On Fri, 1 Apr 1994 dennis%odin-at-odin.morph.med.umich.edu wrote: Snip } My lab is looking for a citation in the literature about the stability of } specimens over time in polymerized Epn blocks. I've checked the literature } but have not had much luck. If anyone knows offhand where this can be find, I } would appreciate the reference.
Hi, I've recently been reading a book called _Artifacts iin Biological Electron Microscopy_ by R.F.E. Crang and K.L. Klomparens, Plenum Press, 1988, and have found much of the info to be very applicable to some of my general problems. There is a section by H.H. Mollenhauer that discusses dehydration and epoxy embedding that might have the info you're looking for. Good luck.
Dwight U. Beebe beebed-at-ere.umontreal.ca Institut de recherche en biologie vegetale Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
Picking up on Cal's idea, would it be possible for you to prepare your particles right on top of the carbon graphite, on Formvar coated grids for support. That way you could photograph them together in the same micrograph and get the highest accuracy measurement of the particle sizes by direct comparison to the carbon lattice image.
The question I have, is can one compare the particles directly to the image of the crystal lattice spacings, or is there a calculation that must be done to correctly use that spacing information? Anyone? Gib Ahlstrtand.
------------ Forwarded Message begins here ------------
Kathy, How about using carbon graphite at 3.4 Angstrom lattice spacing or there is a type of asbestos that gives 9 Angstrom lattice spacing. These could be used at 100KX or above and the mag accuracy of your TEM could then be calculated. Cal ---------- Forwarded Message ends here ------------
-- Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
In reply to Kathy Walters query about resolution measurements, try using a colloidal gold preperation, say a 5nm gold. The makers of the probes send along info which contains a distribution curve of the size of the gold. Hope this helps.
After finding a suitable calibration standard, you could use one of the video measurement systems on the market to do the actual measurements. The standard is used to calibrate the measurement system only, and does not have to be closely related to the size of your sample. This would still give very accurate results, down to a fraction of the size of the standard.
My company makes such a device, called the XR-2000. It has a very large array of measurements, like distance, area, angle, pathlength, etc, and lots of other functions. It is very inexpensive and easy to use.
Your system must use standard video (like Y/C or RGB) for this to work.
If you would like to know more about the XR-2000, private Email to me.
Message-Id: {9404041513.AA02301-at-odin.morph.med.umich.edu} Errors-To: {dennis%odin-at-odin.morph.med.umich.edu} To: microscopy-at-anlemc.msd.anl.gov Cc: dennis-at-odin.morph.med.umich.edu
Greetings! I will teach a comprehensive, semester-long TEM course next Fall and am trying to find a good text. My choice at the moment is: Electron Microscopy. Principles and Techniques for Biologists by J.J. Bozzola and L.D. Russell. Does anyone have other favorite texts? The course will be small (6 graduate students) and will include both theory and practical sessions. Both plants (my specialty) and animals will be used as material. I would also be very interested in hearing directly from people who have taught such a course or one similar to it, for tips and "what works, what doesn't" info. We will also use the RMS Handbook: The Operation of Transmission and Scanning Electron Microscopes by D. Chescoe and P.J. Goodhew, as we have a JEOL 100S for the course and this book has a nice description of alignment procedures which match our instrument. Thanks in advance, any help will be greatly appreciated.
Dwight U. Beebe beebed-at-ere.umontreal.ca Institut de recherche en biologie vegetale Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
} In reply to Kathy Walters query about resolution measurements, try } using } a colloidal gold preperation, say a 5nm gold. The makers of the } probes } send along info which contains a distribution curve of the size of } the gold. Hope this helps.
Colloidal gold is easy to see and measure, but is not all that uniform. As Ed says, the manufacturers send along a size distribution curve. The sizes - diameters and distribution - will vary from batch to batch. One would also want to know how the manufacturer's size distribution was determined. They presumably would also have to measure the sizes microscopically, and the data would not need to be really accurate to suit their purposes.
The Journal of Microscopy has another few new books for review. Interested parties should contact Gillian Wilson at RMS-at-VAX.OX.AC.UK.
The books are:
1. Introduction to Scanning Tunnelling Microscopy. By C Julian Chen. OUP, 1994. 412 pages.
2. Polymerase Chain Reaction. By C R Newton & A Graham. BIOS in association with the Biochemical Society, 1994. 161 pages.
3.Rapid Methods and Automation in Microbiology and Immunology. Edited by R C Spencer, E P Wright & S W B Newsom. Intercept Ltd, France, 1994. 502 pages.
Subject: Time:2:05 PM OFFICE MEMO Re:EM Text Date:4/5/94 Another good text to consider might be "The Principles & Practice of Electron Microscopy" by Ian M. Watt, Cambridge Univ. Press. It is very well written and covers a good range of subjects for a one-term course.
Subject: Time:2:09 PM OFFICE MEMO Re: SEM Screen color Date:4/5/94 I think one justification for using green phosphors is that the human eye has maximum sensitivity in the green range of the visible spectrum.
Additional note: to avoid to thick a formvar which can remain between grid bars, use a quick blast of compressed air directed the grid to vaporize formvar between the grid bars.
On Fri, 1 Apr 1994 wrightr-at-zoology.washington.edu wrote:
} My very first immunolabeling experiment ended when every section } floated off the 25 grids that I was labeling at the time. Since } then, I use a method shown to me by Bonnie Chojnacki. Specifically I } use grids that have been dipped in a dilute formvar solution (about } 0.1% in chloroform) and then dried on filter paper. The grids can } either be dipped individually into the formvar and then placed on } filter paper, or a whole canister can be dumped into the formvar and } each grid retrieved individually onto filter paper. } } } This procedure produces a sticky coat on the grid bars and sections } (epoxy, LR-white, etc) really stay attached throughout all of the } incubations. } } } } } Robin Wright }
The answerto Your questionK about the same information after 30 year depends on what information you want. I wouldnt trust immunocytochemistry of material embedded that long, since we see a difference even after a month (post embed stain of etched epon). However, the structures have not changed and quantitation done will be the same. Do you need a reference if the experience of a great many microscopists says there is no difference?
On Mon, 4 Apr 1994 dennis%odin-at-odin.morph.med.umich.edu wrote:
} Thanks to everyone who responded the my question. However, most of the } citations sent had to do with the stability of sections under beam current, } heat, etc... } } The situation is that a drug company that has provided a grant to us would } like to know for certain that the tissue that has been embedded in epon can be } pulled out from storage thirty years and will still be good. Will the tissue } be sectionable and will it still give the same data as before? } } As I said, most of the people here still have blocks that they did their } undergraduate work in back in the 1960s, and they are still as sectionable as } the day they were pulled out of the oven. In fact, I understand that the } quality of epon actually improves over time because it will have longer to } fully polymerize. } } So I am then simply looking for a citation on something that we already seem } to know, but still have not been able to find it. } } Thanks again, } Dennis Shubitowski } dennis-at-odin.morph.med.umich.edu }
Green phosphor has a greater luminous efficiency, and so can be made much brighter than white phosphor with equivalent electron optics. This makes a green TEM screen useful, but does not make a good argument for a green monitor in a darkened lab. The SE M manufacturer probably chooses a phosphor based on decay time. The green P20 phosphor has a decay time similar to that of the human eye, and so "integrates" the noisy signal optimally.
regards Mark W. Lund, Director MOXTEK, Inc. Orem UT
} I'm looking for a good inexpensive seconhand ultramicrotome similar } or identical to RMC MT-7000 ULTRA or REICHERT ULTRACUT S.
I don't have an Ultracut S., but I do have a Reichert Jung Histocut II that has never been used, and we are now considering selling it. It was purchased for a project that never materialized, and it has never actually cut anything.
As Nestor indicated several times before, and also in the introductory welcome material sent to new subscribers, is important including in your message the suject header. It allows determining nature of message without having to read the content. Given the increased usage of the service, inclusion of the subject header is a nice gesture toward other users. In fact, many more will probably not unsubscribe.
Cesar D. Fermin, Ph.D Tulane Medical School Pathology/SL79 New Orleans, La 70112
Hi, I have not used Image, but have tried to use another image analyser to measure the number of intercepts on a line and the intercept lengths in the past. The general procedure was as follows: 1) Use grey level to differentiate your grains from the grain boundaries and create a bit map of the grains. 2) Decrease your active measurement field until you have only a single rastor line across the screen. 3) If only the "detected" portion of your image is overlayed with a colored "bitmap", then you should have a colored broken line across the screen at this point. 4) Count the objects detected to give you a measure the number of intercepts. 5) Take the maximum dimension of the objects detected to give you a measure of the intercept length. 6) By rotating the image and repeating the procedure, information may be collected in relation to variation with orientation.
Hope this is of assistance. ______________________________________________________________________ Laurie Frederick, A.SC.T. PAPRICAN Corrosion Control Group 3800 Wesbrook Mall The Pulp and Paper Research Vancouver, B.C. Institute of Canada Canada V6S 2L9
I am trying to locate an e-mail address for Dr. Mark E. Welland -at- Cambridge University, Engineering Dept., St. John's College. Last I knew, he was working in Scanning, Tunneling Microscopy. If anyone knows of his address (or, if you're on this list, Mark) please respond directly to my e-mail address. Thanks very much!
Susan K. Smith National Steel Corp. smiths-at-mlc.lib.mi.us
Hi, Sorry to broadcast this, but I deleted the message before I realized I might have an answer. A request was made about a source for a OM-3 motor. I checked with a company I buy equiment from and was told they have several on hand. The company is Marivac, Ltd., 5821 Russell St., Halifax, Nova Scotia, B3K 1X5 Canada. 800-565-5821, FAX 902-455-4007. I have purchased equipment from these folks (Balzers MED 010) and have a service contract with them for my Ultracut. The person I deal with is Chris Cathcart; he operates out of Toronto (416-495-0389), but can be reached via the 800 number. I've been pleased with the quality of the service I get. I have no connection with this company other than as a satisfied customer, standard disclaimers apply, blah, blah, blah. Good luck!
Dwight U. Beebe beebed-at-ere.umontreal.ca Institut de recherche en biologie vegetale Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
Anybody have a good fixative for cross-linking polysaccahridases? I'm trying to do SEM on a "gliding" bacteria strain and find my bacteria being lifted off the agar. I've tried vapor fixation using osmium and then fixing overnight in 2% glut. One colony strain will lift off the agar surface and the other will remain. Any Suggestions?
I have used 50 X 9 mm culture dishes with friction fit lids manufactured by Simport Plastics, Beloeil, Quebec, Canada (514) 464- 1723. The catalogue number was D210-14.
I would be interested in a source of smaller diameter dishes with a larger internal clearance for storing my metallographic samples.
Hope this is of assistance. ______________________________________________________________________ Laurie Frederick, A.SC.T. PAPRICAN Corrosion Control Group 3800 Wesbrook Mall The Pulp and Paper Research Vancouver, B.C. Institute of Canada Canada V6S 2L9
Falcon 35x10mm dishes (available through Fischer) work for embedding cultured cells.
Another possibilty is to grow the cells on culture inserts (available from Millipore, Falcon, and other companies) which are placed into 24 well plates. After fixation (or post-fixation if you prefer), the filter on the bottom of the insert can be removed with a razor blade, and the filters dehydrated with ethanol and embedded. This uses even less reagents that the 35x10mm dishes, and the 24 well plate is easier to manipulate than several round dishes. I've used this technique for culturing fetal kidneys and for endothelial cells.
Hello, Our laboratory is in the process of investigating ion mills. It appears that Baltec, Fischione and Gatan are the principle manufacturers of this piece of equipment. Any comments, suggestions, experiences or thoughts about ion mills would be appreciated. Thanks, John
In answer to Phil Rutledge's query about fixation of "polysaccahridases": The closest thing I know to a fixative for polysaccharides is Alcian Blue. This works reasonably well in most cases where bacterial capsules need to be insolubilised. Try the following refs: Tuffery - 1969, J gen Microbiol 57:41-50 Goldberg & Septier - 1986, Anat Record 216:181-190 Scott - 1972, Histochemie 32:191- (Primary reference) Trachtenberg - 1986, J Ultrastruct Mol Res 97:80-102 Steedman - 1950, Quart. J Microsc Sci 91:477-479 Powell et al - 1992, J Fish Biol 41:813-824 (useful ref) Dellorbo et al - 1992, J Electr Microsc 41:475-479.
Hope this is of some use.
Jan Coetzee Unit for Electron Microscopy Tel:+27-12-420-2075 University of Pretoria Fax:+27-12-342-1738 Pretoria Internet:janc-at-ccnet.up.ac.za 0002 South Africa
Message-Id: {9404111414.AA06580-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: ion mills Orig-Author: {PHELPS-at-ENH.NIST.GOV}:ddn:wpafb ----------------------------------------------------------- Hello, Our laboratory is in the process of investigating ion mills. It appears that Baltec, Fischione and Gatan are the principle manufacturers of this piece of equipment. Any comments, suggestions, experiences or thoughts about ion mills would be appreciated. Thanks, John
Phil Rutledge writes: } Anybody have a good fixative for cross-linking polysaccahridases? I'm } trying to do SEM on a "gliding" bacteria strain and find my bacteria being } lifted off the agar. I've tried vapor fixation using osmium and then } fixing overnight in 2% glut. One colony strain will lift off the agar } surface and the other will remain. Any Suggestions? } Phil,
Here is a well worn ruthenium red staining method for mucopolysaccharides from M.A. Hayat: "Positive Staining for Electron Microscopy", p. 169-170. Whether this will fix polsaccahridASES, or prevent them from sliding off the agar, I cannot say.
A few preliminary notes from Hayat: 1. Maximum contrast obtained when ruthenium red and osmium tetroxide applied together(refering to TEM prep here). 2. Stain can be applied by adding it to fixative solution, water, or buffer solution. 3. Phosphate buffers are not recommended for ruthenium red-osmium tetroxide procedures. Chloride-free cacodylate buffer considered most ideal one to use.
For general purposes, the following method of fixation and staining for acid mucopolysaccharides recommended:
Solutions: A. Aqueous glutaraldehyde (4%) 5 ml 0.2 M Cacodylate buffer (pH 7.3) 5 ml Ruthenium red stock solution 5 ml (1,500 ppm in water)
B. Aqueous osmium tetroxide (5%) 5 ml 0.2 M Cacodylate buffer (pH 7.3) 5 ml Ruthenium red stock solution 5 ml (1,500 ppm in water)
Mix solution B just prior to use. 1,500 ppm is 30 mg ruthenium red in 20 ml water. Fix and stain samples in solution A for 1 hr. at room temperature. Rinse briefly with buffer, then post-fix and stain with solution B for 3 hr at room temperature. Rinse briefly with buffer, then dehydrate and embedd, or critical point dry(in your case) according to standard procedures.
Ruthenium red stain is available from most EM or chemical suppliers.
The above is sort of the mother of all ruthenium staining techniques for EM. Others have since modified its use and here are more recent references:
1. B. Giammara and J. Hanker, Ruthenium Red-osmium bridging with TCH: New techniques to stain biological specimens for light and TEM and to coat them for SEM, Proceedings of the 46th Annual Meeting of the Electron Microscopy Society of America, 1988, pp20-21.
2. M. Jacques and L. Graham, Improved preservation of bacterial capsule for electron microscopy, Journal of Electron Microscopoy Technique 11:167-169 (1989). This paper compares ruthenium-glutaraldehyde staining to glutaraldehyde-amine, which they found to be superior.
The following papers are on non-ruthenium red methods, and involve TCH (thiocarbohydrazide) and tannic acid procedures:
3. R.O. Kelley, R. Dekker and J. Bluemink, Ligand-mediated osmium binding: Its application in coating biological specimens for SEM, J. Ultrastructural Research, 45:254-258 (1973). (The classic reference in applying TCH for SEM)
4. J. Murphy, Non-coating techniques to render biological specimens conductive/1980 update, Scanning Electron Microscopy/1980/I, pp209-220.
-- Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
This Master Class is intended for novices, experienced specimen preparers and scientists involved in the examination of transmission electron microscopy specimens. The course will present the latest materials, tools, and methods for preparing high-quality specimens.
Description
While most aspects of specimen preparation will be covered, emphasis will be placed on preparing cross section specimens of complex composite samples, such as those found in the semiconductor industry; mechanical polishing to transparency; and ion milling. The course will review a variety of modern physical science specimen preparation methods, discussing the advantages, disadvantages, and limitations of each. Experience has shown that a wide range of bulk specimen configurations may be successfully prepared. Discussion will include the preparation of metals, ceramics, thin films and coatings, powders, wires, and difficult (small volume, radioactive, etc.) specimens. Laboratory demonstrations are planned.
Instructors
Ron Anderson; Senior Physicist at the IBM East Fishkill Facility. Vince Carlino; President of VCR Group. George Lane; Materials Product Manager for Bal-Tec, Inc. Jo Ellen Tyson; Applications Specialist for Mager Scientific.
Registration and Attendance
To register, call or e mail Kim Knudtson at 612 626 7594 (kimberly-at-maroon.tc.umn.edu). Enrollment is limited; please respond early to guarantee a space. Disability accommodations will be provided upon request. This publication and material is available in alternative formats upon request.
Tuition
The fee will cover course materials, refreshments, and box lunches. For members of the University community, the charge is $70; for all others, the fee will be $250. University of Minnesota attendees should provide a CUFS number, others should provide information so that they may be billed after the course.
Program Sponsor
The Center for Interfacial Engineering (CIE) is an NSF Engineering Research Center supported by the NSF, the University of Minnesota, and the corporate members of CIE. The CIE Characterization Facility and High-Resolution Microscopy Center provide instrumentation and personnel to facilitate the physical and chemical study of interfaces. These facilities are available to all members of the University community as well as external users. Tours of the labs will be available to interested attendees.
Accommodations
The University of Minnesota is located 10 miles from the Minneapolis-St. Paul International Airport. The Radisson Metrodome Hotel is conveniently located within two blocks of the lecture and laboratory sessions. The telephone number is 612-379-8888. A Days Inn is a 20-minute walk from the University; the telephone number there is 612 623 3999.
Schedule
Day 1, April 28
Registration (8:00 am), 210 Amundson Laboratories held in the High-Resolution Microscopy Center, Shepherd Laboratories
Lectures: Ron Anderson ¥ Strategic plan Initial processing from bulk samples Sawing and grinding, dimpling ¥ Final processing the thin specimen Ion milling/FIB methods Mechanical microthinning
Vince Carlino ¥ Specimen Prep by Dimpling and Ion Milling
George Lane ¥ High Performance Ion Beam Thinning of Solid State Materials
Day 2, April 29
Lectures: Ron Anderson ¥ Other Methods: Microtomy, Cleavage, Replication ¥ Cross-sectional specimen preparation The IBM East Fishkill Method
Demonstrations
¥ Ron Anderson: Tripod polisher
¥ Vince Carlino: VCR Dimpler Ion Beam Sputterer Model IBS/TM200S Ion Milling System
¥ George Lane: RES 010 Rapid Etching System MED 020 Modular High Vacuum Coating System
¥ Jo Ellen Tyson: Reichert Ultracut S/FCS cryo-ultramicrotome Attendees may bring their own samples.
Stuart McKernan stuartm-at-maroon.tc.umn.edu Chemical Engineering and Materials Science OR High Resolution Microscopy Center University of Minnesota, Minneapolis, MN 55455
we charge based on what it costs us: embed.....$6 thick sections (glass slide)....$16 Thin sections........$60 TEM time.............$36/hr TEM negative..........0.65 8X10 print............4.10 Publication quality print.....$5.50 Technician time if needed (microscope operation).......$25/hr
equipment costs included depreciation and service contract
hope this is helpful
On Mon, 11 Apr 1994, Tamara Howard wrote:
} A while back someone asked about the standard charges for a routine TEM } workup on a biological sample...I don't recall ever seeing any answers to that, } and now I am looking for similar information. What is considered to be a } reasonable charge for say one sample, plain old fix, embed, a few grids, and } maybe 2 hours max scope time plus prints? At an academic level, that is - I kno0w } clinical charges can be astronomical. } You can answer by e-mail to tah-at-med.pitt.edu if you do not want to } go through the usegroup on this. } I would really appreciate some help on this one! } Tamara Howard } U. Pittsburgh School of Medicine } Pittsburgh, PA }
I haven't had the chance to actually do it, but according to info. I received at the Bio-Rad Confocal course last month, it should be possible. The relative brightness of Cy3 may mean titrating its molar ratio relative to Cy5 and/or careful selection of filter blocks and PMT filters to prevent Cy3's long trailing slope from comming through the Cy5 signal. Take a look at Brelje et al, Chapter 4, Methods in Cell Biology, vol. 38, edited by Brian Matsumoto.
On Mon, 11 Apr 1994, Michael Cammer wrote:
} If you have a Kr/Ar laser, you can triple label with FITC, Cy3, and Cy5. } } }
} ratio relative to Cy5 and/or careful selection of filter blocks and PMT } filters to prevent Cy3's long trailing slope from comming through the Cy5 } On Mon, 11 Apr 1994, Michael Cammer wrote: } } If you have a Kr/Ar laser, you can triple label with FITC, Cy3, and Cy5.
We have not had a problem with Cy3 being imaged in the Cy5 channel (we image Cy5 only while exciting with the red laser line). However, we have had problems when imaging bright propidium iodide or Texas red with the standard long pass rhodamine filter block; some Cy5 appears to be excited.
We have generous institutional support and I am told that our charges are quite low. We do not have to recover any salary and about half our operational cost are from institutional support.
THESE ARE OUR IN HOUSE RATES. For outside clients I, at least triple the rates
The following charges are in effect at the EM Core .
Standard TEM or SEM sample $65.
Additional similar samples sub- mitted at the same time $55.
Immunolabeling or other cyto- chemical reactions per sample $75.
Negative stain per sample $35.
Partial preparation is also available with price appropriate to our input for example:
Fix and embed only $20/sample
Thin section only $20/block
Use of the microscopes by qualified users is $20 per hour and $1 per photograph. If you want Polaroid you must also supply the film.
For the benefit of the curious Materials Scientist like me, would someone please post a description (short) of what CY3/CY5 is and what it is used for.
I have been having problems with Quetol 651 resin for the past year, after using it successfully in my lab for 2 years. Has anyone else experienced difficulties? Of what sort? I'd like to correspond with someone with a background/knowledge of resin che mistry to possibly diagnose the defect.
Thanks to everyone who gave suggestions to the problem I am having with the fixation of the bacteria and having the cells float off of the agar. Now I'll have to sort through the suggestions and see what works best. Again, thanx to all, Phil Rutledge, Director Center for EM UMBC
Greetings, We just received 176 grams (ampules) of osmium tetroxide. The ampules, we are told, were bought some time back in the 1960's from Fisher, Englehard (in individual wooden cases!), National Lead, and MCB. The integrity of the ampules seems good as evidenced by the translucent greenish color of the osmium. However, the osmium seems to have coalesced into a single mass. Does anyone have any comments, warnings or advice? Considering that this gift is worth over $6K, I'd like to be able to use it.
************************************************* * * * * * Charles J. Butterick * * Electron Microscopy Center * * Department of Cell Biology and Anatomy * * Texas Tech University Health Sciences Center * * Lubbock, Texas 79430 * * USA * * Phone 806 743-2706 voice * * 806 743-2707 fax * * Email hecub-at-ttacs.ttu.edu * * * * * *************************************************
SOMEONE WROTE: In message {199404121522.IAA18262-at-ucsd.edu} writes: } I have been having problems with Quetol 651 resin for the past year, after } using it successfully in my lab for 2 years. Has anyone else experienced } difficulties? Of what sort? I'd like to correspond with someone with a } background/knowledge of resin che } mistry to possibly diagnose the defect. } } Hello,
We've used Quetol for years without trouble. You do not mention what problems you are having so I have no idea what to tell you. Put out another message on the MICROSCOPOY net describing what used to work well for you and what kind of trouble you are having now. That way I think people with experience will be able to zero in on what might work for you, and I will reply then. Thanks.
-- Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
Greetings, We just received 176 grams (ampules) of osmium tetroxide. The ampules, we are told, were bought some time back in the 1960's from Fisher, Englehard (in individual wooden cases!), National Lead, and MCB. The integrity of the ampules seems good as evidenced by the translucent greenish color of the osmium. However, the osmium seems to have coalesced into a single mass in each of the ampules. Does anyone have any comments, warnings or advice? Considering that this gift is worth over $6K, I'd like to be able to use it.
************************************************* * * * * * Charles J. Butterick * * Electron Microscopy Center * * Department of Cell Biology and Anatomy * * Texas Tech University Health Sciences Center * * Lubbock, Texas 79430 * * USA * * Phone 806 743-2706 voice * * 806 743-2707 fax * * Email hecub-at-ttacs.ttu.edu * * * * * *************************************************
Message-ID: {MAILQUEUE-101.940412174341.320-at-bmg.bhs.uab.edu} To: Microscopy-at-anlemc.msd.anl.gov
Does anyone know if there is a User's Group for the Macintosh-based image analysis software IP-LabSpectrum from Signal Analytics Corp.? I would be interested in linking up with users of that particular software platform to exchange ideas, information, scripts ect.
Thanks Kevin McCarthy Assistant Professor Department of Cell Biology University of Alabama at Birmingham Birmingham, Alabama 35294 Phone 205-934-9923/9924 Fax 205-934-7029
I notice from the literature that people setting up microscope-based ion measurement systems to quantitfy Na, Ca or H by fluorescence ratio microfluorimetry usually use a Xenon lamp for excitation of the fluoprophores. We are putting together a system for measuring Ca using Indo on an Olympus IMT-2 but lack a fluorescence source. The mercury lamp is half the price of the xenon, and certainly excites at the wavelengths we require (Indo exc. at 365 =B1 10nm), so we are thinking of going that way rather than the very expensive Xe.
Is the main advatage of Xe then the more constant output over the whole UV range so that dual excitation is more reliable (both excitation intensities the same, whereas with mercury one may fall over a peak and the other not) ? I'd appreciate comments from anyone who has experience in this area. Thanks, Ian Harper iharper-at-eagle.mrc.ac.za
I notice from the literature that people setting up microscope-based ion measurement systems to quantitfy Na, Ca or H by fluorescence ratio microfluorimetry usually use a Xenon lamp for excitation of the fluoprophores. We are putting together a system for measuring Ca using Indo on an Olympus IMT-2, but still lack a fluorescence source. The mercury lamp (100W) is half the price of the xenon, and certainly excites at the wavelengths we require (Indo exc. at 365+/- 10nm), so we are thinking of going that way rather than the very expensive Xe lamp.
Is the main advatage of Xe then the more constant output over the whole UV range so that dual excitation is more reliable (both excitation intensities the same, whereas with mercury one may exc wavelength may fall over/near one of the mercury peaks, whereas the other may not) ? I'd appreciate comments from anyone who has experience in this area. Thanks, Ian Harper iharper-at-eagle.mrc.ac.za
Message-Id: {MAILQUEUE-101.940413083221.320-at-parmly1.parmly.luc.edu} To: microscopy-at-anlemc.msd.anl.gov
} In answer to Phil Rutledge's query about fixation of "polysaccahridases": } The closest thing I know to a fixative for polysaccharides is Alcian Blue. } Jan Coetzee } Ruthenium red is used to demonstrate polysaccarhide coats of cyanobacteria. I don't have a reference to hand, but I believe a receipe is in Dykstra's & Bozzola & Russell's books? Phil Oshel
Hi Ian Mercury sources tend to be much less stable, and are not white enough to provide both wavelengths at comparable intensities for many dual excitation combinations. They can be used for some dual emmission work. We use the same instrument you are considering, and have added the OSP-3 illuminator and photmeter system for rapid spot measurements of ion [] and pH. The system works well, exposes cells to minimal photo-oxidizing irradiation during the measurements, and can be driven from pc-based software. Let me know if I can help
Message-Id: {199404122000543157-at-qmgate.secrc.abb.se} X-Mailer: InterCon Dispatcher/SMTP for QuickMail X-Priority: 4
Looking for someone that has been working with oxidation, hydriding and wear properties of surface coated or surface modified zirconium base alloys (like Zircaloy 2 or 4). The environment is water or steam at ]300!C. I am interested in all topics related to the subject; coating or surface modification methods ( how to get a coating without defects going through the coating?), corrosion tests, investigation of tested samples (LOM, SEM, TEM, SAM, XRD, electrochemical impedance spectroscopy.
Bengt Stridh ABB Corporate Research S-721 78 Vasteras Sweden E-mail: best-at-secrc.abb.se Fax: +46-21-13 41 00 Phone: +46-21-32 30 67
REGARDING Cerius Software Vendor According to a brochure picked up at the latest MRS meeting, the Cerius software is sold through: Molecular Simulations, 16 New England Executive Park, Burlington, MA 01803 ph: (617)-229-9800.
In reply to Charles Butterick's question about old osmium - try calling a vendor of osmium to ask their "proffesional" advice, such as EMS, or Stevens Metallurgical corp.
} Does anyone maintain a list of software suppliers for TEM } applications? I am interested in programs such as MacTempas, } Crystalkit, Desktop Microscopist, etc. Addresses, phone numbers, ftp } site information would all be very useful.
MacTempas and Crystalkit Total Resolution 20 Florida Street Berkeley, CA 94707 Attn: Roar Kilaas 510-527-9100 Fax 510-527-9151
Desktop microscopist Virtual Laboratories 37 Highland Court Ukiah, CA 95482 Attn: Eric Schlienger 707-462-8037
Crisp and ELD Calidris Manhemsvagen 4 S-191 45 Sollentuna Sweden Attn: Sven Hovmoller 46 8 625 00 41
Prism Signal Analytics 440 Maple Ave East, Suite 201 Vienna, VA 22180 Attn: Brian Culhane 703-281-2509
DigitalMicrograph Gatan Inc. 6678 Owens Drive Pleasanton, CA 94588 Attn: Sheri Kurland 510-463-0200
NCEMSS and Interface Tool Telnet from ORAC.LBL.GOV
TCBED from ASU
Image from MSA EMMPDL
That's my list of software sources.
Roseann Csencsits Electron Microscopy Center Argonne National Lab
} In reply to Charles Butterick's question about old osmium -- What exactly could go wrong with a sealed vial of the metal? If the vial seal had been breached, wouldn't the osmium vaporize? I know em makes a person really paranoid about materials, but is there any other reason not to use the stuff?
Tobias Baskin * TEMPORARILY C/O Peter Hepler * Biology Department University of Massachussetts * Amherst * Mass 01003 Tel: 413 - 545 - 2698 FAX 413 - 545 - 3243
In message Charles J. Butterick writes: } Greetings, } We just received 176 grams (ampules) of osmium tetroxide. The } ampules, we are told, were bought some time back in the 1960's from Fisher, } Englehard (in individual wooden cases!), National Lead, and MCB. The } integrity of the ampules seems good as evidenced by the translucent } greenish color of the osmium. However, the osmium seems to have coalesced } into a single mass. } Does anyone have any comments, warnings or advice? Considering that } this gift is worth over $6K, I'd like to be able to use it. } Charles,
We have heard your plea for help and are delighted to be able to respond with our assistance. In our lab, we have over 25 years experience of working with osmium tetroxide and biological systems. Just send us about 25 ampoules(grams), we will test the lot against biological systems we are currently working with, and report the results back to you.
-- Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
Sounds like the osmium has melted at some time and then reformed. After quizzing another EM veteran here, learned that such ampules should still be fine as long as they show that bright green color. Avoid using any vials showing black flecks in the coalesced osmium, as that's a sign of degradation. Similarly, when put into solution, good vials will give a bright yellow-green solution as usual; vials with black specks will probably give solutions with no color or the off-gray color of an old solution.
David Hall Dept. Neuroscience Albert Einstein Col. Med. Bronx, NY
We consider to purchase sample cutters for bulk materials and 3mm slices to prepare transverse TEM samples for HREM. I appreciate for your advice. Welcome companies to send us the price.
Sandy Burany Physics Department Simon Fraser University Burnaby, B.C. V5A 1S6 (604)291-4082 Fax (604) 291-3592 Email burany-at-sfu.ca
Message-Id: {MAILQUEUE-101.940414095946.705-at-prl.pulmonary.ubc.ca} To: microscopy-at-anlemc.msd.anl.gov
We are attempting to demonstrate peroxidase/myeloperoxidase activity in neutrophils in cytospin preparations by using DAB. We find it works well on fresh cytospins, but if cytospins are left for a few days we get no reaction. Any suggestions as to how best to maintain enzyme activity in stored cytospins? Mark Elliott
Re} Peroxidase & Cytospins Why not try fixing the cytospins with aldehyde to retain the peroxidase activity? Neither formaldehyde of gluteraldehyde will affect the enzyme activity but may inhibit proteolysis. The aldehydes will work best on cells that have not been allowed to dry out and will only continue to work if the cells remain hydrated. Leave them in buffer or, if formaldehyde-fixed, in formaldehyde. Glutaraldehyde fixation is only useful if you are using DAB. It will not be useful for fluorescent studies unless the autofluorescence is quenched with sodium borohydride.
Does anyone have experience with a good commercially available Monoclonal or polyclonal antibody against collagen I. Our experiments are performed with rabbits so a polyclonal made in rabbit is useless, or should be conjugated to something (biotin, dig.) Thanks in advance.
with likely the addition of a Hitachi in the next year. each is "Best" for us for what it does and the application for which it is used for.
Let's see what everyone has to say on this one. It should be interesting. I'll just sit back and "listen" for abit.
Remember try to be objective if you are going to respond. This is intentionally not a moderated list and I want to keep it that way.
I'm sure that the EM manufacturers who are also subscribers to this list will be interested in the responses. In that regard, I'll ask them (i.e. any manufacturers) not to respond to this question. If I see something which is not a fair discussion or an objective reply. I will cut the discussion off. If any manufacturer wants to complain please address all mail to me personnally and I will formulate any appropriate notice(s) to the listserver....
Nestor J. Zaluzec ANL EMCenter zaluzec-at-anlemc.msd.anl.gov
For reliability I have been most happy with my Hitachi H-7000 and S-4000. They serve about 99% of the needs we have in a multi-user facility, for mostly biological EM. There are a few times when I wish I had something else for very speialized work, but on the who;e these serve us well. My suspicion is that one could say the same for most of the major manufacturerers' products on the market today.
I agree with Nestor, the best microscope is dependent upon what you wish to do. Even in cameras, the Nikon is not ideal for all applications. However, to join in the foray: We love our Philips 400, 515, and CM-30s.
On Fri, 15 Apr 1994, Larry Hawkey wrote:
} } I have tried to send this message twice but is keeps getting bounced back } to me. } } Some one is asking me about my recommendations on which EM to } buy. So my question to all of you is this: } } Is there in the EM community a sense that there is one } Electron Microscope that is the best that money can buy? } } If some one was to ask about 35mm cameras for instance. I } think that the general consensus is that Nikon is ahead of } the pack. Minolta and Cannon are good and may be a good deal } for the price but if money is not the first concern Nikon is } the first pick. } } I use the JEOL 1200EX II. It is great. I have also used } several of the 100 incarnations. I used the Ziess EM 10 } about ten years ago but I have really only used JEOL for the } past ten years. In addition there seems to be a heavy } concentration of JEOLS in the Duke University community so I } feel that my impression that Ziess is nice but JEOL is best } may only reflect my small world. } } Try to put your biases aside and send me your thoughts. } } Larry Hawkey } Hawkey-at-neuro.duke.edu }
Some one is asking me about my recommendations on which EM to buy. So my question to all of you is this:
Is there in the EM community a sense that there is one Electron Microscope that is the best that money can buy...
Larry Hawkey Hawkey-at-neuro.duke.edu
I will start by saying I am not a TEM user and my answer will support this. I am a microprobe operator and an SEM operator. However, I have supervisory responsibility for support and operation of a lab that has a JEOL 100CX STEM, a JEOL 4000FX 400kV STEM, a Philips EM301 TEM in addition to 2 JEOL 35C SEM's, a JEOL 6400 SEM and a JEOL JXA-8600 EMPA so I am not insensitive to the needs of a TEM user. When we bought our most recent STEM, the 4000FX, we came down to a choice between Philips and JEOL. When we bought our most recent SEM, the 6400, we chose between Philips, CamScan, and JEOL. I would advise any one that in buying a piece of EM equipment there are three considerations: Performance, Cost, and Service. Of the three, cost is the lowest priority. Obviously, if you have limited funds, cost is a consideration up front (i.e. you can't get something for nothing). Once you purchase, what you paid is meaningless. The two remaining criteria will be with you for the life of the instrument. I will let the rest of the EM community steer you on the performance subject but I want to stress that efficent, prompt, compitent service is in my opion as important as performance. The best instrument, when inoperable, is not very useful. Waiting weeks for service is not pleasant, especially when deadlines come and go. Service personel that are not equipped or trained can lead to frustration. Personally, I will sacrifice a liitle performance if the competitor provides better service. Admitedly, I can do this because I do little work that "pushes the envelope". Mosty of my work is routine and not instrument limited. If you are beating back the frontiers of the science, then weigh performance heavily. But always keep in mind that the instrument must be well maintained if you expect to achieve optimum performance on demand. This takes good service.
Message-Id: {9404151852.AA01866-at-odin.morph.med.umich.edu} Errors-To: {dennis%odin-at-odin.morph.med.umich.edu} To: Jay Jerome {jjerome-at-isnet.is.wfu.edu} Cc: Larry Hawkey {hawkey-at-neuro.duke.edu} , Microscopy-at-anlemc.msd.anl.gov, dennis-at-med.umich.edu {Pine.3.89.9404151432.A9297-0100000-at-isnet.is.wfu.edu} "Larry Hawkey" {hawkey-at-neuro.duke.edu} , microscopy-at-anlemc.msd.anl.gov
Reply to: RE} } Who makes the best TEM? Just a note of clarification as to what the person who is looking for a TEM is going to use it for;
The person I am talking to is only interested in Routine Biological TEM, looking at ultra-structure (yes that is still done). Things like counting synapses or looking to see if the Mitochondria are screwed up. Also by best I am interested in compairing The preception that one company just carries a slightly but consistantly better line of EMs.
--------------------------------------
The problem with your question "Who makes the best TEM" is that you haven't specified what you wany to use it for. The best microscope for ultra-high resolution imaging is not necessarily the best for general teaching of students or the best for high performance microanalysis. You need also to factor in the service available. For example, how many engineers does a company have in your area, how many microscopes like the one you are thinking of buying does each engineer have responsibility for (put another way, how much experience does the engineer have on your instrument - a person can be very good, but if yours is the only TEM in his area he will have only limited experience with it). What kind of microscopy do you need to do - fast turn-round screening of biological samples or extensive analysis of materials samples, and so forth. There are good and bad points about all the major manufacturers, and probably each instrument from each maker is the best in its own niche. The analogy with the 35mm camera is a little over simplified.
On Fri, 15 Apr 1994, Nestor J. Zaluzec-ANL EMCenter wrote:
} } Talk about a loaded question. Which microscope is best? } } To state the obvious which I'm sure will be repeated at least } twice or three times in the repies to this question. } } ***It depends on what your trying to do!*** } } CTEM, STEM, AEM, CBED, XEDS, EELS, AES..... } and the list continues, choose your poison. } } At the ANL EMCenter we have: } } JEOL (100cx & 4000EXII) } Philips (EM420T & CM30T) } AEI (EM-7) } VG (603Z) } } with likely the addition of a Hitachi in the next year. } each is "Best" for us for what it does and the application } for which it is used for. } } Let's see what everyone has to say on this one. It should } be interesting. I'll just sit back and "listen" for abit. }
Well, I'm close to retirement so I'll stick my neck out. For biological applications I have used RCA, Siemens, Hitachi, Jeol, Zeiss, and Philips. For ease of use, quality of construction, resolution, and reliability I'll go with the Philips. My work has always been conventional biological TEM.
No, we have not tried cutting the fibers in to disks and perfroming a 3-D reconstruction. I have not heard of this previously applied to composite microstructures, have you? Can you tell me more about this methodology? Can you point me to specific papers in the literature? Is this similar to the type of 3-D reconstructions we read about being used in medical imaging? If so don't I need some features in the image to key off of?
Thanks for your help,
P. Joyce
Dear Peter,
If you consider biological systems to be composite microstructures, then, yes we have a lot of experience with this technique. As applied to your carbon-fiber system, there are several things to consider: 1) Is there enough contrast between the fibers and the matrix? 2) If not, is there a way to add contrast, such as by infiltrating the matrix? 3) Is the thickness of each slice limited by transmissivity or by overlap of features--in the HVEM a spec- imen of several microns can be penetrated, but after about one micron, typical biological specimens have so many features that these can no longer be untang- led? Another possibility if there is low contrast is to find an element in the matrix which is not found in the carbon, and to do element mapping--this is in- herently much lower resolution than imaging--or energy-filtered imaging looking at an edge or the low-loss region. In any event, I presume you have settled on appropriate conditions and obtained images which have then been scanned and converted into digital files. If the sections were thin enough so that the carbon fiber position and orienta- tion do not change appreciably from section to section, you can just stack the images from successive sections and process the resulting file as if it arose from a confocal microscope series or some such. There are many image proces- sing packages which will allow you to view the image volume from various angles with various features highlighted--you do have to tell the program how to iden- tify the features, but at worst, you can do this pixel-by-pixel if necessary. We use SEMPER on our confocal microscope to do this, and some in-house programs in combination with, I believe, MOVIE.BYU to do this for electron micrographs. If the sections can be thicker--e.g. if there are rather few carbon fibers distributed in a non-carbon-containing matrix and if energy-filtered imaging sees the carbon as dark while the matrix is transparent--you should either take stereo pairs and use a pointer to outline the features of interest within the section volume while viewing the stereo pairs, or take a large ser- ies of images at various tilts and do tomographic reconstruction. This may be more work than thin sections, but it has the advantage that features in the middles of sections are not perturbed by the cutting process--which can be a problem with thin sections. For reconstruction using stereo pairs, we use an in-house program, STERECON, and for tomagraphy, we use SPIDER, also an in-house program. SPIDER does reconstructions very much like medical imaging using either Fourier tech- niques, back-projection or projection onto convex sets. Fourier techniques are an old tried-and-true (mostly) method which allows filtration to smooth out noise, and which is well-understood, but which also gives artefacts due to the inevitable missing wedge of information. Back-projection just models the three dimensional object which would give rise to the observed projected views. Fin- ally, projection onto convex sets is a recent method incorporating constraints which, among other things, is used for de-blurring photographs. I don't under- stand this method well enough to try to explain it. Dr. Joachim Frank at our lab is in charge of the image processing end of things, Drs. Bruce McEwen and Michael Radermacher have experience with both programming and using SPIDER, Mr Mike Marko is our resident expert on STERECON and Mr. Ardean Lieth knows about all the programs. A letter (or e-letter) to any of these people should get you more information, and would be better than reading the papers cited for using these programs. I'm sure there are very good write-ups of serial-section techniques, but I am not familiar enough with the literature to reccommend any in particular. All of the programs developed here are available (I'm told the price is reasonable). Dr. Frank is the correct person to tell you about this aspect. I have probably told you more than you wanted to know, but I'd be happy to try to answer any other questions I can. The address for any of the people mentioned is Wadsworth Center for Labs. & Research Empire State Plaza, P.O. Box 509 Albany NY 12201-0509 and the e-mail addresses are username-at-tethys.ph.albany.edu, however, I don't know every one's usernames--try lastname, initial+lastname or initials as gues- ses. Good luck.
Your 'old' osmium should be ok! Concerning the fact that it coalesced into a mass on the bottom of the tube, this is not important: I remember a recipe (by S.Palay) recommending heating the tube (in a water bath) in order to collect all the osmium tetroxide in a single mass at one end of the tube. It worked ok (by the way, there is no point in doing so, if you carefully wash the outside of the ampulla before breaking it ;-)) Cheers! John
*********************************************************** * Jean C. Desclin (John), Associate Prof. of Histology * * Laboratory of Histology - Faculty of Medicine * * Brussels Free University (U.L.B.) * * e-mail: desclinj-at-ulb.ac.be (internet) * * snail mail: route de Lennik 808 * * B - 1070 Brussels Belgium * ***********************************************************
Reply_ RE} } General: Which Scope is Best? Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX: (313)936-3352 jfmjfm-at-umich.edu or John_Mansfield-at-mse.engin.umich.edu IMHO (In My Humble Opinion):
For quality of construction and reliability I would say Philips. In addition they have always been the best scope for CBED.
The one major thing that I dont like about ALL of the newer microscopes is the manufacturer's insistance in making the tilting and translation functions motorized. With these instruments you have no "feel" for the specimen position or tilt and when performing diffraction experiments I like to be able to "tweak" the tilts and sample position manually.
Our lab currently has 2 JEOLs, a 200cx and a 6100. Both instruments are under service contracts with JEOL, and I cannot imagine life otherwise. The contracts may seem expensive at first glance, but when a problem arises the service technicians have always fixed them much more quickley than we could have. The routine maintenance provided by the contracts also helps keep the instruments in good working order even with multiple users. If you have the option, life is much better witha service contract from the manufacturer.
John Phelps NIST, Boulder PHELPS-at-ENH.NIST.GOV
P.S. I promise that I wasn't paid by the service people for the response.
At Argonne National Laboratory, we have maintenance contracts on all of our TEM's, including an old JEOL 100CXII. We simply don't have the time to devote to repair work while the field service techs have the resources to take care of most of the problems that arise. We do some of the repairs ourselves, if they can be done quickly. For instance, we don't ask for help in changing filaments or in fixing a camera if the film gets jammed. We know our limits. One recent repair on our Philips EM420 took a week, and we could not have done it ourselves. Since we have a large base of users, 4 TEMs, 1 STEM, and only 3 staff members, time is very valuable to us.
Russell E. Cook Electron Microscopy Center for Materials Research Materials Science Division Argonne National Laboratory Argonne, IL 60439, USA
Dr. Bogers I talked with our ECM expert for advice on ab's and he suggested a company in Germany called HEYL Goerzallee 253, D-1000, Berlin 37 phone 030-8-17-60 52 FAX 030 8 17 40 49
He also mentionned a company called Collagen Corporation in Palo Alto, California USA, and one in Birmingham Alabama USA which also deals specifically with collagen antibodies. There is also a Developmental Biology Hybridoma Bank in the US which deals with ECM antibodies. He said that any of these may be able to help you and should be fairly good.
IMHO I would not suggest buying a JEOL 100B. This is the microscope that I never could qualify on at ASU, it took a lot of maintenance, and is 20 years obsolete. :)
I would also like to point out that if you want to unsubscribe you must send the message to the listserver, not the list:
listserver-at-anlemc.msd.anl.gov
We should all keep the information that was sent us when we first logged onto this list so that we know how to log off without annoying 1200 listmembers .
regards Mark W. Lund, PhD Director MOXTEK, Inc. Orem UT
I know we are a special case--we have a 1.2 MV HVEM from AEI--but we get along very well without a service contract (} 80% uptime). Our secret is that no ser- vice contracts being available on our beastie, we have had to learn to do it all ourselves. The service people in-house are VERY good--a must. Since we do a lot of development and improvement, we need a person who knows the machine very well, and those people also can service the microscope. If you have a fac-ility which just uses the microscope--as opposed to redesigning it--it is prob- ably just as well to have a service contract, but if you need a machine person or two on hand anyway, you can get along without a contract.
Message-Id: {MAILQUEUE-101.940418160415.32-at-prl.pulmonary.ubc.ca} To: microscopy-at-anlemc.msd.anl.gov
I am sending this again because I am not sure if it was received properly the first time.
Dr. Bogers: I talked with our ECM expert for advice on ab's and he suggested the following companies as good sources. HEYL, Goerzallee 253, D-1000, Berlin 37, Germany Phone 030 8 17 60 52 FAX 030 8 17 40 49
also, a company called Collagen Corporation in Palo Alto California, USA and one in Birmingham, Alabama USA which also deals specifically with collagen antibodies. There is also a Developmental Biology Hybridoma Bank in the US which deals with ECM antibodies . He said that any of these may be able to help you and should be fairly good.
Message-Id: {1994Apr18.101055.761680489-at-ms.sjdccd.cc.ca.us} To: microscopy-at-anlemc.msd.anl.gov (microscopy list)
Dear fellow microscopists, At San Joaquin Delta College, Dept. of Microscopy, Stockton, CA we have an intensive 2 yr hands-on program to train microscopists. We issue certificates in both biological and materials area. We will be graduating 17 students from the program May 28, 1994. If you need an entry level person with 2 yrs intensive experience in TEM, SEM, EDS, OLM, all preps, routine maintenance, report writing, etc. etc. please contact Judy Murphy (murphy-at-ms.sjdccd.cc.ca.us, phone 209/474-5284; fax 209/474-5649) If you want more info about the program or a copy of the Delta Microscopy Newsletter, also contact as above. Our program has been in existence for 24 yrs. and we have graduates all over the United States and several abroad. Hope to hear from those interested. Judy Murphy, San Joaquin Delta College, Dept. of Microscopy, 5151 Pacific Ave., Stockton, CA 95207
JEOL 1200EX Purchased 1989, S/N EM157085-528 Under service contract since purchase Has Tracor Northern EDS (don't know model) Available immediately, shipping negotiable
For more information, contact: melilly-at-aol.com directly, not this list.
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
Interesting and topical question. In my opinion unless inhouse expertise includes a trained microscope service engineer, you are playing russian roulette without a service contract. sooner or later the chamber is going to come up loaded. Over the long term it is not cost effective. However, I have often thought that facilities with good general in house expertise (we fix a lot of minor problems ourself) should be able to negotiate a different kind of contract (with different cost) than a facility that needs a service engineer for every minor problem. Perhaps we need a lobby with the microscope companies.
Having said that, and persuant to Larry Harkey's queries about microscopes,.o Our Philips service engineer in North Carolina is the best I have ever encountered and Philips in general has always provided superior service. However, with any company their are regional differences in the quality of service.
On Mon, 18 Apr 1994, Griffin, Robin wrote:
} } How are the majority of EM labs maintaining their electron microscopes? Are } they using maintenance contracts, in-house repair or something else? I } know how much maintenance contracts cost (EEK!). How well does it work } without maintenance contracts? I would appreciate any opinions about this } very expensive issue. }
How are the majority of EM labs maintaining their electron microscopes? Are they using maintenance contracts, in-house repair or something else? I know how much maintenance contracts cost (EEK!). How well does it work without maintenance contracts? I would appreciate any opinions about this very expensive issue.
How are the majority of EM labs maintaining their electron microscopes? Are they using maintenance contracts, in-house repair or something else? I know how much maintenance contracts cost (EEK!). How well does it work without maintenance contracts? I would appreciate any opinions about this very expensive issue.
Support person for the Electron Microscopy Core Laboratory of the Interdisciplinary Center for Biotechnology Research at the University of Florida.
DUTIES: Preparation and examination of biological specimens for transmission and scanning electron microscopy in a facility that serves the entire university community on a fee for service basis. Collaborative research possible as part of the program. Incumbent is expected to deal directly with the clients on experimental design, execution of the project and preparation of the data for publication, in consultation with the Scientific Director of the laboratory.
QUALIFICATIONS: B.S. or M.S. in a biological science with proven experience in most aspects of biological electron microscopy. Good communication skills, both oral and written. Ability to deal on a one to one basis with investigators from a wide variety of disciplines and backgrounds.
STARTING DATE: July 1, 1994. (Possibly sooner)
APPLICATION DEADLINE: Open until position is filled.
RANK: Appropriate to qualifications (EM Tech., Sr. EM Tech, EM Lab Manager)
SALARY: $17,873.-$22,633.
Inquiries by E-mail or full resumes to the address below
We currently have a Kodak slow-scan CCD system mounted on a 2010; this is a side-mount systems as opposed to the more common Gatan bottom-mount. Does anyone have any comments comparing the technical capabilities of these systems? How much does the placement affect usage, etc...?
Daniel L. Callahan Department of Mechanical Engg. and Materials Science Rice University dlc-at-owlnet.rice.edu
I'm trying for the first time to make some preparation of DNA for TEM. Grids and plates, with some initial problem, seem to be promising. My problem, now, is the elaboration of these pictures. I have seen that I can obtain good digital images with a scanner from the positives, and save them as TIFF files in a PC. I would like to find some PD program (possibly DOS, Windows or Amiga) to elaborate these files: the program should recognize DNA in the image and make some kind of elaboration: I don't know exactly what kind: I think (I hope!) I should be able to do something with these digitalised pictures.
Thank you for your help.
Nanni Iazzetti Dept. Genetics, General and Molecular Biology University of Naples Italy
Re} Best Microscopes I didn't see much use in joining the debate about "best microscopes" until the new data arrived. Buying a microscope for the US is a relatively easy choice and the discussion has covered all points. However, I lived and worked in Nairobi, Kenya for 7 years where we had a Zeiss EM 10A which worked well (and is still working). I think that this was the best choice for an EM in Africa. Although we only saw a service engineer once a year, the microscope was constructed for ease of service and repair. The machine was only used by a few people so a talented local electronics engineer and myself were able to keep this microscope running continually with relatively little effort. The long range support from Zeiss was exceptional and any serious problems were overcome with a few phone calls and faxes. Currently, I work on a Phillips 410 and Jeol 100cx, neither of which I feel capable of fixing when they go wrong. I could fix a Peugeot in Africa but have no idea on where to start with the computerized, fuel-injected cars over here.
Paul Webster Yale University School of Medicine 333 Cedar Street, New Haven, CT 06510.
Even more important, is how much knowledge the use (s) has. For stardard biological observations upt 50K even an old Zeiss S2 wil do the job. Of course, electron coherence, astigmatic correction, and vacum cleanness could drive you crazy, not to say much about frequent aligments. Thus, check your packet book, test your technicians, and determine what you need answered, then have all manufacturers to view and examine the same grid with the problem and make decision.
Cesar D. Fermin, Ph.D Tulane Medical School Pathology/SL79 New Orleans, La 70112
I agree with Nestor, the best microscope is dependent upon what you wish to do. Even in cameras, the Nikon is not ideal for all applications. However, to join in the foray: We love our Philips 400, 515, and CM-30s.
On Fri, 15 Apr 1994, Larry Hawkey wrote:
} } I have tried to send this message twice but is keeps getting bounced back } to me. } } Some one is asking me about my recommendations on which EM to } buy. So my question to all of you is this: } } Is there in the EM community a sense that there is one } Electron Microscope that is the best that money can buy? } } If some one was to ask about 35mm cameras for instance. I } think that the general consensus is that Nikon is ahead of } the pack. Minolta and Cannon are good and may be a good deal } for the price but if money is not the first concern Nikon is } the first pick. } } I use the JEOL 1200EX II. It is great. I have also used } several of the 100 incarnations. I used the Ziess EM 10 } about ten years ago but I have really only used JEOL for the } past ten years. In addition there seems to be a heavy } concentration of JEOLS in the Duke University community so I } feel that my impression that Ziess is nice but JEOL is best } may only reflect my small world. } } Try to put your biases aside and send me your thoughts. } } Larry Hawkey } Hawkey-at-neuro.duke.edu }
There are several short courses offered (1 week) through the Marine Biological Laboratory in Woods Hole, Massachusetts. For information call 508-548-3705 . The next one is in May. Regards, Nina Allen
On Tue, 19 Apr 1994 IDESOUSA-at-BRMVM1.VNET.IBM.COM wrote:
} Is anybody aware of a good short course on optical microscopy? } It is such a vast subject (polarized , UV,IR, confocal,filters, } image analysis, interferometry, ellipsometry etc.)
Might try the following:
1. McCrone Research Institute 2820 S. Michigan Ave. Chicago, IL 60616-3292 (312) 842-7100 [phone] 842-1078 [fax]
*some of their offerings: photomicrography, polarized light microscopy, hotstage microscopy, SEM, TEM, pharmaceutical, forensic, asbestos id, hair and fiber id, wood and pollen id, anb many others, including custom planned on-site courses. I've taken the Applied Polarized Light Microscopy course there, and found it -very- good; although not enough emphasis for materials type. Courses are spread out through the year. Prices for 1994: $1050 (ouch!) for almost all, w/ TEM being higher.
2. Institute for Microstructural Analysis c/o Buehler Ltd. P.O. Box 1 Lake Bluff, IL 60044 (702) 295-4659 [phone]
*courses offered include: image analysis, petrographic prep., basic metallography, advanced materials, ceramics, etc. Prices vary from $700 for 2-day to $1095 (again, ouch!) for 5-day, and higher prices at their Irvine, CA locale. I myself have not been there, but have heard good reports.
3. ASM International Materials Park, OH 44073 (216) 338-5151 [phone]
*courses include: metallographic interpretation, met. techniques, advanced tech. in optical (& electron) microscopy, failure analysis, optical microscopy of ceramics, quantitative metallography, etc... I've taken one course there, and have had several home-study classes. Good all around training. They offer what's called Materials Engineering Institute Extension diploma, and a three-tiered Center for Applied Metallography levels. Prices are similar to Buehler's.
} Could someone suggest books or reference material which is neither } trivial or too technically involved in the physics. (level of a good } metallurgical technician)
Some of my favorites include:
Metallography: Principles and Practice...........Vander Voort, George McGraw-Hill, 1984 (wish it would be updated tho) Polarized Light Microscopy.......................McCrone & Delly Ann Arbor Science Publishers, 1978 (likewise...) -and- many of the Microstructural Science volumes from the proceedings of the annual technical meetings of the International Metallographic Society. -and- many of the ore microscopy books have very detailed and highly useful information.
In addition, the other large met. supply houses besides Buehler, LECO [(616) 983-5531; St. Joseph, MI] and Struers [(216) 871-0071, Westlake, OH] offer tech. tips and misc. publications that are -very- handy and helpful. LECO also offers some training.
} One particular item I would find very handy is a compilation of } uv fluorescence wavelength of common substances. } } Thanks to all!
I'd be interested in such also...
Hope the above is helpful; glad to see another met online! -Rob ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, | Rob Tayloe | MSM Spelunkers Club /-v-\ | | Metallographic Lab. | Missouri Speleological Survey \-v-/ | | Rolla Research Center | Bat Conservation International \-v-/ | | U.S. Bureau of Mines | Missouri Cave & Karst Conservancy | | tayloe-at-rorc.usbm.gov | National Speleological Society #32993 /-v-\ | | (314) 364-3169 x247 | American Cave Conservation Association | ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''
On Tue, 19 Apr 1994 IDESOUSA-at-BRMVM1.VNET.IBM.COM wrote:
} Is anybody aware of a good short course on optical microscopy? } It is such a vast subject (polarized , UV,IR, confocal,filters, } image analysis, interferometry, ellipsometry etc.)
Might try the following:
1. McCrone Research Institute 2820 S. Michigan Ave. Chicago, IL 60616-3292 (312) 842-7100 [phone] 842-1078 [fax]
*some of their offerings: photomicrography, polarized light microscopy, hotstage microscopy, SEM, TEM, pharmaceutical, forensic, asbestos id, hair and fiber id, wood and pollen id, anb many others, including custom planned on-site courses. I've taken the Applied Polarized Light Microscopy course there, and found it -very- good; although not enough emphasis for materials type. Courses are spread out through the year. Prices for 1994: $1050 (ouch!) for almost all, w/ TEM being higher.
2. Institute for Microstructural Analysis c/o Buehler Ltd. P.O. Box 1 Lake Bluff, IL 60044 (702) 295-4659 [phone]
*courses offered include: image analysis, petrographic prep., basic metallography, advanced materials, ceramics, etc. Prices vary from $700 for 2-day to $1095 (again, ouch!) for 5-day, and higher prices at their Irvine, CA locale. I myself have not been there, but have heard good reports.
3. ASM International Materials Park, OH 44073 (216) 338-5151 [phone]
*courses include: metallographic interpretation, met. techniques, advanced tech. in optical (& electron) microscopy, failure analysis, optical microscopy of ceramics, quantitative metallography, etc... I've taken one course there, and have had several home-study classes. Good all around training. They offer what's called Materials Engineering Institute Extension diploma, and a three-tiered Center for Applied Metallography levels. Prices are similar to Buehler's.
} Could someone suggest books or reference material which is neither } trivial or too technically involved in the physics. (level of a good } metallurgical technician)
Some of my favorites include:
Metallography: Principles and Practice...........Vander Voort, George McGraw-Hill, 1984 (wish it would be updated tho) Polarized Light Microscopy.......................McCrone & Delly Ann Arbor Science Publishers, 1978 (likewise...) -and- many of the Microstructural Science volumes from the proceedings of the annual technical meetings of the International Metallographic Society. -and- many of the ore microscopy books have very detailed and highly useful information.
In addition, the other large met. supply houses besides Buehler, LECO [(616) 983-5531; St. Joseph, MI] and Struers [(216) 871-0071, Westlake, OH] offer tech. tips and misc. publications that are -very- handy and helpful. LECO also offers some training.
} One particular item I would find very handy is a compilation of } uv fluorescence wavelength of common substances. } } Thanks to all!
I'd be interested in such also...
Hope the above is helpful; glad to see another met online! -Rob ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, | Rob Tayloe | MSM Spelunkers Club /-v-\ | | Metallographic Lab. | Missouri Speleological Survey \-v-/ | | Rolla Research Center | Bat Conservation International \-v-/ | | U.S. Bureau of Mines | Missouri Cave & Karst Conservancy | | tayloe-at-rorc.usbm.gov | National Speleological Society #32993 /-v-\ | | (314) 364-3169 x247 | American Cave Conservation Association | ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''
the McCrone Institute in chicago has a number of short courses on various aspects of light microscopy - including things that would probably be of interest to you such as polarization, dispersion staining, etc
there are several good, simple books around that are probably at the level you want - check out the Oxford series - I know thee is an flouresence & think there may also be a polarization book - they present the basic theory and are inexpensive - there is also a very practical-oriented book with a yellow and white paper cover whose exact and author escape me ( it is siiting on my bookshelf at home)
if you are not satisfied with your other responses send me a message & I will try to get back to you
We are thinking of purchasing a sapphire knife for our Vibratome so that we can cut high-quality sections. The knives are about $700. Being somewhat poor, we were wondering if the knife is a good investment. What has been people's experience with these knives? Do they cut reliable high-quality sections? Any problems with them? Thanks for any opinions.
-- ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Nancy L Desmond, Ph.D. Department of Neurosurgery University of Virginia Health Sciences Center, Box 420 Charlottesville, VA 22908
We (actually Ed Lachica) have found it an absolute requirement for cutting unfixed chick embryos. Fixed post-hatch brains didn't seem to cut any better. But sectioning unfixed brain, or lightly fixed embryonic brain is helped tremendously.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
On Wed, 20 Apr 1994, Nancy L. Desmond wrote:
} We are thinking of purchasing a sapphire knife for our Vibratome } so that we can cut high-quality sections. The knives are about } $700. Being somewhat poor, we were wondering if the knife is a } good investment. What has been people's experience with these } knives? Do they cut reliable high-quality sections? Any } problems with them? Thanks for any opinions. } } -- } ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ } Nancy L Desmond, Ph.D. } Department of Neurosurgery } University of Virginia } Health Sciences Center, Box 420 } Charlottesville, VA 22908 } } 804.924.5607 (voice) } 804.982.3829 (fax) } ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ }
I am studying Aluminium alloys samples with TEM and especially two kinds of them: Al 3003 (0.12%Cu-1.2%Mn) AL 6061 (0.6%Si-0.27%Cu-1%Mg-0.2%Cr) I will use a double jet electropolisher (Tenupol 2) and I would want to know, if possible: 1-what are the best electropolishing solutions for these alloys (the books are not enough specific ) ? 2-what are the value of current,voltage and what is the temperature used for the solution ? 3-I suppose that we use the classical technique for cleaning but there must be some problems to prevent samples from oxydation which is an important problem for aluminium.Is there a special technique to avoid oxydation ?
NOTE:if no answer is possible, what are at least the best solution and electropolishing conditions for PURE aluminium.
It took nearly three months for BioRad to replace our laser when it failed after only 100 hours of use. So much for 24 hr. service. ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
Does anybody know of a good procedure on the infiltration of yeast cells with LR-White? I dehydrate the pellet normally, starting with 35%ETOH for 10 minutes, 50%ETOH 10 minutes, 75%ETOH 10 minutes, 2 x 100%ETOH 10 minutes, 1:1- 100%ETOH/LR-white 1hour, 1:2- 100%ETOH/LR-White 1hour, 1:3- 100%ETOH/LR-white 1hour, 2x pure LR-white 1hour, bake at 50C for 72 hours. I am still ending up with holes around the cells and some cells aren't being infiltrated with the resin. I have never had problems with infiltration of any type of tissue, bacteria or anything else I've had to embed. This is getting to be pretty frustrating (Jack Daniels-black, here I come). Thanks Phil
Does anybody know the name of the journal that replaced "The Journal of Histochemistry and Cytochemistry" ? I was interested in getting a subscription to it and when I called Elsevier I was told it was discontinued 9 years ago. Someone said it was replaced by another outfit and they weren't sure who the publisher was. Anybody know? Thanks, Phil Rutledge
Message-Id: {MAILQUEUE-101.940422072110.367-at-prl.pulmonary.ubc.ca} To: microscopy-at-anlemc.msd.anl.gov
Phil Rutledge Our library carries a journal by the title of Journal of Histochemistry and Cytochemistry published by the Histochemical Society. It is printed by Williams and Wilkens in Baltimore. Probably is the same one you are looking for. In addition, concerning LR white and yeast infiltration, it was suggested by someone here that you might try using a mixture of osmium and potassium permanganate to fix the tissue, or possibly ruthenium red. You need to permeabilize the cell wall to let the LR white in. Try also to increase infiltration times, or put it under vacuum. Was also suggested you look up papers by C. Bracker on fixation of fungi. Hope this info helps.
I did the studies about Al3003 by TEM. You can find my paper "ATEM study of precipitates in an Al-Mn-Si alloy" publisned on METALLOGRAPHY 21:293-315 (1988). I used 20 HClO4 + 20 HCl + 80 ethyl alcohol, around 25 mA and temperature aroun -25 C. As you can see from my paper I have get lattice image. I am the first author of the paper XIANYING MENG-BURANY. Sandy X. Burany
Apologies to all concerned if this query is a bit dumb! After trawling the literature on HREM imaging theory (particularly the application of the wave-optical Abbe theory of image formation) I am confused as to whether the Fraunhofer diffraction pattern is:
i)a Fourier transform of the object exit wavefunction (being a product of the incident wave amplitude and a transmission function)
OR
ii)as some authors simply state, "the transmission function"
The J Histochem Cytochem is published by the Histochemical Society. The address is: Mount Sinai School of Medicine, 1 Gusatave L. Levy Place, Box 1045, New York NY 10029 Ph: 212-362-1801. You should find this journal in most university libraries. If your library does not get it, talk to your acquisition librarian. It is still the leading histochemical journal.
Denis G. Baskin, Ph.D. University of Washington
On Fri, 22 Apr 1994, rutledge phil wrote:
} Does anybody know the name of the journal that replaced "The Journal of } Histochemistry and Cytochemistry" ? I was interested in getting a } subscription to it and when I called Elsevier I was told it was } discontinued 9 years ago. Someone said it was replaced by another outfit } and they weren't sure who the publisher was. Anybody know? } Thanks, } Phil Rutledge }
} One particular item I would find very handy is a compilation of } uv fluorescence wavelength of common substances.
For ultraviolet(365nm) and blue-violet(400nm) fluorescence of over 600 materials see:
The Particle Atlas, Edition Two, Volume IV, McCrone and Delly (1979). [Out of Print]
The entire six-volume, electronic edition of the Particle Atlas is now available (PAE^2) on CD-ROM from McRI or:
Microdataware 2894 Tribune Ave Hayward CA 94542 TEL 510.582.6624 FAX 510.582.8295
McCrone Research Institute is offering the PAE^2 Version 1.0 for Windows with an educational discount of $550.00, which reduces the price to students to $950.00 if purchased within 90 days of course completion.
For a basic library of microscopy:
"A Basic Microscopy Library", John G. Delly, The Microscope, vol. 34, pp. 11-25 (1986).
This article contains a list and description of books on microscopy for the fields including biomedicine, mineralogy, petrology, petrography, metallography, chemical microscopy, and, of course, general works.
100%ETOH/LR-white 1hour, 2x pure LR-white 1hour, bake at 50C for 72 hours. I am still ending up with holes around the cells and some cells aren't
being infiltrated with the resin. I have never had problems with
infiltration of any type of tissue, bacteria or anything else I've had to
embed. This is getting to be pretty frustrating (Jack Daniels-black,
here I come). Thanks Phil
} } } } } } } } } } } } } } } } } } } } } } } } }
We routinely use LR White for preparing EM sections of yeast in immunolocalization experiments. The critical factor for getting good infiltration is a brief treatment of the fixed, washed cells with 1% sodium metaperiodate. This treatment alters the cell wall so that the resin infiltrates perfectly. After treatment with metaperiodate, a relatively standard dehydration/infiltration schedule can be followed. We also use a temp block (like the ones normally used for restriction digests) to precisely control temperature during polymerization. It seemed to be important to keep the temperature low during polymerization to minimize shrinkage (we used 45-50C for 24 hr).
For details of the procedure see:
Wright and Rine, Methods in Cell Biology 31:473-512.
van Tuinen and Riezman, J. Histochem. Cytochem. 35:327.
Phil wrote: ""Does anybody know of a good procedure on the infiltration of yeast cells with LR-White? I dehydrate the pellet normally, starting with 35%ETOH for 10 minutes, 50%ETOH 10 minutes, 75%ETOH 10 minutes, 2 x 100%ETOH 10 minutes, 1:1- 100%ETOH/LR-white 1hour, 1:2- 100%ETOH/LR-White 1hour, 1:3- 100%ETOH/LR-white 1hour, 2x pure LR-white 1hour, bake at 50C for 72 hours. I am still ending up with holes around the cells and some cells aren't being infiltrated with the resin.""
If you want to try and look at some of your poorly infiltrated yeats. If you have a 200KV instrument, you can cut 1/4 micron sections(maybe the yeast will stay in the plastic and not fall out!); stain with aqueous UA for 45'-1 hr. at 45 degrees, LC 30' and use the 200kv. We do this alot when people send us blocks that are bad.
This is what we do in our facility, with success on yeast: we use LR white, both medium and hard grades; for regular ultrastructure(medium grade)- if we have problems with infiltration: we increase the changes in LR white and make sure each change is accompanied by time on the rotator( -at- 4 RPM)(called rotamix in protocol below) we leave the sample in LR white overnight, on a rotator at 4 degrees. If we have trouble with air bubbles, we reduce stirring to a minimum and only use the rotator for mixing; we polymerize at 55 degrees for only 24 hours. For IEM, we use hard grade -if we have problems with infiltration: we increase the changes in LR white first and accept some yeast cells will not be infiltrated well, in some samples; we always have plenty in each section that are fine, even if we leave the cell wall intact. Note that there are two procedures; if your label is O.K. with procedure b, you'll get better infiltration; but either way gives use plenty of well-infiltrated yeasts cells in each section. If we have trouble with air bubbles, we reduce stirring to a minimum; we polymerize Hard grade at 50 degrees for only 24 hours.
Phil: here's the last half of our protocols; please call or send me e-mail anytime. It should work. Regular ultrastructure 6. Dehydrate : 30% - 5 mins 50% - 5mins 70% - 5mins 100% - three 5min. washes.
7. LR White:100% ETOH 1:1 - 30-mins RT on a gentle Rotamix*** .
8. LR White:100% ETOH 3:1 - 30-mins RT on a gentle Rotamix*** .
9. LR White - 2-3 changes 30-mins each, RT on a gentle Rotamix***
10. Overnight in fridge (-at-4oC), on a gentle Rotamix.
11. LR white - 1 change 1 hr (Warm LR White to RT before change)
12. Place tissue in gelatin capsules, add fresh LR White; seal capsules; allow to settle for 1 hour;
NOTE: if you need to centrifuge material and you have a small sample size, place cells in trimmed beam capsule, seal and centrifuge inside a 12-15ml cenrifuge tube{with a cotton bottom pad}, using a clinical centrifuge; then carefully seal beam capsule inside two 000 gelatin bottoms, that have been filled with LR White. Make sure you seal well with LR White. Use regular mold holders to hold capsules. OR if you need to centrifuge material and you have a large sample size, leave cells in original 00 gelatin capsule(i.e.complete step 12. Then place 00 capsule inside a BEEM capsule that has been cut to accomodate 00 gelatin capsule(i.e. top half is removed); centrifuge inside a 12-15ml cenrifuge tube{with a cotton bottom pad}, using a clinical centrifuge, until sample is spun down.
13. Polymerize at 55-60oC for 24 hours.
*** Use glass vials ; keep out of direct light; Use a rotomix, asLR White needs to be GENTLY stirred. KEEP Oxygen out of LR White; avoid excess air bubbles in resin. The dehydration and embedding are fairly straightforward. USE LR White Medium GRADE Open gelatin capsules to stop all polymerization.
For section stain, start with 2%UA aqueous for 20-45 mins. and LC 2-5 mins. try UA-45'/LC 2' first
FOR IEM Dehydrate : 50% - 30 mins 70% - 2 changes, 30 mins. each
LR White:70%ETOH - 2:1 2 changes 30 mins each ***
a. LR White - 3 changes 30-60 mins each, on a gentle Rotamix. b. LR White - 4-6 changes over three hours, on a gentle Rotamix.
a.Overnight in fridge (-at-4oC), on a gentle Rotamix. b. Place tissue in gelatin capsules, add fresh LR White; seal capsules; allow to settle. SEE NOTE step 8. Polymerize at 50oC for 24 hours.
a.LR white - 1 change 1 hour (Warm LR White to RT before change)
a.Place tissue in gelatin capsules, add fresh LR White; seal capsules; allow to settle for 1 hour;
NOTE: if you need to centrifuge material and you have a small sample size, place cells in trimmed beam capsule, seal and centrifuge inside a 12-15ml cenrifuge tube{with a cotton bottom pad}, using a clinical centrifuge; then carefully seal beam capsule inside two 000 gelatin bottoms, that have been filled with LR White. Make sure you seal well with LR White. Use regular mold holders to hold capsules. OR if you need to centrifuge material and you have a large sample size, leave cells in original 00 gelatin capsule(i.e. complete step 8). Then place 00 capsule inside a BEEM capsule that has been cut to accomodate 00 gelatin capsule(i.e. top half is removed); centrifuge inside a 12-15ml cenrifuge tube{with a cotton bottom pad}, using a clinical centrifuge, until sample is spun down.
9. a.Polymerize at 50oC for 24 hours.
TEST one block for sectioning quality. Polymerize another hour, if necessary.
*** Use glass vials to mix; mix solns. at RT; keep out of direct light; Use a rotomix, as solution is not very miscible & needs to be GENTLY stirred. KEEP Oxygen out of LR White; avoid excess air bubbles in resin. The dehydration and embedding are fairly straightforward.
LrWhite blocks should be stored at -20oC to preserve antigenicity indefinitely. Remove the gelatin and expose block to air in order to stop polymerization.
For section stain, start with 2%UA aqueous for 20-45 mins. and LC 2-5 mins. try UA-20' with no LC first, so you can locate the gold particles easily; adjust stain, as needed for ultrasruture.
JOURNAL OF MICROSCOPY - MAY 1994 ISSUE - VOLUME 174 PART 2
PUBLICATION DATE - 20 MAY 1994
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 61-68
ACCURATE ALIGNMENT OF SETS OF IMAGES
By W. O. SAXTON Department of Materials Science and Metallurgy, Pembroke Street, Cambridge CB2 3QZ, U.K.
Summary Two modifications are described to the conventional procedure of cross-correlation, widely used for establishing the relative alignment of the members of a set of images from which a higher resolution or more interpretable restoration is sought. Both achieve a high and sharp peak in circumstances where the conventional peak is too ill defined to be recognisable; neither involves significant additional computational time. The more general method requires a rough knowledge of the imaging conditions, but a variant applicable to images with axial resolution has no such requirement. In addition, a least-squares procedure is presented for achieving an optimum compromise between many pair-wise displacement measurements, preventing the accumulation of alignment errors across a set of images.
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 69-73
RESOLUTION IN NONLINEAR LASER SCANNING MICROSCOPY
By J. DEITCHE, M. KEMPE & W. RUDOLPH The University of New Mexico, Department of Physics and Astronomy, Albuquerque, NM 87131, U.S.A.
Summary The lateral and depth resolution of nonlinear microscopy was studied systematically. Nonlinear microscopy can be classified into several categories depending on the coherence properties of the process that generates the imaging signal from the illuminating light, on whether a single- or a two-beam geometry is used and whether the optical setup is Type I or Type II. An evaluation of the imaging equations shows that (i) lateral and depth resolution improve with increasing nonlinearity, (ii) the differences between coherent and incoherent imaging diminish, and (iii) nonlinear imaging allows depth discrimination in Type I microscopy.
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 75-84
CRYO AUTOMATED ELECTRON TOMOGRAPHY: TOWARDS HIGH-RESOLUTION RECONSTRUCTION OF PLASTIC-EMBEDDED STRUCTURES
By M. B. BRAUNFELD, A. J. KOSTER, J. W. SEDAT & D. A. AGARD The Howard Hughes Medical Institute and the Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA 94143-0448, U.S.A.
Summary The use of fully automated data collection methods for electron beam tomography allows a substantial reduction in beam dose. The goal has been to develop new protocols for data collection defining optimal approaches for maintaining data self-consistency and maximizing the useful resolution of the reconstruction. The effects of irradiation and post-cure microwaving were examined for a variety of embedding media (Epon, Epox, Lowicryl) in order to quantify beam damage with the goal of identifying the most beam stable embedding medium. Surprisingly, the substantial dose reduction made possible by automated data collection did not result in a significant decrease in specimen shrinkage even for samples stabilized by pre-irradiation. The accelerated shrinkage is a direct consequence of the stroboscopic illumination patterns inherent to automated data collection. Furthermore neither the choice of embedding resin nor microwave post-curing greatly affected shrinkage. Finally, cryogenic data collection was investigated as a means to minimize the effects of secondary radiation damage. Minimal pre-irradiation coupled with low-temperature automated data collection greatly reduces shrinkage and should result in high-quality data for three-dimensional reconstructions.
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 85-92
A NOVEL METHOD FOR MEAN CELL VOLUME ESTIMATION
By P. WEBSTER* & G. GRIFFITHS+ *Yale University School of Medicine, Department of Cell Biology, 333 Cedar Street, New Haven, CT 06510, U.S.A. +European Molecular Biology Laboratory, Postfach 10.22.09, 69012 Heidelberg, Germany
Summary A novel method is described for the estimation of the mean cell volume of cell populations grown in suspension. The cells are filtered on to a nitrocellulose filter to form a cylindrical pellet which is embedded in epoxy resin. Using estimates of pellet height and radius, the number of cells in the pellet and the volume density of cells in the pellet, it is possible to produce an unbiased estimate of the mean cell volume. This method is compared, using cell suspensions of the blood parasite Trypanosoma brucei, with other published methods for mean cell volume estimation. A Coulter channelizer was also used to compare the mean cell volume of living trypanosomes with that of aldehyde-fixed populations, and the values obtained were compared with those obtained with the new method. The estimated mean cell volume of a T. brucei clone was used to derive values from volume densities obtained by point and intersection counts for the absolute volumes of the flagellar pocket, the nucleus and the endocytic organelles containing internalized horseradish peroxidase and transferrin-gold after 30-min incubations at 310K. Estimated values for the cell were also obtained. From known data on the total amount of variant surface glycoprotein molecules per cell and the known packing density of membrane proteins, it is estimated that approximately 80% of the molecules must reside in intracellular compartments. It was estimated that the equivalent of 5% of the surface membrane may be internalized per minute, an amount which is almost the size of the entire flagellar pocket membrane.
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 93-100
THE INFLUENCE OF TISSUE PROCESSING ON QUANTITATIVE HISTOPATHOLOGY IN BREAST CANCER
By M. LADEKARL Stereological Research Laboratory, University Institute of Pathology and Second University Clinic of Internal Medicine, Institute of Clinical Experimental Research, University of Aarhus, Denmark.
Summary Objective grading of breast cancer by morphometry has been suggested for improving the precision of the prognostic prediction. However, the tissue components evaluated might be influenced by variations in the processing, reducing the clinical value. In the present study, the impact of the period of fixation, of the acidity of the fixative and of the embedding medium was investigated by allocating tissue samples from 27 surgical breast cancer specimens systematically randomly to different modes of processing. The volume-weighted mean volume of cancer call nuclei was estimated using the method of point-sampled intercepts on vertical sections. In addition, estimates of the mean nuclear profile area, the nuclear volume fraction, the nuclear profile density and the mitotic profile frequency were obtained. The quantitative histopathological estimates were stable with respect to the investigated variables of the tissue processing. No significant differences were found when comparing the estimates obtained in samples from five tumours fixed in formalin at pH 5.0, 6.0, 7.0, 7.4 and 8.0 respectively. Similarly, no significant correlations between estimates and the period of formalin fixation (24h, 3 days and 3 months) were found in samples from five other tumours. However, the volume-weighted mean nuclear volume of cancer cell nuclei was 13% larger (2p=0.004) and the mean nuclear profile density was 17% smaller (2p=0.04) in hydroxyethyl- methacrylate-embedded samples from 17 tumours as compared to paraffin-embedded samples. Thus, the shrinkage observed in paraffin seems to affect nuclei less than tissue.
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 101-110
METHOD FOR THE STUDY OF THE THREE-DIMENSIONAL ORIENTATION OF THE NUCLEI OF MYOCARDIAL CELLS IN FETAL HUMAN HEART BY MEANS IF CONFOCAL SCANNING LASER MICROSCOPY
By Y. USSON,* F. PARAZZA, * P.-S. JOUK + & G. MICHALOWICZ+ *Dynamique de l'organisation du genome, Universite Joseph Fourier, BP53, 38041 Grenoble cedex 9, France +Medecine Neonatale, Centre Hospitalier Regional Universitaire, Grenoble, France
A series of three-dimensional image analysis tools are used to measure the three-dimensional orientation of nuclei of myocardial cells. Confocal scanning laser microscopy makes it possible to acquire series of sections up to 100 micrometre inside thick tissue sections. A mean orientation vector of unit length is calculated for each segmented nucleus. The global orientation statistics are obtained by calculating the vectorial sum of the nuclear unit vectors. The final orientation statistics are obtained by calculating the vectorial sum of the nuclear unit vectors. The final orientation is expressed by a mean azimuth angle, an elevation angle and a measure of the angular homogeneity. The method is illustrated for two different regions of the myocardium (interventricular septum and papillary muscle) of a normal human fetal heart. This quantitative method will be used to assess and calibrate the information provided by polarized light microscopy.
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 111-119
A NOVEL APPLICATION OF MICROSPHERE PERFUSION AND SCANNING ELECTRON MICROSCOPY TO THE IDENTIFICATION OF PULMONARY ARTERIOLES IN GUINEA PIG AND RABBIT LUNGS
By D. C. WALKER, S. HOSFORD & A. MACKENZIE Christmas Seals Electron Microscopy Laboratory of the Pulmonary Research Laboratory, U.B.C. Pathology, St Paul's Hospital, Vancouver, B.C., Canada V6Z 1Y6
Summary In arterioles of the lung the intravascular blood pressures are lower than in comparable vessels in the systemic circulation and the arteriole walls are thinner. Therefore, it is very difficult to distinguish between arterioles and venules of the same size using scanning electron microscopy. This study describes a novel application of latex microsphere perfusion and scanning electron microscopy which distinguishes between pulmonary arterioles and venules on the basis of endothelial cell morphology. Microspheres, 90 and 45 micrometres in diameter, were perfused into the arterial side of the pulmonary circulation of guinea-pig and rabbit lungs. Scanning electron microscopy of the arterioles on both sides of the lodged microspheres indicated that the endothelial cells are spindle shaped. In contrast, the endothelial cells of equal diameter venules are polygonal. Furthermore, the nuclei of the arteriolar endothelial cells were significantly (P=0.019) narrower than those of endothelial cells in venules of equal diameter. Finally, it was observed that the differences between arteriole and venule endothelial cells persisted distally to the capillaries.
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 121-123
ASTIGMATISM OF A THICK CYLINDRICAL OBJECT IN REFLECTIVE MODE CSLM
By CHANG-GUI WANG, H. RAIKKONEN & M. LUUKKALA Department of Physics, PO Box 9, 00014 University of Helsinki, Finland
Summary The axial response of a basic confocal microscope is determined when the sample is a thick cylindrical or tubular structure. The response from the back wall of the cylindrical sample is split into two separate signals due to basic aberration or astigmatism effects.
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 125-127
Short Technical Note SEVERE SHRINKAGE OF MICROFIL DURING TISSUE CLEARING WITH THE SPALTEHOLZ TECHNIQUE
By J. MOLLER, K. ROBERTSEN & E. S. HANSEN Institute of Experimental Clinical Research, Department of Orthopaedics, University of Aarhus, Randersvej 1, DK 8200 Aarhus N, Denmark
Summary The effect of two commonly used tissue clearing methods on the morphology of vascular casts by Microfil, a silicone rubber injection compound, was investigated. Microfil undergoes extreme shrinkage when the casted tissue is cleared by the alcohol-methyl salicylate clearing technique. No shrinkage is observed when the alternative glycerine clearing method is used. The alcohol-methyl salicylate clearing technique should be avoided in studies employing Microfil.
PLEASE SEND YOUR QUERIES CONCERNING THE JOURNAL TO RMS-at-VAX.OX.AC.UK. WE ARE ALSO PLEASED TO ACCEPT LETTERS TO THE EDITORS BY EMAIL.
Does anybody know where I could find SJ Singer, the man who introduced EM immunocytochemistry by a paper in Nature (London) 1959. I would like to present him by his portrait at a meeting. All advices are welcome. Thanks, Sverker Enestr|m
Has anyone worked with a type of species in the bacteria field known as a marine cytophage type? I'm trying to fix the cells on agar and the cells keep floating up off the surface of the agar. I would like them to stay put. (maybe I need to put them in a training school for bacteria!). I've tried several methods suggested by members of the microscopy board but no success. Hopefully this problem can be taken care of. It's driving me nuts! Thanks, Phil
Message-Id: {9404251801.AA02278-at-odin.morph.med.umich.edu} Errors-To: {dennis%odin-at-odin.morph.med.umich.edu} To: microscopy-at-anlemc.msd.anl.gov Cc: dennis-at-odin.morph.med.umich.edu
Like a dumbbell I forgot to mention I need to do SEM on these little suckers. Fixing them and processing for TEM is no problem. That's easy. Trying to keep the little creatures attached to the agar surface and fixing them without them floating off is a #!$#%$$^&(^*& of a problem. I need to see the surface of the bacteria and how they are oriented in their growth on the agar without losing ANY of the cells. I should have become a mechanic or plumber 27 years ago, EM can be a real -at-$%^%& ! Thanx, Phil
Have you tried attaching them to a millipore filter by passing them from a syringe onto the filter (using millipore syringe fittings) and then fixing them onto the millipore surface by squirting fixative through the filter? This works for suspension culture cells and many small critters. The millipore filters can then be CPD and attached to a stub.
On Mon, 25 Apr 1994, rutledge phil wrote:
} Like a dumbbell I forgot to mention I need to do SEM on these little } suckers. Fixing them and processing for TEM is no problem. That's easy. } Trying to keep the little creatures attached to the agar surface and } fixing them without them floating off is a #!$#%$$^&(^*& of a problem. I } need to see the surface of the bacteria and how they are oriented in } their growth on the agar without losing ANY of the cells. } I should have become a mechanic or plumber 27 years ago, EM can be a real } -at-$%^%& ! } Thanx, } Phil }
Try this sandwich method - it works great for suspensions:
use the thinest layer of agar that you can remove with the colonies intact
- take a 13- 15 mm dameter nylon washer (you can use brass if you are not going to use OsO4) - I suppose you can use a larger diameter washer & filter so long as it will fit in your cpd unit - place an appropriate pore size filter on the washer - place a "spacer" on the filter (for cell suspensions I punch a hole in the blue spacer papres packed with the filters - you might want to try an o-ring) - cover with a second filter - top off the sandwich with another washer
clip the whole thing together with a paper clip (yours may be a triffle thick for that, so you may have to be inventive
we have used relatively short fix/wash/dehydrate times for suspensions with good results
the only catch is taht some of your cell may stick to the top filter (ours do) so you might need to make sure that "chamber" height exceeds the height of your sample and try to keep it right side up during processing
(an easy way to remove small sections of colonies from plates is with a cork borer or a glass pipette)
good luck & please excuse the typos
feel free to contact me if this is unclear
Marcelle A Gillott Univ Wisconsin-Milwaukee Department of Biological Sciences 414-229-4186
First: Thanks to all for their suggestions. Now: Let me go into this problem a little deeper. We have unknown bacteria of marine cytophage species that autofluoresce red and green. We want to look at the surface by SEM to see: 1: surface structures, if any 2: orientation of the bacteria as it grows (there is a definite orientation as seen after some of the cells have floated off) Need: a way to fix the cells without ANY cells floating off the surface Tried: 1: suspensions 2: growing on nucleopore filters 3: growing on coverslips Result: no autofluorescence, no definite orientation Fix: 1: direct addition of glutaraldehyde 2: cutting out pathways in the agar and fixing by letting the agar absorb the fix and fixing the cells from underneath the agar. 3: osmium vapor fixation then glut. Result: Some of the bacteria still float up when washing and dehydrating. Question: Is there a way to crosslink the cells by some fixation technique so they will remain in place on the agar surface?
John Mansfield North Campus Electron Microbeam Analysis Lab University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 08:59
Date:4/26/94 NC EMAL
Macro 'Read AFM File'; {reads header in and searches for the number of Samps/line, uses this value} {as the width and heigth for the image import (therefore supports 128,256 and} {512 squared images} var size, width, height, offset, slices, first, second, third, fourth: integer; icount: integer; temp, one, two, three, four: string; done: boolean; begin; done:= 'false'; width:=8192; height:=1; Offset:=0; slices:=1; SetImport('8-bit'); SetCustom(width, height, offset,slices); Import(''); icount:=1; repeat temp:=chr(getpixel(icount, 0)); if ((temp='S') and (one {} 'S')) then one:=temp; if ( (temp='a') and (one='S') and (two {} 'a')) then two:=temp; if( (temp='m') and (one='S') and (two='a')) then three:=temp; if( (temp='p') and (one='S') and (two='a') and (three='m')) then four:=temp; if (four='p') then done:='true'; icount:=icount+1; until done='true'; first:=(getpixel((icount+8), 0))-48; second:=(getpixel((icount+9), 0))-48; third:=(getpixel((icount+10), 0))-48; fourth:=(getpixel((icount+11), 0))-48; if (first {} 1) then size:=(first*100)+(second*10)+third; if (first=1) then size:=(first*1000)+(second*100)+(third*100)+fourth; Dispose; width:=size; height:=size; offset:=8192; SetImport('16-bit signed,swap bytes'); SetCustom(width, height, Offset); Import(''); end;
Phil, Can you overlay the agar/bacteria with another thin layer of agar then fix as usual. When osmium is added the bugs should turn black (if in colonies) making them visible after emebedding.
Royal Microscopical Society & Oxford Brookes University
26 - 31 March 1995
Location Oxford Brookes University - Oxford's new University, situated 1 mile from the centre of historic Oxford. Full board accommodation will be available at Morrell Hall, 5 minutes walk from the campus.
Cost Registration and full board accommodation will cost no more than œ440.0 pounds sterling. Receptions, the Conference Dinner and a copy of the special edition of the Journal of Microscopy are included in the cost. There are a very limited number of double rooms available.
Delegates To preserve the informal nature of the Botanical Microscopy Meeting, the number of delegates will be restricted to 150. Places at the conference will be allocated on a first come, first served basis with a preference given to those offering to present a poster. (Please note that places will only be guaranteed after payment has been received.)
Booking forms and abstract instructions will be available from June 1994.
Organizing Committee Nick Harris - Durham Chris Hawes - Oxford Nick Read - Edinburgh Peter Shaw - John Innes Institute
Format of Meeting Following the success of Botanical Microscopy 1991 in Durham, the same team are organizing the meeting to be held in Oxford. The scientific programme will be based around presentations from keynote speakers.
A call for abstracts will be made late 1994 and the organizing committee will select suitable papers from these and invite the authors to present them orally. However, posters on any aspect of plant cell biology involving microscopy may be presented.
Social A reception is planned for the opening evening of the meeting and an informal Conference Dinner will be held on the Thursday evening. A number of short tours around Oxford and its environs will be offered on the Wednesday afternoon of the Conference.
Publications Keynote and selected papers will be published as a special edition of the Journal of Microscopy which will be distributed to all delegates upon publication. All manuscripts will be subject to peer review.
Travel Air: Oxford Brookes University is a 60 minute bus ride from Heathrow airport and 130 minute ride from Gatwick airport.
Road: Coaches run from Victoria Bus Station in London every 20-30 minutes. Journey time is approximately 60 minutes.
Rail: There is a good train service from Paddington Station, London to Oxford.
Queries If you would like any further information, please contact Miss Karen Hale at the Royal Microscopical Society, 37/38 St Clements, Oxford, OX4 1AJ. Tel: +44-865-248768. Fax: +44-865-791237. Email: rms-at-vax.ox.ac.uk.
Programme to Include: þMicrotubule and cytoskeletal dynamics
þMicroscopy of living cells - ion imaging, cell-cell signalling
þPlant cell organization - cell walls, meiosis, low temperature techniques
þMolecular mechanisms of plant development - cell cycle, floral development
þPlant microbe-interactions
Keynote Speakers B Gunning - Canberra H Shibaoka - Osaka J Hush - Sydney S Giroy - Penn State K Oparka - Invergowrie K Roberts - Norwich M Parthasarathy - Cornell Z Cande - Berkeley J Doonan - Norwich R Howard - Wilmington
Please complete this coupon indicating your requirements, and return to the Royal Microscopical Society, 37/38 St Clements, Oxford, OX4 1AJ, UK.
I would be interested in attending the 5th International
Does anybody know where I could find SJ Singer, the man who introduced EM immunocytochemistry by a paper in Nature (London) 1959. I would like to present him by his portrait at a meeting. All advices are welcome. Thanks, Sverker Enestr|m
======================================================================= I send this mail again because of local configuration error: "Service unavailable" =======================================================================
Hi, We are considering the purchase of an Energy Dispersive X-ray Analysis (EDX) system for attachment to our SEM within the next year. Our applications are in two areas; paper making and corrosion of materials.
I would like to know if anyone is aware of mailing lists etc., accessable on the Internet, which are specifically interested in EDX. I would like to follow discussions on relative merrits of detector and window types , vacuum requirements for windowless detectors, pulse processor requirements, software options and in particular, read any horror stories associated with a given manufacturer.
If anyone has opinions on what is currently available from recent purchase deliberations or would like to recommend a review paper on current technology or a periodical which they go to for this sort of information it would be greatly appreciated.
My first priority is always dependable service, but having said that, I will be looking for a high quality detector capable of light element analysis, high pulse throughput, good quantitation with ease of use , beam control and image acquisition and replay capabilities.
Thanks for your help.
______________________________________________________________________ Laurie Frederick, A.SC.T. PAPRICAN Corrosion Control Group 3800 Wesbrook Mall The Pulp and Paper Research Vancouver, B.C. Institute of Canada Canada V6S 2L9
I am looking for some help regarding pore sizes in the secondary cell wall of wood. The wood has been partially degraded by fungi, and I want to see how the porosity of the lignin-cellulose substrate has changed. I plan on infiltrating wood with proteins of different molecular weights (my probes), then locating them with immunolabling. I have already decided on a 40000 M.W. (ovalbumin, and MN dependant peroxidase), and a 20000 M.W. (myoglobin) probe. I am still looking for an adequate probe for M.W. 5000, 10000, and 15000. By adequate probe I mean a protein that does not denature easily, and also I can purchase antibodies against. Any help would be appreciated.
Eugene Krueger, Dept of Plant Pathology, University of Minnesota.
-- Eugene Krueger Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
To: MICROSCO:CENTRAL cc: DRStadden:R_D:Armstrong Subj: Spencer Stereoscope ------------------------------------------------------------------
Is anyone acquainted with a good repair service for AO Spencer stereomicroscopes? We have the "gray standard issue", step magnification, ubiquitous model, probably vintage 1959 or so. One of the prisms has come loose, and we've spent too much time already trying to get it EXACTLY back into position. Maybe there's a trick or tool we're missing. Thanks in advance for any help.
I am looking for some help regarding pore sizes in the secondary cell wall of wood. The wood has been partially degraded by fungi, and I want to see how the porosity of the lignin-cellulose substrate has changed. I plan on infiltrating wood with proteins of different molecular weights (my probes), then locating them with immunolabling. I have already decided on a 40000 M.W. (ovalbumin, and MN dependant peroxidase), and a 20000 M.W. (myoglobin) probe. I am still looking for an adequate probe for M.W. 5000, 10000, and 15000. By adequate probe I mean a protein that does not denature easily, and also I can purchase antibodies against. Any help would be appreciated.
Eugene Krueger, Dept of Plant Pathology, University of Minnesota.
-- Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
A question: To detect dim backscattered electron diffraction patterns, one generally uses a SIT (Silicon Intesified Tube) camera behind a phosphor screen. Hamamatsu's model of SIT has a quoted sensitivity limit of 10^-4 lux. Hamamatsu also sells (for a modest premium) an "intensified" CCD which is sensitive down to 10^-5 lux.
We know a SIT camera will work, but imagine the CCD system will work a little better.
Has anyone used an intensified CCD (Either Hamamatsu's or others')? Are there any drawbacks besides the price? Are there better SIT systems available that would rival the intensified CCD?
Thank you, Chris Krenn Graduate Student UC Berkeley Dept. of Materials Science
Are you working with tissue or purified amyloid? If tissue you need to permeablize the tissue with a detergent (such as Triton-X 100). It seems accesability is your problem. Try various concentrations of detergent, 0.1 to 1.0% for various times. Detergents are used after fixation and in all blocking, Ab's, and probe solutions. Hope this helps.
Wayne England has asked: *********************************************************************** I am having a problem getting Unicryl to polymerize properly and am left with a very brittle block or no polymerization at all. I have tried all of the manufacturer's methods (heat and UV covering all the variables) but am not satisfied with the results. Do you find this resin to be normally brittle or am I missing something? Also, does anyone know how this resin is at infiltrating plant material? It's properties sound too good to be true!! *********************************************************************** We have some experience using this polar resin, which has some ad- vantages over Lowicryl K4M but shares the general disadvantages inherent in hydrophilic resins. The labeling intensity is high but more varying than for Epon. I polymerize at -10C by direct UV in the low temperature chamber of Balzers FSU 010 freeze substitution unit for at least 2 days and using the low temperature UV polymerization insert, filled with 10 ml ethanol. The blocks become hard rubber-like and are more easily sectioned than Lowicryl. The ultrathin sections are more resistant to the electron beam than Lowicryl but not as good as Epon. The tecnicians are anxious about hypersensitivity reactions after exposure for some time. What are yours opinion? Sverker Enestr|m Dep of pathology Link|ping, Sweden
Brij 58 is another detergent that works well for immunocytochemistry. It is not as harsh as Triton and preserves a little more ultrastructure. Downside is that it often doesnt permeabilize all cells in a single preparation.
On Wed, 27 Apr 1994 EMLAB-at-opus.mco.edu wrote:
} Monica, } } Are you working with tissue or purified amyloid? If tissue you need to } permeablize the tissue with a detergent (such as Triton-X 100). It seems } accesability is your problem. Try various concentrations of detergent, } 0.1 to 1.0% for various times. Detergents are used after fixation and in all } blocking, Ab's, and probe solutions. Hope this helps. } } Ed Calomeni }
Message-Id: {9404271519.AA00742-at-odin.morph.med.umich.edu} Errors-To: {dennis%odin-at-odin.morph.med.umich.edu} To: microscopy-at-anlemc.msd.anl.gov Cc: dennis-at-odin.morph.med.umich.edu
One of my library patrons is studying corrosion of metal by bacteria. She needs techniques for preparing samples of bacteria on metal for SEM microscopy. She is also looking for methods of staining or gold-labelling to improve SEM sensitivity. The methods need to be applicable to a wide variety of bacteria species. Any information on best SEM conditions - voltage, angle, windows, etc. would also be helpful.
Thanks for your help.
Replies can be made directly to me at smiths-at-mlc.lib.mi.us or you can contact my patron, Cathy Stewart at 313/676-5292.
Susan Smith R&D Tech. Information Center National Steel Corp. Trenton, MI 48183
Message-Id: {MAILQUEUE-101.940427100951.630-at-prl.pulmonary.ubc.ca} To: microscopy-at-anlemc.msd.anl.gov
We inherited a microscope slide awhile back which we would like to replace, but do not know of a source. The slide had a grid of letters on it (black background with white letters) and it was used for finding particular locations of structures on other slides; ie a certain cell on your slide was at position Xy on the reference slide. Ours is getting so beat up that it is going to fall apart any day. I am not sure if it was commercially available, or made by someone. Any help would be appreciated.
Mark Elliott UBC-Pulmonary Research Lab St Pauls Hospital Vancouver
We currently use Agfa Scientia film for (300kV) HREM. It is a little slower (maybe) than Kodak SO163, faster than 4489. It has a slightly finer grain than Kodak SO163, which helps. Two questions: 1) What are people using for faster film. The Agfa film is quite old (i.e. it has been around for at least ten years), and is there anything better at a reasonable price? 2) We have noticed some scratches at times which seem to be a quality control issue in the film. Is this common?
Scanning: the journal of scanning microscopy is one journal that you may want to check. It is published by FAMS Inc., Box 832, Mahwah, NJ 07430-0832 Phone 201/818-1010 FAX: 210/818-0086 Hope this is helpful.
Susan Smith National Steel Corp. Trenton, MI 48183
On Tue, 26 Apr 1994, Laurie Frederick wrote:
} Hi, } We are considering the purchase of an Energy Dispersive X-ray } Analysis (EDX) system for attachment to our SEM within the next year. } Our applications are in two areas; paper making and corrosion of } materials. } } I would like to know if anyone is aware of mailing lists etc., accessable } on the Internet, which are specifically interested in EDX. I would like to } follow discussions on relative merrits of detector and window } types , vacuum requirements for windowless detectors, pulse } processor requirements, software options and in particular, read any } horror stories associated with a given manufacturer. } } If anyone has opinions on what is currently available from recent } purchase deliberations or would like to recommend a review paper on } current technology or a periodical which they go to for this sort of } information it would be greatly appreciated. } } My first priority is always dependable service, but having said that, I } will be looking for a high quality detector capable of light element } analysis, high pulse throughput, good quantitation with ease of use , } beam control and image acquisition and replay capabilities. } } Thanks for your help. } } ______________________________________________________________________ } Laurie Frederick, A.SC.T. PAPRICAN } Corrosion Control Group 3800 Wesbrook Mall } The Pulp and Paper Research Vancouver, B.C. } Institute of Canada Canada V6S 2L9 } } Email: frederick_laurie-at-vanlab.paprican.ca } Tel: 604-222-3200 Fax: 604-222-3207 }
On 27 Apr 1994 MARK-at-prl.pulmonary.ubc.ca wrote of his need for a slide with a special grid on it.
My 1992 McCrone Accessories catalog has a similar slide called an "England Finder" (p/n 313). I would call them and see what they have for you. Their phone nos. are 708-887-7100 and 800-mac-8122.
1994 PACIFIC NORTHWEST EM SOCIETY SPRING MEETING on "Evolution in Microscopy: Interfacing with Computers" Fred Hutchinson Cancer Research Center - (South Lake Union) Pelton Auditorium, Building B 1100 Fairview Ave. N. Seattle, WA 98109 Friday, May 13th
12:55 p.m. Welcoming Remarks Charles Meshul, President, PNEMS VA Medical Center, Portland, OR
1:00 p.m. "The Merging Roles of EM, Microanalysis, & Computers for Characterization of Materials." Nestor Zaluzec, Materials Science Division, Argonne National Lab
1:45 p.m. "The Evolving Role of Hardware in Image Processing: What are the Requirements?" Leonard Pagliaro, Bioengineering, Univ. of Washington
2:15 p.m. Tour of New Fred Hutchinson Facility and Coffee Break
3:15 p.m. "Analysis and Handling of Cosmic Dust." Don Brownlee, Astronomy, Univ. of Washington
4:00 p.m. "Biological Applications of Microscopic Image Analysis." Grace Bartoo, Bioengineering, Univ. of Washington
4:30 p.m. "SEM Photography: Analog/Digital; B&W/Color." David Scharf, Los Angeles, CA
5:15 p.m. Adjourn
5:30 p.m. Social -at- Benjamin's on Lake Union Sponsored by: PNEMS & Corporate Members (EMS/Diatome US, Gatan Inc., JEOL USA Inc., Link Analytical/Oxford Instruments Inc., Palmborg Associates, Philips Electron Optics)
1994 PACIFIC NORTHWEST EM SOCIETY SPRING MEETING on "Evolution in Microscopy: Interfacing with Computers" Fred Hutchinson Cancer Research Center - (South Lake Union ) Rooms B1072 and B1074, Building B 1100 Fairview Ave. N. Seattle, WA 98109 Saturday, May 14th
11:00 a.m. Adobe Photoshop Software Demonstration David Scharf , Los Angeles, CA 90039
12:00 p.m. Lunch
1:00 p.m. PNEMS Business Meeting
1:30 p.m. Video Microscopy Demonstration Gary Crawford, Mideo Systems, Huntington Beach, CA
2:30 p.m. Particle Atlas Electronic Edition CD-ROM Demonstration Steve Shaffer, MicroDataWare, Hayward, CA
3:30 p.m. Real Time Imaging and Electron Diffraction Demonstration Dave Joswiak, Astronomy, Univ. of Washington
4:30 p.m. Adjourn
* This software exchange will consist of the MSA, MAS and EMC Public Domain Library having an excess of 100 megabytes of software (Mac and PC). If you are interested, bring formatted disks to make copies of the available software.
Contact : Mike Rock -at- (206) 685-7073 or Barbara Reine -at- (206) 543-1955 for Information.
PNEMS Spring Meeting Registration Form
This meeting, (May 13 & 14) covering the topic of computers interfacing with microscopes, promises to be exciting and educational. Our speakers representing a wide range of disciplines and interests, all share an expertise in using computers in their microscopic investigation. Please note the correction of the address for the meeting (the NEW Fred Hutchinson Cancer Research Center on South Lake Union, 1100 Fairview Ave. N.) Please fill out, and return, the following form to help us plan for your attending the meeting.
Registration in advance: Students...FREE Others...$ 15.00 Registration on site: Students $ 5.00 Others $ 20.00
Name:_____________________________________
Employer: Company __________________________________________
Chris Krenn asked about experiences with intensified CCD cameras. We have a Pulnix on our high-voltage electron microscope, albeit for a different application. We use it primarily for scanning grids and low dose focussing for beam-sensitive specimens. Under conditions where the illumination is barely visible on our high-brightness screen, but no details are visible, we can clearly see all we need with the video system. We can focus to the nearest setting on the objective fine control (.99 microns per step) in a few seconds, and can focus on the vernier control (1.6 microns full scale) in a few more seconds. This system is great for our applications. There is a trade-off be- tween sensitivity and resolution--especially for the inherently low-contrast images seen at high voltage. We opted for high sensitivity, and, within that constraint, maximized the resolution. We do NOT use the system for collecting ED data, since the intense cen- tral spot would damage the intensifier, even at low-dose conditions. Further- more, it is not at all clear that the CCD gives as good quantitation and sensi- tivity as LoDose x-ray film. The first point may not be a consideration for Chris's application, but the second might. Does anyone know a system with the sensitivity to quantitate small num- bers (1 or 2) of electrons, with the ability to produce a signal proportional to electron number for the more intense reflections (i.e. no saturation)? Since each electron hitting a film grain exposes that grain, it's hard to beat that quantum efficiency. Positional accuracy is less important for us than accurate quantitation, including background subtraction.
"The preparation of samples for [electron] microscopy was still a delicate task requiring skilled human hands; the preparation of a good sample was as demanding a craft as that ever practiced by an artisan -- and took almost as long to learn." Michael Crichton The Andromeda Strain
When this quote first surfaced during lunch a few weeks ago, microscopists were asking:
"Why is specimen preparation for microscopy, whether electron or light, not simple?
"Do I have to be an artisan? After all, I've got microwaves and high tech equipment - and hey, who's got the time?
"Is electron microscopy really only science fiction?
If these and similar questions have troubled you way past midnight, then this year's Spring Symposium may be for you. The application of microwaves is revolutionizing the preparation of biological specimens and, as a bonus, is drastically reducing the time involved. New techniques for minimizing damage in the preparation of materials such as ceramics, polymers, composites and biomaterials are here. Our speakers will be telling us all about these methods.
SPECIMEN PREPARATION
SHERATON INN, MIDWAY I-94 at HAMLINE AVENUE ST. PAUL, MN THURSDAY - MAY 26, 1994
SCHEDULE OF EVENTS
8:00 - 8:30 AM Coffee and Late Registration
8:30 - 8:35 AM Welcoming and Opening Remarks. Dr. Mark Cavaleri, Research Specialist, 3M Analytical & Properties Research Labs, 3M Company, St. Paul, MN.
8:35 - 9:25 AM Preparation of Advanced Materials (ceramics, composits, biomaterials) for the Microscope Dr. Don Zipperian, Manager, Long Range Planning and Development, Buehler, LTD., Lake Bluff, IL.
9:25 - 10:15 AM Specimen Preparation of Polymers for TEM Analysis and Special Applications Ms. Jacqueline Aguilera, Senior Physicist, 3M Company, Corporate Research Analytical, St. Paul,MN.
10:15 - 10:35 AM \ Coffee Break \
0:35 - 11:25 A.M. Quantitative Imaging in the Cereal Industry: Minimizing Specimen Preparation Dr. Gary Fulcher, Professor of Food Science, University of Minnesota, St. Paul, MN
11:25 AM - 1:00 PM 5 LUNCH 6 Box lunches will be provided to participants by MMS and catered by the Sheraton Inn.
1:00 - 1:10 PM Short Business Meeting Ratification of Bylaws, Election of Officers (read Bylaws below)
1:10 - 2:00 PM Rapid Microwave Fixation of Biological Specimens for Light & Electron Microscopy Dr. Gary Login, (Part One), Departments of Pathology at the Harvard School of Dental Medicine, Harvard Medical School, and the Beth Israel Hospital, and the Charles A. Dana Research Institute, Boston,MA.
2:00 - 2:50 PM Impervious Biological Specimens: Techniques and Tricks Ms. Virginia Lindley, Dept. of Agronomy, University of Arizona; Consultant for Industrial and Federal Agencies; President of Arizona EM Society.
2:50 - 3:10 P.M. \ Coffee Break \
3:10 - 4:00 P.M. A Toolkit for Calibrating and Standardizing Microwave Fixation and Staining Dr. Gary Login, (Part Two).
------------------------------------------------------------------------------- Please make your reservation in advance, POSTMARKED NO LATER THAN MONDAY, MAY 23, if you plan to attend the Symposium. Symposium Fee: $20.00 current regular MEMS/MSOM members 93/94, $30 non-member(confers regular membership), $10.00 student members 93/94, $15.00 non-member students(confers student membership). Fill out the form near the end of this newsletter and send it in as directed(prefered) or pay at the door. -------------------------------------------------------------------------------- - REGISTRATION FOR MEMS/MSOM SPRING SYMPOSIUM, MAY 26, 1994, MIDWAY SHERATON (if your registration includes a new membership, fill out and include MMS Membership Form, below). Please postmark your reservation no later that Monday, May 23.
Name__________________________Phone____________Affiliation______________________ __ # Individual Members -at- $20.00 each = $_______. # Student Members -at- $10.00 each = $________. Total Amount Enclosed = $________. Above cost for current 93/94 MEMS/MSOM members only. Non-members and renewals please include membership dues and form(see above).
Make out your check to MMS and mail it together with this form to: Dwight Erickson, MMS Treasuresr, 3M Center, Bldg. 251-1A-03, Saint Paul, MN 55144. Late reservations may pay at the door.
------------------------------------------------------------------------------- MMS(MEMS/MSOM) Membership Form: 1993-94 All microscopists are urged to support their Society at one of the membership levels offered below. The more dues-paying members we have, the more likely we are to attract sustaining corporate member- ships which form the financial backbone of our Society. Often, supervisors will support MMS member- ships out of their project budget because they recognize that it is a very inexpensive way to maintain and increase the skills of their microscopists. If you have been a member over the years and recognize the of MMS to the community of microscopists it serves, consider upgrading your membership this year to the patron or sustaining level. Thank you. Name_______________________________ Dr____ Mr____ Ms____ Phone ( )__________ Affiliation_________________________________________Position____________________ _ Address_____________________________________________________ ZIP__________ Describe your areas of interest; state manufacturer and model of instrumentation below: Bioscience___________________________________________________________________ Materials Science ______________________________________________________________ SEM____________________ TEM______________________ X-ray_____________________ Are you an MSA Member?_______ MAS Member?_______ Other Professional groups?___________ Basic $10___ Patron $25___ Sustaining $100___ Student $5___ Due by Dec 31, '93. Make checks payable to MMS and mail to our treasurer: Dwight Erickson, MMS Treasurer, 3M Center, Bldg. 251-1A-03, Saint Paul, MN 55144.
-- Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
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Bill Tivol open the discusion about CCD cameras to the possible alternatives to phtographic film recording.
Did you consider the electron images plates (Fuji / Kodak / JEOL)? As far as I know (from the litterature), the DQE is about the same or better than for photographic films. The linearity is better, the dynamic range larger and they are suitable for electron diffraction. If somebody is interested I can look in my files to find more details and some references to papers (Ultramicroscopy ... a couple of years ago). Of course the complete system is a quite expensive investment(although compared to a microscope....) and seems to be available only for JEOL microscopes.
It would be nice to hear how they perform in practice from people who use them daily.
__________________________________________________________________ Philippe Buffat Ecole Polytechnique Federale de Lausanne (EPFL) Institut Interd=E9partemental de Microscopie Electronique Address: EPFL-I2M, Batiment MX-C, CH-1015 Lausanne, Switzerland Phone: +41(21)693 29 83 (Central European Time) Fax: +41(21)693 44 01 ______________________ Eudora 2.0.2 __________________________________
I also had regular problems with scratches on Dupont Cronar film. I have seen no scratches on 4489.
On Wed, 27 Apr 1994, L. D. Marks wrote:
} We currently use Agfa Scientia film for (300kV) HREM. } It is a little slower (maybe) than Kodak SO163, faster than } 4489. It has a slightly finer grain than Kodak SO163, which } helps. } Two questions: } 1) What are people using for faster film. The Agfa film } is quite old (i.e. it has been around for at least ten years), } and is there anything better at a reasonable price? } 2) We have noticed some scratches at times which seem to } be a quality control issue in the film. Is this common? } } Thanks } } Laurie Marks } Northwestern University
} From: IN%"pataky-at-bcu.ubc.ca" } Subj: Cryostat help! } } Return-path: {BIOSCI-REQUEST-at-net.bio.net} } Received: from net.bio.net by gnv.ifas.ufl.edu (PMDF V4.2-11 #3240) id } {01HBOOXBZ5DS8X4B47-at-gnv.ifas.ufl.edu} ; Wed, 27 Apr 1994 22:18:06 EST } Received: (from daemon-at-localhost) by net.bio.net (8.6.8.1/8.6.6) id TAA23358 } for cytonet-list; Wed, 27 Apr 1994 19:04:06 -0700 } Received: (from news-at-localhost) by net.bio.net (8.6.8.1/8.6.6) id TAA23346 for } cytonet-arpanet; Wed, 27 Apr 1994 19:04:05 -0700 } Date: Wed, 27 Apr 1994 23:50:46 +0000 (GMT) } From: pataky-at-bcu.ubc.ca (Dave Pataky) } Subject: Cryostat help! } To: cytonet-at-net.bio.net } Message-id: {pataky-270494154951-at-steevlab.generes.ca} } Content-transfer-encoding: 7BIT } Followup-To: bionet.cellbiol.cytonet } NNTP-Posting-Host: steevlab.generes.ca } } Anybody out there know why Tissue-Tek, presumably designed for embedding } samples for cryostat cutting, is so $##-at-***%* annoying to work with? The } problem: I'm cutting chick embryonic brain tissue, anywhere from 10-40 } microns, at -20 and no matter what I try the sections won't stop curling } up, rolling into tubes as soon as I lift the antiroll plate. I've tried } adjusting the blade angle, the "anti-roll" (that's a joke) plate, the } temperature, speed of cutting, new blade (disposable), nothing works. } Ideally I'd like to see nice flat sections that stay that way, preferably } "ribboning" off the blade so I can mount several at once. Know any tricks } or alternatives to Tissue Tek which may work better? Any ideas how to deal } with static electricity? (sometimes the sections stick to the "anti-roll" } plate). Feels like I'm battling the antichrist (and losing!), } } } -- } Dave Pataky } Dept of Zoology, UBC } pataky-at-bdc.ubc.ca } Brain can be particularly difficult. I suggest cryoprotecting in 20% sucrose with 3% PEG MW 400. Cut at -25. Don't expect to get a ribbon. We are usually satisfied with one section at a time. Rarely do we get two. I have a nice handout on cryosectioning that I picked up at a meeting. Give me your FAX # or mailing address and I will send a copy.
We were using SO163 but switched back to 4489. The SO163 seemed to have emulsion problems (much grosser defects than mere scratches, we had big chunks of emulsion fall out). We could never identify anything we were doing wrong and it seemed to be specific to certain Kodak lot numbers. We have not had problems since switching back to 4489 but sometimes need the faster film. Anyone else having problems with SO163 or should we try it again? We can't find a supplier of the Agfa film.
On Thu, 28 Apr 1994, Rodney L Kuehn wrote:
} } I also had regular problems with scratches on Dupont Cronar film. I } have seen no scratches on 4489. } } On Wed, 27 Apr 1994, L. D. Marks wrote: } } } We currently use Agfa Scientia film for (300kV) HREM. } } It is a little slower (maybe) than Kodak SO163, faster than } } 4489. It has a slightly finer grain than Kodak SO163, which } } helps. } } Two questions: } } 1) What are people using for faster film. The Agfa film } } is quite old (i.e. it has been around for at least ten years), } } and is there anything better at a reasonable price? } } 2) We have noticed some scratches at times which seem to } } be a quality control issue in the film. Is this common? } } } } Thanks } } } } Laurie Marks } } Northwestern University } } } }
Mime-Version: 1.0 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable To: microscopy-at-anlemc.msd.anl.gov
Bill Tivol open the discusion about CCD cameras and the possible alternatives to phtographic film recording.
Did you consider the electron images plates (Fuji / Kodak / JEOL)? As far as I know (from the litterature), the DQE is about the same or higher than for photographic films. The linearity is better, the dynamic range larger and they are suitable for electron diffraction. If somebody is interested, I can look in my files to find the references of some (old) papers (Ultramicroscopy ... a couple of years ago). Of course the complete system is a quite expensive investment(although compared to a microscope....) and seems to be available only for JEOL microscopes.
It would be nice to hear how they perform in practice from people who use them daily. Yours
__________________________________________________________________ Philippe Buffat Ecole Polytechnique Federale de Lausanne (EPFL) Institut Interd=E9partemental de Microscopie Electronique Address: EPFL-I2M, Batiment MX-C, CH-1015 Lausanne, Switzerland Phone: +41(21)693 29 83 (Central European Time) Fax: +41(21)693 44 01 ______________________ Eudora 2.0.2 __________________________________
We have been using Kodak SO163 without difficulty for years and we now use it in our JEOL JEM-4000EXII. We started using it because it is faster than 4489 and it can be pushed. The slightly larger grain does not seem to be a problem for our HREM users, but most do want increased film speed.
Russell Cook Electron Microscopy Center for Materials Research Argonne National Laboratory Argonne, IL 60439
Can someone offer suggestions for photographing Bacillus subtilis at the optical microscope level? These bacteria are small, motile and contain refractile bodies. In a fluid media there is a lot of Brownian motion and the refractile bodies create diffraction rings. We were thinking of using methyl cellulose. Can anyone suggest the proper viscosity to use? Does anyone know of a book or book chapter describing photographing bacteria?
I am just getting started trying to do low-dose HREM at high (1 MV) voltage. I have been told that SO163 developed in undiluted D-19 for 12 min is the best way to go, and I plan to try it as soon as the film arrives. Mean- while, I have been working at high mag (200kx-400kx) with Dupont LoDose. It is at least an order of magnitude more sensitive than SO163, but the grain size is also an order of magnitude larger (makes sense, equal sensitivity per grain and all that). Other drawbacks are the blue backing for LoDose and the tendency to fog and to show arrowhead-like marks from static electric discharges when very dry films are separated. Working in total darkness is also required with Lo- Dose. Murray King, at our lab, did a systematic study of the developing and fixing conditions for 4489 and LoDose to produce the best combination of sens- itivity and low fog. As a result we use 4 min in D-19 (4489) or GBX (LoDose) and 5 min fix. I'd be interested in anyone else's results with SO163. Although tedious, I believe such systematic studies are very valuable. If this particular wheel has not already been invented, I'll probably undertake the study in my copious free time (to quote from T. Lehrer).
Chris Krenn asks about using SIT cameras for detecting backscattered electron diffraction patterns.
As I understand this problem, you may not need the full sensitivity of the SIT (10^-4). A good CCD camera with exposure control may do the trick. A sensitive camera would have about .5 lux sensitivity, or less, with a nod given to the noise floor, at 1/60 sec exposure. Integrating on the chip for under a second would give around 10^-2 lux, with more available at longer times. This may be enough.
This camera would cost, maybe, $1400, or around 8% of your SIT.
My company makes a device called the OMNEX which can control these cameras, as well as do real time averaging, memory functions, measurements, digital contrast control, psuedocolor, zoom, frame storage, and lots of other things. We have a lot of people using it for applications where a little longer exposure time is all that's needed. (We also have people using it with SIT cameras for all of its other functions.)
Good luck. Send me a note if you would like any more info.
Bill Tivol posted a message regarding films for low-dose HREM. Perhaps I can suggest to use CCD camera (if one can afford it) to do a much better job. I have been doing low-dose HREM for several years now, and after taking the pains and frustrations with films, we started using slow-scan CCd camera 3 years ago. We are very pleased with the performence of the camera, and able to record low-dose HREM images with much confidence. I think CCD cameras offers the following advantages over conventional films: (1) Digital recording, i.e. on-line evaluation of image quality and fine tuning microscope operating conditions; (2) Very high sensitivity (low-dose). I am going to give a talk on low-dose HREm in the upcoming MSA meeting. If anyone is interested, please send me an email.
Ming Pan CSSS, Arizona State University panm-at-csss.la.asu.edu
Laurie asked for help on EDX selection, in this case windows:
Disclaimer: MOXTEK makes beryllium windows and ultra-thin windows for Si(Li) detectors, and supplies about half of the Si(Li) windows used world wide. I honestly tried to be objective in the following, but needed to warn you! I would be very interested in any comments, anecdotes, etc. from the microscope community on this subject.
I just wrote a chapter on thin windows for light element analysis for the book that Dave Williams and Dale Newbury are editing for the MAS. The book is entitled "X-ray spectrometry in electron beam instruments." It should be out this fall. A few items from my paper may be helpful:
If you are interested in light element analysis you should buy the spectrometer as a whole instrument for light element analysis. There is a lot more to light element sensitivity than the window (detector, FET preamp, processing electronics, software). It is possible to buy the best window, but still not get the best detection limits.
Windowless detectors have generally not lived up to their potential because of icing problems. A layer of ice can absorb as much as a thin window. If your microscope is very clean and scrupulously maintained vacuum-wise, however, this may be a good option. (This subject needs more text to treat fairly: manufacturers have done a good job to provide de-icing cycles, etc. to solve these problems.)
It is essential to maintain a steady regimen of standards testing to know the condition of the detector with all Si(Li) detectors. Two major things that can go wrong are detector icing and loss of vacuum (which will warm the detector, causing an increase of noise.)
Sometimes, to obtain better sensitivity at Be and B, a thin window is used that does not have an aluminum light blocking layer. Consider whether you need to block light from the instrument, room, or sample before selecting one of these. Even with aluminum coatings ultra-thin windows leak more light than beryllium windows do.
Since you are putting the spectrometer on an existing SEM it is a good idea to discuss your selection with both the electron microscope company and the spectrometer company. The biggest failure mechanism of thin windows is impact of particles on the window during venting. Some microscope models never have a problem, and some have a big problem. There are 'fixes' for the venting systems of problem microscopes.
The traditional reason for using something like dry nitrogen is degassing from the walls of a vacuum system, which is the major vacuum limitation (plus to some extent) of an unbaked system. If one uses dry nitrogen, then the initial chemisorbed layers tend to be quite nitrogen rich; nitrogen desorbs easily. If instead one uses air, then water and hydrocarbons (car exhaust fumes, people's breath etc) chemisorb on the walls and only very slowly leave. I must admit that with a modern microscope there is probably not that much of an effect since pumps have come a long way in pumping speed/$. However, if you are really concerned with UHV systems or are fighting contamination problems nitrogen (with very low hydrocarbon content) is probably going to help. I am not sure that the water level will be very relevant for most people.
Addendum: My mailer ate part of my last message. Leaks are also important, but one should worry more about backstreaming from roughing pumps in my experience.
Subject: Time:4:10 PM OFFICE MEMO Re: Backfill gases Date:4/29/94 This is a topic that I have discussed in some detail in a book on "Vacuum Methods in Electron Microscopy" that will be pub- lished early in May as the latest volume in Audrey Glauert's series PRACTICAL METHODS IN ELECTRON MICROSCOPY. Briefly, the purpose of backfilling with a dry gas is to reduce the amount of water vapor that is adsorbed onto the surfaces inside the vacuum system when the system is let up to atmospheric pressure. In even a moderately humid environment, or under conditions of prolonged exposure to the atmosphere, a surface can become covered with up to a hundred molecular layers of adsorbed water. Because water molecules are highly polar they adsorb to most surfaces quite tenaciously, and then desorb only very slowly when the system is pumped down again. This greatly prolongs the time required to pump down to an operating vacuum. Furthermore, unless a system can be baked out at temperatures above 200#161#C, the desorption of water also makes it very difficult to reach pressures much below the upper end of the 10-7 Torr range. You are correct, however, in concluding that there is little benefit to admitting a dry gas into the photographic chamber of an electron microscope, simply because the water evolved by the film will quickly coat the surfaces with water anyway. The same applies to bell jars, the specimen chambers of SEMs, electron guns, and other systems which are left standing open to the atmosphere for long periods of time. However, in situations where only a small hole will be opened in a vacuum system, such as when an aperture manipulator is being serviced, it is beneficial to use the dry gas, and to plug the opening loosely with aluminum foil and to maintain a slow flow of dry gas through the system while it is at atmospheric pressure. The primary concern in selecting the gas to use is that it be free of both water vapor and oil vapor. Gas pumped with an oil-sealed compressor will quickly lead to a very high rate of hydrocarbon contamination on the specimen. Other than that, nearly any dry gas will work, even dry air. Dry nitrogen is so commonly used because it is so readily available. As described in my book, it is even possible to capture the nitrogen that boils off from a Dewar flask in a plastic balloon and to use it for this purpose.
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Thanx for the info about the image plate and CCD talks at MSA 94. I'll be at both. In both cases, the delay in readout, etc. is nothing compared to the time it takes to scan a negative with 10*10 micron pixels and to prepare the file to be small enough so that our computer doesn't choke on it. For HREM, digitization via CCD is really the way to go, and we intend to go that way if the next renewal of our grant allows us to purchase the equipment. For quan- titation of ED patterns, I don't know whether the CCD might not be overloaded at the center spot--our intensified CCD can be damaged by trying this. From the viewpoint of "making every electron count", can either method sense a single electron--actually, if each electron produces n photons, where n} } 1, this sensitivity is not as tough as it might first appear. There might be an interesting idea in trying a thicker phosphor or YAG for ED and a thinner (thus better resolution) one for HREM. Has anyone investigated this yet? In parti- cular, for our 1.2 MV electrons this makes a lot of sense to me.
Re the discussion of water-cooled xma detectors, does anyone make or use a de- tector cooled by a Peltier device? I used such a detector many years ago for proton detection. There, the detector was a silicon junction detector and was cooled in order to reduce noise; whereas, the LN cooling of Si(Li) detectors is essential to preserve the drift profile.
Alex King asked about the reasons for the use of a dry gas for airing the vac- uum in a microscope. Others have answered the why. The grade we use is the "HP nitrogen"; the UHP seems to be no better and much more $.
The detector in an EDX system is cooled to liquid nitrogen temperatures to lower the noise due to thermally generated carriers. A second reason the detector is cooled is to keep the lithium from drifting around, but modern systems can be warmed if the detector is biased.
The systems that seem to be water cooled are actually cooled with thermoelectric coolers to cryogenic temperatures. The water is used to carry away the heat from the TE coolers, which is considerable. The TE coolers cannot get the detector to 77K, which is why these systems are noisier than the LN cooled systems.
regards Mark W. Lund, PhD Director MOXTEK, Inc. Orem UT
On 27 Apr 1994 MARK-at-prl.pulmonary.ubc.ca wrote of his need for a slide with a special grid on it. I was unable to reach him with e-mail.
My 1992 McCrone Accessories catalog has a similar slide called an "England Finder" (p/n 313). I would call them and see what they have for you. Their phone nos. are 708-887-7100 and 800-mac-8122.
Manny Olds oldsma-at-iia.org
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