, OK Ugly Bug contest which is sponsored by the Microscopy Society there. Thanks. Barbara Hodges bbddhodges-at-aol.com
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With regard to my question on the model of dye sub printer for the EM photos, eighteen people have sent me replys. Most of them suggested to use Epson printer, 870, 900 or 1270 not only due to the price, but also due to the image quality. Dr. John Mackenzie also kindly recommended to use both of Epson 900 for the resolution and 870 for the archiving because of fading problem of 900. But some of them wrote that dye sub is better than ink jet printer. They were using Codonics, Fuji Pictography, Tektronics, or Kodak 8670 dye sub printers.
I am thinking to buy Epson 900 and small Kodak dye sub printer 4270. 4270 is not so expensive, and prints only 4 inch wide photos, good for EM photo only. Many of its users are for passport size personal photographs. I will see how good is resolution of 4270.
Epson 900 sprays 3 picoliters of color dots, smallest one that I've ever known, but 10 picoliters of grey-scale dots, somewhat large one. Epson 870 sprays 4 picoliters of color dots, but unknown for grey scale. I do not know how much difference 3 and 4 picoliters dots would make. Epson 870 is photo version and has various capability of photography. Epson 900 is for office color printing. If the difference is insignificant, 870 would be much better. Any comments?
Thank all of you who have kindly posted suggestions and comments.
Best regards,
Jondo Yun Kyungnam University Division of Advanced Materials Electron Microscopy Laboratory 449 Weolyeong-dong Masan, 631-701, Korea
Gary M. Easton wrote: } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Listers, Anyone out there know of a good(hopefully easy!) way to } remove carbon tracking deposits on a ceramic electon gun? Thanks in } advance. Gary M. Easton Scanners Corporation Gary, The concentrated HCl is great but the metal parts become a real problem. Try Bon Ami if the surface is glazed. It shouldn't scatch. If it's not glazed then be more aggressive with Comet or Ajax.
Bon Ami also works wonders on a yellowed specimen chamber if you can work with just the chamber in a sink. It beats acetone or alcohol hands-down.
} } I am interested in finding out more about the Oklahoma City, OK Ugly Bug } contest which is sponsored by the Microscopy Society there. Thanks. Barbara } Hodges bbddhodges-at-aol.com
Barbara -
The Oklahoma Microscopy Society website is http://www.ou.edu/research/electron/oms/, and the popular Ugly Bug contest has its own (very entertaining) site: http://www.ou.edu/research/electron/oms/uglybug/ Paige Johnson {paige.l.johnson-at-iolok.com} chairs the OSM outreach program.
Caroline Schooley Project MICRO Coordinator Microscopy Society of America Box 117, 45301 Caspar Point Road Caspar, CA 95420 Phone/FAX (707)964-9460 Project MICRO: http://www.msa.microscopy.com/ProjectMicro/PMHomePage.html Intertidal invertebrates: http://www.fortbragg.k12.ca.us/AG/marinelab.html
Postdoctoral Positions in Materials Science University of Kentucky
Two post-doctoral researcher positions are available at the University of Kentucky, as detailed below. The research programs will make extensive use of the university's Electron Microscopy Facility, which houses two SEMs, two TEMs and one AFM. Most of the research will be conducted on a JEOL 2010F Field Emission TEM outfitted with an Oxford energy dispersive x-ray spectrometer (EDS), Gatan electron energy loss imaging filter and acquisition hardware and software for spectrum imaging.
1. The first position will be in the area of carbon-based materials under the auspices of the NSF-sponsored Center for Advanced Carbon Materials. The structure and chemistry of chemically-modified carbon nanotubes and metal-doped diamond-like carbon films will be investigated and correlated with physical property measurements. Most experimentation will involve the use of high-resolution electron microscopy (HREM) and electron energy loss spectroscopy (EELS).
2. The second position will be in the area of nanocrystalline oxide materials for sensor applications. In collaboration with the Chemistry Department at the University of Kentucky, we are studying the crystallization kinetics of metal-oxide precursors which have been designed at the molecular level to facilitate low-temperature synthesis routes. Phase and particle analysis of various oxide materials will be assessed quantitatively by TEM and XRD. Dopant segregation and the effects on dielectric behavior will also be studied.
Qualified candidates will have Ph.D. in Materials Science, Physics or any related field and practical experience in analytical electron microscopy. The salary will be commensurate with qualifications and experience. Please send applications, including two references to:
Professor Elizabeth Dickey Department of Chemical and Materials Engineering University of Kentucky A254 ASTeCC Bldg. Lexington, KY 40506-0286 ecdickey-at-engr.uky.edu
The University of Kentucky is an equal opportunity employer.
Could you tell me if agarose is PAS positive? I stained a dinoflagellate using agarose embedding technique and it was negative, however, others in my field tell me it should have stained positive. Can you clarify?
Sue Tyler, Biologist MD Dept. of Natural Resources 904 S. Morris Street Oxford, MD 21654 410-226-5193
Does anybody knows an efficient and quick way to get spare parts for Edwards Auto 306 coater?. It seems that EPROM in the control unit is dead and the instruments is out of control (after switching on the display diodes light randomly). An official Edwards' representative in Poland told us that it would take months for them to get the needed parts from the Edwards headquarters.
Leszek Kepinski
Institute of Low Temperature and Structure Research, Polish Academy of Sciences, P.O.Box 1410, 50-950 Wroclaw, Poland
Does anybody know an efficient and quick way to get spare parts for Edwards Auto 306 coater?. It seems that EPROM in the control unit is dead and the instruments is out of control (after switching on the display diodes light randomly). An official Edwards' representative in Poland told us that it would take months for them to get the needed parts from the Edwards headquarters.
Leszek Kepinski
Institute of Low Temperature and Structure Research, Polish Academy of Sciences, P.O.Box 1410, 50-950 Wroclaw, Poland
Greetings, I have PAS stained samples embedded in agarose and then in plastic and there was never an agarose reaction.
However, this info is simply anecdotal. I have no idea is PAS is "supposed" to stain agarose, and I don't do PAS "properly" (just perioidic acid and then Schiff's). Did you have a positive control? Sometmes the Schiff's reagent goes bad. I recall placing a drop on a piece of white tissue paper and if it went pink in a moment or two then the reagent was still ok.
Hope this helps even a little, Tobias
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
On Wednesday, September 20th, there was an accident at the New York-Presbyterian Hospital that might be of some interest to members of the list. It was to me since my wife works just outside the accident area.
For those that get the New York Times, it was on page B12; it was on page 2 of the New York Daily News.
In summary, a supervisor was killed by asphyxiation while working alone in a poorly ventilated trailer located outside the hospital. He was part of a team installing a new MRI at the hospital. For reasons not clear, one of the liquid nitrogen tanks connected by a valve to the room started leaking, filling the trailer space and causing the asphyxiation. Six others were treated for dizziness and other minor injuries. Two of the six injuries resulted from attempting to rescue the supervisor as they went into the room before it had been ventilated. At that time, it was not known that the room had been filled with nitrogen and that the oxygen had been displaced.
I cannot determine if the trailer was a temporary facility or part of the new installation that was being done. The nitrogen tanks were outside the trailer and the trailer where the supervisor was working was on the order of 15 x 40. It is not clear from the reports why the supervisor was working alone or how long he had been working in the trailer. Also, there is no indication of whether the trailer was part of the installation or a temporary facility.
Just thought you all would be interested.
Tony Mitchell, Ph. D. 7 East Willow Street Beacon, NY 12508
I have watched the replies to your question, and have noted that for the most part they reflect my experience. Perhaps you could try to begin your dehydration at very low %, e.g. 30% or even 10% EtOH, and do a more gradual (10,20,30,40,50,60,70,80,90,90,100,100,100) series. This has worked for some of the cultured cells I have prepared for SEM over the years. Other little nuances have included a very slow bleed in for the initial CO2 step in the CPD, and then watch the flow during the process. A final issue is watching the temperature the CPD cycles at for final drying. Sometimes the CPD tends to run a little hot, and that can also affect the final results. Hope one or more of these are of use.
Roger Moretz, Ph.D. Dept of Toxicology Boehringer Ingelheim Pharmaceuticals On Tue, 19 Sep 2000 14:16:25 -0500, Alan Burns wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } I am having trouble obtaining good SEM preparations of cultured endothelial } cell monolayers. The monolayers show evidence } of artefactual cracking along cell borders. The cracks appear whether the } cells are grown on glass coverslips or transwell filters. I have tried } various } fixatives (buffered glutaraldehyde alone, glutaraldehyde followed by osmium } and uranyl acetate), different dehydrating agents (ethanol or acetone), } followed by critical point drying (CPD). I have even tried } tetramethylsilane (Ted Pella) in place of CPD. The only time I get good } images (i.e., } no cracks) is when a small portion of the monolayer has inadvertently } detached from its substrate during tissue processing. The cells on this } small flap look marvelous (no cracks). I believe, in the absence of } substrate adhesion, the cells on the flap shrink uniformly during dehydration } when surface tension forces exert their effects. Does anyone know how to } prepare cell monolayers for SEM that leaves them intact and free } from artefactual cracking? } } Thanks. } } Alan Burns, Ph.D. } Assistant Professor } Cardiovascular Sciences } Department of Medicine } Baylor College of Medicine }
_______________________________________________________ Say Bye to Slow Internet! http://www.home.com/xinbox/signup.html
RSSL's Microscopy Laboratory has an extremely varied and busy working environment and its members of staff are committed to providing a first class microscopy service. A major proportion of work concerns Foreign Body Analysis, largely for the pharmaceutical and food sectors, for which the Laboratory is a market leader and enjoys a good reputation amongst its customers. Light and scanning electron microscopy imaging and Research and Development accounts for most of the remainder of the work. In house techniques available include LM (using different contrasting methods), SEM, cryo-SEM, X-ray microanalysis, FT-IR microspectroscopy, image analysis and freeze fracture/etching; TEM is undertaken off site.
Due to expansion and subsequent staff development, an opportunity has arisen in to assist in the Foreign Body Identification service that runs from the Microscopy Laboratory. The position also supports ongoing research projects, particularly in both chocolate and sugar confectionery project areas. Other tasks include provision of support in maintenance of laboratory equipment including the electron and light microscopes, digital imaging and dark room facilities. On occasions, work into the evenings and at the weekends might be required and this post carries a requirement to share in a rota to undertake out of hours analyses as part of RSSL's comprehensive Emergency Response Service.
We are looking for a candidate who is educated to degree level or equivalent with a minimum of 4 years microscopy experience in food science, biology or materials science. Although training will be given, experience in X-ray microanalysis of bulk samples and/or of FT-IR microspectroscopy is highly desirable. Experience or knowledge of general equipment maintenance and a willingness to become involved in this; be an excellent team player with the ability to work independently; be customer focussed and able to work under pressure, as well as capable of providing written reports of long and short term projects.
If you are a self starter, looking for an interesting and challenging role and believe you can make a positive contribution to our already successful team, we would be interested to hear from you.
Please send your CV with a covering letter including your salary expectations to Jane Bienkowski, Human Resources Officer, Reading Scientific Services Lld, Lord Zuckerman Research Centre, PO Box 234, Whiteknights, Reading, RG6 6LA or e:mail jane.a.bienkowski-at-rssl.co.uk or fax to 0118 986 8932.
Hi Phil, We have odd bitz for the above. Let me know the dimensions of the feed thro and I'll have a rummage in the box. Chris Smith, IACR-Rothamsted, UK. chris.smith-at-bbsrc.ac.uk
Take the chance to learn about the developments in scanning electro microscopy! A course on "Scanning Electron Microscopy - Imaging and Microanalysis", SEM 2000, will be given at Chalmers University of Technology, Gothenburg, Sweden, October 17 - 19, 2000.
This course will be given in collaboration with four microscope manufacturers (Hitachi, Jeol, Leo, Philips/Fei) and suppliers of equipment for EDX, WDX, EBSP and CL.
The aim of this 3-day intensive course is to give a theoretical background in the morning sessions and experimental insights in the afternoons. The lectures and the demonstrations will be given by application specialists from the different companies representad at the course, and also by researchers working with different applications in SEM. The demonstrations and lab classes will be carried out on equipment brought to Chalmers specifically for this course.
Detailed information about SEM 2000, including course programme and registration form, is posted on: http://fy.chalmers.se/microscopy This web-site will be continuously updated until the course starts.
Looking forward to seeing you at SEM 2000!
Birgitta Castedal, Lena Falk and Mats Halvarsson Course Organisers
____________________________
Associate Professor Lena Falk Department of Experimental Physics, Chalmers University of Technology, SE-412 96 Göteborg, SWEDEN
Your description does not fit the classic EM fields interference pattern, as other responses had pointed out. The high level of the stray fields in the room is bad, yet it will not necessary cause a problem. Your problem could be resulted from a combination of conditions. JEOL, however, has a reason for recommending the mu-metal shield. You can perform a quick and easy test to see whether external electromagnetic fields are responsible for the problem. Assuming that the SEM column is clean (especially the final aperture!), and focusing circuit operates properly. 1. Obtain an image in continuous LIVE (no integration!) mode on the monitor screen with contrast enough vertical features, so distortion of the vertical lines is clearly visible. Magnification may vary, but you will need at least several 1000s. This distortion (if present), will be a sawtooth pattern, with the period smaller at the slowest beam scan speed, larger at higher speeds, and becoming a single "wave period" at the fastest speed which is a TV rate. The later will be an image "floating" back and forth horizontally on the screen. If the described pattern is not obtainable, the problem is unlikely to be caused by stray AC fields. 2. Change accelerating voltage at least 50%. Focus the image. Make sure that you have exactly the same sample feature in the image. Do not change any other settings. If the described distortion is caused by external electromagnetic fields, the amplitude of the sawtooth pattern (slow scan) or the amplitude of "floating" (TV mode) will change in approximately reverse proportion to the accelerating voltage value, i.e. at 10 kV will be twice of what it was at 20 kV, and so forth. 3. If the distortion will stay the same, then the source of your problem could be, for example, the mechanical vibrations. Those are caused by AC electric motors (vacuum pumps, air compressors, elevators, etc.) most of the time, vibration frequencies being harmonics of the AC line frequency. So the distortion will look very similar, except its amplitude will not be a function of accelerating voltage. Also, interference affecting the electronic circuits (ground loops, for instance), may look the same and will not be a function of the accelerating voltage. 4. Ground loops are the most frequent problem in my experience. Their influence could be inconsistent, which makes the problem difficult to identify. For example: the AC line conduit was used for the ground. Everything was fine until big air-conditioning unit was installed behind the wall. Now, a strong 60Hz AC field is measured every time when aircond. motor kicks in. Then expensive mu-metal shield was installed, and... made no difference. The reason: new unit was connected to the same power line. Existing ground loop was OK before due to low power consumption of the SEM. It is greatly increased now, and ground loop badly affects electronics of the SEM. The SEM column itself, at the same time, was sufficiently shielding external fields. 5. In any case, try self-made soft Iron shield before spending big $$ on it.
Good luck.
Vitaly Feingold Scientific Instruments and Applications 2773 Heath Lane, Duluth GA 30096 (770)232-7785 ph. (770)232-1791 fax -----Original Message----- } From: Mark West {mwest-at-ifcsun1.ifisiol.unam.mx} To: microscopy-at-sparc5.microscopy.com {microscopy-at-sparc5.microscopy.com}
Hi Again, I received many replies to my question of late regarding the composition of formalin vs. formaldehyde. All of them were pretty consistent except for the most recent which touched on a very important, related issue, safety. As I think that it is particularly important, I'm forwarding to the whole list (with the permission of the author - who's name I've withheld). Be careful out there. Kristen
"Kristen,
I didn't follow the thread on this topic and what has been said. However, the comment about being "pretty much covered with it while doing some mass field fixes" caught my eye. In case no one else mentioned it, exposure to formaldehyde is thought by some pulmonology specialists, especially those researching Idiopathic Pulmonary Fibrosis (IPF), suspect that formaldehyde exposure may trigger the disease. IPF is fatal and has no treatment except lung transplant. I pass this on as one who has experienced the type of exposure you describe, the diagnosis of IPF, and the double lung transplant that is something that very, very few people are fortunate enough to get. And even with a lung transplant, the five year life expectancy is only 48%. PLEASE, please be very careful with lab chemicals and teach those you work with to do likewise.
} My exposure to formalin/formaldehyde occurred during my undergraduate and } graduate years, not only during standard class preparations but also in } the field when we went collecting algae at freshwater and more so marine } habitats. It was there when we were exposed to a lot of it; I remember my } fingers getting "fixed", my eyes watering and stinging, you know the } feeling. The fact is that the disease occurred about 20 years later. Yes } the story has a "happy" ending, so far, but for reasons other than getting } a rare double lung transplant and doing fine after 3 years. But that is } another story! Yes, many people who handle chemicals in the lab don't get } IPF, but don't take the attitude that nothing can happen to you. There } are many other ways that chemical exposure can damage your } health. Remember, take care of your body as it is the only one you get } and damn few people get spare parts! :-) } } You may pass this along if you want, if it helps one person it's worth it."
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I have a problem with Digital Micrograph 2.1 related to image file sizes.
Sometimes DM saves an image at 1.2MB while at other times (in fact, the very next image) may be saved at 1MB or even less. Unfortunately, when we do image analysis on these images the measurements will be off since the number of pixels varies. I have tried saving in various formats and nothing seems to change the events.
What gives here? Why does the program save in such differing file sizes and so randomly? I can not figure out any logic to this....... It is driving me crazzzzzzzzzzzzzzzyyyyyyy.
Thank you,
JB #################################################################### John J. Bozzola, Ph.D., Director Micro-Imaging and Analysis Center 750 Communications Drive - MC 4402 Southern Illinois University Carbondale, IL 62901 U.S.A. Phone: 618-453-3730 Fax: 618-453-2665 Email: bozzola-at-siu.edu Web: http://www.siu.edu/departments/shops/cem.html ####################################################################
A response directed to Paul Webster, c/o this invaluable and open forum:
I would like to continue with the topic of immunogold labeling for TEM, relating to your suggestion:
"One possible step you could put in is to first try out your whole preparation and immunolabeling protocol at the LM level. Use the same reagents and same preparation protocols as you plan to use for EM and expose to silver enhancment for a longer time so that you can see the signal by LM. If you see a signal then you will be confident that you will see a signal when you go to examine the EM experiment."
How reliable is LM for predicting TEM immunogold labeling results?
I have tried this approach before, with mixed results. It sounds like a great idea, but in attempting to "modify" the EM protocol into a LM pilot experiment, it becomes a completely different beast. During primary fixation, EM requires glutaraldehyde to preserve ultrastructure. You suggested that glut not be used until AFTER immunolabeling, but won't the damage to the fine structure already be done? I was under the impression that glut "postfixation" is intended primarily to crosslink gold antibodies in place after they have bound to primary antibody.
Also, silver and gold enhancement reagents come in LM and EM formulae; usually, EM versions are less acidic. Does a longer LM enhancement accurately predict the action of a brief EM enhancement? Perhaps EM formulae can be used to directly monitor enhancement with the light microscope. However, because optimal EM enhancement is invisible by LM, one will need to empirically determine EM enhancement times for each cell system. Doesn't this argue against spending too much time with LM experiments, when EM also includes osmium, uranyl acetate, dehydration, and resin infiltration?
I still use pilot LM experiments, but more for their complimentary benefit, rather than for their predictive value.
Routinely, I see a discouraging signal to noise ratio at the LM level. However, at the EM level noise is restricted to the first couple of resin sections of a monolayer culture, and subsequent sections contain beautiful results. I interpret this as nonspecific binding of antibodies to the poly-L-lysine and lamin-coated glass substrate. In this example, routine LM (DIC optics without optical sectioning) does not accurately predict EM performance.
Sincerely,
Michael Plociniak Research Technician Albert Einstein College of Medicine Neuroscience Dept. Rose Kennedy Center, 529 Bronx, NY 10461 (718) 430-3509 plocinia-at-aecom.yu.edu
Thanks everyone who responded to my request for labs in the DFW area that would consider "renting" space on their scopes. I've sent the information on to the researcher who is looking for space and he and I were impressed by the many places that were gracious enough to consider letting outsiders use their equipment.
Whenever I use Schiffs reagents for student lab classes I test it first by adding just a couple of drops of neutral buffered formalin (the diluted form = 3.8% formaldehyde) to about 10ml in a clear tube and they can then watch the colour develop over a few seconds. If was also 'water white' to begin with and with no hint of pink we use it. It hasn't failed me so far and the students get to see a dramatic colour change before they do the method.
Hope this helps?
At 08:30 2000-09-25 -0500, Tobias Baskin wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Med vänliga hälsningar/With best regards
Gareth
"A wise person is certain of few things - a fool of everything"
"The foolish reject what they see, not what they think; the wise reject what they think, not what they see." Huang Po
Gareth Morgan MPhil MSc FIBMS, Institutionen för Mikrobiologi, Patologi och Immunologi(IMPI), H5, Karolinska Institutet, Avdelningar för biomedicinsk laboratorievetenskap och biomedicinska ämnen, Box 12 773, S 11 296, Stockholm Sverige
In celebration of the acquisition of a new JEOL 3010 transmission electron microscope with environmental cell capabilities, the Naval Research Laboratory Stennis Space Center will be conducting a day long symposium on November 2, 2000. Outstanding microscopists from around the country will join local experts to discuss marine environmental and materials science applications of TEM. On Friday morning, November 3, participants are invited to visit informally with NRL researchers in the new Microscopy Facility.
In addition to the opportunities for exploring and sharing insights during the period of formal presentations, transportation will be provided Thursday evening to the French Quarter in New Orleans. Here participants may enjoy interacting with each other in a setting famous for its music, its food, and its stimulating atmosphere.
We are also strongly soliciting contributed papers. Although a primary area of emphasis is expected to be environmental cell and in-situ microscopy, submissions of a wide variety of ‘frontier’ microscopy results are welcome!
Invited speakers include: Larry Allard - Oak Ridge National Laboratory -- “Aberration Correction for High Resolution Imaging and Advanced Analytical Electron Microscopy†Frances Ross - IBM Watson Research Center Renu Sharma - Arizona State University Judith Yang - University of Pittsburgh
Other speakers planning to participate include: Tyrone Daulton – NRL -- “Application of High-Resolution and In-Situ TEM to Solving Problems in Diverse Fields of Study†Yoko Furukawa – NRL -- “Ultrastructure of Aquatic Sediments and its Effect on Early Diagenesis†Dawn Lavoie – NRL -- “Preparation Techniques and Quantitative Characterization of Clay Sediments using TEM†Brenda Little – NRL -- “Environmental Electron Microscopy Investigations of Microbiologically Influenced Corrosion†Eric Stach – NCEM -- “In-situ TEM of Dislocations in Thin Filmsâ€
This symposium is co-sponsored by the Southeastern Microscopy Society.
We anticipate there will be approximately 16 presentations at the Symposium. Arrangements are underway to develop a special section of Microscopy and Microanalysis devoted to the papers presented. Finished manuscripts are due October 15. The total number of participants will be limited to a maximum of 60.
REGISTRATION Please register early by completing and returning the form below; the registration deadline is October 13.
Sincerely, your organizers: Bhakta Rath, Co-Chair - Naval Research Laboratory Eric Stach, Co-Chair - National Center for Electron Microscopy, Lawrence Berkeley National Laboratory Matthew Hulbert, Coordinator - Research Dynamics
______I wish transportation to the French Quarter Thursday evening.
______I do not wish transportation to the French Quarter.
If you wish to give a presentation, please provide its title below.
Return registration information to Matthew Hulbert. E-mail is preferred -- matthew_hulbert-at-hotmail.com If you choose to use US mail, the address is 1332 Pembroke Drive, West Chester, PA 19380.
Most standard histology books will give a procedure for digestion of a sample as negative control. If you need the procedure please let me know and I will e-mail it to you.
Pamela A Marcum Histology/Microscopy Product Development Manager Polysciences, Inc.
-----Original Message----- } From: Tobias Baskin [mailto:BaskinT-at-missouri.edu] Sent: Monday, September 25, 2000 9:30 AM To: Microscopy-at-sparc5.microscopy.com Cc: STYLER-at-dnr.state.md.us
Greetings, I have PAS stained samples embedded in agarose and then in plastic and there was never an agarose reaction.
However, this info is simply anecdotal. I have no idea is PAS is "supposed" to stain agarose, and I don't do PAS "properly" (just perioidic acid and then Schiff's). Did you have a positive control? Sometmes the Schiff's reagent goes bad. I recall placing a drop on a piece of white tissue paper and if it went pink in a moment or two then the reagent was still ok.
Hope this helps even a little, Tobias
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
In celebration of the acquisition of a new JEOL 3010 transmission electron microscope with environmental cell capabilities, the Naval Research Laboratory Stennis Space Center will be conducting a day long symposium on November 2, 2000. Outstanding microscopists from around the country will join local experts to discuss marine environmental and materials science applications of TEM. On Friday morning, November 3, participants are invited to visit informally with NRL researchers in the new Microscopy Facility.
In addition to the opportunities for exploring and sharing insights during the period of formal presentations, transportation will be provided Thursday evening to the French Quarter in New Orleans. Here participants may enjoy interacting with each other in a setting famous for its music, its food, and its stimulating atmosphere.
We are also strongly soliciting contributed papers. Although a primary area of emphasis is expected to be environmental cell and in-situ microscopy, submissions of a wide variety of ëfrontierí microscopy results are welcome!
EARLY REGISTRATION WOULD BE GREATLY APPRECIATED!!!
Invited speakers include: Larry Allard - Oak Ridge National Laboratory -- ìAberration Correction for High Resolution Imaging and Advanced Analytical Electron Microscopyî Frances Ross - IBM Watson Research Center Renu Sharma - Arizona State University Judith Yang - University of Pittsburgh Other speakers planning to participate include: Tyrone Daulton ñ NRL -- ìApplication of High-Resolution and In-Situ TEM to Solving Problems in Diverse Fields of Studyî Yoko Furukawa ñ NRL -- ìUltrastructure of Aquatic Sediments and its Effect on Early Diagenesisî Dawn Lavoie ñ NRL -- ìPreparation Techniques and Quantitative Characterization of Clay Sediments using TEMî Brenda Little ñ NRL -- ìEnvironmental Electron Microscopy Investigations of Microbiologically Influenced Corrosionî Eric Stach ñ NCEM -- ìIn-situ TEM of Dislocations in Thin Filmsî
This symposium is co-sponsored by the Southeastern Microscopy Society.
We anticipate there will be approximately 16 presentations at the Symposium. Arrangements are underway to develop a special section of Microscopy and Microanalysis devoted to the papers presented. Finished manuscripts are due October 15. The total number of participants will be limited to a maximum of 60.
REGISTRATION Please register early by completing and returning the form below; the registration deadline is October 13.
Sincerely, your organizers: Bhakta Rath, Co-Chair - Naval Research Laboratory Eric Stach, Co-Chair - National Center for Electron Microscopy, Lawrence Berkeley National Laboratory Matthew Hulbert, Coordinator - Research Dynamics
REGISTRATION INFORMATION Name: ______________________________________________________ Organization: ______________________________________________ Address: ___________________________________________________
___________________________________________________________ e-mail Address: ____________________________________________ __I wish transportation to the French Quarter Thursday evening. __I do not wish transportation to the French Quarter. If you wish to give a presentation, please provide its title below. Return registration information to Matthew Hulbert. E-mail is preferred -- matthew_hulbert-at-hotmail.com If you choose to use US mail, the address is 1332 Pembroke Drive, West Chester, PA 19380. _________________________________________________________________________ Get Your Private, Free E-mail from MSN Hotmail at http://www.hotmail.com.
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Have any other microscopists had problems with Peltier coolers failing on water cooled CCD cameras?
In my facility I have three such cameras all plumbed into the microscope water system and connected, by the camera manufacturer, into wall sockets. On one of the three cameras I have had the Peltier cooler fail four times in two years. After the latest failure the manufacturer asked if the camera had been run without water. I told him not intentionally but when we have power cuts and the power is restored, the camera comes back on but the microscope (and water) does not. The camera can therefore run for some time without water cooling. Power failures are not uncommon at this university!
Many e-mails to the manufacturer have resulted in no response to my questions about reliability of the Peltier coolers. The manufacturer is treating the failure as an out of warranty issue but I believe there has been something wrong with this camera since it was installed.
Any experiences of listservers with Peltier cooler failures would be appreciated. Please e-mail me directly.
Regards
Alan W Nicholls, PhD Electron Microscopy Service Director Research Resources Center - East (M/C 337) Room 100 Science and Engineering South Building The University of Illinois at Chicago 845 West Taylor St Chicago, IL 60607-7058
Alan W Nicholls, PhD Electron Microscopy Service Director Research Resources Center - East (M/C 337) Room 100 Science and Engineering South Building The University of Illinois at Chicago 845 West Taylor St Chicago, IL 60607-7058
Just this morning we were discussing the same problem with our Gatan CCD cameras on our HD-2000, one of which is retractable and used for TEM imaging, and the other of which is at the end of the GIF for energy-filtered imaging. These cameras are connected in series for the water flow, and have very tiny water lines which can easily clog, even with recirculated water. There is no provision by the manufacturer to provide for a safety cut-off of the Peltier cooler in the event of loss of water flow. We have recently experienced a burn-out of the TEM camera due to a clog in the line, which resulted in 2 weeks of downtime for repair (nicely done for free by Gatan), but still no response to queries about some sort of protection. So we have decided to find an appropriate water flow sensor, and to install it in an appropriate place in the water line, and to connect it to the camera controllers to provide a power cut-off when the water flow diminishes. I'll post the details of the solution when it is completed.
Larry
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Dr. Lawrence F. Allard Senior Research Staff Member High Temperature Materials Laboratory Oak Ridge National Laboratory 1 Bethel Valley Road Bldg. 4515, MS 6064 PO Box 2008 Oak Ridge, TN 37831-6064
I am having trouble processing bacteria in liquid culture for TEM/SEM. Too much of sample is being lost because I only have a small table top microfuge. The culture won't spin down into a hard enough pellet. Does anyone have a different protocol for processing bacteria in liquid culture? Please reply to capri-at-zianet.com Kristine Fambrough
Dear Alan, My experience with Peltiers does not involve a CCD camera, but it may be instructive nonetheless. When we wanted to provide a constant temperature for the chemicals in the darkroom--which required sometimes heating the water and at other times cooling it--we decided that Peltiers were the best solution. We bought a commercial thermoregulating unit which sent current of the appropriate polarity through the Peltiers to adjust the temperature to the desired setpoint. The Peltiers failed at an expensive rate, and we found that the thermoregulator was such that it provided a specific current, which it turned on and off in a duty cycle (the greater the temperature difference, the greater fraction of time the current was on). The sudden changes in current were the cause of the Peltiers' demise. Our shop designed and built a regulator which operated in such a way that the current was not turned on and off, but was proportional to the temperature difference, and the Peltiers worked properly for many years. We were also warned that moisture in the Peltiers could lead to an electrochemical process which caused corrosion and subsequent failure. Check that the controller does not vary the current rapidly and that the Peltiers are protected from condensation in your CCD setup.
Bill Tivol Wadsworth Center Albany NY (518) 473-7399 WFT02-at-health.state.ny.us
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John, for all high resolution work and image analysis, I always collect images as a ".gat" (with a Digital Micrograph 3.3.1 on our system, I get 2.1 MB file). I have similar problem with exporting files to users not having DM (not able to open gatan files); either tiff or jpeg files are only about half the original size. I get better resolution if I go through the "File/Export" option rather than just "File/Save As/". Does anyone have a more elegant way? Thanks, Alice.
Alice Dohnalkova Environmental Microbiology Battelle, PNNL MS P7-50 Richland, WA 99352 (509) 372-0692
} From: John J. Bozzola [mailto:bozzola-at-siu.edu] Sent: Monday, September 25, 2000 3:40 PM To: microscopy-at-sparc5.microscopy.com
I have a problem with Digital Micrograph 2.1 related to image file sizes.
Sometimes DM saves an image at 1.2MB while at other times (in fact, the very next image) may be saved at 1MB or even less. Unfortunately, when we do image analysis on these images the measurements will be off since the number of pixels varies. I have tried saving in various formats and nothing seems to change the events.
What gives here? Why does the program save in such differing file sizes and so randomly? I can not figure out any logic to this....... It is driving me crazzzzzzzzzzzzzzzyyyyyyy.
Thank you,
JB #################################################################### John J. Bozzola, Ph.D., Director Micro-Imaging and Analysis Center 750 Communications Drive - MC 4402 Southern Illinois University Carbondale, IL 62901 U.S.A. Phone: 618-453-3730 Fax: 618-453-2665 Email: bozzola-at-siu.edu Web: http://www.siu.edu/departments/shops/cem.html ####################################################################
Kristine There was a thread on this a few months ago, the concensus of which was that for TEM most people advocated solidifying a spun- down bacterial suspension using agar or agarose before getting too far into the process. You might want to consider whether this should be done pre-fix or post-fix. I would advocate the latter. Anyway, this produces a chunk of material that can be handled through teh solution changes more conveniently than a cell suspension. However, many people found that a pellet solidifed in this way would infiltrate rather poorly if left in the bottom of the microfuge tube / eppendorff, etc. The best solution is probably to detach it and perhaps chop it up a little to minimize specimen size, providing reagents and solvents free access to all surfaces. Best wishes Chris Jeffree
} From: "sghoshro-at-nmsu.edu"-at-sparc5.microscopy.com Date sent: Tue, 26 Sep 2000 09:21:58 -0600 (MDT) To: microscopy-at-sparc5.microscopy.com
The original question by Tom Phillips has stirred up a great discussion on this list. Even though we're getting a little away from the original subject, I wanted to throw in my views with respect to the issue of antibodies working in LM and not in EM. I'm afraid it will get kind of lengthy, but I hope it is useful.
When immunodetection at the LM level works, but not at the EM level, I prefer to follow a logical procedure to find where the bottle necks are. Basically there may be two differences in the approaches: 1. the reagents used, 2. the specimen preparation method.
To make things simple, let us assume the labeling at the LM level works reliably. Then we are left with a situation where we need to know whether the antigens (epitopes) are still recognizable and available in the EM specimen and whether the reagents for EM detection will do the job they are supposed to do. How do we get an answer to these questions?
The first thing one should do is to check the reactivity of the reagents for EM with the primary antibody of interest. This does not necessarily have to be checked on the specimen. It is even better not to do that, because there are too many other parameters different from the LM approach. A simple dot-spot test can do the trick and give a conclusive answer in about two hours. In practice: a dilution series of primary antibody (monoclonal or affinity purified) is applied to a strip of nitro-cellulose. The strip is blocked and incubated with the immunogold reagent. If applicable (with small particle conjugates) the strip can be silver enhanced. If antigen is available, the dot-spot test can even be used to get a rough idea about the influence of fixatives. A dilution series of antigen (or an enriched cell lysate) is applied to the nitro-cellulose strip, the strip can be treated with fixative (exactly the same concentrations, time and temperature as intended to be used for specimen fixation), then the strip is washed with glycine, blocked, incubated with primary antibody, gold conjugate and silver enhanced. A comparison of different strips (fixed with several different fixatives and unfixed) gives somewhat of an indication whether a particular fixative is worthwhile to try. It is not an absolute test, as the environment of the antigen may play a role in the effect of fixation as well (cross-linking, densely packed structures). Should anyone need information how to do this in practice, please get in touch with us. If antigen is not available, you could use cryostat sections or even paraffin sections that worked at the LM level and run the same test. Make sure that the gold and silver enhancement reagents being tested here are the same reagents that will be used for EM labeling.
What about the influence of specimen preparation? To some extent the issue of fixation has been addressed above. If reactivity of the immunogold and silver enhancement reagents had been tested fine and EM specimens had been prepared the same way as for LM (only thinner of course, at least in the end), the approach should work. Unfortunately, the specimen preparation for EM often differs a lot from the one for LM. So let us start with the EM sample preparations that are similar to those for LM, because logically those should be the most suited, at least from the point of view of antigen preservation and presentation. In ultrathin cryosections the influence of the embedding material is avoided, so antigens will not be as masked as they are in resin or acrylate embedded specimens. I don't know if there still is the same consensus about the validity of this technique as there was some years ago, but we always found that cryo ultra microtomy was the approach to most likely give a positive result.
Another approach, that has been addressed in a few postings recently, is to use pre-embedding labeling. In the time period before ultra small gold conjugates became available, pre-embedding immunolabeling with the classic gold conjugates was only feasible after harsh detergent treatments which were often applied during chemical fixation. As a result much of the cytoplasm and organelles were either destroyed or washed out. The state of ultrastructural preservation was far from ideal and this has imprinted the pre-embedding approach with a negative image for a long time. However, with the ultra small gold conjugates becoming available, harsh detergent treatments of the sample as a trade off for immunoreagents to penetrate were no longer necessary. Even only a pre-treatment with sodium borohydride, which was applied to quench aldehyde groups, was sufficient to make some types of single cells accessible for ultra small gold reagents.
I don't want to state that these two approaches would be the only ones I would consider useful. But they are good steps to start with. A great advantage of the combination of ultrathin cryosections and ultra small gold reagents is that these reagents penetrate with greater ease into the section’s interior, compared to larger gold conjugates, detecting not just antigens exposed on the surface.
For me cryosectioning also worked with plant material like soy beans, but then again they were not the dormant dry seeds, these were sprouts. I am sure they are a lot easier to cut than the dry ones. Teaching workshops at botany departments I have learned how hard it is to get even an acceptable level of ultrastructural preservation and good immunolabeling, although I got the impression that high pressure freezing can be a very good starting point.
To be able to use ultra thin cryosections you need a cryo ultra microtome. For pre-embedding you don't need a lot of fancy equipment. The results some of us are getting with pre-embedding immunolabeling are really very encouraging, especially with Fab2 and single Fab fragment-ultra small gold reagents and efficient and homogeneous silver enhancement reagents being available. I don’t think I should be the one doing a lot of talking here since it is not my work. But I hope that researchers who have done this or are experienced with pre-embedding will jump into the discussion. Hong, why don't you tell us? And yes, I realize, useless for internal antigens in plant tissue. But for cultured cells and animal tissue it can be great.
I wholeheartedly agree with Paul Webster that it is a must for an investigator to try to understand the ins and outs of a method and that ‘we must stop being the "black box" of science’. That is the only way to understand the results and to make progress. When I was still associated with the University in Utrecht I experienced very often that non-EM people have a tendency to look upon EM as a way to make the picture to fit the story. As a sort of confirmation of the biochemical or molecular data they already have fixed in their minds. You're lucky (!) if you can confirm their beliefs, and they're happy to have a nice demonstration supporting their ideas. IEM is a scientific method that not just adds up to the results that are there, but my own experiences convinced me that it may provide results that have been overlooked with other, biochemical or whatever analysis methods. I learned something from my PhD professor that I still value very much: "don't try to get the results that you or somebody else wants you to get, but try to understand the results you're actually getting".
In spite of the fact that most of you will have fallen asleep by now as a result of this lengthy posting, I would like to make a few more comments.
Don't compare apples with pears! Use the same reagents for LM and EM. Protocols for LM and EM should be comparable as much as possible, including the use of buffers, blocking reagents, dilutions.
I am reluctant about using cell fractionation in some instances: you might only get to see what you want to see. This may degrade the labeling to a confirmation of a mental picture, and prevent you to find labeling in places you didn't expect before and which may not be present in your fraction-specimen anymore. And also, if you already know where the antigen is, why would you bother to do immuno EM? But often this so-called knowing is just an idea based on data acquired up to that point in time. Immuno EM may reveal additional data that shed a new light on the question.
Secondary antibody conjugates vs. protein A: theoretically secondary antibodies give less resolution, true. Is there anyone who actually made the comparison and found one to be different from the other in that respect? I mean not in molecular labeling: molecules or viruses attached to a film on grid and labeled for specific epitopes, but labeling in or on a section?
To end this message: unfortunately, sometimes it happens that even if you've checked carefully and think it should work, sometimes it doesn't. Those cases are rare and although they are most annoying they are also the most interesting cases from a technological/methodological point of view, because they can teach us something we didn't understand before.
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} From: John J. Bozzola, bozzola-at-siu.edu
{I have a problem with Digital Micrograph 2.1 related to image file sizes.
{Sometimes DM saves an image at 1.2MB while at other times (in fact, {the very next image) may be saved at 1MB or even less. Unfortunately, {when we do image analysis on these images the measurements will be {off since the number of pixels varies. I have tried saving in various {formats and nothing seems to change the events.
{What gives here? Why does the program save in such differing file {sizes and so randomly? I can not figure out any logic to this.......
Fun with Digital Micrograph :-(
Actually in DM2.X and lower when you "Save As.." you are in effect saving a screen resolution image and the size of the image you save is dependent on how much you have zoomed the image on the screen. So if you zoomed out once and the image was 512X512 on screen (with a 1024 camera) then your image would get saved as a 512X512 image and you lost half of your resolution. What I always did was to save the image in DM format first without touching anything in the image (contrast, brightness, zoom, etc). That way I knew I had a pristine image that had not been messed up in anyway and I could go back to that when needed. In order to save a tiff or whatever, I would make sure that my zoom was 1:1 and then I would save it in the format needed.
In DM3.X they modified that slightly. If you hit "Save as.." and save it in DM format it is OK, if you "Save as.." in any other format it saves just as I mentioned above. However, they now have an export menu item that saves the image in original resolution in whatever file type you want. I always tell my students to ignore the "Save as.." feature and just use export. Gatan has also removed some of the bugs in the auto save feature. If you have the extra $$$$$$$$$ it is probably worth upgrading to DM3.X just note that they changed their DM image format somewhat but new DM can read old DM images but not the other way around.
Norm Olson
******************************************* Norm Olson Senior Research Electron Microscopist Department of Biological Sciences Lilly Hall of Life Sciences Purdue University West Lafayette, IN 47907
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Hi, Alan
} we have power cuts and the power is restored, the camera comes back on but } the microscope (and water) does not. The camera can therefore run for some } time without water cooling. Power failures are not uncommon at this } university! }
I think that all equipment like this should be set up so that when the power goes off, the equipment stays off when the power comes on again.
Devices to acheive this are common in the world of electricians, they call them "zero-voltage switches" (at least down here they do).
Get your electrician to install one for the mains socket from which the camera derives its power.
Mine have saved me from complications many times.
cheers
rtch
Ritchie Sims Phone : 64 9 3737599 ext 7713 Department of Geology Fax : 64 9 3737435 The University of Auckland email : r.sims-at-auckland.ac.nz Private Bag 92019 Auckland New Zealand
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Reply to: RE: Can LM predict immunogold TEM? Hi Michael,
I read your comments with some dismay. I may have misunderstood, but the impression I get is that you feel there will be such large differences between LM labeling and EM labeling that you don't think it worthwhile to check your antibodies by LM first. If there were such differences then the experiment should not be performed in the first place. The LM result should be the same as the one you see by EM. I would not expect anything less.
One point of my previous posting was to suggest that immunolabeling experiments can be carefully designed so that LM screening is possible before moving onto the EM level. I think that the reason my antibodies work for EM is that I first tested them by LM to make sure I would get a result.
I do not think that glutaraldehyde is a prerequisite for good morphology, or that samples have to be treated with osmium tetroxide, uranyl acetate either. What I was suggesting is that it should be possible to screen samples by LM before moving onto the EM level. This will confirm that immunoreagents will react with the sample, will give an idea of the best dilutions to use, and will give information on the relative number and ditribution of antigens. All this should be possible using the same samples for LM and EM.
As an example, suppose I wanted to look for a mast cell antigen in nasal mucosa. I know from my biology lessons that there will be few mast cells in this tissue so I know I will have to search for them before I can label them. Now I make a choice between pre- and post-embedding labeling. I know that the antigen I want is going to be inside the granules of the mast cells, which are inside the tissue. For this reason, I decide that pre-embedding labeling may not be the best approach. I choose to label sections. Now, I have to decide whether I want to cryosection or to embed in resin. As I am lucky enough to have a cryoultramicrotome in my lab, and as I know from experience that I usually get higher sensitivity on cryosections, I choose to prepare the samples this way. If I want to perform immunolabeling on the samples, I will choose not to fix in glutaradehyde, which gives the tissues an autofluorescent glow (and which can also be a useful counterstain, when used properly). I therefore choose to fix with formaldehyde (which, btw, is a gas and which when dissolved in water becomes formalin, and is actually converted to methylene glycol CH2O + H2O = HO CH2 OH). I also know that at concentrations above 2% the methylene glycol begins to polymerize to form polyoxymethylene glycol, and that these polymers are what react with and crosslink proteins in biological material. My choice of fixative might then be an initial immersion fix in 4% buffered formaldehyde for perhaps 1 hr, followed by a second immersion fix in 8% buffered formaldehyde for 24 hr. I would make sure not to use phosphate buffered saline, but something with a better buffering capacity (such as 100mM phosphate buffer).
Once fixed, I would cut off a small portion of the tissue (carefully so as not to crush the sample), infiltrate with sucrose and prepare cryosections, mount them onto glass coverslips and label them with my antibody and a fluorescent secondary. I could also visualize the bound antibody by reacting with protein A gold and then silver enhancing the bound gold. I have no concern about whether the silver enhancer will work at the EM level because I know I will be able to easily detect the 5nm protein A-gold at the magnifications I use.
Once I had found a part of the tissue containing the mast cells, I would then trim down the block, cool the ultramicrotome and cut thin sections for EM. These I would label with antibody and protein A-gold, confident that the sections contained mast cells and that the immunoreagents would be working.
If I found that the antibody gave unusual results, suggesting either a migration or extraction of antigen, I would take part of the tissue still in aldehyde and embed it in Lowicryl. For this I have the choice of PLT (progressive lowering of temperature) or freeze substitution. As I know that freeze substitution generally gives increased sensitivity, I would probably use that. I would cryoprotect small pieces of sample in sucrose (above 1.6M) and freeze by immersion in liquid nitrogen (only because I do not have a high pressure freezer), and put the frozen blocks in cold methanol (to increase contrast I may add 1% osmium tetroxide or uranyl acetate to the substitution medium). While still cold, I would gradually replace the methanol with unpolymerized Lowicryl until I was in 100% resin and could polymerize it by exposure to UV light, still at low temperature.
The sectioning step would be the same as above. I would first prepare sections for LM to screen the tissue for mast cells and for immunolabeling. Once I had both, I would thin section and look at the sample by EM. If this routine is kept standard then the level of success will increase as the level of frustration drops.
As for your comment that you see background in the first few sections of your EM labeling, I presume that this is outside the cells. It is possible that this background is also there in your LM preparations but because it is only in a thin layer, the LM may not have the sensitivity to register it. If you are getting a high signal to noise ration at the LM level but not by EM, it would be a good idea to figure out why this is. The result may affect the way you interpret your EM pictures. I notice that many antibodies when tested by LM appear to be very specific with no background, but when used at the EM level, are unusable because of the large amount of background they produce. If the LM is carefully performed and the results examined before they have been exposed to the process I have called "Photoshop purification of antibodies", the background can be picked up there too. Only with very few antibodies have I noticed that no background is detected by LM or by Western blotting, but that it comes up in the EM. Usually this is a result of the high concentrations I use the antibodies at, compared to what is used for other detection emthods. The offending signal, which is still specific, has been diluted out. This background can be eliminated by careful preparation and purification of antibodies.
By all means expose the labeled sections to a fixation step after the labeling protocol is complete. It is such a small part of the process as to be negligible. I would be interested, however, in any quantitative results that actually show this step to retain gold particles when compared with labeling protocols that leave it out.
Regards,
Paul Webster.
Disclaimer: Other than a satisfied user, I have no affiliation with or interest in Lowicryl resins or Adobe Photoshop. All opinions are my own and are freely given. How they are used is not my responsibility.
Paul Webster, Ph.D House Ear Institute 2100 West Third Street Los Angeles, CA 90057 phone:213 273 8026 fax: 213 413 6739 e-mail: pwebster-at-hei.org http://www.hei.org/htm/aemi.htm
Michael Plociniak wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } A response directed to Paul Webster, c/o this invaluable and open forum: } } I would like to continue with the topic of immunogold labeling for TEM, } relating to your suggestion: } } "One possible step you could put in is to first try out your whole } preparation and immunolabeling protocol at the LM level. Use the same } reagents and same preparation protocols as you plan to use for EM and } expose to silver enhancment for a longer time so that you can see the } signal by LM. If you see a signal then you will be confident that you will } see a signal when you go to examine the EM experiment." } } How reliable is LM for predicting TEM immunogold labeling results? } } I have tried this approach before, with mixed results. It sounds like a } great idea, but in attempting to "modify" the EM protocol into a LM pilot } experiment, it becomes a completely different beast. During primary } fixation, EM requires glutaraldehyde to preserve ultrastructure. You } suggested that glut not be used until AFTER immunolabeling, but won't the } damage to the fine structure already be done? I was under the impression } that glut "postfixation" is intended primarily to crosslink gold antibodies } in place after they have bound to primary antibody. } } Also, silver and gold enhancement reagents come in LM and EM formulae; } usually, EM versions are less acidic. Does a longer LM enhancement } accurately predict the action of a brief EM enhancement? Perhaps EM } formulae can be used to directly monitor enhancement with the light } microscope. However, because optimal EM enhancement is invisible by LM, } one will need to empirically determine EM enhancement times for each cell } system. Doesn't this argue against spending too much time with LM } experiments, when EM also includes osmium, uranyl acetate, dehydration, and } resin infiltration? } } I still use pilot LM experiments, but more for their complimentary benefit, } rather than for their predictive value. } } Routinely, I see a discouraging signal to noise ratio at the LM level. } However, at the EM level noise is restricted to the first couple of resin } sections of a monolayer culture, and subsequent sections contain beautiful } results. I interpret this as nonspecific binding of antibodies to the } poly-L-lysine and lamin-coated glass substrate. In this example, routine } LM (DIC optics without optical sectioning) does not accurately predict EM } performance. } } Sincerely, } } Michael Plociniak } Research Technician } Albert Einstein College of Medicine } Neuroscience Dept. } Rose Kennedy Center, 529 } Bronx, NY 10461 } (718) 430-3509 } plocinia-at-aecom.yu.edu } } transmission and scanning electron microscopy } } } } } RFC822 header } ----------------------------------- } } Received: from ultra5.microscopy.com [206.69.208.10] by mailhouse.hei.org } with ESMTP } (SMTPD32-6.00) id AC422BAD01DC; Tue, 26 Sep 2000 11:34:42 -0700 } Received: (from daemon-at-localhost) } by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) id SAA03897 } for dist-Microscopy; Mon, 25 Sep 2000 18:47:37 -0500 (CDT) } Received: from no_more_spam.com (sparc5 [206.69.208.10]) } by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) with SMTP id SAA03893 } for "MicroscopyFilteredEmail-at-msa.microscopy.com"; Mon, 25 Sep 2000 } 18:47:07 -0500 (CDT) } Received: from post.aecom.yu.edu (post.aecom.yu.edu [129.98.1.4]) } by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) with ESMTP id SAA03886 } for {Microscopy-at-sparc5.microscopy.com} ; Mon, 25 Sep 2000 18:46:53 -0500 (CDT) } Received: from amca (amca.aecom.yu.edu [129.98.90.216]) } by post.aecom.yu.edu (8.9.3/8.9.3) with SMTP id TAA26413 } for {Microscopy-at-MSA.Microscopy.com} ; Mon, 25 Sep 2000 19:40:58 -0400 (EDT) } Message-Id: {3.0.6.32.20000925193749.0094c470-at-mailserver.aecom.yu.edu} } X-Sender: plocinia-at-mailserver.aecom.yu.edu } X-Mailer: QUALCOMM Windows Eudora Light Version 3.0.6 (32) } Date: Mon, 25 Sep 2000 19:37:49 -0400 } To: Microscopy-at-sparc5.microscopy.com } From: Michael Plociniak {plocinia-at-aecom.yu.edu} } Subject: Can LM predict immunogold TEM? } Mime-Version: 1.0 } Content-Type: text/plain; charset="us-ascii" } Errors-to: Microscopy-request-at-sparc5.microscopy.com } X-RCPT-TO: {pwebster-at-hei.org} } X-UIDL: 257555315 } Status: U } Paul Webster, Ph.D House Ear Institute 2100 West Third Street Los Angeles, CA 90057 phone:213 273 8026 fax: 213 413 6739 e-mail: pwebster-at-hei.org http://www.hei.org/htm/aemi.htm
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I need some help. If anyone out there has a Philips 420 TEM, I would appreciate some info on hooking up the main transformer (i.e. from the installation manual or anything else that I might use), our electrian cann't figure out the wiring for hooking it up. Any help would be greatly appreciated. Thank You, Chuck
Hello, I have just changed the Ar tank that is used on my PIPS. I now have a hugh amount of contamination from the grid on my TEM samples. The only thing that has changed is the tank. I made sure the guns are aligned and the flow adjusted properly.
Has anyone else had this happen after changing the tank and is there some advice for me?
I have a bunch of microslide storage boxes, the plastic kind that hold 100 glass slides. They have been used, but are in good condition . I don't need them any more, let me know if you would like some or away they go.
Jonathan Krupp Microscopy & Imaging Lab University of California Santa Cruz, CA 95064 (831) 459-2477 jmkrupp-at-cats.ucsc.edu
Mark: I can't comment on a mu-metal shield, but have used field-cancelling systems on SEMs and FIBs to great effect. I can't remember what we paid for ours, so I don't know if such a system would cheaper than the shield. But if you're curious try this URL: http://www.spicerconsulting.com/. This company is in the UK, but I think they have people in the Americas.
Mark West wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Hi, } } A question about SEM interference. We have high electromagnetic field } levels (about 5 times the Jeol recommended limits) and our SEM isn't much } use above 10 000x mag (a Jeol 5410 low vacuum) - the focus is fuzzy and } astigmatism hard to correct. I've had water and air pipes removed from the } room, and we've been everywhere measuring fields but the fields and the } poor performance don't change. Changing the room is not an option (to the } people in charge of the institute!). Jeol recommended, ruling out a room } change, that we fit a mu-metal shield around the column. I have contacted } the company that makes the mu-metal and the thing would cost about } US$6000.00 and a hassle to ship to Mexico, so I don't want to go ahead and } find that we're in the same place with our performance. } } Does anyone have experience with fitting a mu-metal shield to their } microscope (especially a scanning EM) and resolving (or not resolving!) } interference problems. I'd be very interested in hearing about your } experiences with this stuff. } } Thanks, } } Mark } } PS - the serial sections are coming along nicely (not perfect,but heading } that way!), but I've still got a lot of ideas to try yet. Many thanks. } } ******************************************** } Mark West, } Unidad de Microscopia Electronica, } (Electron Microscopy Unit) } Instituto de Fisiologia Celular, } Universidad Nacional Autonoma de Mexico, } 04510 Mexico D.F. } } tel (unidad/lab) *(525) 622 5610* } (casa/home) (525) 619 3020 } Fax (525) 616 2282 } ********************************************
In one of his recent postings, Dr. Webster suggested using cell fractionation for immuno-localization. This pointed to an alternative path to the usual route I take for immunolabeling. I of course understand that in some circumstances, this approach might be the only way to acquire valid data. However, on the other end of the spectrum, there are also situations, perhaps a majority, in which the localization is only relevant when the cellular and tissue organization is intact. I work a lot with brain tissue, in which the membrane integrity is essential for identifying different neuronal elements, and the labeling technique is often intended to help figure out the relationship among those elements. In this case, cell fractionation is certainly not suitable, and even in intact tissue, the morphology can only be compromised to a certain point. Inconveniently, this point may well be beyond the threshold that allows antigen survival or antibody access.
Fortunately, technology has advanced so much thanks to the relentless efforts of many. With the availability of ultrasmall gold conjugates and silver enhancement reagents for EM, the detectability of antigens in fixed, wet tissue with excellent ultrastructure is no longer unreachable. I routinely use 4% paraformaldehyde plus 0.2% glutaraldehyde to perfuse brain and then post fix it with the same fixative for 1 hour. The ultrastructure quality obtained with this fixation is such that there is no severe dilation of the ER and Golgi, very little swelling of mitochondia, and most membranes are still intact. Yet, with this morphology, I have labeled epitopes in the cytoplasm, nuclei, both inside and outside of Golgi and ER, and on both sides of the cell membranes. Too bad one is not allowed to post images on this listserver.
There are two stages involved in the final preservation of ultrastructure during pre-embedding immuno-labeling. The initial fixation determines the starting point of ultrastructure quality and is limited by the tolerance level of the antigens. Subsequently, the immunolabeling procedure itself is responsible for preventing deterioration of the ultrastructure. The latter is closely related with reagents to be used, and has been the focus of reagent improvement for the last few years. Using a single Fab conjugate allows less harsh permeablization of the tissue. A near neutral pH silver enhancement reagent reduces the harm of pre-osmium enhancement on morphology.
Will it work at the EM level if it does at the LM level? Pre-embedding labeling with ultrasmall gold conjugates and silver enhancement has definitely made it easier to carry LM success to the EM level.
Thank you.
Hong
============= Hong Yi Emory Neurology Microscopy Core Laboratory Emory University School of Medicine 6215 Woodruff Memorial Research Building 1639 Pierce Drive Atlanta, GA 30322 Tel: (404) 727-8692 Fax: (404) 727-3157 Email: hyi-at-emory.edu
.. I am just curious: What's the good thing about DM? "Everbody" seems to be complaining about this "image format"(?). - What's the advantage of DM compared to TIFF?
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Have you thought about filtering your bacteria on polycarbonate filters? This method would obviously only work for the SEM.
There was a thread a while ago and I think I copied it from the archives where I searched for the the keyword "filters". Quite a few people were discussing how to prepare bacteria for SEM and nearly all suggested filters.
A typical protocol was: filtering suspension of bacteria through 0.2 um polycarboante filter, remove filter from housing and place into 2.5% glutaraldehyde in buffer, follow with buffer washes, optional post fixation with OsO4, gradient alcohol series (3 x 100 % EtOH) and then either 3 washes in HMDS (hexamethyldisilizane) followed by air drying or critical point drying.
I am working with leukemia cell cultures and pellet the cells for TEM (my cells are easier to pellet) and filter them on polycarbonate filters for SEM.
Regards
Claudia Dr. C. Hayward-Costa School of Life Sciences Kingston University Penrhyn Road, Kingston upon Thames Surrey KT1 2EE, UK 44(0)208 547 2000 x 2240 Email: c.hayward-at-kingston.ac.uk
I am looking at Field Emission SEMs with a view to a lasting and meaningful relationship, and am reminded of the problem of verifying manufacturer's claims about resolution. I dimly recall having been here before, but it is such a long time since I had the luxury of considering SEM purchase that it has become buried in my deep subconscious, alongside other strongly repressed issues like full costs recovery, the Oedipus complex, resentment of academic payscales and Bo Derek.
The more I reflect on resolution the more it looks like a can of worms. For example, it has recently become apparent that some FESEMs can "resolve" 1nm gold on a featureless carbon film under conditions where the manufacturer is only claiming a lesser resolution. This situation must be analogous to the situation where 15nm gold can be imaged by reflection light microscopy, or stars are imaged by eye or telescope. In other words the individual point sources of light (or secondaries) are seen, but are represented by the imaging system as objects larger than their true size. Most manufacturers seem to use gold particles on carbon or mag tape as resolution test objects. I have reservations about this, partly because of the effect described above, which will result from the massive contrast between gold and carbon, but also because it is not a realistic test of performance in the context of low-kV operation and light-element materials and biological samples.
I would therefore be grateful for your experience and advice on some of the following issues:
How should resolution be defined?
Is there a rigorous way of measuring the resolution achieved in a given SEM image?
Is there a rigorous way of establishing the fundamental resolving power of the SEM imaging system? (I think these two questions are different)
What would you recommend as a test object for testing the practical resolution of the sytem in imaging of light-element samples?
Consider the following conditions:
Field width on specimen = 1µm Magnification = 200k at display width of 200mm (8") At a digital image size of 1024x768 pixels (typical basic FESEM display resolution) the pixel size is therefore about 1nm Presumably at this magnification even if the SEM is resolving 1nm (let's assume it is) the display system is inadequate to demonstrate the fact.
Question: How many pixels would be required to image and resolve the 1nm data in this image? The answer to this question is presumably influenced by the criteria employed for establishing the resolution (point to point? peak width at half height? etc) and presumably dictates either the minimum magnification or the minimum digital resolution necessary to test resolution at the approximately 1nm current limit of FEGSEM performance.
I would be grateful for your comments Chris
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Dr Chris Jeffree University of Edinburgh Biological Sciences EM Facility Daniel Rutherford Building King's Buildings EDINBURGH EH9 3JH Tel: +44 (0) 131 650 5345 FAX: +44 (0) 131 650 6563
My thanks to everyone for their suggestions about how best to troubleshoot my slow dye sub printer. I can't say that I have gotten a conclusion yet, but I have learned enough to see that taking the printer off the SEM's computer is my best bet. It prints fine from every computer I have tried EXCEPT the SEM one and after some problems I've had with that one, it seems wise to separate this function from that system. That done, all is fine.
Thanks again to all here who were so generous of their time and ideas.
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A very basic point that is almost always overlooked when discussing SEM "resolution," is the difference between "resolution" and "resolving power."
An SEM might have a resolving power of 1nm or so based on the observation of gaps between gold particles on a featureless carbon film, as you point out. Fine--if your job is to look at gold on C.
For the actual 3-D samples most everyone looks at, EVERY micrograph has a DIFFERENT resolution, closer to 10nm, because of secondaries generated by back scattered electrons, edge effects, ... etc. If you want resolutions closer to the instrument's resolving power you have to look at thin TEM specimens without sharp edges and angles.
In our semiconductor lab we have several FESEMs advertised to have resolutions of 1nm that can't resolve 4 and 5 nm layers in a film stack. There is no question that these instruments are superior to non-FESEMs and that today's SEMs are feature-filled and wonderfully reliable--just don't expect 1nm resolution in real images and remember the difference between resolution and resolving power.
So if every instrument has one "resolving power" and a different "resolution" for each picture, to answer your question: the only test of "resolution" for you is to take your bread-and-butter samples to each manufacturer and compare the resulting images. There isn't any theoretical formula that will provide a more meaningful answer.
Ron
Ron Anderson, IBM, Hopewell Jct., New York, USA. anderron-at-us.ibm.com
IBM Analytical Services; http://www.chips.ibm.com/services/asg
We haven't changed the Ar tank yet, so I have not observed the problem you describe. However, we do get Cu contamination periodically in the PIPS due to buildup of contamination on the bottom surface of the shutter (facing the sample). This is easily fixed by removing the shutter and cleaning it with very rough, 320 grit or coarser, grinding paper. The timing may be coincidental with the change of Ar tank, but just in case check that the presure is set correctly in the tank regulator (8 psi) and the gas control valves; a higher pressure may have increased the Ar in the beam and the sputtering from the shutter to the sample. The most common source of contamination from the grid is the grid itself: grid thickness varies, and when it is larger than nominal the grid "shadows" the sample at low milling angles sputtering grid material onto the sample. I hope this helps.
Augusto Morrone Seagate Technology Augusto_A_Morrone-at-notes.seagate.com
"Leah Wajdyk" {leah.wajdyk-at-onsemi.com} on 09/26/2000 05:55:59 PM
To: Microscopy-at-sparc5.microscopy.com cc:
Hello, I have just changed the Ar tank that is used on my PIPS. I now have a hugh amount of contamination from the grid on my TEM samples. The only thing that has changed is the tank. I made sure the guns are aligned and the flow adjusted properly.
Has anyone else had this happen after changing the tank and is there some advice for me?
There is a group here that would like to use digital microscopy to characterize sizes of vesicles obtained by fractionation of myocytes. The problem is to get them to stick to glass coverslips in a non-selective way. Because we had it on hand, we tried poly-L-lysine and letting the vesicles settle overnight at 4C. The proportion of material that remained on the coverslips was very small. I have a few questions: 1) Is it possible that different populations (with different membrane proteins etc.) would adhere differentially to the positively-charged surface? 2) Does anyone have experience with different substrates (e.g. Cel-Tak, silane, albumin) that would be more appropriate. 3) Should we abandon this approach and use the more reliable (?) negative stain?EM route.(I worry about shrinkage artifacts due to air-drying and heavy-metal stains) Any advice will be gratefully considered and references appreciated. Thanks in advance. Regards,
Andrew Ochalski, Microscopy Technician, Dept. of Biology, University of Ottawa Room 108, Gendron Bldg. 130 Louis Pasteur, Ottawa, Ontario CANADA K1N 6N5 613-562-5800 x6343 FAX: 613-562 5486
An additional consideration for any low angle ion milling is to change to Mo grids. They have a much lower sputter rate. They are also thinner, allowing a much lower milling angle on the grid side of the specimen. Using 1 mm diameter grids, and with the feature of interest cnetered in the hole, we routinely mill as low as 3 degrees into the grid hole. They are particularly suitable for specimens which require extended low angle milling, on the order of 2-3 hours.
Phil
Philip L. Flaitz IBM Analytical Services, Hopewell Junction, NY http://www.chips.ibm.com/services/asg Ph.......(914) 892-3094, FAX -2003 flaitz-at-us.ibm.com
The Digital Micrograph (DM) format is not a image file format for say in the way that TIFF, PICT, JPEG, GIF, etc. are file formats (some of which use lossy compression). Digital Micrograph, in addition to saving the image (with no reduction in data), saves a lot of experimental header information, for instance calibrations, images size, keywords, magnification, electron energy, TEM operator name, image annotations etc. Many of these can be entered in the Object Info dialog. Also, DM can save multiple images in a single DM file.
Tyrone Daulton
Gunnar Glasser wrote:
} Hi, } } .. I am just curious: What's the good thing about DM? "Everbody" seems to } be complaining about this "image format"(?). - What's the advantage of DM } compared to TIFF? } } Dipl.Ing.(FH) G.Glasser } Elektronenmikroskopie } Max Planck Institut fuer Polymerforschung } Ackermannweg 10 } 55128 Mainz, GERMANY } phone: ++49 +6131 379195 } fax : ++49 +6131 379100 } web: http://www.mpip-mainz.mpg.de/~glasser } } Disclaimer: The above statement is mine alone and does not implicitly represent the position of the MPI-P or the MPG!
-- _____________________________________________________ Tyrone L. Daulton Director: Marine Geosciences - Electron Microscopy Center Physicist and Senior Electron Microscopist
Naval Research Laboratory Marine Geosciences Division (Code 7400) Stennis Space Center, MS 39529-5004
The Mid-Atlantic Microbeam Analysis Society and the NIST Surface and Microanalysis Science Division will be meeting at the National Institute of Standards and Technology Gaithersburg, MD on
Thursday, October 19, 2000 10:15 am to 3:00 pm NIST Administration Building Lecture Room D
10:30 am Energy-Filtered TEM Jim Bentley Oak Ridge National Laboratory 2000-2001 MAS Tour Speaker
1:00 pm Electron Microprobe Dating of Minerals John Hanchar Department of Earth and Environmental Sciences The George Washington University
For more information, contact John Henry Scott 301-975-4981, johnhenry.scott-at-nist.gov, or Ryna Marinenko 301-975-3901, ryna.marinenko-at-nist.gov Don't forget to bring your MAMAS dues ($5.00)
Hope to see you there, -- John Henry
John Henry J. Scott Bldg 222/Rm A113 NIST Microanalysis Research Group 100 Bureau Drive Stop 8371 (301) 975-4981; (301) 417-1321 FAX Gaithersburg, MD 20899-8371
------------------------------------------------------------------------ The Microscopy ListServer -- Sponsor: The Microscopy Society of America
{Subject: .... again: Digital Micrograph??? {From: Gunnar Glasser, glasser-at-mpip-mainz.mpg.de {.. I am just curious: What's the good thing about DM? "Everbody" seems to {be complaining about this "image format"(?). - What's the advantage of DM {compared to TIFF?
It is a proprietary format, no other common program can use it. In an earlier post I say that I save everything in Digital Micrograph format and that is my archival image. If you have properly calibrated the mag on your camera and you spend time putting in keywords, etc, these become a permanent part of the image and you can always go back and get that image. I always try to be as verbose as I can with information. For example, my image name may be: 000927rhinovirus.1 which has today's date and a short sample name. This also allows me to sort files by a name that can be chronologically based. In the tags and keywords I have the pixel size (based on mag), sample name, collaborator's name, sample prep conditions, etc., etc. You can also measure items automatically without having to calculate in your mind that x pixels in the image equals y nm in the sample. You cannot do that with a TIFF or whatever. After I have saved my image in DM format, then I will resave it again, if I want to, in any other format but I always have the untouched original to fall back to.
Norm Olson
******************************************* Norm Olson Senior Research Electron Microscopist Department of Biological Sciences Lilly Hall of Life Sciences Purdue University West Lafayette, IN 47907
by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) id JAA09576 for dist-Microscopy; Wed, 27 Sep 2000 09:53:32 -0500 (CDT) Received: from no_more_spam.com (sparc5 [206.69.208.10]) by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) with SMTP id JAA09572 for "MicroscopyFilteredEmail-at-msa.microscopy.com"; Wed, 27 Sep 2000 09:53:02 -0500 (CDT) Received: from mercury.uwe.ac.uk (mercury.uwe.ac.uk [164.11.132.23]) by ultra5.microscopy.com (8.9.1b+Sun/8.9.1) with ESMTP id JAA09565 for {Microscopy-at-sparc5.microscopy.com} ; Wed, 27 Sep 2000 09:52:46 -0500 (CDT) Received: from fas655.uwe.ac.uk ([164.11.205.145]) by mercury.uwe.ac.uk (2.0.4/SMS 2.0.4-devel) with SMTP id PAA21211; Wed, 27 Sep 2000 15:46:23 +0100 (BST)
Claudia, do you notice any difference in your cell cultures between HMDS and CPD?
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Dear Kristine } } Have you thought about filtering your bacteria on polycarbonate } filters? This method would obviously only work for the SEM. } } There was a thread a while ago and I think I copied it from the } archives where I searched for the the keyword "filters". Quite a few } people were discussing how to prepare bacteria for SEM and } nearly all suggested filters. } } A typical protocol was: filtering suspension of bacteria through 0.2 } um polycarboante filter, remove filter from housing and place into } 2.5% glutaraldehyde in buffer, follow with buffer washes, optional } post fixation with OsO4, gradient alcohol series (3 x 100 % EtOH) } and then either 3 washes in HMDS (hexamethyldisilizane) followed } by air drying or critical point drying. } } I am working with leukemia cell cultures and pellet the cells for } TEM (my cells are easier to pellet) and filter them on polycarbonate } filters for SEM. } } Regards } } Claudia } Dr. C. Hayward-Costa } School of Life Sciences } Kingston University } Penrhyn Road, Kingston upon Thames } Surrey KT1 2EE, UK } 44(0)208 547 2000 x 2240 } Email: c.hayward-at-kingston.ac.uk }
---------------------------------------- Patton, David Email: David.Patton-at-uwe.ac.uk "University of the West of England"
The encoding of the image information in DM is the TIFF format, with the extra header info listed below. It is possible on files containing single images to strip the header info and open them with Photoshop.
Craig Wall
*%*#*%*#*%*#*%*#*%*#*%*#*%*# Disclaimer: The above statements are mine alone and do not implicitly represent the position of the Goodyear Tire & Rubber Co. Craig G. Wall, Ph.D. The Goodyear Tire & Rubber Company 142 Goodyear Boulevard Akron, Ohio 44305 (330)796-2335 (330)796-3304 FAX
The Digital Micrograph (DM) format is not a image file format for say in the way that TIFF, PICT, JPEG, GIF, etc. are file formats (some of which use lossy compression). Digital Micrograph, in addition to saving the image (with no reduction in data), saves a lot of experimental header information, for instance calibrations, images size, keywords, magnification, electron energy, TEM operator name, image annotations etc. Many of these can be entered in the Object Info dialog. Also, DM can save multiple images in a single DM file.
Tyrone Daulton
Gunnar Glasser wrote:
} Hi, } } .. I am just curious: What's the good thing about DM? "Everbody" seems to } be complaining about this "image format"(?). - What's the advantage of DM } compared to TIFF? } } Dipl.Ing.(FH) G.Glasser } Elektronenmikroskopie } Max Planck Institut fuer Polymerforschung } Ackermannweg 10 } 55128 Mainz, GERMANY } phone: ++49 +6131 379195 } fax : ++49 +6131 379100 } web: http://www.mpip-mainz.mpg.de/~glasser } } Disclaimer: The above statement is mine alone and does not implicitly represent the position of the MPI-P or the MPG!
-- _____________________________________________________ Tyrone L. Daulton Director: Marine Geosciences - Electron Microscopy Center Physicist and Senior Electron Microscopist
Naval Research Laboratory Marine Geosciences Division (Code 7400) Stennis Space Center, MS 39529-5004
Hi, anybody has experiences on small angle(or 90 degree) cleavage method of making cross section TEM samples with MgO substrates? Any information would be appreciated.
---------- } From: Norm Olson {nho-at-bilbo.bio.purdue.edu} } To: MSA {Microscopy-at-sparc5.microscopy.com} } Subject: Fwd: .... again: Digital Micrograph??? } Date: September 27, 2000 11:35 AM }
} } } {Subject: .... again: Digital Micrograph??? } {From: Gunnar Glasser, glasser-at-mpip-mainz.mpg.de } {.. I am just curious: What's the good thing about DM? "Everbody" seems } to } {be complaining about this "image format"(?). - What's the advantage of } DM } {compared to TIFF? } } It is a proprietary format, no other common program can use it. In an } earlier post I say that I save everything in Digital Micrograph format } and that is my archival image. If you have properly calibrated the mag } on your camera and you spend time putting in keywords, etc, these become } a permanent part of the image and you can always go back and get that } image. I always try to be as verbose as I can with information. For } example, my image name may be: } 000927rhinovirus.1 which has today's date and a short sample name. ................After I have saved my image in DM format, then } I will resave it again, if I want to, in any other format but I always } have the untouched original to fall back to. } } Norm Olson }
For what it's worth, our NORAN Voyager system has its own proprietary image format called a .grey (theoretically readable only by Voyager systems), which also incorporates important information such as pixel size, magnification, date, and whatever other notes you wish to include. I'm told by one of my colleagues that with a little creative backstripping of headers, and a foreknowledge of the size of the file, you can open these things in Photoshop, or CorelDraw or whatever. So far I haven't really had to do anything like that - our images all get stored both as .greys, and as .tiffs. With the Voyager software, you can always make a new .tiff from a .grey, but you can't make a .grey from a .tiff. These proprietary image formats can be a bit of a pain sometimes, but at least they do allow the inclusion of all that extraneous information. In our lab, when a job is finished, the client gets a CD containing all their images in both formats and I make a duplicate for myself, as an archive. Theoretically, the client can't look at the .greys. The client's CD then becomes an off-site archive copy for me, should mine turn out to be a dud (hasn't happened yet, but I remain pessimistic). If the client is from "outside", there may be ownership/confidentiality issues involved, so I always check to see if they mind me keeping an archive for them. So far, they've all been happy to have me keep a copy, just in case. But then, I'm not doing any work for military or spy agencies, or even big corporations, or anybody like that.
F.C. Thomas Geological Survey of Canada (Atlantic) Bedford Institute of Oceanography Dartmouth, Nova Scotia Canada B2Y 4A2
"William F. Tivol" wrote: } My experience with Peltiers does not involve a CCD camera, but it may be } instructive nonetheless. When we wanted to provide a constant temperature for } the chemicals in the darkroom--which required sometimes heating the water and at } other times cooling it--we decided that Peltiers were the best solution. We } bought a commercial thermoregulating unit which sent current of the appropriate } polarity through the Peltiers to adjust the temperature to the desired setpoint. } The Peltiers failed at an expensive rate, and we found that the thermoregulator } was such that it provided a specific current, which it turned on and off in a } duty cycle (the greater the temperature difference, the greater fraction of time } the current was on). The sudden changes in current were the cause of the } Peltiers' demise.
Sorry, but that seems not to be the case.
http://www.tellurex.com/resource/txfaq.htm
"Can I use pulse-width modulation to control my Peltier device if I keep the voltage at VMax or below? Yes, and this is one of the most electrically-efficient ways to control voltage to your device—although you must observe some precautions."
And they suggest 2kHz PWM frequency.
I think your temperature regulator supplied too much voltage or current to the peltier, causing overheating and destruction. Just a guess.
} We were also warned that moisture in the Peltiers could lead to an } electrochemical process which caused corrosion and subsequent failure.
This is valid. Water inside peltier will cause both mechanical trouble (freezing inside peltier), and corrosion problems etc..
To the original poster: I _really_ think you should think of some way to make certain that there either is water flow _always_ to the peltier, or that power to peltier (and camera?) is switched off at power/flow failure. The latter (switch off) should be easier. The peltier _WILL_ die from overheating if there is not sufficient cooling. It might kill your camera by overheating it as well.
If you use just water (not glycol or something) as coolant in recirculators, perhaps you could add redundancy by adding a couple of magnetic valves to the peltier cooling lines so that while there is mains current, the valves lets recirculator water to flow through peltier, and otherwise let water from water mains to flow through peltier and to sink. Something like this:
water mains } ----------- valve1 -----o | o--------} peltier in | recirculator out } ------ valve2 -----o
recirculator in {------- valve3 -----o | o-------- { peltier out | sink {------------------ valve4 -----o
Where: valve1: normally open (NO = conducts when coil NOT powered) valve2: normally closed (NC = conducts when coil IS powered) valve3: normally closed valve4: normally open
Just connect all valve coils in parallel and to circuit below.
The idea is to switch on the system (ie. recirculator flow) with the button. With relay switched on, contacts 1 and 2 are closed and power goes to valves. Next, when mains power goes off, relay is nolonger switched on, and no power goes to valve coils (thus mains water cooling). User has to press button again to put relay on - return of mains power won't do this.
Total cost for valves etc. about 100usd or less.
Of course, if you are using glycol or some other chemical, it might well be that regulations forbid you from mixing piping of water mains with your glycol piping, because of danger of contaminating water mains.
STILL, I really think it would be electrically trivial to make certain that the camera does NOT switch on when there is no coolant flow (or simpler yet, no mains - just use circuit above with camera connected instead of valve coils). A simple pressure or flow switch at coolant line, and power to camera via the pressure/flow switch will be easy.. This should be pretty fool-proof protection for the camera in _any_ case of coolant flow failure. I think sooner or later you _will_ have coolant failure without mains failure (recirculator pump failure or something), and this should be anticipated in your design. Of course, you could as well add flow/pressure switch NOcontact in series with relaycoil in circuit above.
} Date: Wed, 27 Sep 2000 10:04:02 -0500 } From: Andrew Ochalski {aochalsk-at-science.uottawa.ca} } To: Microscopy-at-sparc5.microscopy.com } Subject: Sticking vesicles to glass coverslips } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } } Dear Listmembers, } } There is a group here that would like to use digital microscopy to characterize sizes of } vesicles obtained by fractionation of myocytes. The problem is to get them to stick to } glass coverslips in a non-selective way. Because we had it on hand, we tried poly-L-lysine and } letting the vesicles settle overnight at 4C. The proportion of material that remained } on the coverslips was very small. I have a few questions: } 1) Is it possible that different populations (with different membrane proteins etc.) would adhere } differentially to the positively-charged surface? } 2) Does anyone have experience with different substrates (e.g. Cel-Tak, silane, albumin) that would } be more appropriate. } 3) Should we abandon this approach and use the more reliable (?) negative stain?EM route.(I worry } about shrinkage artifacts due to air-drying and heavy-metal stains) } Any advice will be gratefully considered and references appreciated. Thanks in advance. } Regards, } } Andrew Ochalski, } Microscopy Technician, } Dept. of Biology, } University of Ottawa } Room 108, Gendron Bldg. } 130 Louis Pasteur, } Ottawa, Ontario } CANADA } K1N 6N5 } 613-562-5800 x6343 } FAX: 613-562 5486 } Not sure if this will help, but it's worth a try. Many years ago, we did SEM of red blood cells and needed to have them stuck to a substrate. We treated glass coverslips with collagen, added the RBCs, and glued them down with glutaraldehyde. They stuck very well.
Coverslips Chip one edge of a cover slip (we used 9 x 9 mm) in a specific corner so that you will be able to know which side is up if it gets flipped over. _______ | | | | | c |______|
You can do this by holding the cover slip between thumb and forefinger of one hand and gently dragging the tips of an old pair of EM forceps across the edge at a downward angle. We had about 70% sucess without breaking.
Acetone clean cover slips and let air dry. Put them into a moist chamber (Petri dish with wet filter paper) and add a large drop of 1% aqueous collagen. Note the orientation of your chip mark on the cover slip. Don't let the drop flow over the sides, but just sit there and rounded up.
Incubate 30 min.
Remove drop of collagen with Pasteur pipette for future use.
Wash coverslips well with dH2O. Can use right away or store dried for future use.
Add membrane prep, cells, etc., without letting drop run over the sides. (Surface tension will allow you to cover the glass with a rounded-up drop.) Do not add too much sample; leave enough room to add more fluid later. Let sit 30 min.
Then add to the suspension a drop of concentrated glutaraldehyde. We added 50 ul of 25% glut in PBS to the cell suspension without letting the drop of suspension flow over the coverslip edge. Do this very slowly so as not to disturb the stuck material. (The glut is a di-aldehyde that links things together; in your case, hopefully, the material to the cover slip. Immunologists use this method to link antigens or antibodies to solid substrates.)
Let sit another 30 min, then GENTLY wash off the suspension. (We then critical point dried for SEM, but you can do your routine prep from here.)
Collagen This stuff comes as a flaky powder. Weigh out 0.1 gram and add to 10 ml dH-at-O. Stir overnight in the cold (cold to minimize bacterial growth). Filter through Whatman or any kind of filter paper to remove undissolved chunks. Keep refrigerated. Can autoclave if long term storage desired.
Good luck.
Sara E. Miller, Ph. D. P. O. Box 3712 Duke University Medical Center Durham, NC 27710 Ph: 919 684-3452 FAX: 919 684-3265
Department of Cellular & Structural Biology The University of Texas Health Science Center at San Antonio
The Department of Cellular and Structural Biology is recruiting faculty for tenure track positions at the Assistant, Associate and Full Professor level. The Department is particularly interested in recruiting qualified applicants with interests in cell and molecular biology, aging, cancer, immunology or neurobiology, and who utilize optical imaging approaches as a main research tool. The ability/willingness to teach in one of the Department's professional and/or graduate courses is also required.
Interested individuals should submit their curriculum vitae, a statement of research and teaching interests and experience, and three letters of recommendation to:
Dr. Ellen Kraig Chair, Cellular & Structural Biology Faculty Search Committee The University of Texas Health Science Center 7703 Floyd Curl Drive Mail Code 7762 San Antonio, Texas 78229-3900
Applications received prior to December 1, 2000 will be considered.
Further information about the Department and its optical imaging facility can be found at: http://www.uthscsa.edu/csb/csbhome.html
The University of Texas is an Equal Employment Opportunity/Affirmative Action Employer.
Dear sirs Respectfuly,i beg to let you now that i am a 26 year old Iranian researcher.Since 1993 to present i have been ivolved in research activities.I am the director of an Iranian research institute .One of the main activities of whichis to operate departments of electron microscopes.Also i am responsible electron microscope department of the Isfahan university of medical scienceis(one of the most reputed universities of Iran).Meanwhile i am in close relations with other electron microscope departments all through the country. Unfortunately hoewer, in Iran ,in spite of the existence of a great number of electron microscopes,the science of electron microscopy is not very developed and most of the electron microscope departments are eather inactive or have little activities.Therefore i intend to stablish the first center for development of electron microscopy technology in Iran by the assistance of the private sector.In this connection i will very much appreciate receiving assistance from your center.I intend to pass a complementary course in the field of electron microscopy at your center and then developing the above mentioned center in one of the cities or international free zones of Iran under support and assistance of your center .The programs of this center will include accepting electron microscopy students from Iran or your country, instruction of applied electron microscopy,instruction of electron microscopy courses at different Iranian universities,helding electron microscopy workshops,operating electron microscopy departments,supplying electron microscopy equipments and consumption electron microscopy materials,performing different electron microscopy techniques,praviding expanded electron microscopy services to scientific researche, industrial and medical diagnosis departments. With regard to the acceptance that receives the activities of our institute in the field of electron microscopy it seems that in the future the activities of the center for development of electron microscopy technology will be welcomed in a similar way by both public and private sectors. Therfore i would like to ask you to advise me in the above mentional fields.port of my executive and research cactivities are as follows: 1.Director of Isfahan's Sinapooyesh institute of research . 2.Responsibility of electron microscope department of Isfahan university of medical scienceis. 3.Responsibility of processing system of electron microscope department at Shahid Beheshti university of medical scienceisof Tehran. 4.Membership of the Iranian national standard commitee. 5.Iam the first provider of nanotechnology in Iran. 6.Receiving the appreciation medallion of the twelve th Kharazmi research festival in Iran. 7.Recieving the first prize of the national young researchers festival in Iran. 8.Reseiving the special prize at the national congress of the security of communications of Iran. 9.Instruction of electron microscopy courses at MSc and phD levels in reputed universitesof Iran. 10.Participaton in several national and international research congresses and presenting several scientific articles and activities. Meanwhile,from 1993 to 1995 i have studied in the field of medical diagnosis and then from 1995 to 1998 i have learnt electron microscopy techniques at Isfahan university of medical scienceis .since 1998 to present i am pursuing my studies in the field of electronic engineering. Thank you very much in advance for your sincer assistance and advice in this regard. sincerely yours Iman moradi
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There is No advantage to digital micrograph over other formats. It is a format supported by Gatan by force feeding it to users of their software. You can jump through some hoops and get it to export images in other formats though.
\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\ Gordon Ante Vrdoljak Electron Microscope Lab ICQ 23243541 http://nature.berkeley.edu/~gvrdolja 26 Giannini Hall gvrdolja-at-nature.berkeley.edu UC Berkeley phone (510) 642-2085 Berkeley CA 94720-3330 fax (510) 643-6207 cell (510) 290-6793
On Wed, 27 Sep 2000, Gunnar Glasser wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Hi, } } .. I am just curious: What's the good thing about DM? "Everbody" seems to } be complaining about this "image format"(?). - What's the advantage of DM } compared to TIFF? } } } Dipl.Ing.(FH) G.Glasser } Elektronenmikroskopie } Max Planck Institut fuer Polymerforschung } Ackermannweg 10 } 55128 Mainz, GERMANY } phone: ++49 +6131 379195 } fax : ++49 +6131 379100 } web: http://www.mpip-mainz.mpg.de/~glasser } } Disclaimer: The above statement is mine alone and does not implicitly represent the position of the MPI-P or the MPG! } }
Do any of you have experience with the GATAN DigiScan hardware and Digital Micrograph for converting a non-digital sem to a digital sem? We have a JEOL T200 we are considering for conversion. Your responses are greatly appreciated.
Sincerely, Ken Tiekotter, Adjunct Professor The University of Portland Department of Biology 5000 N. Willamette Blvd. Portland, OR 97203
Tyrone is correct in saying that some of the file formats in circulation (for example JPEG) use lossy compression and one should be careful with them. TIFF, however, is not necessarily one of them. TIFF usually allows to select compression options, which can include lossless compression (LZW). Also, TIFF is a flexible format where additional information can be stored in so-called "tags" (hence the name: Tagged Image File Format). There are public tags (like image size, etc), but each software can implement private tags, which are just ignored by other software. That way it is possible to incorporate additional information into the file itself without making it proprietary. That's the strategy we have pursued. TIFF, apparently like DM, can also store multiple pages in one file. Other formats, like BMP, only encode the actual image data and one needs additional mechanisms like a database to keep track of other elements like keywords.
Michael Bode, Ph.D. Soft Imaging System Corp. 1675 Carr St., #105N Lakewood, CO 80215 =================================== phone: (888) FIND SIS (303) 234-9270 fax: (303) 234-9271 email: mailto:info-at-soft-imaging.com web: http://www.soft-imaging.com ===================================
-----Original Message----- } From: Tyrone L. Daulton [mailto:tdaulton-at-nrlssc.navy.mil] Sent: Wednesday, September 27, 2000 8:24 AM To: Microscopy-at-sparc5.microscopy.com
The Digital Micrograph (DM) format is not a image file format for say in the way that TIFF, PICT, JPEG, GIF, etc. are file formats (some of which use lossy compression). Digital Micrograph, in addition to saving the image (with no reduction in data), saves a lot of experimental header information, for instance calibrations, images size, keywords, magnification, electron energy, TEM operator name, image annotations etc. Many of these can be entered in the Object Info dialog. Also, DM can save multiple images in a single DM file.
Tyrone Daulton
Gunnar Glasser wrote:
} Hi, } } .. I am just curious: What's the good thing about DM? "Everbody" seems to } be complaining about this "image format"(?). - What's the advantage of DM } compared to TIFF? } } Dipl.Ing.(FH) G.Glasser } Elektronenmikroskopie } Max Planck Institut fuer Polymerforschung } Ackermannweg 10 } 55128 Mainz, GERMANY } phone: ++49 +6131 379195 } fax : ++49 +6131 379100 } web: http://www.mpip-mainz.mpg.de/~glasser } } Disclaimer: The above statement is mine alone and does not implicitly represent the position of the MPI-P or the MPG!
-- _____________________________________________________ Tyrone L. Daulton Director: Marine Geosciences - Electron Microscopy Center Physicist and Senior Electron Microscopist
Naval Research Laboratory Marine Geosciences Division (Code 7400) Stennis Space Center, MS 39529-5004
The prices I have heard for field canceling systems has ranged from ~US$15k to $30k. A list of suppliers is available at "www.jcnabity.com/links.htm#Magnetic Field Cancellation".
Joe _________________________________________ Joe Nabity, Ph.D. JC Nabity Lithography Systems E-Beam Lithography using Commercial SEMs & STEMs PO Box 5354, Bozeman, MT 59717 USA Voice: (406) 587-0848 FAX: (406) 586-9514 E-mail: info-at-jcnabity.com Web: www.jcnabity.com
In a message dated 9/27/2000 3:30:27 AM Mountain Daylight Time, r-holdford-at-ti.com writes:
} Mark: I can't comment on a mu-metal shield, but have used field-cancelling } systems on SEMs and FIBs to great effect. I can't remember what we paid for } ours, so I don't know if such a system would cheaper than the shield. But } if } you're curious try this URL: http://www.spicerconsulting.com/. This } company } is in the UK, but I think they have people in the Americas. }
I have surplus a LKB Ultratome V microtome in full working order. This would be available free to an academic institution (you pay shipping) or for a reasonable cost (offers?) to a commercial lab. It is an older instrument but still produces excellent thin sections at room temperature. We have replaced it with a new instrument with cryogenic capability.
Obviously, the closer you are to Cambridge, Massachusetts, the easier shipping would be!
Contact me directly if you have interest or any questions.
Tony Garratt-Reed.
** Anthony J. Garratt-Reed ** MIT Room # 13-1027 ** 77 Massachusetts Avenue ** Cambridge, MA 02139-4307 ** USA ** ** Phone: (+) 1-617-253-4622 ** Fax: (+) 1-617-258-6478 **
I am interested in synchronising Hela cells and collecting samples in each stage of Mitosis. Does anybody have a protocol and the times at which each stage is reached after release from G1?
Ken Blight Senior Scientific Officer Electron Microscopy Unit Imperial Cancer Research Fund London England
In an early posting I wrongly suggested that the Ar pressure in the PIPS should be set at 8psi:
"...The timing may be coincidental with the change of Ar tank, but just in case check that the presure is set correctly in the tank regulator (8 psi) and the gas control valves; a higher pressure may have increased the Ar .."
Neal Zimmermann, who works in my lab, corrected me: the pressure should be set to 25psi as stated in the Gatan PIPS manual. Fortunately we had it set right, just my memory lapsed.
Augusto
Augusto Morrone Seagate Technology Augusto_A_Morrone-at-notes.seagate.com
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Reply to: RE: Immuno LM/EM Hi Jan,
Thanks for joining in. I was beginning to feel a bit lonely out here. It is interesting, however, to read the many off-line relplies I have been getting about this posting. Obviously many people are struggling with there attempts to understand the many different choices available for immunolabeling cells and tissues. I thank you for the support you have given.
I agree with your posting almost entirely and hope that we might have made our point by now. I have a few extra points to make which will probably end this thread for good (mostly from boredom, I guess).
First, I think we both agree that we should have reliable immunoreagents. I have preferences in the reagents I use, but I know there are other choices available. Use whatever works.
You express a reluctance to using cell fractions, something that someone off-line also disagreed with. I am not saying use poor morphology to get a result, I suggest using the best morphology that gives the result. In some instances the use of purified fractions of organelles has provided important results. Perhaps the number of specialized organelles in a cell system is so low that it is impossible to find sufficient profiles of them in the thin sections we use. If we can positively identify these organelles by some other unique marker, then it is a simple task to section the fraction and apply two antibodies (one being the identifier, one being the experimental). This gives the information we look for and thus allow us to continue, hopefully to label sections of whole cells at a later date. This is just part of the data collection process for the whole project. Again, use what works.
I was being negative about secondary marker antibodies to add a little spice to my posting. I know that many people use these markers but I did want them to question their reliability. There is a tendency to condemn the primary antibody without thinking there may be other causes for signal to be absent. Maybe there is a need for careful monitoring of the gold probes during storage.
I do not know of one published work that has extensively examined the effect of storage on colloidal gold probes (same antigen system, same preparation protocols, antibody-gold compared with protein A-gold, systematic counting of gold particles etc.). If anyone knows of such a study, I would be interested to hear of it. I often use unconjugated secondary antibodies as a bridge for protein A-binding. It is clear that this extra step increases sensitivity and also adds more gold particles to the section (supported by published results from Jan Slot, I think). Although the gold is not so closely associated with the structures I expect to label, I still get results from this sort of labeling.
The problems I have with the use of pre-embedding techniques are the same I had when I first used them in the 1980's (and my morphology was pretty good in those days too). It was always difficult to quantify the result because I could never be completely sure that all the antigens had been totally accessible to the antibodies or visualization reagents. This problem may not be due to size of the particles used, or with permiabilization (which do play a role), but may also be due to other factors that cannot be controlled for, such as biological activity within whole mounts. I am also reluctant to use pre-embedding labeling as a routine protocol because I cannot yet see a way to easily perform multiple labeling experiments. I look forward to being enlightened.
The possible lack of total accessibilty will also make it difficult to prove a negative result (how can I be sure that the vesicle lumen not labeled is because the antigen is not there, and not because the antibody or gold probe is not there?). I know these things are also problems with on-section labeling, but the methods for on section labeling are a little better understood. For this problem I would prepare thinner sections or count the gold particles. It may not improve things but I would be sure there is some possibility for labeling to occur. Limitations are easier to live with when we understand them a little more. Having siad all that, I realize that for some systems, pre-embedding is the way to go. If it works, use it.
I strongly support your advice that we should let the result drive our work and accept that what we find may not fit in with the favorite theory of the lab. The protocols, reagents and equipment available to us for the study of morphology and immunocytochemistry are the most sophisticated we have ever had. For this reason we have the ability to provide completely unambiguous quantitative results that are difficult to ignore. Learning to stand by them is our next step.
Finally, in an attempt to spice things up and to keep this thread going in some form, here are some extra comments to consider. Remember my question many years ago on this listserver when I asked if EM was a science or an art? All of the inital replies were that EM is an art. Only when I asked why we published in scientific journals did I get a few slightly more scientific replies. They didn't convince me too much though. I still think that many EM people see themselves as artists able to perform wonderful things (i.e. miracles) with complicated machines. We are wrong to think this. We are also wrong to think that we are able to provide every researcher who walks through the door with the result they need to finish their grant/paper/report. With the tools available, we can provide unbiased results that will have biological meaning only in the context of what else is known about the system. For immunocytochemical experiments at least, we should really be active participants in the whole project. Non-microscopists need to read this stuff and think about what they expect from EM too.
Best regards,
Paul Webster.
Paul Webster, Ph.D. Associate Scientist & Director Ahmanson Advanced Electron Microscopy & Imaging Center House Ear Institute 2100 West Third St. Los Angeles, CA 90057
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CENTRAL INSTITUTE FOR THE DEAF, St. Louis, Missouri
announces the following:
New Frontiers in the Amelioration of Hearing Loss
A Conference in Honor of James D. Miller
CID is now accepting poster submissions and registration for this conference in St. Louis, MO, March 22-25, 2001.
The conference will offer two expert seminar tracks, one for cellular and molecular biologists and one for scientists studying applied technologies and methods for the rehabilitation of children and adults with hearing loss. The speakers include some of the most active and progressive researchers working today.
In addition to serving as a forum for cutting-edge research, the conference is designed to bridge the information gap between basic and applied research related to hearing and deafness. Scientists from different tracks will be provided with helpful information during a primer session held before each expert seminar track. Social sessions will bring together diverse professionals to foster new perspectives and ideas. Attendees will have the opportunity to tour new CID campus facilities, including the Fay and Carl Simons Center for Biology of Hearing and Deafness and CID's new "quiet" oral school.
For poster guidelines, preliminary program and registration materials, please contact Sarah Uffman, CID Research Department, 4560 Clayton Avenue, St. Louis, MO 63110, or visit our web site at www.cid.wustl.edu { {http://www.cid.wustl.edu/} http://www.cid.wustl.edu/} .
Jaclynn M. Lett, Research Assistant
Faye and Carl Simons Center for the Biology of Hearing and Deafness Central Institute for the Deaf 4560 Clayton Avenue St. Louis, MO 63110
Mitosis is quite short, so if you get the cells reasonably synchronized (they pass through mitosis in a Gaussian distribution), you can probably catch most if not all stages if you time your collection right.
See our paper in Methods in Enzymology for a synchronization protocol:
Brady, R.C, M.J. Schibler and F. Cabral. 1986. Isolation of mitotic spindles from mammalian cells. Meth. Enzymol. 134: 217-225.
Matthew J. Schibler Ph.D. UCLA Brain Research Institute 1524A Gonda (Goldschmied) Center for Neuroscience and Genetics Los Angeles, CA 90095-1761
-----Original Message----- } From: ken blight [mailto:blight-at-icrf.icnet.uk] Sent: Thursday, September 28, 2000 8:16 AM To: EM information site
I am interested in synchronising Hela cells and collecting samples in each stage of Mitosis. Does anybody have a protocol and the times at which each stage is reached after release from G1?
Ken Blight Senior Scientific Officer Electron Microscopy Unit Imperial Cancer Research Fund London England
The answer I gave Kristine was purely theoretically - because I read about it.
Unfortunately our CPD is out of service since years and so far I have not used HMDS because I still have a few mls of PELDRI II.
Sorry, I can't give you an answer.
Regards
Claudia
Dr. C. Hayward-Costa School of Life Sciences Kingston University Penrhyn Road, Kingston upon Thames Surrey KT1 2EE, UK 44(0)208 547 2000 x 2240 Email: c.hayward-at-kingston.ac.uk
My apologies for using network bandwidth for this, but I can't possibly respond to all the e-mail and phone messages individually. However, the microtome I offered yesterday has already found a good home where it will be productively used by students. Many thanks to all of you who responded.
Tony.
** Anthony J. Garratt-Reed ** MIT Room # 13-1027 ** 77 Massachusetts Avenue ** Cambridge, MA 02139-4307 ** USA ** ** Phone: (+) 1-617-253-4622 ** Fax: (+) 1-617-258-6478 **
Some colleagues are trying to fix plant tissue for LM and possibly confocal microscopy using ETOH and acetic acid--they asked me if I knew what % of glycerol to include to keep the samples from being too brittle. Does anyone know a good protocol for glycerol fixation? They don't want to go the glutaraldehyde &/or osmium route. I remember seeing something about glycerol fixation in an ancient book on plant microtechnique but the book disappeared long ago...
Thanks for bailing me out again, Margaret -- Margaret Dienelt Plant Pathologist
Electron Microscopy Lab FNPRU, National Arboretum ARS, USDA
B. 010A R. 238 BARC-W 10300 Baltimore Avenue Beltsville, MD. 20705
Thanks to all who replied to my query on staff salaries.
To summarize the findings: The ranges that I reported for industry are similar for university facilities, although the universities tend to be in the lower end of each range. The people in higher cost of living regions don't seem to be making that much more than those living in other regions. The actualy salary offerings vary with duties required and experience (as they should), so one should not use just the numbers to compare salaries.
The (sad) bottom line is: Administrators (the person(s) above the lab director) think that staff salaries are adequate.
Thanks, Lucille
******************************************************************* Lucille A. Giannuzzi, Ph.D.
Associate Professor, Mechanical, Materials, and Aerospace Engineering Director, UCF/Cirent Materials Characterization Facility University of Central Florida 12443 Research Parkway, Suite 305 Orlando, FL 32826 email lag-at-mail.ucf.edu phone (407) 275-4354,5,6 fax (407) 275-4321 -------------------------------------------------------------------- "Good judgement comes from experience.
Experience comes from making bad judgement."
Mark Twain ********************************************************************
I am looking for a replacement part for an LKB Huxley ultramicrotome Mark 2. Does anyone know of an LKB Dealer or possibly have in their lab a spare Suspension Strip Hinge (LKB Part No. W52271-30)? This piece is made of polypropylene rod with 2 thin indented areas in the rod.
Thanks a lot for all the responses. However, it seems that not everbody has the same opinion regarding the possibility of cleaving samples with MgO substrate for x-section TEM. Does anybody have sucessful experices on this? Thanks a lot.
} } } Margaret Brannigan {brannign-at-asrr.arsusda.gov} 09/29 8:31 AM } } } ------------------------------------------------------------------------ The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Hi,
Some colleagues are trying to fix plant tissue for LM and possibly confocal microscopy using ETOH and acetic acid--they asked me if I knew what % of glycerol to include to keep the samples from being too brittle. Does anyone know a good protocol for glycerol fixation? They don't want to go the glutaraldehyde &/or osmium route. I remember seeing something about glycerol fixation in an ancient book on plant microtechnique but the book disappeared long ago...
Thanks for bailing me out again, Margaret -- Margaret Dienelt Plant Pathologist
Electron Microscopy Lab FNPRU, National Arboretum ARS, USDA
B. 010A R. 238 BARC-W 10300 Baltimore Avenue Beltsville, MD. 20705
On Fri, 29 Sep 2000, Margaret Brannigan wrote: } Hi, } } Some colleagues are trying to fix plant tissue for LM and possibly } confocal microscopy using ETOH and acetic acid--they asked me if I } knew what % of glycerol to include to keep the samples from being too } brittle. Does anyone know a good protocol for glycerol fixation?
Steve Ruzin's Plant Microtechnique book gives a brief mention of softening dried specimens in "10% glycerin-ethanol-water; probably 10% glycerol in 50% EtOH" and gives the reference as Peterson et al, 1989(Comparative leaf anatomy of the annual Muhlenbergia. Nord. j. Bot. 8:575-583)
Lots of people have asked me for results of the survey. My initial numbers below were obtained from an industry (not university) inquery. These numbers are higher than what my staff is actually receiving: (an I am therefore trying to fix this).
$30's-40's: for starting engineers with little to no experience that require training mid $40's to mid $50's: for an experienced engineer mid $50's to mid $70's: for a very skilled engineer who can trouble shoot to the component level independently without any back-up help.
Summary of Responses: (I have removed any reference to individual names of people/locations, etc.)
Based on what I have seen, and as reported by the ACS, your ranges are quite acceptable.
} From what I have deduced from my commercial EM engineers, I think you're right on the money. We don't have in house engineers that do this kind of servicing. The in house guys get the middle range that you listed.
Your figures are pretty much on the mark. What you might consider is how technicians (not engineers) fit into the picture. They can be at pay scales $10K-$20K less overall yet still do a very good job. Plus, they tend to stay on the premises much longer than engineers, who seem to seek greater rewards more frequently.
Your numbers look good to me, though I would add supervisory responsibilities and lab management for someone in the upper range.
I am employed at XXX and my job is to service the electron microscopes on campus. I repair at the component level and have not required manufacturer help. I have many instruments I am responsible for. I worked 11+ years for YYY in service and have been here over 4. My salary is low 50's. Even though my job does not require me to do so I work on other things as well. I put a new motor in a print processor today. I often work on microtomes. I know I saved a department about 6K a couple weeks ago by replacing a component in their power supply. After inspection ZZZ would have sold them a new power supply. I fixed a Horse treadmill last year. I love it here. A whole lot of fun.
These salaries seem to be in line (maybe a little higher) with what universities pay technical persons doing these kinds of jobs. In my case, I reluctantly transitioned from industry after 19 years and went from 70's to low 40's. My observation is this, if you are trying to find good people with experience then most likely you would need to offer salaries near the upper end of these ranges. Keep in mind that technology changes, and even experienced people need training and interaction with vendors. I believe this investment will pay off as you are able in time to eliminate expensive service contracts on all instruments. Salary is just part of the package that will attract good people. I believe a good technical person will flourish in the academic environment if they have a good salary, are free from benefit concerns, and have the resources to do the job well.
As a coincidence, one of my technologists pulled your request for survey response off the web to tell me that I was under paying him. I presume that your classification of "engineer" refers to technical degree level people. I manage a group of 6 technical people with about ten state of the art level SEM's, TEM, EMP's and XRD equipment for a very long term contract. Because we reside near Silicon Valley we must reach deeper for starting rates here where entry level housing is about $300K. We cannot compete with the high tech firms that offer stock options, signing bonuses, etc. I just lost a materials scientist who about doubled her salary at a firm in Fremont. Obviously, our starting rates are a little higher than you indicate for engineers/scientists as well as technicians with little difference for degree status. I don't think that we have started anyone in our organization lower than the mid $40's in the past couple of years. Otherwise, we work with a similar salary range for non senior level professionals without significant supervisory responsibility.
We have service contracts on all machines so much of our staff is engaged in teaching etc.. and minor maintenance. In any case the $s sound about the same for us as well... except the third category for which we do not have anyone. But benefits (health, 403b, facilities etc..) are of great interest to most which varies so widely across institutions
I get 65K, and probably should get a hell-of-alot more. But then again, I am such a push-over.
In our Characterization Facility most of the user training and class teaching is done by PhD staffmembers with the title "research associate" or "senior research associate" (both faculty rank, non-tenure track positions). At many universities the analogous staff are called "assistant research professor" or "associate research professor". Some training is done by "junior" or "senior" level "scientists" with bachelors or masters degrees; these are "civil service" staff and members of the state workers union. Our instrument service and some administrative tasks are done by the above people, or by our technician, altogether ranging from bachelors to PhD degrees in various sciences (materials science, physics, chemistry, geology). I'm not sure how this translates to your "staff engineers". Our pay ranges have always struck me as pitifully low, relative to what our staff could make in analogous positions in industry. Here they are: PhD staff: low $40k's to low $60k's including very experienced people throughout the range. bachelors to masters staff: low $30k's to high $40k's. All of these people are capable of independent troubleshooting. Most, however, are not electrical or mechanical engineers. Thus much of our service is contracted to the instrument companies. We are funded almost entirely by our revenue, so down time is very detrimental and in many cases it makes no sense to try to service things ourselves at the level of the guts of electronics. Of course we do our own troubleshooting to identify the problematic instrument component, but in most cases we do not service things in the sense of an electrical engineer. No doubt it is difficult to make comparisons given the diversity of scenarios at the various universities plus industry. I applaud you for attempting.
we have three people who work in the areas you have discussed. They train students and faculty to use the instruments, and they maintain eleven microscopes and sample prep equipment. They also perform administrative tasks. They all have more than 15 years experience with microscopes. They are in the $50K to $60K salary range. Because of their many years of service and the high quality of their work, I consider their pay to be at least 15% lower than it should be. If possible, I would be interested in the results of the other responses.
I am looking for a replacement part for an LKB Huxley ultramicrotome Mark2. Does anyone know of an LKB Dealer or possibly have in their lab a spare Suspension Strip Hinge (LKB Part No. W52271-30)? This piece is made of polypropylene rod with 2 thin indented areas in the rod.
Betty Loraamm Univ. of South Florida Dept. of Biology SCA 110 4202 E. Fowler Ave Tampa, FL 33620-5150 (813)974-2676 Fax:(813)974-3263
My posting to this list server from a few weeks ago stating our problems with the Denton Bench Top Turbo may have been taken out of context. Denton is resolving our problems with the carbon coater. There customer service has been excellent. I want to offer my apologies for any misrepresentation I may have caused them.
Harry Ekstrom Materials Laboratory (602) 231-2744 e-mail: harry.ekstrom-at-honeywell.com
I have been carrying our quantitative analysis of a lead titanate like oxide using a Microspec WDX-2A-3PC on a scanning electron microscope. I have modified the normal experimental procedure and the reproducability improved from 3-5% to 1%. Are there any further modifications of experimental procedure that might further improve the reproducability by another factor of 2?
I am using a compound standard similar to all of the unknowns. All samples are polished well. Count times are long enough to get statistics well better than 1% for each of the 5 elements. Data is collected from each element on standard and then on sample, and thus beam current in the SEM is stable to better than 1%.
I am talking about reproducability of the k ratios being accurate to better than 1%, not about the accuracy of the measured composition. I think the compound standard will produce the best accuracy one can do.
Hello everyone, I am an undergraduate at Miami University, and could use some protocol advise to best achieve the results I am looking for. I have already obtained a protocol for general drosohpila SEM sample preparation, but my specific area of interest is the eye. I was wondering what the best method for acquiring a clean cross-section would be. If you have done this, or could give me some helpful hints it would be greatly appreciated. Thanks, Thomas Litzinger
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