an exiting new position created for our major expansion in the micro-electronics industry.
If you have - Experience in defect review/yield improvement, - Background in lithography and CD measurement, - The ability to communicate, train, and assist users of equipment, and work in a fledging highly motivated group, - An enquiry mind with imagination and the drive to see your ideas implemented, - PhD in a related area, or equivalent experience, - Preferably experience with scanning electron microscopy, - And you would like to have your home base in the Netherlands,
then please send your C.V. without delay to the undersigned.
Renumeration and conditions of employment are attractive for the right person.
Mr. J. Jackman Philips Electron Optics Building AAE-1 P.O. Box 218 5600 MD Eindhoven The Netherlands Tel. +31 40 766548 Fax. +31 40 766164 E-mail: jjn-at-eo.ine.philips.nl
Last year we had a failure of our SUTW (super ultra thin window) on our TEM EDS detector. This is our second failure of a SUTW, although not on the same machine (a SEM) and not from the same manufacture. After talking to several people, it has become apparent that the reliability of the SUTW is not very good, although manufactures claim otherwise.
I am curious what other users experiences with SUTW windows are. In particular what are the typical down times when a failure occurs, do you take any special precautions or modify your operation procedure, would you put a SUTW (instead of a standard window or turret detector) on an SEM for instant which is vented frequently using nitrogen? What was the reason for the failure?
Does anyone have experience on P4 SPM MDT scanning tunneling microscopes and atomic force microscopes? These devices are produced by someone at an institute that used to be a part of Russian Academy of Sciences.
mail me or this listserver if you have ever heard about them, and i'll be grateful!
Can anyone tell me wich concentration PBS should be used in immune-incubations for LM and EM? We got a little bit confused because some books mention to use a 0.1M PBS solution, and some mention to use 0.01M PBS. I can imagine that using a ten-fold solution can give problems. Wich concentration is the best (immuno-fluorescence LM and immuno-EM)?
Please let me know ! Thanks
Luc Analbers
*************************************************************************** * Luc Analbers * E-mail: Analbers-at-med.ruu.nl * *************************************************************************** * Utrecht University * LLL * * Medical Faculty * LLL * * Dept. Medical Physiology & * LLL A * * Sportsmedicine * LLL AA AA * * PO-box 80043 * LLL AA AA * * Zip: 3508 TA * LLLLLAAALLLAAALLL * * Utrecht The Netherlands * LLLLLAAALLLAAALLLL * * Tel: 030 - 538911 * AAA AAA * * Fax: 030 - 539036 * AAA AAA * ***************************************************************************
Aloha! What is known about the interaction between glutaraldehyde and polysaccharides? Does glut bind to polysaccharides in a way that would cause fluorescence (beyond that which binds to the small amount of protein that is present in the sample)? From what I understand, it is known how it binds to protein and that it binds to glycogen, but that it doesn't fix soluble polysaccharides, is that right? Could anyone help us out with the chemistry beyond that? Thanks in advance!
And a Happy New Year to you all!
Tina Weatherby Carvalho Biological EM Facility University of Hawaii
I have been asked to post this by the head of my research group (John Steeds). He would be grateful if you could pass it on to anyone who might be interested.
Any replies should go direct to him at J.W.Steeds-at-bristol.ac.uk
A post doctoral research assistantship is available for research on the microstructure and processing of unique metastable Ti-Mg alloys produced by vapour mixing and condensation. The material is made at the Defence Research Agency (Farnborough, UK) and close collaboration will be required with research workers at this Research Agency.
In the as-deposited state, the material is a porous, supersaturated solid solution with a nanoscale microstructure. The research will involve hot vacuum pressing and thermal treatments to eliminate porosity and obtain an optimum dispersed phase microstructure.
The hot working will be carried out in the Mechanical Engineering Department in an Instron servo controlled press equipped with a high vacuum chamber. The microstructural studies will require the use of high spatial resolution transmission electron microscopy and associated analytical techniques in the Physics Department. Some hardness and mechanical testing will be carried out in collaboration with DRA (Farnborough) to relate the microstructure to the mechanical properties.
The person to be appointed should be able to provide evidence of proven ability at relating microstructure, processing and properties in these novel alloys whilst working in an interdisciplinary team as well as considerable experience of advanced transmission electron microscopy techniques.
For more information please contact Professor J.W. Steeds by e-mail at
Quick question! I am getting small bubbles in my formvar ciated grids. How do I get rid of these bubbles? I am not overtly introducing moisture onto my glass slides am I am using EM Sciences prepared 0.25% formvar from a new bottle!!!
I don't want to make a project out of this, so input is appreciated.
Hello Jaime and all, In the way of a suggestion, you might try a new source for your formvar solution. I had the same problem after about 20 years of coating girds with no problem. It turned out the powder (I make my own solution) I was getting was contaminated with water. I found this out when I called the supplier and asked for a bottle from a new lot and all they had was that one lot. So I ordered from a new source and the problem was resolved. It, of course, took about 2 months of hair pulling and gnashing of teeth to work through all this. I hope you can resolve your problem more easily.
Sandra Zane Sandra F. Zane, EM Tech. Biol. Dept. UNCC Charlotte, NC 28223 sfzane-at-unccvm.uncc.edu Fax (704) 547-3128
Let me clarify my question on the interaction between glutaraldehyde and polysaccharides, and the fluorescence with confocal microscopy---
As I understand it, the people who asked me about this are actually rather happy that their specimen (whatever it is) is fluorescing so well and giving great photos. But they would like to be able to explain it. I gather they think they don't have all that much protein in their sample and so would like to explain it away by glut binding to polysaccharides. Is this plausible? Or can someone come up with another explanation?
Thanks, again, for musing on this!
It's 79 F, clear, sunny, and the surf is up. Happy New Year!
Tina Weatherby Carvalho Biological EM Facility University of Hawaii
Dear Robin, There is unlikely to be damage to the eye due either to EM use per se or to radiation--in the first place, eye tissue is not fast-growing like in- testinal epithelium, and in the second place, the radiation levels should be low. At our HVEM, for example, there is { 0.25 mr/hr (usually { { 0.25 mr/hr), and the column is monitored and interlocked to shut off the beam if the level exceeds 0.75 mr/hr at any of three positions. The most serious eye safety problem, IMHO, is exposure to the chemicals used in specimen preparation. Yours, Bill Tivol
Tina, It sounds like you are fixing plant tissue, which often is autofluorescent. An easy way to check, of course, is to look at unfixed tissue. As this is the main point of your question it seems pointless to go too deeply into gluteraldehyde chemistry. If you are interested, though, I will put you in contact with an expert ( he wouldn't forgive me for making his name too public). Gluteraldehyde reacts very rapidly with amines to form numerous products. The well known reactions are with primary amines but sulfhydral groups from cysteine and imidazole side chains of histidine also help in the cross-linking process. Theoretically, primary amino groups on amino lipids should also react with gluteraldehyde. As far as I understand it, there is little chance of gluteraldehyde cross-linking carbohydrates. That they are retained in fixed material is probably due to the cross-linking of nearby proteins which hold them in place. The best visual demonstration I saw was the addition of gluteraldehyde to homogenates of different tissues that were stirring in beakers. Liver and striated muscle gelled the instant gluteraldehyde was added. Brain gelled after a couple of hours and apple leaves were still stirring when we returned the next day! Hope this is of help to you. If it makes you feel better, in New Haven it is dark before 5 pm, there is a light dusting of snow on the ground and it is cold (below zero). What's it like in the Twin Cities anyone?
The subject of ultra thin window failures is one of extreme interest to me, since MOXTEK is the largest supplier of these windows. I am afraid that an informal survey on the Net will give warped data, since usually only those that have problems respond. We have supplied over 5000 ultra thin windows. We have a very good idea of failure rates over the first year, which is our warantee period to the EDX manufacturer. We also have some visability beyond that, because we often do failure analysis on windows for EDX manufactures as a service.
The data vary due to the fact that some microscopes are harder on windows than others. The most common failure in the field is due to particles striking the window during venting. The second most common failure is touching the window with fingers, stages, samples. etc. Very rarely does the window fail in such a manner as to be traced to defects in the manufacture. All this being said we have data showing over 7 years mean time before failure from one EDX manufacturer, and a total of 5% returns over the history of SUTW shipments from another manufacturer.
I would love to get anecdotal information from people that have had problems with x-ray windows to help us in making the window more reliable. These windows are by very nature extremely fragile. Only by the most careful manufacture, testing, and installation could the high reliability of these windows have been reached.
We are beginning a new project with bone tissue. Samples are being prepared for TEM and we have a couple of procedures for decalcification. We have not found any procedures for detecting the endpoint of the decal. process.
I would grateful for any assistance regarding decal. endpoint detection and additional decal. procedures.
Thanks for your help,
John
John S. Gardner Microscopy Lab 128 WIDB Brigham Young University Provo, UT 84602
Some time ago, Dr. Lisa Detter-Hoskin asked about edge wick experiments on a paper laminate material. She invited "any other thoughts about evaluating the morphology".
We have found that Scanning Probe/Atomic Force Microscopy yields interesting information about the surface structure of wood (and many other) fibers. Recently in our laboratory we have begun to supplement the topographic information of AFM by developing methodology for point chemical analysis. In fact, one of our early successes was in distinguishing lignin from cellulose on wood pulp fibers.
If this type of information interests you, please contact me.
DON CHERNOFF 317-251-1364 ADVANCED SURFACE MICROSCOPY FAX: 317-254-8690 6009 KNYGHTON RD. E-MAIL: echernof-at-ucs.indiana.edu INDIANAPOLIS IN 46220 Toll free: 800-374-8557 ASM is an independent analytical service and contract research laboratory.
Gernot Fuchs asked about the NanoScope III height formula, in order to scale bitmap data for display by the NS3. I have studied the NS3 data format in detail, in order to create a software package called "GP3 - Enhancement Software for NanoScope III" (by the way, this software is available for sale).
Here are some specific answers to Fuchs' questions: -The factor (Zmax * 2) is used because Zmax = 220V in the data file parameter list, but the total Z range of the piezo is 440V. -Fuchs may have neglected an important parameter from the "image list". In version 2 software, this is "\Z scale". In version 3 software, this is "\Z scale height". It is necessary to supply an appropriate value for this parameter in the file header in order for NS3 to display the data correctly. Some experimentation might be necessary to select the correct value for Fuchs' purpose--I only considered this problem going in the opposite direction.
DON CHERNOFF 317-251-1364 ADVANCED SURFACE MICROSCOPY FAX: 317-254-8690 6009 KNYGHTON RD. E-MAIL: echernof-at-ucs.indiana.edu INDIANAPOLIS IN 46220 Toll free: 800-374-8557 ASM is an independent analytical service and contract research laboratory.
We are trying to classify muscle fibers on semithin sections embedded in Agar 100 for further analyze in the electron microscope. Normally one uses toulidine blue as staining but we would like to classify the fibers of different (type IIA, IIB, I,... etc) at the light microscopic level to be able to cut out the right fiber for EM. Is there a staining method for epoxy sections that is useful for this purpose? There is some polychromatic stains but I cant recall the reference. Anyone having any ideas?
SWEDISH UNIVERSITY AGRICULTURAL SCIENCES Hans Ekwall Dept. Anatomy & Histology Box 7011, S-750 07 Uppsala, SWEDEN
We are contemplating the purchase of a liuid helium cooling stage for a TEM with a side-entry goniometer. What companies besides Oxford Instruments and Gatan manufacture LHe stages? Would those of you who use such stages please give me your opinions about your equipment? Specifically, what are the lowest temperatures reached routinely, and what is the degree of difficulty in using the equipment?
Russell Cook Electron Microscopy Center for Materials Research Argonne National Laboratory Argonne, IL 60439 (708)252-7194
I am about to buy a dye sublimation printer and hesitate between the 3 following products. 1) Tektronix 440 (that has replaced phaser IISDX) 2) Kodak XLS8600 (that has replaced the kodak color Ease) 3) Codonics NP1600 (based on the kodak XLS8600) Our electron microscopy images are all made of grey levels and I would like to use the new monochrome ribbons that are cheaper than the coloured one. More precisely, my idea would be to work with monochrome ribbons most of the times and from time to time to use coloured ribbons. * The Tektronix seems too be well adapted to do this because it has a special ribbon tray. However, its black ribbons make green-grey pictures * The Kodak monochrome ribbon gives more grey pictures, but the exchange of the ribbon could be more precarious as there is no special ribbon tray. * Codonics will sell the monochrome ribbons in France only in April ? (Although the kodak monochrome ribbons that should be the same are already available ?)
Has anyone any comments or experience on that ? Has anyone found problem running one of the three previous printers (especially the Codonics with its special image conversion software) ?
We want to sell,l and will let go as pair to best offer a 1) TEM Lynx (Leica) Tissue Processor and a 2) Grid Leica Ultra Stainer. If interested, and your offer is serious (that is not a couple hundred bucks) I will compile offers for a week and then contact the winner and runner up. Both items were purchased new in 1990 for about $15,000 and were hardly used, mainly because the work load is not sifficien and when use we end up wasting solutions. I will include some solutions with instruments.
Please respond directly to me {Fermin-at-TMC.Tulane.edu} , NOT to the user list.
************************************************************ *Cesar D. Fermin, Ph.D \|*|/ Fax (504) 587-7389 * *Tulane Medical School /|*|\ Answ. Mach.(504) 584-2618 * *Pathology/SL79 \|*|/ Secretary (504) 584-2436 * *New Orleans, La 70 112 /|*|\ Lab (504) 5841 * *Fermin-at-TMC.Tulane.edu -} Director of Morphological Services* ************************************************************
} We are trying to classify muscle fibers on semithin sections embedded in } Agar 100 for further analyze in the electron microscope. Normally one uses } toulidine blue as staining but we would like to classify the fibers of } different (type IIA, IIB, I,... etc) at the light microscopic level to be } able to cut out the right fiber for EM. } Is there a staining method for epoxy sections that is useful for this purpose? } There is some polychromatic stains but I cant recall the reference. } Anyone having any ideas? } } } SWEDISH UNIVERSITY AGRICULTURAL SCIENCES } Hans Ekwall } Dept. Anatomy & Histology } Box 7011, S-750 07 Uppsala, SWEDEN } } E-mail Hans.Ekwall-at-ah.slu.se } Voice: +46 18 672141 Telefax: +46 18 672852 }
A relatively simple and fast procedure for polychromatic staining of epoxy sections has been published by J.Tolivia et al in Histochemistry 101: 51-55. I have not used it myself. Ask me for details if this reference is not readily available to you.
Greetings!
Herman Meekes Biological Sciences ______________ ______________ University of Missouri ---__ \ / __--- 109 Tucker Hall ------__\---/__------ Columbia, MO. 65211 \( )/ Tel: 314-882-0171 V Fax: 314-882-0123 / \ e-mail: hmeekes-at-biosci.mbp.missouri.edu /___\
I can't contribute to the glut-polysacc. discussion, but since you asked for a weather report... a very mild winter here so far, but went skating last night under the stars; temp -6 F, wind chill below -20. Cold nose, cold toes, but had the ice all to myself. Warm breezes are nice, but Winter's great too. Need more snow, though. Thanks for the review of glut reactivity.
======================================================================= Chris Frethem (612)624-4652 (voice) Cell Biology & Neuroanatomy (612)624-8118 (FAX) U of MN, Minneapolis e-mail: frethem-at-lenti.med.umn.edu
On 4 Jan 1995, Paul Webster wrote:
} Tina, } It sounds like you are fixing plant tissue, which often is autofluorescent. } An easy way to check, of course, is to look at unfixed tissue. } As this is the main point of your question it seems pointless to go too deeply } into gluteraldehyde chemistry. If you are interested, though, I will put you } in contact with an expert ( he wouldn't forgive me for making his name too } public). } Gluteraldehyde reacts very rapidly with amines to form numerous products. The } well known reactions are with primary amines but sulfhydral groups from } cysteine and imidazole side chains of histidine also help in the cross-linking } process. Theoretically, primary amino groups on amino lipids should also } react with gluteraldehyde. } As far as I understand it, there is little chance of gluteraldehyde } cross-linking carbohydrates. That they are retained in fixed material is } probably due to the cross-linking of nearby proteins which hold them in place. } The best visual demonstration I saw was the addition of gluteraldehyde to } homogenates of different tissues that were stirring in beakers. Liver and } striated muscle gelled the instant gluteraldehyde was added. Brain gelled } after a couple of hours and apple leaves were still stirring when we returned } the next day! } Hope this is of help to you. } If it makes you feel better, in New Haven it is dark before 5 pm, there is a } light dusting of snow on the ground and it is cold (below zero). } What's it like in the Twin Cities anyone? } } } } }
Hair is a difficult tissue to infiltrate and embed. We've been using an extended infiltration protocol with Embed 812. Cutting thicker than usual sections (150 nm) gives marginal results. Has anyone worked with various hair samples lately? We're looking for better infiltration and less wrinkling of the cortex and medulary areas. We'd also like to get TEM pictures of whole cross and longitudinal sections. Any ideas???
--
Darryl 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
Jamie- 0.25% may be a little thin, I am used to using 0.75% formvar just my 2 cents -Mike
On Wed, 4 Jan 1995, Jaime Dant wrote:
} } } } [A } } Quick question! } I am getting small bubbles in my formvar ciated grids. How do I get rid of } these bubbles? I am not overtly introducing moisture onto my glass slides } am I am using EM Sciences prepared 0.25% formvar from a new bottle!!! } } I don't want to make a project out of this, so input is appreciated. } } Thank you for your time. } } Jaime A. Dant } jaime-at-borcim.wustl.edu } } Sorry about the typo, thats formvar coated grids. }
Just because eye tissue is not fast growing, and there is no exaggerated risk of tumors, don't neglect the possibility of cataracts. The susceptibility varies with individuals, but radiation is a definite risk factor. --John Mardinly Intel Materials Tech. Dear John, It is true that radiation can be a risk factor, and it should be mon- itored for an EM instrument. In our case, with { 0.25 mr/hr, a full shift's use of the HVEM (assuming that the beam is only on 50% of the time) will de- liver { 1 mr. Over the course of a year, this adds up to { 500 mr, the limit for non-radiation-workers. If other EM's produce as little radiation as the HVEM, the exposures will also be below the allowed limit. Of course, expos- ure to any radiation should be kept to "as low as reasonably achievable", in the words of the NCRP. I would still guess that there are many other sources of eye damage more significant than radiation exposure from an EM. Yours, Bill Tivol
Fixing shark tissue shouldn't be too difficult should it? I used 4% formaldehyde (from paraformaldehyde, in 100 mM PO4 buffer, pH 7.4) which works well for MDCK cells but swelled up the shark cells. My guess is the osmolarity is too low and needs to be closer to 1000mOm. Does anyone have any advice and/or recipes? I use this fixative to prepare cells for cryosection immunocytochemistry so that I can do light and electron microscopy on the same tissue pieces (yes 4% FA for EM doe work). I have no objection to using gluteraldehyde, acrolein or any other legal substance if it works.
Thank you to all who responded to the weather question. Brief summary: Colder than here in Minneapolis and Madison (wind chill -20F, quelle surprise). Wonderful in Sidney, Australia (how is the surf?), and only two more weeks before they get to see the sun again in Tromso, Norway. Thanks in advance, Paul Webster, Yale School of Medicine.
We have a Balzers freeze-fracture 301, not exactly the latest model but working very well, and have struck a problem with the supply of crystals for the thickness monitor, which is the model QSK 113. Balzers evidently no longer supply them -- has anyone else had this problem and found another source?
Thanks, Sally ---------------------------------------------------------------------- Sally Stowe Australian National Univ. Facility Coordinator Canberra, AUSTRALIA ANU Electron Microscopy Unit Ph 61 6 249 2743 RSBS, Box 475 Email stowe-at-rsbs-central.anu.edu.au FAX 61 6 249 4891
Message-ID: {MAILQUEUE-101.950106095638.480-at-anat.uct.ac.za} To: microscopy-at-aaem.amc.anl.gov
New Year greetings to everyone,
Weather in Cape Town Hot and windy typical for this time of year. For the surfers out there SURF IS UP.
Getting to the query regarding Hair.
We routinely look at hair samples (albino and normal). We use a standard Spurr resin which gives quite a good result. I do extend the infiltration time by not putting in accelerator and allowing the resin to infiltrate overnight at 40 degrees C. I have also obtained reasonable results with Molleneur's Epon / Araldite mixture slightly modified with the same extended time in the oven for the initial infiltration. Infiltration with the accelerator is also as long as possible at 40 degrees before embedding when using the Epon / Araldite mix.
Using this method I can get good cross and longtitudinal sections.
Hope you have some success with these.
Peter _______________________________________________________________
-at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at- -at- -at- -at- Peter D. G. Richards -at- -at- Dept Anatomy and Cell Biology -at- -at- UCT Medical School -at- -at- Observatory -at- -at- 7925 -at- -at- RSA -at- -at- Tel: 021-406 6285. -at- -at- Internet: retep-at-anat.uct.ac.za -at- -at- -at- -at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at-
I am working on imaging polished cross sections of portland cement particles by BE and XR imaging in the SEM. The cement powder is mixed with a resin (L.R. White) and the resin is cured. Cement particles are typically finer than 100 microns. As about half of the polished section is resin I am having difficulty with the operating conditions necessary (12kV, 5nA) burning the resin. Does anyone have suggestions for a better embedding medium? The ideal material would be stable under the beam, not allow plucking of fine particles during polishing, be fairly hard, and not be hazardous to handle or for disposal. Thank you in advance for your suggestions.
In message Fri, 6 Jan 1995 06:23:40 -0500, "SALLY STOWE" {STOWE-at-rsbs-central.anu.edu.au} writes:
} We have a Balzers freeze-fracture 301, not exactly the latest model } but working very well, and have struck a problem with the supply of } crystals for the thickness monitor, which is the model QSK 113. } Balzers evidently no longer supply them -- has anyone else had this } problem and found another source? } } Thanks, } Sally } ---------------------------------------------------------------------- } Sally Stowe Australian National Univ. } Facility Coordinator Canberra, AUSTRALIA } ANU Electron Microscopy Unit Ph 61 6 249 2743 } RSBS, Box 475 } Email stowe-at-rsbs-central.anu.edu.au FAX 61 6 249 4891 } } ------------------------------------- -------------------------------- } - } ***************
I have even an even older Balzers BA360 that I use for teaching. If you have not thrown away the used quartz crystals that had stopped functioning due to heavy deposition of platinum/carbon on them, you can remove the deposits by carefully "rubbing" them out with an unused pencil eraser (the one on the back of pencils work fine) and reuse them!
************************************************************************* M.V. Parthasarathy Professor of Plant Biology Section of Plant Biology 228 Plant Science Building Cornell University, Ithaca, NY 14850 Telephone: (607) 255-1734 Fax: (607) 255-5407 E-mail: mvp2-at-cornell.edu ***********************************************************************
You might try Streuers "EPOFIX" resin, it is avail. through most EM vendors, it cures very hard, and is recomended for non-biological materials. -Mike
On Fri, 6 Jan 1995 STUTZ-at-ENH.NIST.GOV wrote:
} I am working on imaging polished cross sections of portland cement particles } by BE and XR imaging in the SEM. The cement powder is mixed with a resin } (L.R. White) and the resin is cured. Cement particles are typically finer } than 100 microns. As about half of the polished section is resin I am } having difficulty with the operating conditions necessary (12kV, 5nA) burning } the resin. Does anyone have suggestions for a better embedding medium? The } ideal material would be stable under the beam, not allow plucking of fine } particles during polishing, be fairly hard, and not be hazardous to handle } or for disposal. Thank you in advance for your suggestions. }
I am working on imaging polished cross sections of portland cement particles by BE and XR imaging in the SEM. The cement powder is mixed with a resin (L.R. White) and the resin is cured. Cement particles are typically finer than 100 microns. As about half of the polished section is resin I am having difficulty with the operating conditions necessary (12kV, 5nA) burning the resin. Does anyone have suggestions for a better embedding medium? The ideal material would be stable under the beam, not allow plucking of fine particles during polishing, be fairly hard, and not be hazardous to handle or for disposal. Thank you in advance for your suggestions.
These vary in hardness, cure rate, clarity etc. so you can select the one that best fits your needs. If you provide your name and mailing address, I'll be please to send you specifications on these materials along with prices. I'll also send information on our other materials for cutting, grinding, polishing etc for metallography and electron microscopy.
Thank you!
David Henriks South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673
In my previous post I mentioned that we have supplied over 5000 ultra thin windows. This should have been 'over 4000 ultra thin windows.' It just seems like like a lot more....:) With my integrity back intact, Mark Lund MOXTEK, Inc Orem UT
} I am about to buy a dye sublimation printer and hesitate between the 3 following} products. } 1) Tektronix 440 (that has replaced phaser IISDX) } 2) Kodak XLS8600 (that has replaced the kodak color Ease) } 3) Codonics NP1600 (based on the kodak XLS8600)
We did a month long evaluation of several dye-sub printers for our facility, and finally received shipment of the Seiko ColorpointPSF dye-sub last week. Our original budget was in the $7,000 range, however, once the ethernet option, memory, extral printer trays, etc were factored in, the printer camein at just over $9,000.
For our evaluation we had color confocal images as well as b&w scanned gels. We sent out the files to the various resellers for sample prints. To do an apples to apples comparison. Incidentally, Codonics was the only organization with an ablitiy to ftp image files, however, their connection appeared to be a SLIP connection requiring a tremendous amount of patience when ftp ing.
We received samples from all three manufactures as well as Seiko. Now, we do know from previous postings that the quality of the printer driver often determines the quality of the hardcopy prints. There are a tremendous number of settings when printing to the dye-sub including, linear inks, color ramps, etc, things catering more to the pubishing industry. However, as we did not have the time to get into all that, we just took whatever each manufacture printed at face value. The bulk of our printing needs are gels and images from a phosphorimager.
The interesting thing is that although the Codonics was the pricier printer with rave reviews from almost everyone, we found the Seiko noticeably better for color. For b&w, the Seiko exhibited truely dark blacks. Tecktronix & Condonics prints had either a brownish or greenish tint. The Seiko also has the added advantage of printing on cheaper thermal paper. In evaluating dye-sub to purchase try to get sample prints of your own image files. Codonics sells their printer exclusively while the other manufacturers use resellers.
Feel free to email me questions directly regarding our dye-sub pruchase. There is a reseller based in New York who was tremendously helpful in obtaining the various sample prints. Our core facility is seriously considering the purchase of another dye-sub, the Itochu Pictograph, the Cadillac (or Lexus) of dye-subs.
._____________________ | Peter J. Hahn | ------------- | Thomas Jefferson University Jefferson Cancer Institute Confocal Facility JCI-BLSB-915 233 South 10th Street Philadelphia, PA. 19107 tel : 215-955-4770 | fax : 215-923-1098 | hahnp-at-jeflin.tju.edu | -------------+-----------------+----+------+
WORKSHOP ON ELECTRON MICROSCOPY OF MATERIALS (organizers: Gareth Thomas & David Barber)
A workshop on Electron Microscopy will be held at the Hong Kong University of Science and Technology between the 19th and 25th of February 1995. The aim is to present the principles of scanning and transmission microscopy, diffraction and microanalysis, specimen preparation and applications. In addition to lectures there will be daily lab sessions on microscopy, analytical methods and specimen preparation techniques.
The HKUST e.m. facility is part of a new well-equipped research centre, with several scanning and transmission microscopes from Philips and JEOL available for demonstration and hands-on use. The workshop is being supported by many of the major manufactures including JEOL, Philips, Gatan, Oxford Instruments, and South Bay Technology. Various ancillary pieces of equipment will be for use by the participants and specialists from the companies will be on hand to discuss any problems.
In addition, once the week of lectures and hands-on practice is finished, you can relax with a free trip in a motorised Chinese Junk around beautiful Hong Kong!
As the Workshop is limited in numbers, please be prompt in applying.
For further information and registration, please contact:
Miss Alice Yuen, HKUST RandD Corporation Ltd, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
Hello Netters, Since there is a discussion on epoxy for SEM, I thought I'd broaden the discussion a bit. Are there any epoxies available for making TEM cross-sections (from semiconductor materials in this case) that cure quickly and are stable during ion milling and under the electron beam? I currently use 'Devcon' - a 5 minute epoxy - but it tends to evaporate under the ion beam and isn't particularly stable in the electron beam. The best glue I've ever used is good old Araldite, which you can buy at any hardware store, but takes a long time to cure. Araldite 'rapid' seems to have the same problems as Devcon. What else is available?
Richard Beanland Dept. of Mat. Sci. and Eng. University of Liverpool, P.O. Box 147, Liverpool L69 3BX England.
One of my researchers wants to identify nerve fibers in whole mounts of Drosophila muscle preparations. Any suggestions for selectively labeling nerves? References from the 1980s use cobalt filling and/or silver nitrate, but note that muscle fiber will also take up the stain.
We would like to use both TEM and SEM. Backscatter mode for silver impregnated nerves looked interesting, but not if both nerve and insect muscle take up the label.
suggestions in this regard would be appreciated. Thanks in advance
steve
---------------------------------------------------------- Dr. Steven Barlow EM Facility/Biology Dept. San Diego State University San Diego CA 92182-0057 phone: (619) 594-4523 fax: (619) 594-5676 email to sbarlow-at-sunstroke.sdsu.edu
We are beginning a project where we will be looking at the structure of different regions of several bones that are subject to stress fractures. We are planning to bleach the samples to remove the soft tissue and then use acetone to remove any fat. We were wondering if it is necessary to Critical Point Dry the samples or just allow air drying. Are there any known changes that occur in bone material when allowed to air dry? In addition, is fixation necessary when looking at just bone? I would appreciate any comments. Thanks
Mike
--------------------------------------------------------------------------- | Michael Dunlap | lab (916) 752-0284 | | Facility For Advanced Instrumentation | fax (510) 422-2282 | | University of California | mrdunlap-at-ucdavis.edu | | Davis CA, 95616 | | ===========================================================================
There are several tests, which I found in the book: "The Preparation of Decalcified Sections" by Edward B. Brain, pub. by Charles C. Thomas, 1966. Precipitation with calcium oxalate and calcein fluorescence seem to be the best alternatives to pin pricking or X-ray. Techniques for oxalate and calcein appear in pp. 138-141. I use oxalate pptn.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
On Wed, 4 Jan 1995, gardnerjs wrote:
} Hi everyone: } } We are beginning a new project with bone tissue. Samples are being } prepared for TEM and we have a couple of procedures for decalcification. } We have not found any procedures for detecting the endpoint of the decal. } process. } } I would grateful for any assistance regarding decal. endpoint detection and } additional decal. procedures. } } Thanks for your help, } } John } } John S. Gardner } Microscopy Lab } 128 WIDB } Brigham Young University } Provo, UT 84602 } } Phone: 801-378-2202 } } }
We are beginning a project where we will be looking at the structure of different regions of several bones that are subject to stress fractures. We are planning to bleach the samples to remove the soft tissue and then use acetone to remove any fat. We were wondering if it is necessary to Critical Point Dry the samples or just allow air drying. Are there any known changes that occur in bone material when allowed to air dry? In addition, is fixation necessary when looking at just bone? I would appreciate any comments. Thanks
Mike
--------------------------------------------------------------------------- | Michael Dunlap | lab (916) 752-0284 | | Facility For Advanced Instrumentation | fax (510) 422-2282 | | University of California | mrdunlap-at-ucdavis.edu | | Davis CA, 95616 | | ===========================================================================
See the many fluorescent markers for neurons made by Molecular Probes, Eugene, Oregon. They have a free very informative voluminous catalog and reference guide to all their hundreds of fluorescent probes.
Marc C. Brande, M.S. SD3D Email List:3D Imaging San Diego 3D Imaging Group To subscribe/unsubscribe:listserv-at-bobcat.etsu.edu 3840 Camino Lindo To post a message:sd3d-at-bobcat.etsu.edu San Diego, CA 92122 Email:BRANDE-at-SDSC.EDU Voice:619-587-4830
On Mon, 9 Jan 1995, Steve Barlow wrote:
} One of my researchers wants to identify nerve fibers in whole mounts of } Drosophila muscle preparations. Any suggestions for selectively labeling } nerves? References from the 1980s use cobalt filling and/or silver } nitrate, but note that muscle fiber will also take up the stain. } } We would like to use both TEM and SEM. Backscatter mode for silver } impregnated nerves looked interesting, but not if both nerve and insect } muscle take up the label. } } suggestions in this regard would be appreciated. Thanks in advance } } steve } } ---------------------------------------------------------- } Dr. Steven Barlow } EM Facility/Biology Dept. } San Diego State University } San Diego CA 92182-0057 } phone: (619) 594-4523 } fax: (619) 594-5676 } email to sbarlow-at-sunstroke.sdsu.edu }
I know others were discussing this but I have not kept up: What do people consider to be the best but not hugely expensive film recorder for recording digital images to 4 * 5 film at about 2000 lines resolution? Details: we want to make digital composite images of up to 16 objects (bivalve shells) each digitally recorded on an ETEC SEM at about 2000 line original resolution (we do not yet have this digitizer, wanting to get the recorder specifications determined first, we are aiming toward the 4PI company, any experience with them) We also want to print color images, (probably to 35 mm film?) from beta max 3/4 inch tape (we do not yet have the beta tape reader but think we have a knowledgeable source (any recommendations welcome) We want the very highest resolution for both color and spatial detail but realize that beta is only about 700 lines original (recorded on beta tape from 3 chip high resolution cameras) We make the b/w composite SEM images by enlarging 4 * 5 negatives to the correct size, cutting out the images, pasting to black background special paper, make a tmax or plus x 4 * 5 photographic negative, also photograph text letraset independanly, combine the negative and make prints in the darkroom, no digital component. We want to get rid of all the darkreoom except the final prints from a negative (we need multiple copies) and also to be able to do the color images from a beta tape through a macintosh via photoshop (we have photoshop and a mac that probably does not have enough memory, we plan to upgrade when we know what input and output requirements are needed. Sorry abou the length but any help would be appreciated. The old XL 7000 kodak film recorder was recommended by a user but is no longer mad I like the Harris b/w dry silver printer but we need negatives to make multiple copies and it does not do color AND? it may not be high enough resolution for the composite pictures? any comments? If I get good returns of info, I will post a summary to the group. Alan Pooley Marine Science SEM lab rutgers univ 908 932 8959 x 225 pooley-at-ahab.rutgers.edu
Message-Id: {9501100024.AA15042-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: EPOXIES Orig-Author: {"John Mardinly, 5-2346, Page 322-6490, SC2-24" {JMARDINLY-at-MATTEC.intel.com} }:ddn:wpafb ----------------------------------------------------------- Another epoxy that is excellent and particularly suited for rough surfaces is Varian Torr-Seal. The stuff is made to patch high vacuum systems that will be baked up to 250C. It is strong and extremely resistant to beam damage.
Message-Id: {9501100023.AA15025-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: TEM Epoxies Orig-Author: {"Dr. R. Beanland" {beanland-at-liverpool.ac.uk} }:ddn:wpafb ----------------------------------------------------------- Hello Netters, Since there is a discussion on epoxy for SEM, I thought I'd broaden the discussion a bit. Are there any epoxies available for making TEM cross-sections (from semiconductor materials in this case) that cure quickly and are stable during ion milling and under the electron beam? I currently use 'Devcon' - a 5 minute epoxy - but it tends to evaporate under the ion beam and isn't particularly stable in the electron beam. The best glue I've ever used is good old Araldite, which you can buy at any hardware store, but takes a long time to cure. Araldite 'rapid' seems to have the same problems as Devcon. What else is available?
Richard Beanland Dept. of Mat. Sci. and Eng. University of Liverpool, P.O. Box 147, Liverpool L69 3BX Englan
I am intending to stain epoxy sections of kidney biopsies for electron microscopy and am in need of a good receipe for a PTA staining method.
------------------------- Romuald Wroblewski, Ph.D. Associate Professor Department of Pathology Karolinska Institute voice/fax:+46-8-7293597 -------------------------
I am intending to stain epoxy sections of kidney biopsies for electron microscopy and am in need of a good receipe for a PTA staining method.
------------------------- Romuald Wroblewski, Ph.D. Associate Professor Department of Pathology Karolinska Institute voice/fax:+46-8-7293597 -------------------------
To all of thise interested in where to buy Epotek 353 ND:
You can get Epotek 353 ND from:
Epoxy Technology 14 Fortune Drive Billerica, MA 01821
TEL 800-227-2201 FAX: 508-663-9782
You can also buy it from us in smaller quantities - albeit a higher price!
P/N 0714-010 price: $50/8 oz kit
The only advantage in buying from us is that you can combine it with your other supply orders and we also can ship it to you the same day. Otherwise, you can get a lot more for your money buying it direct from Epoxy Technology.
I hope this helps!
Best regards-
David Henriks South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673
We are finalizing plans for a new central microscopy laboratory. In part, these plans are modeled after those furnished by R A Harris (University of California, Davis) where areas are compartmentalized i.e. cryo microtoming in separate room, etc). For general health reasons we have restricted solvent usage to one room within and isolated from this larger room. We are experiencing some resistance to this concept. Can any of you lend some support to this concept? Or tell us why it is not a good idea. Please contact me via email, fax or phone with your welcome comments. As ever, thanks in advance for your comments.
L E Porter Phone (216) 796-1620 Head of Microscopy Fax (216) 796-3304 The Goodyear Tire & Rubber Company EMail LPORTER-at-GOODYEAR.COM Dept 415A 142 Goodyear Blvd Akron, OH 44305 USA
Epoxy Technology 14 Fortune Drive Billerica, MA 01821
TEL 800-227-2201 FAX: 508-663-9782
You can also buy it from us in smaller quantities - albeit a higher price!
P/N 0714-010 price: $50/8 oz kit
The only advantage in buying from us is that you can combine it with your othe r supply orders and we also can ship it to you the same day. Otherwise, you can get a lot more for your money buying it direct from Epoxy Technology.
I hope this helps!
Best regards-
David Henriks South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673
I think I'm jumping into the middle of a conversation, but here are my two cents. I've used Torr-Seal for making cross sections in the past and had reasonably good luck. It takes about 24 hrs. to cure and stands up well in the ion mill; I, however, am not sure about the particle issue mentioned. My sample was well characterized before XTEM.
I still like LR White Hard Grade resin for microtoming particulate material and free standing optical thin films for XTEM. My samples are typically ceramics and metals. LR White is stable in the electron beam assuming it is C-coated and can be cured very quickly with an accelerator or in an oven. Must be mindful of potential brittleness.
Jim Heuer GE (510) 862-4501 heuerj-at-vncpo1.ne.ge.com
I think I'm jumping into the middle of a conversation, but here are my two cents. I've used Torr-Seal for making cross sections in the past and had reasonably good luck. It takes about 24 hrs. to cure and stands up well in the ion mill; I, however, am not sure about the particle issue mentioned. My sample was well characterized before XTEM.
I still like LR White Hard Grade resin for microtoming particulate material and free standing optical thin films for XTEM. My samples are typically ceramics and metals. LR White is stable in the electron beam assuming it is C-coated and can be cured very quickly with an accelerator or in an oven. Must be mindful of potential brittleness.
Jim Heuer GE (510) 862-4501 heuerj-at-vncpo1.ne.ge.com
We are interested in cross-sectioning devices on silicon with a thin layer of polyimide. This is primarily for optical or SEM imaging, not TEM. The polyimide layer is on the order of 10 micrometers thick (and possibly an order of magnitude higher). My limited microtomy experience has been that cleaving the polymer introduces substantial dimensional distortion. Does anyone have a good idea for this? We are considering trying a "standard" mechanical polish but have serious reservations because of the polymer. We also know it can be done, having seen good pictures (lacking, of course, specimen prep information).
Any suggestions will be welcome.
Daniel L. Callahan Department of Mechanical Engg. and Materials Science Rice University dlc-at-owlnet.rice.edu
} } Happy New Year } } I am intending to stain epoxy sections of kidney biopsies for } electron microscopy and am in need of a good receipe for a PTA staining } method. } } ------------------------- } Romuald Wroblewski, Ph.D. } Associate Professor } Department of Pathology } Karolinska Institute } voice/fax:+46-8-7293597 } ------------------------- } } Hi Romuald, I think you should try a modification of Stempak & Ward 's (1964) receipt which involves a methanolic solution of Uranyl Acetate. Just replace the UA by PTA and carefully follow their method, which is described in J. Cell Biol. 22 pp. 697-701. I have used this with success for staining proteinaceous structures embedded in Araldite. It cannot be used for material fixed in Permanganate, but this should not affect many people nowadays! Hope you have success with it. Jim Chalcroft
I just realized that you have a materials microscopy lab. Sorry - I know some of my ideas and experience is not applicable. Hope some of it is.
Good luck with the new lab.
Dan
On Tue, 10 Jan 1995, LE Porter wrote:
} We are finalizing plans for a new central microscopy laboratory. In part, } these plans are modeled after those furnished by R A Harris (University of } California, Davis) where areas are compartmentalized i.e. cryo microtoming } in separate room, etc). For general health reasons we have restricted } solvent usage to one room within and isolated from this larger room. We } are experiencing some resistance to this concept. Can any of you lend some } support to this concept? Or tell us why it is not a good idea. Please } contact me via email, fax or phone with your welcome comments. As ever, } thanks in advance for your comments. } } L E Porter Phone (216) 796-1620 } Head of Microscopy Fax (216) 796-3304 } The Goodyear Tire & Rubber Company EMail LPORTER-at-GOODYEAR.COM } Dept 415A } 142 Goodyear Blvd } Akron, OH 44305 } USA } } } }
On Tue, 10 Jan 1995, ROBERT K. NAUMAN wrote: } Can anyone provide me with a source for a new 450 Watt Xenon Lamp? } Thanks! } Bob Nauman } Baltimore, MD 21201
May contact Ralph Maxwell in Massachusetts -at- (508) 692-4973. He operates a company called Remtron that we used to retrofit our old B&L Research I Metallograph from carbon arc to Xenon. He sells the OSRAM XBO 450W OFR for about $480 per bulb. If this is not the correct type you need, I'm sure that he can help ya find the exact type. Also, if memory serves me, (haa haa!) I think OSRAM may have a facility in New York or somewhere in the east; altho' the bulbs I have are German-made.
Hope this is of help, -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 | ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''
necessary to know what kind of examination is to be done. We have examined bone and tooth here at our facility at the U. of P. dental research department and for quick examination of both these mineralized tissues air-drying has been sufficient. However, we also first examine samples at 10x to 100x in a dissecting microscope while fresh from the source for micro-fractures to determine if any small cracks we may see in the SEM have been caused by the drying method or were there at the beginning. To minimize crystal metamorphosis, the mineral sample is immersed in LN2 and lyophilized. For a moderately rigorous drying procedure, we do a slow dry out of Freon 113 and omit critical point drying. This is done by treating the mineral as any soft tissue--Karnovsky's fixative, followed by an ethanol drying series and, after two exchanges in 100% ethanol, samples are immersed in Freon 113. The container with the bone and freon (usually the bottom of a 30 mm petri dish) is covered with parafilm with a few needle holes in it and allowed to air-dry (a few hours). An excellent reference is "Methods of Calcified Tissue Preparation" Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017. Particularly, Chapter 7 by Alan Boyde.
I have no knowledge of the design plans you mention, but I assume that the solvent usage room has special ventilation and/or hood space. I would also assume that all the tissue embedding, grid preparation, slide staining, solution preparation, and tissue fixation (implying the presence of transmission and dissecting light microscopes) would be done in this room. If so, the only objection I can think of would involve the occasional use of small amounts of solvent (ethanol, acetone and xylene) needed for equipment cleaning, slide coverslipping, and possibly section mounting in the microtomy area. The necessary amounts of these things would be very small and it is doubtful that their presence and use in the microtomy room would constitute a hazard. If a large number of slides need to be coverslipping at one time, it is, of course, necessary to do it in a highly ventilated environment - but coverslipping an occasional individual slide shouldn't require the inconvenience of moving to another room.
If you plan to separate working with tissues (fixed or otherwise) to another room, you may generate many inconveniences unless small amounts of certain chemicals (primarily fixatives, eg. aldehydes) are allowed.
I have worked as a microscopy technologist for approximately 17 years in a number of settings. I am aware of the safety issues but, also know that if you place unrealistic restraints on people, they will find a way around them sooner ar later. The best solutions seem to me to usually involve a certain amount of compromise.
Dan
On Tue, 10 Jan 1995, LE Porter wrote:
} We are finalizing plans for a new central microscopy laboratory. In part, } these plans are modeled after those furnished by R A Harris (University of } California, Davis) where areas are compartmentalized i.e. cryo microtoming } in separate room, etc). For general health reasons we have restricted } solvent usage to one room within and isolated from this larger room. We } are experiencing some resistance to this concept. Can any of you lend some } support to this concept? Or tell us why it is not a good idea. Please } contact me via email, fax or phone with your welcome comments. As ever, } thanks in advance for your comments. } } L E Porter Phone (216) 796-1620 } Head of Microscopy Fax (216) 796-3304 } The Goodyear Tire & Rubber Company EMail LPORTER-at-GOODYEAR.COM } Dept 415A } 142 Goodyear Blvd } Akron, OH 44305 } USA } } } }
If you are using a Nikon power supply, Nikon recommends that you DO NOT USE Osram bulbs. For some reason it drastically shortens the life of the power supply (i.e. shorter than the life of the bulb!). In our case our Nikon supplier suggested Ushio bulbs.
We specifically have a 75 watts system and the same may not be true of the 450 watt systems but you may want to find this information out before you fry your power supply.
(I have nothing against Osram, I regularly use Osram bulbs for many other applications)
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
keeping the solvents "restricted" to the prep lab is a good idea, however in reality it is sometimes necessary to us solvents (chloroform, acetone, ethanol) in thet microtomy or microscope labs. -Mike
Is anyone knowledgeable about Tracor Northern NS-880 EDS x-ray analysis = system? Specific question: How do you boot from the tape drive when the = system has had a floppy drive but it is missing?
Any information would be greatly appreciated. Dr. N.K. Smith
} Does anyone know where to buy freon 113 in gal. size? Our freon pump } cooling the Cameca MBX probe needs periodic refill. } } Thanks in advance! } } Vincent } } PS. I really doubt that the heavy molecules like freon 113 can fly } high to the ozone layer. Most likely they "sink" to the ground level } and help fighting the ozone created by industrials and autos(?).
Heuer Jim P. writes:
} Try "Fluorinert" Electronic Liquid (FC-77) from 3M Co. (213) 726 6361. } About $300 for 11 lbs. (3/4 gal.)
I'd suggest a different 3M material, SF-2, which is used as a blanket material for vapor phase soldering. It might be a better replacement, but that all depends on what you mean by a Freon pump. Is the material used as a heat transfer medium or is it evaporated? If the material is used in a closed system, the FC-77 would be an adequate choice.
Tom Ostertag ostertag_tom-at-mn15-gw.mavd.honeywell.com
We have a user with a nasty problem that we haven't been able to dent. They are trying to do some immunocytochemistry on HeLa cells growing on glass coverslips. They fix in either acetone or 2% paraformaldehyde. The paraformaldehyde fixed monolayers are subsequently permeabilized with acetone, triton x-100 or saponin (problem is the same regardless). At this point there is no autofluorescence at any wavelength. When they incubate the cells in a high quality donkey anti-mouse IgG coupled to Cy5 (Jackson) there is a very high level on non-specific adhesion. We have tried the following blocking agents: normal Donkey serum, BSA, gelatin, non-fat dry milk, or all of the above simultaneously. Pretreating the sections with the blocking agents has no effect. Using other anti-mouse antibodies coupled to FITC or rhodamine or a Goat anti-rabbit serum give the same problem. I should point out we routinely use these antibodies in my own laboratory following the same protocols and do not get any background binding. We get the same result when we personally run up the cells so we can't blame it on the technician. Is there something weird about HeLa cells or something else I am missing? Do HeLa cells have IgG receptors? Your helpful advice will be gratefully received.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
Fellow Researchers, If anyone knows of research being done on bowel disorders I have a sick daughter and her doctors are at the brain-storming stage after a year of exploring. This is a personal request, please contact me through my E-Mail address. I appreciate any help at this point. Thnaks.
Someone here in our lab also uses HeLa cells, and stains tehm for coxsackie virus using monocloanl antibodies. He has not had any problems with sticky secondaries. He did say that HeLa cells can possess/express Fc receptors. These might be isotype specific depending on the "strain" of HeLa cells you have. Otherwise can't think of any other reason, but what concentration of blocking agents did you try?
Mark Elliott Pulmonary Research Lab UBC Vancouver Canada
Are you using Fab fragments for your secondary antibodies? Generally, if we have problems it is because we are using the whole antibody. Also going to affinity purified or the highest specificity we can obtain usually gives the best results. The addition of serum (ie goat serum for goat anti-mouse IgG) throughout the incubation is also helpful.
_____________________________________________________________________ | | | | James V. Jester | Dept Ophthalmology | | jester-at-crnjjsgi.swmed.edu | UT Southwestern Medical Center | | TEL (214)648-7215 | 5323 Harry Hines Blvd | | FAX (214)648-2382 | Dallas, TX 75235-9057 | |__________________________________|__________________________________|
Does anyone know the name and contact information for a person at UC = Irvine who had three Tracor Northern NS-880 x-ray analysis systems? This = is as per John Liska, formerly with Tracor Northern--Noran. Information = is greatly appreciated,,,Dr. N.K.R. Smith
In regards to the response concerning xenon bulbs, I'd like toprovide the following information.
Nikon's 75 watt xenon power supply is usually shipped with Osram 75 watt arc lamps and that is the recommended supplier, although technically there is no difference between Osram and Ushio.
Nikon's 100 watt xenon power supply sold outside the USA is shipped with Ushio 100 watt xenon arc lamps. I believe only ushio manufactures this type of 100 watt arc lamp.
In Nikon's 100 watt mercury power supply either Osram or Ushio 100 watt mercury arc lamps of the same specification are usable and performance is the same.
450 watt xenon bulbs were typically used in Microprojectors used to project microscope slide images on to a projection screen for use in teaching, CCTV has now replaced this technique.
Regards,
Stan Schwartz Manager, Biomedical Instruments Nikon Inc. Instrument Group 1300 Walt Whitman Rd. Melville, NY 11747 516-547-8529 Fax 516-547-0306 E-mail NIKONBIO-at-aol.com
We use CD ROM for archival image/data storage. CD Writer's have come down in price quite abit lately. What I originally paid $5K for is now available for ~$2K and the unit is twice as fast. You may want to seriously look into these instead of tape. Blank CD's cost ~$15 and hold ~650Mbytes.
There are several vendors on the market. I am currently using Pinnacle Micro, but there are at least 2 other manufacturers out there.
A postdoctoral position is available (immediately) exploiting the unique capabilities of a UHV-HREM (2 Angstroms and 6x10-11 torr) at Northwestern. Connected to this instrument are a number of analytical (XPS, Auger, SEM) and a GaAs deposition chamber. The position would involve working on projects involving GaAs deposition, as well as number of other projects involving small particles and metal-semiconductor surfaces.
Experience with TEM (HREM preferred) plus surface science techniques are important.
Send C.V. plus other material to:
L. D. Marks Department of Materials Science and Engineering Northwestern University Evanston, IL 60208 ldm-at-apollo.numis.nwu.edu
We want to set up a computer-based index to our EM lab negative files. This is a request for your experiences and recommendations of software. We would like to store and be able to search thumbnail views of each image as well as assignable fields and keywords. The images themselves will be kept as negatives, positives, slides and other formats as separate files. Commercial packages which manage images themselves are reported to suffer in searching speed, power and reliablity (A.Abernathy, "Managing your media", MacUser Sept. 1993, Pp. 190-206). Perhaps a database program that allows thumbnails to be imported would be best. Your comments invited.
jacob Jacob Bastacky, MD 1-116 Lawrence Berkeley Laboratory EMail: sjbastacky-at-lbl.gov University of California Phone: (510) 486-4606 Berkeley, California 94720 Fax: (510) 486-4750
I for one would greatly appreciate the names of the other two companies (with telephone numbers if possible). Also, what is your opinion and experience with the Pinnacle Micro? Any recommendatins?
On Fri, 13 Jan 1995, Nestor J. Zaluzec-Argonne Nat. Lab. wrote:
} Keith } } We use CD ROM for archival image/data storage. } CD Writer's have come down in price quite abit lately. } What I originally paid $5K for is now available for } ~$2K and the unit is twice as fast. You may want } to seriously look into these instead of tape. } Blank CD's cost ~$15 and hold ~650Mbytes. } } There are several vendors on the market. I am } currently using Pinnacle Micro, but there are } at least 2 other manufacturers out there. } } Nestor Zaluzec } ANL }
Multiple Write sessions on a single CD-ROM have been possible for several years. The problem is that many inexpensive CD ROM readers donot support Multi-session formated disks. So if you intend to send/give the data to someone with a standard format CD reader (i.e. the run-of-the-mill ~$200-300 variety) you will have to cut them a new disk. This generally is not a problem assuming that you already have the CD writer.
Since the whole reason for a visitor to come to a user facility is to do an experiment and leave with the data I see no conflict with not "filling" up the disk. For example if you are in a user facility and a visitor comes to do some experiments collecting a few hundred Mbytes of data, then writing a single CD with just that few 100Mb on it is usually more effective than writing tens of floppies or several Syquests. The logic to a CD vs a removable/rewritable media (Syquest,MO, etc..) is that everyone can generally afford to buy a $200 CD reader but not everyone can afford to buy a ~$900 MO reader. This way a "user-facility" can support many type of users and they do not all have to go out and buy expensive drives. On the flip side if the user and the facility both have compatible media then using that media is reasonable (since I happen to have lots of different drives and media I can accomodate most users)
For local storage issues, the approach I tend to take is to have a local disk server (2.5 Gb partitioned into 3-650 MB segements plus a scratch disk) which is used to temporairly store data . When a disk partition is full then it is written to CD (usually 2 copies) and then cleared. If an individual user wants to archive things this procedure can be also used. In this case, we download his/her data overnight to an empty partition and then write the data and give the user back the finished CD. Then wipe the partition for the next user.
Generally I tend to avoid multi-session CD's, but have used them on occassion. You just have to carefully label the disk. Also when you mount a multi-session CD you get alot of "virtual" disk icons mounted on your desktop. One per CD session. So if you get into the habit of writing small partitions to your CD then you will find lots of icons cluttering up your desktop. My real desk is bad enough so I try to avoid the problem propagating to the screen of my workstation.
Hope this proves useful...
Nestor
--------------------- P.S. Someone asked for information on phone numbers for manufacturers of CD writers I pulled this out of MacWorld Magazine.
Pinnacle Micro RCD-1000 CD writer: 800-553-7070
Most CD writer's are sold direct by the manufacturer I don't recall seeing very many advertised in the mail-order catalogs. So you may have to do some hunting. I'm positive that there are at least 2 other manufacturers out there of ~$2K writers. I seem to recall Sony and Chinon, but don't take that as Gospel.
BTW for the record I have no financial interest in Pinnacle Micro Systems.
I use Claris Works database to maintain my records and include film numbers for each record or subject. Any search for an image is slowed by division into years but I have done retreivals rapidly by using one criterion for search. Good Luck-- Kate Connolly
Like Nestor: We have stopped using tape completely,(anyone want a Jetstore 5000!) having found that we actually needed our backup on occasion. We are using the Pinnacle RCD1000 currently, it cost -at-$2000 to go in a pc, this included an Adaptec SCSI board. Generally we generate about 1 Gig/mo of images from a variety of platforms, Confocal, EM, and LM. However I would like to make a few comments on the viability of CD's as a principle storage media
1: They are an absolute boon in a multiuser environment. We give users a CD and they are gone forever, we do not have to coddle their data for the next 20 years or try and find it on a tape It doesn't matter what platform they use,(PC/MAC/SGI) the CD works. However this is tempered by two problems. a: The recordable CD's are not as robust as ones made by commercial presses and do not like being scratched. b: The namespace of the platform chosen is important. For example we use SGI's to analyse all our confocal data however the data may be stored on a Novell server and accessed through NFS. When the data is archived to CD, only the DOS name space is recorded, which means that some of the UNIX names get truncated to 8 letters and then become useless
2:Cd's are geat if you have 650meg to back up. One of the biggest problems we faced when we started using CD's was how to organize the data. By project? by date etc. The problem is that unlike a tape or disk where you can keep adding on forever with a CD you loose a lot of space when the disk is multisessioned. Effectively the FAT takes up 20MEG and every time you add something to the disk in a new recording session you have to rewrite the FAT.... To get round this problem as we ideally we would like to make single user archives we have a large online resevoir of space (13MEG) and use 650MEG Flop-opticals as an intermediate storage device.
3:You need to buy a 1 Gig drive along with the CD. It is possible to make a CD with a virtual image which then pulls all the data from the vaious directories and drives etc. However this is dangerous... A much better solution is to plant a decent sized hard drive in the machine you will be archiving and make a single, large ISO compliant file which is written directly to the disk.
4:When recording you cannot expect the pc to do anything else. This includes running a screen saver. I estimate that the happy Xmas Screen saver we had this year cost us about 5 CD's.
5:It is worth thinking about a Juke box player to go with it. We are using a Pioneed 604X Takes 6 disk cassettes, costs about $1000. Trouble is that if you want to use it over a net then you must buy multidisk support software/hardware. Microtest make either diskport (a hardware soln) or diskserve (a software soln). Either work well.
Generally we are extremely happy with this media, We have Flop-opticals, DAT and non-DAT backup devices. CD's are really the only way to go though they are cheap ($15/650meg) relatively fast and perhaps most importantly now that almost all PC's have CD players in them, distributable!
---------------------------------------------------- Simon Watkins Ph.D Director Structural Biology Imaging Center Scaife 840 University of Pittsburgh Pittsburgh PA 15261
} We want to set up a computer-based index to our EM lab negative files. } This is a request for your experiences and recommendations of software. } We would like to store and be able to search thumbnail views of each } image as well as assignable fields and keywords. The images themselves } will be kept as negatives, positives, slides and other formats as separate } files. Commercial packages which manage images themselves are reported to } suffer in searching speed, power and reliablity (A.Abernathy, "Managing } your media", MacUser Sept. 1993, Pp. 190-206). Perhaps a database program } that allows thumbnails to be imported would be best. } Your comments invited.
The new version of HiJack Pro solves many of these problems in a cost effective ($90) fashion. It works differently to the earlier version and keeps a central database of images which can be accessed very rapidly. It works accross servers, allows multiple indexes, does thumbnails, and updates itself automatically, comes with a montaging and editing program. We love it
---------------------------------------------------- Simon Watkins Ph.D Director Structural Biology Imaging Center Scaife 840 University of Pittsburgh Pittsburgh PA 15261
Hi, I am currently looking into switching to digital image archiving and have found two referances which may be of assistance to others in the same position:
- There is a review of cd-rom drives in "PC Computing", Nov., 1994. This supplies name and phone number for several manufacturers as well. - Kodak offers a CD-ROM drive guide which lists CD-ROM drives, controller cards and device driver software combinations which are compatabile with Kodak Photo CD. Not all combinations are equal. I have publication DCI-249 dated 8/94 which lists combinations for PC and Mac. I don't know what is available for Unix.
Hope this is helpful. Laurie
______________________________________________________________________ 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
14th International Congress on X-ray Optics and Microanalysis August 29 - Spetember 2nd 1995 GuangZhou, CHina
This congress will be held, 39 years after the first UK congress, in GuangZhou, a city only two hours by train from Hong Kong. The program will include the following topics: X-ray Optics, Sources and Microscopy; X-ray Photoelectron Spectroscopy; Analytical Electron Microscopy (AEM); Scanning Electron Microscopy; Electron Probe Microanalysis; Auger Electron Microscopy; Ion Probe and Secondary Ion Mass Spectroscopy; Laser Probe; Scanning Probe Microscopies (STM, AFM); and Confocal Microscopy.
Contributed papers prepared in 2 or 4 camera ready pages with text and figures inside a frame of 144mmx210mm will be published in Proceedings available to each participant at the Congress. A comprehensive Trade Exhibition will also be scheduled.
The deadline for contributed papers is April 30th, 1995, and the early registration fee, thru June 30th, is US$350. The accomodation rate (including three meals) in a three star hotel is US $50-85 per day. The organizing committee will provide arriving participants with transportation from GuangZhou International Airport and the railroad station on Aug.29th.
Second circulars, registration forms and contributed paper format are now available from
On the subject of CD ROMS writers, I looked through a British computer magazine and found advertisments for a few manufacturers, of the ones likely to be available in the US try Philips (UK price ~$3000) or Yamaha who manufacture a quad-speed writer (~$5000). However prices in the UK tend to be much higher than in the US, so they could be considerably less over there. I hope this is of use to somebody!
Doug Arrell +------------------------------------+ | Dr Douglas Arrell | | Mechanical Performance and Joining | | Institute for Advanced Materials | | 1755 ZG Petten | | Netherlands | | {ARRELL-at-JRC.NL} | | Tel. (+31) 2246 5287 | | Fax (+31) 2246 1917 | +------------------------------------+
I am in need of optical crystallographic properties for FeCl3 and FeCl3 - nH20. Winchell's Optical Properties of Artificial Minerals has the +2 valence state of iron, but not the +3 state.
The June 94' issue of MacUser has a good article on CD-ROM writers listing a number of different companys and MacUser's ideas on the pros and cons of each. Included is MacUser's commemnts on different software that is available for the writers. I beileve PC Week had a simular article.
Mike
--------------------------------------------------------------------------- | Michael Dunlap | lab (916) 752-0284 | | Facility For Advanced Instrumentation | fax (510) 422-2282 | | University of California | mrdunlap-at-ucdavis.edu | | Davis CA, 95616 | | ===========================================================================
John Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, 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: 4:39 PM
Date:1/16/95 URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL
Three software packages have been added/modified in the Microbeam Analysis Society Software Library (MASSL) in recent months. The MASSL resides on freebie.engin.umich.edu username: anonymous password: your email address in the form user-at-domain. Directory: /pub/MSA+MAS/MASSL New files in: 1. /pub/MSA+MAS/MASSL/xphi A Full abstract was not included by the author of this program. Here is a summ ary of the information that he supplied in printed form.
Xphi is a PhiRhoZ correction program. For a description see: "An Accurate Computer Correction Program for Quantitative Electron Probe Microa nalysis", Claude Merlet, Mikrochim. Acta 114/115 363-376 (1994). Install the program by exploding the auto-unzip file xphiz.exe into a directory on your hard drive (say, c:\xphi). Install the fonts, FIX8X14.FON and FIX8X16.F ON that come with the program in the Windows sytem directory (e.g. in c:\windows \system). The program is called xphi.exe, add the program to your program manag er listing so that you may double click it to run it from the windows shell.
2. /pub/MSA+MAS/MASSL/shrli A program called Simply Shrli, designed to run on the PC. This from the readme file. SIMPLY : WHAT IS IT FOR ? ************************* SIMPLY is a set of programs dedicated to Transmission Electron Microscopy in
Materials Sciences. It allows you to BUILD structures, DRAW and HANDLE cells, CALCULATE, DISPLAY and HELP to INDEX diffraction patterns, CALCULATE and DIS-
PLAY High Resolution IMAGES. SIMPLY runs "SHRLI", (c) M.A.O'KEEFE, (1980). .../...
SIMPLY first appeared as: "SIMPLY: a package for the SIMulation and disPLaY of HREM images on PCs", T. EPICIER, M.A.O'KEEFE, communication at 33rd Ann. Meeting of the French Electron Microsc. Soc. (SFME), Villeurbanne-Lyon - F., june-july 1993. It has been demonstrated during the "Computers Open lab", at the 13th Intern. Congress on Electron Microscopy (ICEM 13), Paris - F., 17-22 July 1994.
3. /pub/MSA+MAS/MASSL/KAKER Two abstracts here in the requested format (MANY THANKS Henrik!!!). Title : Edax Keywords : XEDS,SEM Computer : IBM PC or compatible Operating System : MS-DOS Programming Language : Turbo Basic,GWBasic Hardware Requirements : EDS Edax 9100,RS232C Card Author(s) : Henrik Kaker Correspondence Address : SEM/EDS Laboratory,.Metal d.o.o.,Koroska c.14 : 62390 Ravne,Slovenia,E-mail:kaker&ctklj.ctk.si Abstract:
Program for connection energy dispersive spectromter Edax 9100 with IBM PC or compatible computer via line printer port of the Edax 9100.Program allows transfering intensity and spectral data to PC and processing this data with standardless programs for bulk and thin film samples.
Documentation: EDAX.DOC
Source Code: Yes
and
Title : EPMA Database Keywords : EDS, WDS, SEM, EPMA Computer : None Operating System : None Programming Language : None Hardware Requirements : None Author(s) : Henrik Kaker Correspondence Address : SEM/EDS Laboratory,.Metal d.o.o.,Koroska c.14 : 62390 Ravne,Slovenia,E-mail:kaker&ctklj.ctk.si Abstract:
Database of 681, 826 and 40 published entries for performance testing of EPMA programs. Database EPMA40.DAT present collection of published data from different manufactures of EDS and WDS hardware and software and is suitable for testing standardless (no-standard) analysis programs.
Documentation: EPMA.DOC
Source Code: No
Two other points of information. A. Some of the archived files have been found to be corrupted and even our backups are no good. If anyone has good copies of the following files, please send me copies. They are under pub/MSA+MAS/MASSL 1) ~/ECPORI/ECPORI.SIT 2) ~/FINDCSL/FINDCSL.SIT B. The MASSL and the Electron Microscopy and Microanalysis Public Domain Libraries will be merged into one library, however this will only occur when Nestor Zaluzec get chance to sit down and organise the files. So, please be patient. Many thanks.
Dear Em specialists, since a few months we are dealing with a stupid problem: every time we prepare samples for classical em investigation we sometimes have contrast and sometimes not. The procedure we uses is: 3% glutaraldehyde fixation (from 1 hour to overnight), wash in 0.1 M cacodylate (approx 30 min) buffer, osmiumtetroxide fixation (1 or 2% in caco for approx 1h), again a wash in caco (30 min), dehydration in a ethanol series (70, 90, 2x100 every step 30 min) then a 2X10min incubation in propylene oxide, an overnight impregnation in 1:1 epon (lx): propylene oxide and finally an embedding in lx at 60 degrees for 60 hours. After sectionning, the sections are contrasted for 2 to 3 min with 5 % uranylacetate in water and finally 1 min lead citrate. Every time we use exactly the same procedure but the contrast varies from perfect to absent. We have already tried to change the fixatives and contrasting solution , prepare fresh, change lot, change companie but nothing helped. Also we changed the incubation times but now ower inspiration is gone so ... who can help us solving this irritating problem? Thanks for your help. Annette bakker
Annette wrote: } since a few months we are dealing with a stupid problem: every time we } prepare samples for classical em investigation we sometimes have } contrast and sometimes not. The procedure we uses is: } 3% glutaraldehyde fixation (from 1 hour to overnight), wash in 0.1 M } cacodylate (approx 30 min) } buffer, osmiumtetroxide fixation (1 or 2% in caco for approx 1h), again a } wash in caco (30 min), dehydration in a ethanol series (70, 90, 2x100 } every step 30 min) then a 2X10min incubation in propylene oxide, an } overnight impregnation in 1:1 epon (lx): propylene oxide and finally an } embedding in lx at 60 degrees for 60 hours. After sectionning, the } sections are contrasted for 2 to 3 min with 5 % uranylacetate } in water and finally 1 min lead citrate. Every time we use exactly the } same procedure but the contrast varies } from perfect to absent. We have already tried to change the fixatives and } contrasting solution , prepare fresh, change lot, change companie but } nothing helped. Also we changed the incubation times but now ower } inspiration is gone so ... who can help us solving this irritating problem? } Thanks for your help. } Annette bakker
Hello Annette! I think we all have had more or less problems getting optimal contrast in our specimens but there are also the ugly precipitates which can terrify us. We poststain with UAc (2.5% ethanol solution) for 10 min or with 5% water solution of UAc for 20-30 min, followed by staining with fresh Pb-citrate for 2 min. I think your staining periods are too short. The staining results are also depending on what tissue you work with. UAc reacts prin- cipally with nucleic acids and proteins. Pb-citrate increases the general contrast of membranes and also stains nucleic acid, glycogen and proteins due to the presence of carboxyl and sulfhydryl groups. Good luck and feel free to contact me for further discussions. Sverker Enestr|m, M.D., Ph.D. Department of Pathology Link|ping university, Sweden
Kem Rogers asked about image enhancement for cytochemistry
Here is my two cents worth, As an ardent image processor enhancer, I have no apriori bias against using image enhancement. However, in cytochemistry if you are comparing two things, I think both images should be taken under the same conditions and processed equivalently. Kem mentions processing to remove background, and under the circumstances he outlines this seems reasonable. However, I think background substraction should be done with great care. One mans background could be another mans signal. If you have measured background under conditions where no signal is possible and then subtract it uniformly from all images that seems reasonable. However, I have had people ask me to remove this defect or that "small area of background staining". I think that begins to constitute scientific fraud. Thats my humble opinion.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Dept. of Pathology * * Bowman Gray School of Medicine of Wake Forest University * * Medical Center Blvd * * Winston-Salem, NC 27157-1092 * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
concerning your staining problem: 1) try doing an "en bloc" stain with 2-4% UA, after OsO4 and your buffer wash, and just before your dehydration step. simply immerse your tissue in UA for 30-90 min (keep at 4 degrees C) then proceed with dehydration 2) when staining your sections with UA and Pb citrate, staining times should be 10-15 min. in each stain
Annette, I agree with Dr. Sverker. The staining times are too short. I stain for 25 min in uranyl acetate and 15 min in Pb citrate. These times may seem excessive but we have never had a problem with contrast. Additiionally, more than one rinse is suggested by my sources (at least 3/15 min each) in rinse buffer to remove excess glutaraldehyde. This should eliminate any interaction between unreacted glutaraldehyde and OsO4 which will cause peppering and general visual noise in your sections.
Hope this is helpful!
Dirk W. Domaschko Marshall University Huntington, WV Domaschk-at-musom01.mu.wvnet.edu
In addition to the sticky from cellophane tape, thick sections which are notoriously difficult to keep in place can be stuck to grids using unpolymerized epoxy resin. Dip one side of the grid to the top of a drop of epoxy, use compressed air to blow away excess resin from the open spaces, adhere the section and cure the resin in the oven.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Dept. of Pathology * * Bowman Gray School of Medicine of Wake Forest University * * Medical Center Blvd * * Winston-Salem, NC 27157-1092 * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
I'm not allowed to give the name of my reference, but "word has it" that the pH of the lead citrate is very important: if it is } 12, there will be little staining, and it may in fact bleach the sections (and lots of ugly contamination if much {12). Something to be checked each time the lead citrate is made. Also, small, unobvious changes in pipette bore, or how the pipette is held can cause significant changes in drop size, therefore in amount or concentration of solution delivered. Phil Oshel U Illlinois Center for Electron Microscopy oshel-at-ux1.cso.uiuc.edu At 10:18 AM 1/18/95 +0100, Willem.Jacob wrote: } Dear Em specialists, } since a few months we are dealing with a stupid problem: every time we } prepare samples for classical em investigation we sometimes have } contrast and sometimes not. The procedure we uses is: } 3% glutaraldehyde fixation (from 1 hour to overnight), wash in 0.1 M } cacodylate (approx 30 min) } buffer, osmiumtetroxide fixation (1 or 2% in caco for approx 1h), again a } wash in caco (30 min), dehydration in a ethanol series (70, 90, 2x100 } every step 30 min) then a 2X10min incubation in propylene oxide, an } overnight impregnation in 1:1 epon (lx): propylene oxide and finally an } embedding in lx at 60 degrees for 60 hours. After sectionning, the } sections are contrasted for 2 to 3 min with 5 % uranylacetate } in water and finally 1 min lead citrate. Every time we use exactly the } same procedure but the contrast varies } from perfect to absent. We have already tried to change the fixatives and } contrasting solution , prepare fresh, change lot, change companie but } nothing helped. Also we changed the incubation times but now ower } inspiration is gone so ... who can help us solving this irritating problem? } Thanks for your help. } Annette bakker } } } } Phil Oshel oshel-at-ux1.cso.uiuc.edu Center for Electron Microscopy Univ. of Illinois (Urbana) (217) 244-3135
We operate 7 electron microscopes (4 scanners, 2 TEM's, 1 microprobe), optical photomicroscope, 2 ultramicrotomes, materials science specimen prep equipment, high vacuum coater, ion beam coater, darkroom, etc - pretty much the generic general EM lab. At present we use a paper-based (dog-eared diary) booking system for scheduling users on the equipment.
I know that there are many labs that use electronic booking systems. Is this worthwhile, or does it cause more problems than it solves? Nearly all our users have access to ethernetted PC's. Information on pros and cons, problems, solutions, which software etc would be most useful. Our LAN superviser is especially interested in being pointed in the right direction for sources of software that will do the job.
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
I implimented this type of approach back around 1990, and have found it to be very useful. (My source code is very simple since it is designed for VT100 emulation across the ethernet, so I would not recommend it.) Apart from simple security issues such as making sure that authorized (safe) people only use the microscopes, the biggest advantage is speed. Noone has to spend hours collating the pages, and you do not have to worry about honesty -- how many people write down all the hours that they use? The major issue is not the software (trivial to write) but arranging electronic locks for the hardware such that they can only be used with an appropriate password (different for each user). We got an electronics technician to build a simple card for our TEM to control the beam. I would be interested in hearing of any more general solutions.
Reply... RE} EM Lab Scheduling 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 URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html
Here at Michigan our lab serves users from many departments (nuclear, materials, chemical , electrical, civil and mechanical engineering, physics, chemistry, geology, pediatric cardiology and pharmacy to mention a few) and they are spread out all over the city of Ann Arbor (about a five mile radius). Since there are University computer sites all over campus and many people have Macintosh computers or can at least get access to them and there is a campus-wide Appletalk network, we us a simple scheduling system based on a collection of Hypercard stacks. Each microscope, or other instrument that is schedulable, has a stack associated with it. These stack are stored on a server in our lab and are accessible from anywhere on campus. Each stack is a basic calendar with the days of the week listed in four hour block for the day time (~8-5) and 8 hour blocks in the evening and at night. Clicking on any one block of time allows the user to book that entire block or part of it. The user needs a user name and password and trained users are issued these ids that are stored within the stack. users can book up to 1 week in advance normally but I have the option of overriding that. When the stack reaches a certain size it is archived and the current data is copied to a new stack. It is not a really secure system but it works well for us. If anyone wants a copy of any of the stacks to modify for their own purposes, I will make them available on freebie.engin.umich.edu. The proviso is that any improvements should be made available to everyone, us especially!. OK? Just my 2 cents worth. Jfm.
I am interested in the discussion of light element analysis using a Kevex Quantum detector. From my discussions with Kevex, it seems that the software available has a hard time compensating for matrix effects when light elements are mixed with high atomic number elements. I was wondering how other people deal with this. Our system runs on RT11, so people who have TSX systems will have to bear with me. It appears that the answer is standards, standards, and more standards. Let's hear it... Randy Nessler
A couple of questions on CPD: 1. How important is it to use CO2 from a cylinder furnished with an ejector tube?
2.Can one use a conventional cylinder turned upside-down?
3.Am I right in assuming that cooling the CPD-chamber down to { 0 C, should condensate the CO2 to a liquid?
4.What grade (purity) should one use? I have always used "Medical Grade", obviously at a higher cost than "Technical Grade"
Discussion would be appreciated.Alan Hall Unit for Electron Microscopy University of Pretoria, Pretoria Tel +27-12-420-3297 South Africa Fax +27-12-420-3266
Could anyone please direct me to sources of Anopore disc filters or Flotronics filters for use in support of particulate samples for SEM viewing. A Canadian or US supplier would be helpful. Thanks Carolyn J. Emerson Biology Dept. Memorial University St. John's Newfoundland Canada cemerson-at-kean.ucs.mun.ca
I have someone interested in doing some reflection electron microscopy. Not being the surface type, I am unable to assist. If anyone out there is remotely interest in doing some REM please contact me directly for more details.
Thanks for your time.
Jeff --------------------------------------------------------- U U | | U U | Jeff Shield | U U | Department of Materials Science and Engineering | U U U U | University of Utah | U U U U | Salt Lake City, UT 84112 | U U U U | 801/581-3179 | UUUUU U | Fax: 801/581-4816 | U U | | U U --------------------------------------------------------- UUUUU
Of making many books there is no end, and much study wearies the body. -Eccl 12:12
AMRAY has been very good about supplying parts for our 1000A (still our workhorse 4-6 full days per week). Their telephone number is 800-225-1462. We've also had to replace some of these switches after some years.
} We are trying to repair the push-push type switches in the control } panel of our AMR 1000 SEM. The failure is mechanical rather than electronic. } The switches have "Master Specialties Co." writen on them and are in the } 12-4 series. Any line on where we can beg or buy at least two such switches } would be greatly appreciated. } Thanks in advance. } "For want of a nail...." } Tom Hanley } 706 568-2075 } thanly-at-uscn.bitnet
Jacob Jacob Bastacky, MD 1-116 Lawrence Berkeley Laboratory EMail: sjbastacky-at-lbl.gov University of California Phone: (510) 486-4606 Berkeley, California 94720 Fax: (510) 486-4750
Reply... RE} CD ROM image archive 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 URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html
While we are on the subject, has anyone seen the Pinnacle Micro drive for $1995 that is a double speed reader and also a CD-R drive? Sounds really nice!
You can read about reflection microscopy (which visualizes zones of cellular attachment to glass in epi-illuminated specimens by surface reflection interference) in chapter 5 in ELECTRONIC LIGHT MICROSCOPY, edited by David Shotton, Wiley-Liss Inc., 1993. Modern REM makes use of video-enhanced contrast technic, well described by David Shotton.
*************************************** Petra M Nederlof, Ph.D. Max-Planck-Institute for Biochemistry Dept. Structural Biology Am Klopferspitz 18 a D-82152 Martinsried (M=FCnchen) GERMANY
I have a Tektronix Phaser II SDX printer that I want to put on my campus network. I bought the Ethernet adaptor card but my network administrator is telling me it's not possible to put the printer on our network. We support Novell and TCP/IP. Has anyone put this printer on a network using either of these protocols? If so, can you give me any words of wisdom that might help me get the printer on the net?
Steven J. Eppell Center for Cardiovascular Biomaterials Case Western Reserve University
MICROBEAM ANALYSIS SOCIETY STUDENT SCHOLARSHIPS TO MAS-1995 (Breckenridge, CO) and IUMAS-1 (Sydney Australia)
All students (and faculty) involved in microanalysis-related research, should note a remarkable opportunity for travel to two forthcoming microanalysis conferences. The Microbeam Analysis society is offering student scholarships to the 29th Annual MAS meeting in Breckenridge CO (August 6-11, 1995). Student papers must be submitted, in standard MAS-MSA format to Dr. E. Etz, NIST, Division 837, Bldg. 222, room Q-113, Gaithersburg, MD 20899 by March 15, 1995. The best submitted papers will be awarded funds towards attending the annual meeting in Breckenridge. Any more information about the program can be obtained from Dr. Etz at etz-at-gapnet.nist.gov
What makes this scholarship offer extraordinary is that the best three papers given by student scholarship winners at the Breckenridge meeting will be awarded a minimum of $500 towards the cost of attending the 1st International Union of Microbeam Analysis Societies Conference in Sydney, Australia, February 5-9, 1996. These scholarships are only open to student members of MAS, and student application forms for MAS are available in past issues of Microbeam Analysis, the MAS journal. Student membership is a great bargain at $2.50, and doesn't require that the advisor be a MAS member - although if you aren't, I ask that you consider joining.
I will mail you more information with appropriate details about the meetings and the paper format etc., but if you have any immediate questions, please don't hesitate to contact me by email (dbw1-at-lehigh.edu).
we connercted our printer up for multiple users via the ethernet connection -first check to see that the installed tag is next to the ethernet port (if not you need a bourd installed) -connect to the ethernet port -load the phaser utilities, the network utilities, and the drivers -there are a few entries (words/code #'s/and commands) needed to load it all and get it going -if all else fails call Tektronix
good luck Mike
On Mon, 23 Jan 1995, Steven J. Eppell wrote:
} I have a Tektronix Phaser II SDX printer that I want to put } on my campus network. I bought the Ethernet adaptor card but } my network administrator is telling me it's not possible to } put the printer on our network. We support Novell and TCP/IP. } Has anyone put this printer on a network using either of } these protocols? If so, can you give me any words of } wisdom that might help me get the printer on the net? } } Steven J. Eppell } Center for Cardiovascular Biomaterials } Case Western Reserve University } } sje-at-po.cwru.edu }
Steven} I have a Tektronix Phaser II SDX printer that I want to Steven} put on my campus network. I bought the Ethernet adaptor Steven} card but my network administrator is telling me it's not Steven} possible to put the printer on our network. We support Steven} Novell and TCP/IP. Has anyone put this printer on a Steven} network using either of these protocols? If so, can you Steven} give me any words of wisdom that might help me get the Steven} printer on the net?
Steven} Steven J. Eppell Center for Cardiovascular Biomaterials Steven} Case Western Reserve University
Steven} sje-at-po.cwru.edu
If you are trying to print from a Unix workstation you can use the standard BSD print spooling mechanism to print to a network node (the IP address assigned to the Ethernet adaptor. If this is a Novell network and you need to print from a Sun Unix workstation you can use a package from Puzzle Systems called SoftNet Utilities that allows bidirectional printing.
-- Kevin Burton Noran Instruments voice: (608) 831-6511 x317 2551 West Beltline Highway, Room 532 FAX: (608) 836-7224 Middleton, WI 53562 email: kburton-at-noran.com
All subscribers Please remember to change your default address of "microscopy" to
Microscopy-at-aaem.amc.anl.gov
ANLEMC is dead, and mail is aliased to the new site.
Unfortunately the alias does not always work so some messages to microscopy do not always come through.
--------------------------- As for your Tek IISDX....
You should likely talk to Tektronics people on your network problem. I have a IISDX which is on an Appletalk/Ethernet connection and works fine. There are special drivers which you must load for using the printer on Mac, PC, or Sun workstations. You should have gotten these with your hardware. You could also run a direct line between the printer and your workstation.
On the Mac side you simply, plug-in and run. Virtually no setup time is required, except to copy the drivers from the disks to the system folder and connect the printer to the net. Then simply use the "choozer" to locate the printer on the appropriate zone on your networks. (This is the network connection I use)
On the PC side you will have to install the Windows drivers and then Mount the printer as a network printer. However, I cannot help with Novell nets.. How do you normally connect network printers? You must have laserprinters on your Novell network and I would guess that the procedure would be similiar. My version of the IISDX is NOT IP aware (it's about 2 years old) so TCP/IP is not an option here.
Just a warning. Putting a dye sub on a network can also be expensive if you have users that are not careful. People often forget that they were using a dye-sub printer and when they want to print their next manuscript/email note sometimes send it to a printer assuming they are still connected to a standard text printer, without checking to see that the have "de-selected" the dye sub. Hope you have some ideas on how to limit access otherwise you may end up having lots of expensive text printed out on your dye sub.
I use the IISDX printer for literally all my image hardcopy now. Haven't made a print from TEM negatives in nearly 2 years. Of course, some of my instruments are entirely digital (VG HB603Z) and hence there is no film.
Message-Id: {9501232034.AA26017-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Light element EDS Orig-Author: {randy nessler {rnessler-at-emiris.iaf.uiowa.edu} }:ddn:wpafb -----------------------------------------------------------
I am interested in the discussion of light element analysis using a Kevex Quantum detector. From my discussions with Kevex, it seems that the software available has a hard time compensating for matrix effects when light elements are mixed with high atomic number elements. I was wondering how other people deal with this. Our system runs on RT11, so people who have TSX systems will have to bear with me. It appears that the answer is standards, standards, and more standards. Let's hear it... Randy Nessler
An eductor tube ie a syphon tube (not ejector) is usual to get the liquid phase of co2 out of the tube. Ris at Univ Wisc used a small cylinder upside down, not very pratical for a large cylinder. I don't think it practical to get the cylidar down to a low enough temp to get liquid out, I forget the liquification temperature but it must be close to -70 C ie close to dry ice temp? Quality is important, I have had cylinders that had water mixed with the co2.... bad scene!! I order extra dry co2 (I believe, its written down where I order, not here) MOST important! be sure that the syphon/eductor tube is really there! Test by letting gas out at a good rate, the valve should start to frost up within 5-10 seconds at at a reasonable flow. about 20 percent of cylinders have broken or missing eductor tubes! so beware. A 'condenser valve' is available, I think from Fullam? (mine is ~12 years old) this will bleed some c02 out while cooling an internal cold finger that will recondense gaseous co2 passing through the valve to the cpd back to liquid... provided the room temp is not too high and the cylinder is not too empty etc. Also only about 30 of the 50 pounds of co2 can be gotteon out as liquid even with a condensing valve. Cold water around the chamber also helps. Hope this helpes people. alan pooley marine sci sem lab rutgers univ
John Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, 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: 4:46 PM
Date:1/23/95 URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL
Hi there, we are in the process of converting our JEOL 4000EX from freon to SF6 and want to know where we can get the said gas at a reasonable price. At the moment it looks like we are looking at between $1000 and $1700 for a single charge and so I would appreciate it if anyone could give me pointers for a source with a more reasonable cost Please reply direct to me and I will summarize to the list if there is sufficent interest. Thanks John Mansfield.
Ta muchly - hooked up and ready to go Brendon J. Griffin Centre for Microscopy and Microanalysis The University of Western Australia Nedlands, WA, AUSTRALIA 6907 ph 61-9-380-2739 fax 61-9-380-1087
we're wanting to get back into the lucifer yellow business after a few years - filling cells in slices, observing the fills with fluorescence, then immunostaining with antibody against LY and through to DAB. In the past, we used a gift antibody. Are there commercial Ab's out there that also do a good job? I have heard that there can be some loss of fine detail in the immuno procedure, and want to know if anyone has strong preferences as to LY antisera.
------------------------------------------------------------------ |Glenn Holm *mime mail ok* Internet:karuzis-at-wccf.mit.edu | |M.I.T Dept. of Brain + Cog. Sci. This VAX doesn't do NeXTmail | |Cambridge, MA 02139 "Real Neuroscientists don't do gels!" | ------------------------------------------------------------------
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John In my recent experience, SF6 is available from all of the major industrial gas suppliers (ie Selox) for about the same price...~$700 per 100 lb. That was enough for about 3 charges on our JEM 1210. Your 4000 must have a BIG tank. It took about 6 weeks for our SF6 to arrive. Obviously there is some primary supplier, but I don't know where. Good luck!
buddy
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
A few months ago, I put a message on the board looking for a Freeze Fracture machine, didn't get any good leads, so I thought it was time for another try:
We would like to obtain a freeze fracture machine in good working order (one of the Balzers or Cressington machines would do fine). We have money to pay for the machine, though not enough for a new one (sound familiar!). If you or someone you know would be interested in selling a machine to a good home please let me know
Thanks. ---------------------------------------------------- Simon Watkins Ph.D Director Structural Biology Imaging Center Scaife 840 University of Pittsburgh Pittsburgh PA 15261
The price for SF6 depends greatly on the purity. Commercial purity (99.7%) is generally available around $650-700 for a cylinder (115lb capacity). Instrument grade (99.99%) is near $1100 per cylinder. Our prices are from AGA (about a year ago), although I don't think you'll find significant price differences from other vendors.
The microscope manufacturers recommend instrument grade (of course!), although we successfully ran our H9000NAR on commercial grade.
Cheers, Henk
} } Hi there, } we are in the process of converting our JEOL 4000EX from freon to SF6 and } want to know where we can get the said gas at a reasonable price. At the } moment it looks like we are looking at between $1000 and $1700 for a single } charge and so I would appreciate it if anyone could give me pointers for a } source with a more reasonable cost } Please reply direct to me and I will summarize to the list if there is } sufficent interest. } Thanks } John Mansfield.
Henk Colijn colijn.1-at-osu.edu OSU Campus Electron Optics Facility ------------------------------------------------------------------- Research Law #1 If observations disagree with theory, the observations are obviously in error.
I surveyed sources of freeze drying units last year when finishing up my book on "Vacuum Methods in Electron Microscopy". At that time, these devices were available from: Agar Scientific (UK) Fx:0279-815106 Bal-Tech Fx: 508-374-7070 Denton Vacuum Fx: 609-424-0395 Edwards High Vacuum Fx: 505-658-7969 Electron Microscopy Sciences Fx: 215-646-8931 Emitech Fx: 713-893-8443 Energy Beam Sciences Fx;413-789-2786 Fisons FX: 415-961-8656 GeneVac (UK) FX: 0473-461176 Microfield (UK) FX:0865-821459 VG Microtech (UK) FX: 0825-768343 I don't know about the degree of automation offered, but perhaps this will give you a start on sorting things out. Good Luck!
--------------------------------------
------------------------------------------------------------------------ | David O'Neil tel: (902) 426-8258 | | National Research Council of Canada fax: (902) 426-9413 | | Institute for Marine Biosciences _____ _____ | | 1411 Oxford St. | | __/\__ | | | | Halifax, Nova Scotia B3H 3Z1 | | __/\\ //\__ | | | | Canada | | \ \\ // / | | | | | | /___ ___\ | | | | email: oneild-at-imb.lan.nrc.ca | | /__ __\ | | | | | | || | | | | |_____| |_____| | ------------------------------------------------------------------------
------------------ RFC822 Header Follows ------------------ Received: by mse.engin.umich.edu with SMTP;24 Jan 1995 12:59:48 -0400 Received: by truelies.rs.itd.umich.edu (8.6.9/2.2) with X.500 id MAA14718; Tue, 24 Jan 1995 12:58:43 -0500 with SMTP id MAA14681; Tue, 24 Jan 1995 12:58:39 -0500 To: Microscopy-at-AAEM.AMC.ANL.GOV
This is a follow-on to the 'pre-announcement' that I posted several weeks ago. The position is now real.
Materials Science and Engineering Laboratory National Institute of Standards and Technology
We are presently expanding our capabilities within the Materials Science and Engineering Laboratory at NIST in the area of HREM, and have an opening for a highly qualified Ph.D. scientist. Candidates must have extensive hands-on experience in high resolution imaging, be well versed in imaging and diffraction theory, and have exper- ience in image simulation. Experience in PEELS and EDS is essential, as is some background in materials science. Strong interest in development and implementation of new methodology is necessary. The position will include extensive work with other staff members of MSEL. For permanent employment, US citizenship is required.
Interested parties should submit a curriculum vitae and the names of three references by MARCH 15, 1995 to:
Dr. F.W. Gayle NIST Building 223, Room B164 Gaithersburg, MD 20899
Ed, I tried to respond to your request for print processor information, but my attempt bounced back with "host unknown" message. Please contact me privately and send e-mail address and I'll try again.
--
Gib Ahlstrand, MMS Newsletter Editor 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
Good day all, Thanks to all the contributors who posted (and are still posting) responses to my question on electronic lab work scheduling. Shall try to post a summary of the responses.
Another query: We need to put together a cooling stage for freeze drying of small samples. One way of doing this could be by using Peltier-effect cooling. These devices are used in various pieces of equipment but I have not been able to find someone who makes them. Could anyone help by supplying references to manufacturers of Peltier-effect devices? Thanks
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
I was looking for such a unit for years. I am currently using unit which was build in our workshop for 10 years ago and still going strong. The unit have rotary and turbo-pumps. For cooling Neslabs cryocool and liquid nitrogen are used. Temperature of specimens down to -150C and condenser, down to -180C. Using a data acquisition card from Strawberry Tree + Mac I can use controll and register temperatures at 4 different sites and register and display vacuum. Freeze-drier is used for both SEM-preparations and Low Temperature Vacuum Embedding. I will present the latest results from this system at SEM-meeting in Houston, Texas in May 1995. Costs of total unit are about USD 10 000 INCLUDING pumps, acq-card (not crypcool).
Regards
Romuald
------------------------- Romuald Wroblewski, Ph.D. Associate Professor Department of Pathology Karolinska Institute voice/fax:+46-8-7293597 -------------------------
I thought I would poll the EDS, WDS, EELS and other users about the proper disclaimer to place in reports issued internally or externally.
Most of our analysis is concerned with mineral fillers and those elements heavier than neon dispersed in an organic matrix. In some cases we will examine the organic matrix or we ash the organic matrix and examine the ash. The ash is either pressed on to a carbon block or on to a conductive double-sided tape. We believe that a realistic blanket statement of detectable for semi-quantification mode is "between neon to neptunium if present in concetrations of approximately 0.5%" but we feel a need to add some weasel words about the sample.
The question: Which best describes the limitations of our analysis by our samples? 0.5% by weight or 0.5% by volume, or is there a better phrase? Or are we just fooling ourselves?
I thought I would poll the EDS, WDS, EELS and other users about the proper disclaimer to place in reports issued internally or externally.
Most of our analysis is concerned with mineral fillers and those elements heavier than neon dispersed in an organic matrix. In some cases we will examine the organic matrix or we ash the organic matrix and examine the ash. The ash is either pressed on to a carbon block or on to a conductive double-sided tape. We believe that a realistic blanket statement of detectable for semi-quantification mode is "between neon to neptunium if present in concetrations of approximately 0.5%" but we feel a need to add some weasel words about the sample.
The question: Which best describes the limitations of our analysis by our samples? 0.5% by weight or 0.5% by volume, or is there a better phrase? Or are we just fooling ourselves?
Just a note. It is clearly inappropriate to use the Microscopy Listserver to send out Test messages to see if your Email server is running. There are somewhere over 2000 people receiving these messages please be intelligent. If you are having an Email problem then send a message to only one person. If you are really in a bind then just send a message to me and I will respond when I get a chance, but not to the world!!
Nestor Your Friendly (& Lately Tired) Neighborhood SysOp
I am planning to teach a short course in biological SEM for novices and need to find a textbook for the students. I am considering Postek et al., 1980 published by Ladd. Would any of you out there recommend anything more recent that would be appropriate for grad students, staff, faculty, post-docs. Any input willl be appreciated.
Although my course is intended for local consumption, iot would be open to anyone, so if there are interested parties wanting to spend a week in Florida in May, just E- mail me at the address below
I posted this note yesterday on several newsgroups, but just found out about this listserver today (1-25-95). I am looking for some help...
My group is responsible for examining uranium-metal spent nuclear fuel (N-Reactor, Single-Pass Reactors) which has degraded over time while in water storage at the Hanford, Washington site. There is a possibility that some of the uranium may have reacted with water to form uranium hydride which still may be present in some of the fuel. One of our tasks is to determine if this scenario is real. Thus, we will be examining several fuel elements to look for evidence of hydrides. We have (and will use) optical microscopy and electron microscopy (AFM, SEM, TEM) in our efforts.
Because the fuel is highly radioactive, and has a potential for pyrophoric reactions, our first efforts will be to cut, section, polish, and optically examine the fuel in a radiation hot cell using an argon cover gas blanket over all operations. However, when we get to the point of specifying etchants or etching techniques to examine the uranium, no one is sure how best to bring out the features of uranium hydride in uranium metal. We will try cathodic etching first, largely because this is what has been traditionally done to examine uranium at our lab. However, we aren't sure what to expect from the hydride (if it exists). Our literature searches have drawn a blank with regard to microscopy techniques for uranium hydrides.
So, I would like to ask, has anyone had experience optically examining uranium hydrides? If so, could you recommend a procedure? We also have the possibility of hiring a consultant if the right person or lab anywhere in the world) is identified to assist us.
I have a question for those people involved in light microscopic immunolabelling and trying to quantify the amount of stainig. Can anyone do it reliably?? We have someone in the lab who wants to determine if the staining obtained with 6 different antibodies co-localize within the tissue. They are staining paraffin-embedded sections and are staining one antibody/section. They want to know if there is a way to do this using an imaging system of some sort. IMHO I don't thinnk it can be done easily. If anyone has any trhoughts/ideas they would be GREATLY APPRECIATED.
Thanks Mark Elliott, PhD Pulmonary Research Lab UBC Vancouver Canada
Quantitative fluorescence microscopy can be tricky. Please refer to the chapter by Jesse E. Sisken (Fluorescence Standards) in Methods in Cell Biology, vol. 30, edited by L.Taylor and Y. Wang (1989; Academic Press) for recommendations.
************************************************************************* M.V. Parthasarathy Professor of Plant Biology Section of Plant Biology 228 Plant Science Building Cornell University, Ithaca, NY 14850 Telephone: (607) 255-1734 Fax: (607) 255-5407 E-mail: mvp2-at-cornell.edu ***********************************************************************
Try using silver paint, we use it & it seems to be working well. -Mike
On Wed, 25 Jan 1995 tsu-at-cae.wisc.edu wrote:
} } Hi, microscopist, } } Does anyone know if there's any conductive glue by which we can attach } our TEM samples to Cu/Au grids? M-bond is what I currently use and has } been suspected to cause some shifting of images especially for tiny } samples. Any information or comment will be appreciated. } } I-Fei Tsu } ASC-MRG } UW-Madison } }
Does anyone know if there's any conductive glue by which we can attach our TEM samples to Cu/Au grids? M-bond is what I currently use and has been suspected to cause some shifting of images especially for tiny samples. Any information or comment will be appreciated.
I am trying to prepare TEM thin foils from Mo-47.5Re (at.%) using a twin-jet electropolisher, without success. I have tried cold sulfuric acid/methanol solutions and perchloric acid/methanol solutions. These produce a pebbly surface with minimal electron transparent areas.
Does anyone out there have experience producing TEM thin foils from Mo-Re? I would greatly appreciate any suggestions! _ _ O-O J (a sideways smiley) Thanks, Dan Schwartz ~
I spoke to Bernie Kestel at Argonne National Lab and he gave the following solution that has worked well for him:
Material: Mo 30a/o Re Equipment: South Bay Technology Model 550 Single Vertical-Jet ElectroPolisher Electrolyte: 12% Sulphuric Acid 83% Methanol 5% Butyl Cellosolve Temperature: -50C Voltage: 190V Current: 50-75 ma Pump Speed: Slow
Since the work was done with a SBT Single Jet System, he suggested that you change the temperature to perhaps -40C and use a somewhat lower voltage for a twin jet system.
Of course, my solution would be to buy a South Bay unit, but I do have a vested interest in that! We also publish a bibliography of technical reports on sample preparation and distribute copies of the papers free of charge. Most of the reports deal with TEM sample preparation - primarily electropolishing, dimpling and Tripod Polishing.
Best regards-
David Henriks South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673
Message-Id: {m0rXYnF-0000zMC-at-pegasus.cc.ucf.edu} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Electropolishing Mo-Re Orig-Author: {schwartz-at-mrvx03.mdc.com}:ddn:wpafb ----------------------------------------------------------- My Fellow Microscopists:
I am trying to prepare TEM thin foils from Mo-47.5Re (at.%) using a twin-jet electropolisher, without success. I have tried cold sulfuric acid/methanol solutions and perchloric acid/methanol solutions. These produce a pebbly surface with minimal electron transparent areas.
Does anyone out there have experience producing TEM thin foils from Mo-Re? I would greatly appreciate any suggestions! _ _ O-O J (a sideways smiley) Thanks, Dan Schwartz ~
This may be a simple minded question, but I could use some advice. I just mixed some Epon Araldite type embedding plastic and it got really dark in color after adding the BDMA. I was using an Epon 812 substitute, DDSA, Araldite 502, and BDMA. It is a slightly different recipe than we have used before and our plastic is usually a pleasant golden color, this stuff was like dark caramel in color. The color came from the interaction of the BDMA and DDSA, or at least that is the way it looked after mixing the BDMA with the other components individually.
Does anyone have a suggestion to explain the dark color and any free advice about this phenomenon in general.
In a related question, can anyone say what the useful shelf lives of the various embedding components might be. Some of ours are older, someone buys a lot and never uses it up, should we toss everything and start over or can we hold on and use what we have?
Thanks for your help.
Jonathan Krupp Microscopy and Imaging Lab University of California Santa Cruz, CA 95060 emlab-at-ucsco.ucsc.edu (408) 459-2477
I DON'T THINK THE AGE OF YOUR RESIN COMPONENTS HAS ANY INFLUENCE ON THE DARK COLOR. OUR RESIN BATCHES ARE TURNED-OVER RAPIDLY, AND WE HAVE NOTICED A DARK COLOR IN OUR SPURR IN COMPONENTS THAT WERE RECENTLY ACQUIRED. WE CALLED ONE OF THE SUPPLY COMPANIES AND WERE TOLD THAT AN ALTERED PH IN ONE OF THE COMPONENTS WAS RESPONSIBLE. SINCE OUR SPURR RECIPE AND YOUR RESIN RECIPE DO NOT HAVE ANY COMPONENTS IN COMMON, PERHAPS THIS IS A GENERAL RESIN PROBLEM. ANY COMMENTS FROM RESIN ENGINEERS?
MARGE
Marge Hukee EM Core Facility hukee.margaret-at-mayo.edu Mayo Foundation (507) 284-3148 ---------------------------------------------------------------------------- --
About to enjoy a break.....please unsubscribe, and thanks.
Ron Oldfield School of Biological Sciences Macquarie University NSW Australia 2109 email: roldfiel-at-rna.bio.mq.edu.au phone: (612) 850 8173 fax: (612) 850 8116
Mats Karlsson (at Dept. of Pathology, Orebro Medical Center Hospital, Orebro, Sweden) et al. have recently described the methodological approach to quantify immunostained objects in histological sections by computerized image analysis in: Pathology, Res. and Pract. 1995 (in press), using frozen sections. Intra- and interindividual variations were less than 5% and the correlation of reproducibility was high. If you are interested, contact me directly.
================================================================== Sverker Enestrom M.D., Ph.D. Department of Pathology University of Linkoping, Sweden Phone: +46 13 22 15 20 Fax: +46 13 13 22 57 ==================================================================
Does anyone has experience how to get channeling patterns on JEOL Jem 2000FX electron microscope equipped with ASEA20 in STEM/BEI mode? Please some hints, advices.
Thank You.
Peter Makroczy Technical University of Kosice Dept.of Materials Science Park Komenskeho 11 043 85 Kosice Slovak republic
Message-Id: {9501271212.AA08568-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Electropolishing Mo-Re Orig-Author: {schwartz-at-mrvx03.mdc.com}:ddn:wpafb ----------------------------------------------------------- My Fellow Microscopists:
I am trying to prepare TEM thin foils from Mo-47.5Re (at.%) using a twin-jet electropolisher, without success. I have tried cold sulfuric acid/methanol solutions and perchloric acid/methanol solutions. These produce a pebbly surface with minimal electron transparent areas.
Does anyone out there have experience producing TEM thin foils from Mo-Re? I would greatly appreciate any suggestions! _ _ O-O J (a sideways smiley) Thanks, Dan Schwartz ~
Does anyone has experience how to get channeling pattern on JEOL Jem 2000FX electron microscope equipped with ASEA 20 in STEM/BEI mode. Please some hints, advices.
Thank You.
Peter Makroczy Technical University of Kosice Dept. of Materials Science Park Komenskeho 11 043 85 Kosice Slovak republic
Way back when, I was using EPON-ARALDITE resin components that were at least 10 years old at the time. No problems with using these. I do not recall having any problems with a dark color. What % of BDMA are you using? I used 2%(v/v). The only problem I can forsee in using a dark batch of resin would be that your background of your sections will not be as light as they should be. Best of luck.
It is hard to understand why people are still attempting to 'quantitate' imunohistochical reactions which rely on a final step in which the 'quantity' of stain is not related to the amount of antigen present. In order to quantitate anything there has to be a quantitative relationship which can be measured relating what is seen in the section to the amount of material detected. In other words you cannot quantiate the standard immunoperoxidase techniques used in immunocytochemistry. This does not negate their importance, but means that one should not attempt to do image analysis on this material.
For a complete discussion of these limitations see the bottom of page 6 in the Gareth Griffiths book on "Fine Structure Immuno-cytochemistry, Springer-Verlag, 1993, ISBN 3-540-54805-X. The following is a quote from this page "..., Fig 1 shows a striking example of a little appreciated fact, namely, how the immunoperoxidase method can often give results that, though qualitatively correct, may be quantitatively misleading. Most people who have worked extensively with any of the HRP techniques become aware of these problems and may be frustrated by the presence of many variables that cannot, even empirically, be completely controlled. Even if only qualitative data are required, it is very difficult in practice to find the right compromise between acceptable fine structure preservation and specific labelling. This does not belittle the historical importance of the immunoperoxidase techniques..."
Herbert K. Hagler, Ph.D. Microscopy and Imaging Service Center Dept of Pathology, UT Southwestern Medical Center 5323 Harry Hines Blvd., Dallas, TX 75235-9072 phone (214) 648-3890 fax (214) 648-3925
please, unsubscribe ------------------------------------------- Giovanni Valdre' Dipartimento Di Scienze Mineralogiche Piazza di Porta S. Donato 1 I-40126 Bologna, Italy Tel. +39 51 243556 FAX +39 51 243336 email gvaldre-at-geomin.unibo.it -------------------------------------------
Please send copies of Temhelp letters or questions to me. We perform all types of EM analyses and can probably help with a variety of techniques/comments.
Thank you very much.
Regards,
Mark W. Rigler, Ph.D. Materials Analytical Services Norcross, Georgia 1800-421-8451
Many different epoxies from various manufacturers, with widely varying ages have all given me exceptionally dark pot mixtures from time to time. There are only two things that I've been able to narrow it down to. One is accelerator, BDMA, DMAE or DMP-30 that has been opened too long. Water contamination has been suggested by mfrs. I now toss the accelerator when a bottle of epoxy (Epon, DER 736, etc.) is emptied. Some suppliers are worse than others. We quit dealing with Polysciences entirely, because of irregularities in the final blocks. (color is minor, it's when things won't get hard, and kits arrive missing components that things are serious). The other correlation on block color is delay in mixing. If you delay, you get coloration. Delay too long, or mix with a stir bar so slowly that you get layer formation, and the upper layer will get very dark and form lumps. Just my $.02.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
looking for feedback (mostly negative) from users - I have demo'd the systems and have a few "satisfied customer" refs from sales and of course each has annon. "horror stories" about the competition Hi everyone
I am in the process on deciding on a purchase of cryo-fixation and ultramicrotomy equipment and would appreciate some feedback from users -
I have demo'd both the Reichert Ultracut S and the RMC 7000 with their cryo systems and they seem pretty comparable, each having its own plusses and minuses -
This instrument will be used for both teaching and research - also it will msot likey be a very long time before we purchase another one
I would appreciate any positive and/or NEGATIVE comments you might have on these instruments - particularly your experience regarding reliability and service and support after the sales (I will not be taking out a service contract)
We are also contemplating the purchase of a jet freezer from either Bal-Tec or RMC (they bought out the Life-cell system) & would appreciate any comments from users about those instruments as well
Responses which are sent directly to me (as opposed to those posted to the list) will remain confidential
Thanks
Marcelle Gillott, Ph.D. Director, EM Laboratory University of Wisconsin-Milwaukee
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 181-187.
In situ analysis of microbial consortia in activated sludge using fluorescently-labelled, rRNA-targeted oligonucleotide probes and scanning confocal laser microscopy
M. Wagner, B. Assmus, A. Hartmann, P. Hutzler & R. Amann Lehrstuhl fur Mikrobiologie, Technische Universitat Munchen, Arcisstrasse 21, D-80290 Munchen, Germany
SUMMARY
Activated sludge flocs are complex consortia of various micro- organisms. The community structures of samples taken from municipal sewage treatment plants were characterized using fluorescently labelled, 16S and 23S rRNA-targeted oligonucleotide probes in combination with confocal scanning laser microscopy (CSLM). In comparison with conventional epifluorescence microscopy, CSLM considerably improved the capability to visualize directly the spatial distribution of defined bacterial populations inside the sludge flocs. Analyses could be performed at high resolution undisturbed by problems such as autofluorescence or limited spatial resolution in thick samples. In addition, CSLM was used to analyse some structural properties of paraformaldehyde-fixed activated sludge flocs, such as floc size and homogeneity. Typical floc sizes were found to be in the range between 5 and 50 micrometre. Whereas most of the flocs were completely colonized by bacteria, there were also examples of flocs containing gas bubbles or particles in the interior.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 188-194.
Scanning interference and confocal microscopy
R. Juskaitis & T. Wilson, Department of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, U.K.
SUMMARY
The form of the interference term image in scanning confocal and scanning conventional interference microscopes is identical in all respects including optical sectioning. This observation is used to obtain confocal images and surface profiles from conventional scanning interference microscope images.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 195-203.
Time- and wavelength-resolved spectroscopy in two-photon excited fluorescence microscopy
S. Andersson-Engels, I. Rokahr & J. Carlsson Department of Physics, Lund Institute of Technology, PO Box 118, S-221 00 Lund, Sweden
SUMMARY
Two-photon excited fluorescence spectroscopy has been performed at a microscopic scale in combination with normal, white light microscopy. This gave simultaneously a spectral resolution of 20nm and a temporal resolution of 20ps, from a volume element less than 5 micrometre in all three dimensions. The sample was excited with light from a continuously mode- locked Ti:sapphire laser that was focused on the sample in a fluorescence microscope. A polychromator and streak-camera were used for detection. The method has been used on tissue, plant and paper samples. It has also been demonstrated how substances naturally occurring in the samples can be identified from their spectroscopic properties and the spatial distribution of these substances can be observed.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 204-210.
Intracellular localization of the antitumour drug adriamycin in living cultured cells: a confocal microscopy study
S. Meschini, A. Molinari, A. Calcabrini, G. Citro & G. Arancia Department of Ultrastructures, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome, Italy
SUMMARY
The intracellular distribution of the anthracyclinic antibiotic adriamycin in living cultured cells has been investigated by confocal microscopy. In human melanoma cells (M14), adriamycin was localized inside the nuclei. When adriamycin-treated M14 cells were allowed to recover in a drug-free medium, a complete efflux of the drug from the nucleus was revealed. In recovered cells, a weakly fluorescent signal was observed in the perinuclear region. When M14 cells were recovered in a medium containing colcemid, a microtubule depolymerizing agent, the drug transport from the nucleus to the cell periphery appeared to be inhibited, suggesting that the microtubule network is strongly involved in drug transport mechanisms. In multidrug- resistant (MDR) cells the intracellular location of adriamycin was shown to be noticeably different from that of the parental wild-type cells. In particular, in resistant human breast carcinoma cells (MCF-7), adriamycin appeared to be exclusively located within the cytoplasm, whereas the nuclei were shown to be completely negative. When adriamycin treatment was performed in association with MDR revertants, such as Lonidamine (inhibitor of the energy metabolism) or verapamil (inhibitor of the P-glycoprotein efflux pump), a marked enhancement of the cytoplasmic signal was observed in resistant cells. Under these conditions, adriamycin appeared concentrated in the perinuclear region, but the nuclei were still negative. Confocal microscopy proved to be a useful method for the study of the intracellular transport of fluorescent substances, such as anthracyclinic antibiotics, and for the investigation of the multidrug resistance phenomenon in tumour cells.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 211-221.
A versatile tilting device for fluorescence microscopes
J. Bradl, M. Hausmann, B. Schneider, B. Rinke & C. Cremer Institut fur Angewandte Physik, Albert-Uberle Strasse 3-5, D-69120 Heidelberg, Germany
SUMMARY
A tilting device for biological specimens (rotation angle up to 2 pi) especially fluorescence-labelled cell nuclei, was developed. It consists of a quartz glass capillary and a mounting adaptor for the microscope stage. The applicability of the device was tested for several epifluorescence and confocal scanning laser microscopes. The axis of rotation is perpendicular to the optical axis of the microscope. The capillary can be tilted around its axis at any desired angle or in equiangular steps. This can be done manually or by remote control using a stepping motor. The three-dimensional (3-D) image-forming properties of the capillary system were experimentally examined using an inverse confocal scanning laser microscope. The results were compared with measurements obtained from the same microscope with the standard stage for plane slides with cover glasses. The measured point spread function suggested that, in spite of aberration effects, the optical arrangement used allows a gain in the 3-D resolution by tilting the object. A low-cost, fully-automated 3-D imaging system was built on the basis of a conventional epifluorescence microscope with a cooled black-and-white CCD camera. The system was operated by a personal computer. The online visualization ('movie') of rotating objects indicates the feasibility of the system.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 222-225.
P. E. Hanninen, E. Soini & S. W. Hell Department of Medical Physics, University of Turku, Center for Biotechnology, Tykistokatu 6, FIN-20521 Turku, Finland
SUMMARY
Two-photon excitation fluorescence imaging is feasible with continuous wave lasers. Images of biological specimens are obtained by employing photon counting in conjunction with an increasing recording time. The approach allows two-photon three-dimensional imaging of fluorescently-labelled specimens with inexpensive lasers.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 226-230.
Refractive-index-induced aberrations in two-photon confocal fluorescence microscopy
H. Jacobsen, P. E. Hanninen, E. Soini & S. W. Hell Department of Medical Physics, University of Turku, Center for Biotechnology, Tykistokatu 6, FIN-20521 Turku, Finland
SUMMARY
The effect of refractive index mismatch on the image quality in two-photon confocal fluorescence microscopy is investigated by experiment and numerical calculations. The results show a strong decrease in the image brightness using high-aperture objectives when the image plane is moved deeper into the sample. When exciting at 740nm and recording the fluorescence around 460nm in a glycerol-mounted sample using a lens of a numerical aperture of 1.4 (oil immersion), a 25% decrease in the intensity is observed at a depth of 9 micrometre. In an aqueous sample, the same decrease is observed at a depth of 3 micrometre. By reducing the numerical aperture to 1.0, the intensity decrease can be avoided at the expense of the overall resolution and signal intensity. The experiments are compared with the predictions of a theory that takes into account the vectorial character of light and the refraction of the wavefronts according to Fermat's principle. Advice is given concerning how the effects can be taken into account in practice.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 231-237.
The tetrahedral tip as a probe for scanning near-field optical microscopy at 30nm resolution
U. C. Fischer, J. Koglin & H. Fuchs Westfalische Wilhelms Universitat, Physikalisches Institut, Wilhelm-Klemm-Strasse 10, 48149 Munster, Germany
SUMMARY
The tetrahedral tip is introduced as a new type of probe for scanning near-field optical microscopy (SNOM). Probe fabrication, its integration into a scheme of an inverted photon scanning tunnelling microscope and imaging at 30nm resolution are shown. A purely optical signal is used for feedback control of the distance of the scanning tip to the sample, thus avoiding a convolution of the SNOM image with other simultaneous imaging modes such as force microscopy. The advantages of this probe seem to be a very high efficiency and its potential for SNOM at high lateral resolution below 30nm.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 238-244.
Studies of porphyrin containing specimens using an optical spectrometer connected to a confocal scanning laser microscope
O. Trepte, I. Rokahr, S. Andersson-Engels & K. Carlsson Physics IV, The Royal Institute of Tech, S-100 44 Stockholm, Sweden
SUMMARY
A spectrometer has been developed for use with a confocal scanning laser microscope. With this unit, spectral information from a single point or a user-defined region within the microscope specimen can be recorded. A glass prism is used to disperse the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit keeps the detector at 277K, thereby allowing integration times of up to 60s. The spectral resolving power ranges from 350 at 400nm to 100 at 700nm. Since the entrance aperture of the spectrometer has the same size as the detector pinhole used during normal confocal scanning, the three-dimensional spatial resolution is equivalent to that of normal confocal scanning. Light from the specimen is deflected to the spectrometer by a solenoid controlled mirror, allowing fast and easy switching between normal confocal scanning and spectrometer readings. With this equipment, studies of rodent liver specimens containing porphyrins have been made. The subcellular localization is of interest for the mechanisms of photodynamic therapy (PDT) of malignant tumours. Spectroscopic detection is necessary to distinguish the porphyrin signal from other fluorescent components in the specimen. Two different substances were administered to the tissue, Photofrin, a haematoporphyrin derivative (HPD) and delta-amino levulinic acid (ALA), a precursor to photoporphyrin IX and haem in the haem cycle. Both are substances under clinical trials for PDT of malignant tumours. Following administration of these compounds to the tissue, the potent photosensitizer and fluorescent compound photofrin, or protoporphyrin IX, respectively, is accumulated. For our study Wistar/Furth rats were injected either with Photofrin or with ALA 3-5h before they were killed. The organs were removed directly after and snap-frozen in carbon dioxide ice with isopentane. No further staining or fixation procedures were adopted.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 245-253.
Modelling of inclined and curved surfaces in the reflection scanning acoustic microscope
W. Weise, P. Zinin & S. Boseck Institute for Materials Science and, Structure Research, Physics Department, University of Bremen, 28334 Bremen, Germany
SUMMARY
An expression is derived for the output signal when an inclined plane surface is imaged by the reflection scanning acoustic microscope, which is modelled as a spherical transducer. This expression is applied to model non-planar surfaces. The accuracy of this approach is tested for perfectly reflecting spherical surfaces. The influence of inclination on V(z) curves is considered when Rayleigh waves occur.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 254-261.
Scanning force microscopy on live cultured cells: imaging and force-versus-distance investigations
D. Ricci & M. Grattarola Dipartimento di Ingegneria Biofisica, ed Elettronica, Universita degli Studi di Genova, Via Opera Pia 11a, 16145 Genova, Italy
SUMMARY
Extensive measurements with the scanning force microscope (SFM) on living cells in their native liquid environment are described with the purpose of critically assessing the extent of the interaction between the SFM tip and the (soft) cell materials and the effect of such interaction on topographic information. Images are obtained under various force conditions and systematically correlated with force-versus- distance curves. As a result, detailed indications about tip indentation are given, thickness estimates deduced and identification of submembranous cytoplasmic structures suggested.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 262-275.
In vivo analysis of angiogenesis and revascularization of transplanted pancreatic islets using confocal microscopy
F. A. Merchant, S. J. Aggarwal, K. R. Diller & A. C. Bovik Biomedical Engineering Research Program, ENS 612, University of Texas, Austin 78712-1084, U.S.A.
SUMMARY
A technique to measure angiogenesis and revascularization in pancreatic islets transplanted at the renal subcapsular site in the rat has been developed. In vivo imaging of the microcirculation of transplanted pancreatic islets was conducted using a confocal scanning laser microscope (CSLM) to achieve optical sectioning through the graft in order to perform a computer reconstruction of the three-dimensional neovascular morphology. Individual islets were harvested by enzymatic digestion of excised pancreas from Fischer 344 rats. Isolated islets were cultured for 24h, and approximately 300- 350 islets were transplanted at the renal subcapsular site of the left kidney in an anaesthetized rat. Six to 14 days post- transplantation, the animal was anaesthetized and prepared for in vivo imaging of the microvasculature on a Zeiss LSM-10. Optical contrast of the microvasculature was enhanced by the administration of fluorescein-labelled dextran into the circulating blood. The transplant site was identified and serial sections were obtained through the vascular bed at varying z-intervals. Complementary fluorescence video images were also obtained via a silicon intensifier tube camera mounted on the CSLM. At completion of the imaging procedure, the kidney was returned into the body cavity, the area was sutured and the animal was allowed to recuperate for subsequent examinations. Image processing algorithms, such as grey-level thresholding, median filtering, skeletonization and template matching, were applied to compute the vessel density and diameters and extrapolated to measure 3-D vessel lengths and the tortuosity index of the neovasculature.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 276-280.
Optoelectronic detector probes for scanning near-field optical microscopy
H. U. Danzebrink Physikalisch-Technische Bundesanstalt, Bundesallee 100, D-38116 Braunschweig, Germany
SUMMARY
A brief explanation of the optoelectronic probe concept and a comparison between the implementation of passive waveguide probes and optoelectronic probes in scanning near-field optical microscopy (SNOM) is presented. The first probe realizations using cleaved semiconductor crystals and the work at present in progress using microfabricated Si pyramids are described. These crystals with evaporated metal electrodes forming a slit aperture with subwavelength dimensions work as metal-semiconductor-metal photodetectors. Their optical detection behaviour is investigated by measuring the intensity distribution of a laser focal point. Measurements where the external bias voltage is changed show that it is possible to modify the detection behaviour of the device because of the varying depletion widths. The last part of the article describes a concept where pyramidal probes should function simultaneously as senors for scanning force microscopy (SFM) to measure topography and as optoelectronic probes for scanning near-field optoelectronic microscopy (SNOEM).
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 281-286.
Imaging in the far-red with electronic light microscopy: requirements and limitations
C. Cullander School of Pharmacy S926, University of California, San Francisco, CA 94143-0446, U.S.A.
SUMMARY
The acquisition of simultaneous dual confocal images with red and far-red light has both advantages (e.g. lower autofluorescence) and limitations. An understanding of these requisites is necessary to acquire high-quality images and to avoid the misinterpretation of experimental data. The poor detection of far-red light mandates a high optical transfer efficiency for the system, thus the transmittance of the objective lens and its axial and lateral chromatic aberration in the far-red are important factors for consideration. This technical note is an attempt to 'demystify' the process of filter set design for confocal microscopy by discussing the considerations that went into the construction of a filter set for use with the reagents cyanine 3.18 (Cy3) and cyanine 5.18 (Cy5), and thus to encourage users to look beyond the multipurpose designs available commercially. The 568-nm laser line exciting Cy3 is at its emission maximum, which limits the collectable Cy3 fluorescence. High-transmission optical filters with sharp bandpass cutoffs are thus desirable for maximum light throughput. Light path mirror efficiency rapidly degrades above 700nm, but the loss of this portion of the Cy5 emission spectrum is acceptable since the fluorophore is very bright, and these very long wavelengths are also likely to introduce aberration. While resolution is decreased with far- red light, there is also greater penetration and less scattering, and it is thus possible to obtain high-quality images from deeper within the specimen. Although only one make and model of confocal microscope (the Bio-Rad MRC-600) is considered, similar considerations pertain to the design of filter sets for any confocal microscope that accommodates user-installed filters.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 287-299.
Simultaneous confocal recording of multiple fluorescent labels with improved channel separation
K. Carlsson, N. Aslund, K. Mossberg & J. Philip Physics IV, The Royal Institute of Tech, S-100 44 Stockholm, Sweden
SUMMARY
Confocal microscopes are often used to study specimens labelled with fluorophores. A commonly used method for simultaneous recording of the distribution of multiple fluorophores is to divide the fluorescent light emitted by the specimen into different wavelength regions using dichroic and bandpass filters. These different wavelength regions are then distributed to multiple detectors. However, fluorophores often result in considerable cross-talk between channels. A new technique, intensity-modulated multiple-beam scanning (IMS) microfluorimetry, can be used to reduce this cross-talk substantially. The IMS technique is implemented with two laser beams of different wavelengths, intensity-modulated at different frequencies, which illuminate the specimen simultaneously. The two laser wavelengths predominantly excite one fluorophore each. Fluorescent light from the specimen is divided into two wavelength regions (red and green) which are detected by two photomultiplier tubes. The output signals from the photomultiplier tubes are connected to lock-in amplifiers. The effect of using modulated laser beams, in combination with lock-in amplifiers, is strongly to reduce the cross-talk between channels. The performance of the IMS technique using various types of specimen is compared with the results obtained using the conventional multi-detector design.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 1-6.
In vivo determination of fibril orientation in plant cell walls with polarization CSLM
J.-P. Verbelen & D. Stickens Department of Biology, University of Antwerp (UIA), Universiteitsplein 1, B-2610 Wilrijk-Antwerpen, Belgium
SUMMARY
Congo Red fluorescence is used to detect cellulose in the wall of plant cells. The orientation of the cellulose fibrils is determined by using polarized light for excitation. The absorption characteristics of Congo Red make this approach a method of choice for applications with any standard confocal scanning laser microscopy (CSLM). The semi-quantitative character of CSLM observations combined with the non-toxicity of the stain allow a very fast and reliable assessment of cellulose orientation in the wall of living plant cells.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 7-17.
Three-dimensional reconstruction of the human renal glomerulus
K. Preston Jr, B. Joe, R. Siderits & J. Welling Kensal Corporation, 5055 East Broadway (Suite C206), Tucson AZ 85711, U.S.A.
SUMMARY
The capillary bed of human renal glomerulus is one of the more complex capillary structures in the human body. This paper illustrates three-dimensional reconstruction of the capillary bed from serial sections. It shows that, although traditional methods of three-dimensional rendering by computer fail to handle the complexities of the capillary structure, new methods based on filtering using three-dimensional mathematical morphology are capable of revealing previously unseen details. This is done at the expense of eliminating fine structure (small capillaries). An error analysis allows the degree to which fine details are lost to be estimated.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 18-30.
Quantitative water mapping of cryosectioned cells by electron energy loss spectroscopy
S. Q. Sun, S.-L. Shi, J. A. Hunt & R. D. Leapman Health & Human Services, Public Health Service, National Institutes of Health, Building 13 Room 3W13, Bethseda MD 20892, U.S.A.
SUMMARY
A direct technique based on electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) has been developed to map subcellular distributions of water in frozen-hydrated biological cryosections. Previously, methods for water determination have been indirect, in that they have required the cryosections to be dehydrated first. The new approach makes use of spectrum-imaging, where EELS data are collected in parallel at each pixel. Several operations are required to process the spectra including: subtraction of the detector dark current, deconvolution by the detector point-spread function, removal of plural inelastic scattering and correction for the support film. The resulting single scattering distributions are fitted to standard reference spectra at each pixel, and water content can be determined from the fitting coefficients. Although the darkfield or brightfield image from a hydrated cryosection shows minimal structure, the processed EELS image reveals strong contrast due to variation in water content. Reference spectra have been recorded from the major biomolecules (Protein, lipid, carbohydrate, nucleic acid) as well as from vitrified water and crystalline ice. It has been found that quantitative results can be obtained for the majority of subcellular compartments by fitting only water and protein reference spectra, and the accuracy of the method for these compartments has been estimated as plus/minus 3.5%. With the present instrumentation the maximum allowed dose of 2000e/nm2 limits the useful spatial resolution to around 80nm plus/minus 5% precision at a single pixel. By averaging pixel intensities a value of 56.8% with a precision of plus/minus 2.0% has been determined for the water content of liver mitochondria. The water mapping technique may prove useful for applications to cell physiology and pathophysiology.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 31-42.
Hybrid scanning transmission electron/scanning tunnelling microscope system for the preparation and investigation of biomolecules
H. F. Knapp, R. Wyss, R. Haring, C. Henn, R. Guckenberger & A. Engel Maurice E Muller Institut fur, Hochauflosende Elektronenmikroskopie, Universitat Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
SUMMARY
A hybrid scanning transmission electron/scanning tunnelling microscope vacuum system is introduced, which allows freeze drying and metal coating of biological samples and their simultaneous observation by scanning transmission electron microscopy and scanning tunnelling microscopy (STM). Different metal coatings and STM tips were analysed to obtain the highest possible resolution for such a system. Bovine liver catalase was used as a test sample and the STM results are compared to a molecular scale model.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 43-52.
Backscattered electron imaging of the undersurface of resin-embedded cells by field emission scanning electron microscopy
R. G. Richards & I. Ap Gwynn AO/ASIF Research Institute, Clavadelerstrasse, CH-7270 Davos Platz, Switzerland
SUMMARY
In this study backscattered electron (BSE) imaging was used to display cellular structures stained with heavy metals within an unstained resin by atomic number contrast in successively deeper layers. Balb/c3T3 fibroblasts were cultured on either 13mm discs of plastic Thermanox, commercially pure titanium or steel. The cells were fixed, stained and embedded in resin and the disc removed. The resin block containing the cells was sputter coated and examined in a field-emission scanning electron microscope. The technique allowed for the direct visualization of the cell undersurface and immediately overlying areas of cytoplasm through the surrounding embedding resin, with good resolution and contrast to a significant depth of about 2 micrometre, without the requirement for cutting sections. The fixation protocol was optimized in order to increase heavy metal staining for maximal backscattered electron production. The operation of the microscope was optimized to maximize the number of backscattered electrons produced and to minimize the spot size. BSE images were collected over a wide range of accelerating voltages (keV), from low values to high values, to give 'sections' of information from increasing depths within the sample. At 3- 4keV only structures a very short distance into the material were observed, essentially areas of cell attachment to the removed substrate. At higher accelerating voltages information on cell morphology, including in particular stress fibres and cell nuclei, where heavy metal were intensely bound, became more evident. The technique allowed stepwise 'sectional' information to be acquired. The technique should be useful for studies on cell morphology, cycle and adhesion with greater resolution than can be obtained with any light-microscope- based system.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 53-67.
Tomographic reconstruction of the cross-sectional refractive index distribution in semi-transparent birefringent fibres
T. C. Wedberg & W. C. Wedberg Physics Department, University of Bergen, Allegt 55, N-5007 Bergen, Norway
SUMMARY
Optical diffraction tomography (ODT) is used to reconstruct the complex refractive index distribution in cross-sections of semi-transparent, birefringent fibres. The selected fibres were polymer and animal fibres of either circular or non- circular cross-section with average thicknesses in the range 8-110 micrometre. This choice of samples was made to illustrate the imaging capabilities of ODT, and also to demonstrate some potential applications of the technique. The images representing the reconstructed refractive index distributions have a spatial resolution of about 2 micrometre, and show noticeable image contrast for refractive index variations of about 0.001. The ODT reconstructions compare well with refractive index information provided with the samples, and with scanning electron micrographs of cross- sections of the same fibre samples. From these results it appears that ODT can be used to reconstruct the complex refractive index distribution in cross-sections of semi- transparent birefringent fibres.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 68-76.
Computer simulated high-resolution transmission electron microscopy (HRTEM) in tourmaline
E. A. Ferrow Avdelningen for Mineralogi och Petrologi, Geologiska Institutionen, Lund Universitet, Solvegatan 13, 223 62 Lund, Sweden
SUMMARY
The contrast distributions observed in high-resolution transmission electron microscopy (HRTEM) images of tourmaline depend on the types and magnitudes of the exchange components present and on the degree of atom overlap along the direction of observation. Furthermore, the fractional atomic coordinates in tourmalines are valid only for the specific specimen refined. These properties make the interpretation of experimental HRTEM images of tourmaline using image simulation if not impossible at least extremely difficult. A correct interpretation of experimental HRTEM images of tourmaline is possible provided the structural refinement data on the same crystal are available. Nevertheless, it is possible to interpret the experimental HRTEM images of tourmaline if the composition of the structural model chosen during image simulation approximates the composition of the specimen studied by electron microscopy. A good control of the composition of the specimen studied and an appropriate choice of a structural model for image simulation are therefore as important as properly controlling specimen thickness, specimen tilt, beam tilt and objective lens defocus.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 77-84.
Confocal microscopy in the analysis of the etched nuclear particle tracks in polymers
J. Jakes, P. Gais & H. Schraube Institut fur Strahlenschutz, GSF-Neuherberg, Postfach 1129, D-85758 Oberschleissheim, Germany
SUMMARY
The possibility of the morphometric analysis of etched tracks, induced by protons and alpha particles in the organic polymer allyl diglycol carbonate (CR-39), using the confocal scanning laser microscope (CSLM), was studied. The detectors were investigated in two groups of irradiation experiments, namely: (a) irradiated with mono-energetic neutrons of energy 1.2MeV, (b) exposed to the alpha radiation from 222Rn and its progeny. Both groups were irradiated at normal incidence. Radiation- induced latent tracks were electrochemically etched, and their morphometric parameters were investigated in the reflection mode by using the 488nm spectral line of an argon ion laser. A constant number of up to 200 optical sections in Z-scan mode was taken through each selected etched track at vertical spacings of 0.642 micrometre. Successive reconstructions of Z- sections were used to determine the following parameters: the mean radius of the opening channel, the maximum diameter and the length of the track, and the angle of the track wall to the surface of the sample. The results show that tracks produced by alpha particles differ from those induced by protons. The radius of the opening channel of alpha-particle- induced tracks ranges from 7.9 to 11 micrometre, whereas for protons the same parameter ranges between 2.0 and 3.8 micrometre for a specific electrochemical etch procedure.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 85-89.
A simple method for overcoming some problems when observing thick reflected biological samples with the confocal scanning laser microscope
C. Rumio, M. Morini, J. R. Miani, I. Barajon & P. Castano Institute of Human Anatomy, Via Mangiagalli 31, 20133 Milano, Italy
SUMMARY
A simple device is described, which allows the range of depth of scanning to be reduced when observing thick reflecting biological specimens with a confocal scanning laser microscope (CSLM). Thick histological sections of human skin and rat brain stem were mounted between two coverslip ('sandwich style') and the optical tomography was performed from both sides by turning the 'sandwich' upside-down. The samples were impregnated using standard Golgi-Cox, 'rapid Golgi' or other silver methods. The ability to turn the sandwich upside-down is particularly useful when the reflective structure inspected is deep inside the section, i.e. near the lower surface of the specimen, or when it is opaque to the laser beam of excessively reflective.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 90-92.
Thick section preparation using a silicon-rubber-based sealant
H. Cox, C. Walker & C. V. Howard Department of Orthopaedic & Accident Surgery, Royal Liverpool University Hospital, Prescot Street, PO Box 147, Liverpool, L69 3BX
SUMMARY
A method has been developed, using a silicon-rubber-based sealant, which allows 2-3-mm-thick specimens to be maintained in a protected fluid environment for a number of months, without risk of dehydration. Following this, the specimen can be retrieved, stained, embedded and sectioned further. For example, 2-mm-thick sections of fixed unstained bone are easily examined by means of epi-illuminated polarized light and fluorescence microscopies using either conventional or confocal optics. The method could easily be extended to other tissues, for example brain tissue.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
A method to compensate for light attenuation with depth in three-dimensional DNA image cytometry using a confocal scanning laser microscope
A. Liljeborg, M. Czader & A. Porwit Physics IV, The Royal Institute of Tech, S-100 44 Stockholm, Sweden
SUMMARY
A method to compensate for attenuation of detected light with increased depth of the collected optical section, and its application in three-dimensional (3-D) DNA image cytometry is described. The method is based on studying the stack of 2-D histograms that ca be formed from each consecutive pair of sections in a stack of optical serial sections. An attenuation factor is calculated interactively and a new compensated section series is computed. Formalin-fixed paraffin-embedded rat tissue was stained with propidium iodide. Each cell nucleus is extracted by thresholding and its total intensity is calculated. The coefficient of variation (CV) of the total intensity of all cells in each stack is computed. For comparison the CV of the same cells is computed in the uncompensated stacks. This study shows a significantly lower CV for the compensated data, thus contributing to the accuracy of DNA quantification in 3-D DNA image cytometry.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
Microfractography of granitic rocks under confocal scanning laser microscopy
M. Montoto, A. Martinez-Nistal, A. Rodriguez-Rey, N. Fernandez-Merayo & P. Soriano University of Oviedo, Department of Geology, Group of Petrophysics, 33005 Oviedo, Spain
SUMMARY
Scanning laser microscopy, in the confocal mode (CSLM) has been applied to a granitic rock to characterize its fissure space. The technique provides a unique three-dimensional picture of the rock microfractomography. CSLM is unique in observing fine details of the fractographic network (connectivity, tortuosity, etc.), its geometry and its relation to other rock-forming components. The fractographic images with standard fluorescence microscopy are compared with those obtained with CSLM. The examples presented emphasize the advantages of CSLM: three- dimensional visualization of the microfractographic network, crack connectivity, automatic evaluation of direction and slope of fissures. These studies are related to the migration of radionuclides in the geosphere. The relations between potentially water-conducting open fissures and the rock- forming minerals provide a means of modelling the 'radionuclide retardation mechanism', a security factor in their definitive storage in rock masses.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
PHOEBE, a prototype scanning laser-feedback microscope for imaging biological cells in aqueous media
T. L. Wong, S. L. Sabato & A. Bearden Division of Neurobiology, Department of Molecular & Cell Biology, 229 Stanley Hall, University of California at Berkeley, Berkeley CA 94720-3206, U.S.A.
SUMMARY
Based on the principle of laser-feedback interferometry (LFI), a laser-feedback microscope (LFM) has been constructed, capable of providing an axial (z) resolution of a target surface topography of approximately 1nm and a lateral (x,y) resolution of approximately 200nm when used with a high- numerical-aperture oil-immersion microscope objective. LFI is a form of interferometry in which a laser's intensity is modulated by light re-entering the illuminating laser. Interfering with the light circulating in the laser resonant cavity, this back-reflected light gives information about an object's position and reflectivity. Using a 1-mW He-Ne (wavelength=632.8nm) laser, this microscope (PHOEBE) is capable is obtaining 256x256-pixel images over fields from 10x10 micrometre to 120x120 micrometre in approximately 30s. An electrochemical feedback circuit holds the optical pathlength between the laser output mirror and a point on the scanned object constant; this allows two types of images (surface topography and surface reflectivity) to be obtained simultaneously. For biological cells, imaging can be accomplished using back-reflected light originating from small refractive-index changes (} 0.02) at cell membrane/water interfaces; alternatively, the optical pathlength through the cell interior can be measured point-by-point by growing or placing a cell suspension on a higher-reflecting substrate (glass or silicon wafer). Advantages of the laser-feedback microscope in comparison to other confocal optical microscopes include: simplicity of the single-axis interferometric design; the confocal property of the laser-feedback microscope (a virtual pinhole), which is achieved by the requirement that only light that re-enters the laser meeting the stringent frequency, spatial (TEM00), and coherence requirements of the laser cavity resonator mode modulate the laser frequency; and the improved axial resolution, which is based on interferometric measurement of optical amplitude and phase rather than by use of a pinhole as in other types of confocal microscopes.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
Imaging periodic surface relief structures
J. T. Sheridan & T. O. Korner TP 680, Institute for System Engineering and, Informatics Science R&D, Joint Research Centre JRS/CCR, I-21020 Ispra (VA), Italy
SUMMARY
Because of shadowing, multiple scatter and polarization effects, the interpretation of images of grating with fine periods, isolated deep structures, and multiple scattering volume objects is seriously complicated. In this paper a review of methods used to model such effects is presented. Periodic surface relief gratings are of particular current importance because of the possibility of producing calibration samples using them. Several examples which illustrate electromagnetic volume effects are examined. General trends which help in validating the use of Fourier-transform-based scalar transmittance theory are then indicated. The angular spectrum approach, which can be used , together with a scatter function generated using the rigorous electromagnetic theory, to calculate coherent, partially coherent and confocal images of volume objects, is also discussed.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
Application of confocal laser microscopy and three-dimensional Voronoi diagrams for volume and surface area estimates of interphase chromosomes
R. Eils, E. Bertin, K. Saracoglu, B. Rinke, E. Schrock, Y. Usson, M. Robert-Nicoud, E. H. K. Stelzer, J.-M. Chassery, T. Cremer & C. Cremer Institut fur Angewandte Physik, Albert-Uberle Strasse 3-5, D-69120 Heidelberg, Germany
SUMMARY
This study demonstrates the use of Voronoi tessellation procedures to obtain quantitative morphological data for chromosome territories in the cell nucleus. As a model system, chromosomes 7 and X were visualized in human female amniotic fluid cell nuclei by chromosomal in situ suppression hybridization with chromosome-specific composite probes. Light optical serial sections of 18 nuclei were obtained with a confocal scanning laser fluorescence microscope. A three- dimensional (3-D) tessellation of the image volumes defined by the stack of serial sections was then performed. For this purpose a Voronoi diagram, which consists of convex polyhedra structured in a graph environment, was built for each nucleus. The chromosome territories were then described by three morphological parameters, i.e. volume, surface area and a roundness factor (shape factor). The complete evaluation of a nucleus, including the calculation of the Voronoi diagram, 3-D visualization of extracted territories using computer graphic methods and parameterization was carried out on a Silicon Graphics workstation and was generally completed within 5 min. The geometric information obtained by this procedure revealed that both X- and 7-chromosome territories were similar in volume. Roundness factors indicated a pronounced variability in interphase shape for both pairs of chromosomes. Surface estimates showed a significant difference between the two X- territories but not between chromosome 7-territories.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
H. Heinzelmann, B. Hecht, L. Novotny & D. W. Pohl Institut fur Physik, Universitat Basel, Klingelbergstrasse 82, 4056 Basel, Switzerland
SUMMARY
Near-field optics (NFO) opens the door to light microscopy techniques with resolutions well beyond the diffraction limit. The richness of optical investigations is now applicable on a near-molecular level. Among the novel scanning near-field optical microscopy (SNOM) schemes, the most prominent representatives are aperture SNOM and scanning tunnelling optical microscopy (STOM or PSTM). New experimental and theoretical work has to be performed to study the phenomena specific to NFO. One such example is the angular dependence of light emission in aperture SNOM. The detection of radiation at angles greater than the critical angle of total internal reflection alpha=arcsin(1/n), where n is the sample refractive index, can represent a microscopy scheme that combines the respective advantages of both aperture SNOM and STOM. Recent experiments have demonstrated the expected exponential dependence of light intensity on gap width (for fixed emission angle). The decay length as a function of alpha is in agreement with the Fresnel description of the evanescent field when total reflection occurs at an interface. These investigations were additionally motivated by calculations based on the multiple multipole method.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
Automated correction of linear deformation due to sectioning in serial micrographs
T. Jansson, T. Gustavsson, M. Rydmark, C.-H. Berthold, R. Pascher & T. Skoglund Department of Applied Electronics, Chalmers University of Technology, S-412 96 Goteborg, Sweden
SUMMARY
This paper describes an objective and automatic method for detection and correction of sectioning deformations in digitized micrographs, as well as an evaluation of the method applied to light and electron microscopic images of semi-thin and ultra-thin serial sections from brain cortex. The detection is based on matching of image subregions and the deformation model is bi-linear, i.e. two first-order polynomials are used for modelling compression/expansion in perpendicular directions. The procedure is applicable to prealigned serial two-dimensional sections and is primarily aimed at three-dimensional reconstruction of tissue samples consisting of a large number of cells with random distribution and morphology.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
Comparison of three-dimensional imaging properties between two-photon and single-photon confocal fluorescence microscopy
Min Gu & C. J. R. Sheppard Physical Optics Department, School of Physics, The University of Sydney, NSW 2006, Australia
SUMMARY
The imaging performance in single photon (1-p) and two-photon (2-p) fluorescence microscopy is described. Both confocal and conventional systems are compared in terms of the three- dimensional (3-D) point spread function and the 3-D optical transfer function. Images of fluorescent sharp edges and layers are modelled, giving resolution in transverse and axial directions. A comparison of the imaging properties is also given for a 4Pi confocal system. Confocal 2-p 4Pi fluorescence microscopy gives the best axial resolution in the sense that its 3-D optical transfer function has the strongest response along the axial direction.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
Double pulse fluorescence lifetime imaging in confocal microscopy
M. Muller, R. I. Ghauharali, K. Visscher, T. D. Visser & G. J. Brakenhoff Department of Molecular Cytology, University of Amsterdam, Plantage Muidergracht 14, 1018 TV Amsterdam, The Netherlands
SUMMARY
A theoretical analysis of a new technique for fluorescence lifetime measurement, relying on (near steady state) excitation with short optical pulses, is presented. Application of the technique to confocal microscopy enables local determination of the fluorescence lifetime, which is a parameter sensitive to the local environment of fluorescent probe molecules in biological samples. The novel technique provides good time resolution, since it relies on the laser pulse duration, rather than on electronic gating techniques, and permits, in combination with bilateral confocal microscopy and the use of a (cooled) CCD, sensitive signal detection over a large dynamic range. The principle of the technique is discussed within a theoretical framework. The sensitivity of the technique is analysed, taking into account: photodegradation, the effect of the laser repetition rate and the effect of non-steady-state excitation. The features of the technique are compared to more conventional methods for fluorescence lifetime imaging.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
Near field imaging: some attempts to define an apparatus function
D. Courjon Laboratoire d'Optique PM Duffieux, Associe au CNRS URA-214, UFR des Sciences et des Techniques, 25030 Besancon Cedex, France
SUMMARY
Near-field microscopy is a promising new tool capable of imaging details smaller than the wavelength. The mechanism of imaging is analysed and an overview of the apparatus functions which could be used to define an image quality criterion is given.
Apologies for sending such a long file at once. Please note that summaries will be posted less frequently as Gillian Wilson is now on maternity leave.
Gillian Wilson Executive Editor The Royal Microscopical Society Journal of Microscopy & 37/38 St Clements Proceedings of the RMS Oxford OX4 1AJ UNITED KINGDOM rms-at-uk.ac.ox.vax OR rms-at-vax.ox.ac.uk Telephone +44 (0)865 248768 Fax +44 (0)865 791237
I apologize for not being complete regarding the URL I gave on the SCIL-Image software. Here is a more precise specification of the path towards the information.
URL: http://www.tn.tudelft.nl
select : Pattern Recognition Group - Software Developments - SCIL-Image
Hope this saves you time.
-- Kees van der Wulp
******************************* ATTENTION *************************** * * * Change in INTERNET address ! * * * * From : vanderwulp-at-mbl.tno.nl * * To : vanderwulp-at-voeding.tno.nl * * * *********************************************************************
TNO - Nutrition & Food Research INTERNET : vanderwulp-at-voeding.tno.nl Division : Toxicology VOICE : +31 15 843101 Department : Genetic Toxicology FAX : +31 15 843989 PO-Box 5815 General TNO Info : http://www.tno.nl 2280 HV RIJSWIJK (NL) THE NETHERLANDS
Groeten, Gert van Antwerpen. TNO Institute of Applied Physics. P.O.Box 155, 2600 AD Delft, The Netherlands. Phone: +31 15-692226, Fax: +31 15-692111 Email: antwerp-at-tpd.tno.nl
-- Kees van der Wulp
******************************* ATTENTION *************************** * * * Change in INTERNET address ! * * * * From : vanderwulp-at-mbl.tno.nl * * To : vanderwulp-at-voeding.tno.nl * * * *********************************************************************
TNO - Nutrition & Food Research INTERNET : vanderwulp-at-voeding.tno.nl Division : Toxicology VOICE : +31 15 843101 Department : Genetic Toxicology FAX : +31 15 843989 PO-Box 5815 General TNO Info : http://www.tno.nl 2280 HV RIJSWIJK (NL) THE NETHERLANDS
1,4-Diazobicyclo(2,2,2)octane (Dabco) delays fading of immunofluorescence preparations Langanger, Gabriele; De Mey, Jan; Adam, Hans Mikroskopie 1983; 40 (7-8): 237-41 Language: German
Fading of immunofluorescence during microscopy. A study of the phenomenon and its remedy Johnson G D; Davidson R S; Mcnamee K C; Russell G; Goodwin D; Holborow E J J Immunol Methods 55 (2). 1982. 231-242
Regards, Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
I encountered the identical problem of dark caramel colored epon mix after addition of DMP-30, Substitution of the standard DDSA component with a new bottle of "Specially Distilled DDSA" corrected the problem and produced the golden colored resin mix. I was also told (see Marge Hukee's reply) the problem was a function of the pH of the resins
---------------------------------------------------------- Dr. Steven Barlow EM Facility/Biology Dept. San Diego State University San Diego CA 92182-0057 phone: (619) 594-4523 fax: (619) 594-5676 email to sbarlow-at-sunstroke.sdsu.edu
Because I still miss the original message and only received the additional message I posted (Re : 2D software for Unix), I repost the original correc- ted message to the microscopy list. I apologize for this 2nd message if you DID receive the first one somehow.
Dear Norm,
Regarding your question,
} } I ask for comments, recommendations, advice, for 2D image analysis software } for a UNIX platform (SGI Indy R4000). } } If there is interest I'll post a summary. Thanks. }
I can give you 2 URL's, leading to the same SCIL page, that provide you with a brief description of SCIL-Image (version 1.3), as well as an e-mail address of Mr. G. van Antwerpen whom I gave your address. He will take care of providing you with more information on SCIL-Image.
I am using SCIL-Image on my Personal IRIS 4D/35 for a couple of years now. It is an extensive 2D multi-layered image processing package (} 200 ip commands). It can be used on various platforms (Sun, HP, SGI, IBM, PC's, Mac's) and can be run from a mouse driven menu(1), a commandline interpreter (2) and from compiled routines (3) which makes it run very fast. It is a combination of a Standard C Interpreter Language (part 1) and an extensive Image processing library (part 2). It incorporates a complete program flow control mechanism and enables you to create and mix C- statements (and complete programs) with image processing commands. You can also easily extend the package with your own library of user created (compiled or C-source) routines and incorporate them into the mouse driven menu. I run it from my X-Terminal (Tektronix XP 27 and XP 356, colour terminals) in interactive as well as batch mode. I heard from mr. van Antwerpen that you can evaluate the full package for a period of 3 months at a rate of about US$ 250.= However for these kind of things please contact him, for I am only a user of the package.
URL: http://www.tn.tudelft.nl
select : Pattern Recognition Group - Software Developments - Scil-Image
or URL: http://galaxy.ph.tn.tudelft.nl:2000/Software/scil.html
e-mail address G. van Antwerpen : antwerp-at-tpd.tno.nl
All people interested in more info can get it from him.
Good luck,
-- Kees van der Wulp
TNO - Nutrition & Food Research INTERNET : vanderwulp-at-voeding.tno.nl Division : Toxicology VOICE : +31 15 843101 Department : Genetic Toxicology FAX : +31 15 843989 PO-Box 5815 General TNO Info : http://www.tno.nl 2280 HV RIJSWIJK (NL) THE NETHERLANDS
We have developed a new method for fast data transfer from a JEOL microscope connected to a LINK eXL computer system to DOS based personal computers.
In our institute we are using a JEOL JSM 6400 SEM equipped with an energy dispersive detector driven by a LINK eXL computer running the GENIE operating system.
In former days we experienced great problems transferring images captured from the screen and stored in tiff format to DOS based computers, since we neither posses any GENIE image processing software nor do we have network connectivity on the Link system.
That's why we have developed a unique hardware/software solution which allows users to transfer tiff files at the rate of approx. 1 sec. per file (262 KB) from the Link computer to the DOS computer.
The software is capable of viewing the contents of GENIE formatted floppy disks, hard disks, MO's and changeable devices (e.g. Syquest drives) and copying selected images and spectra to the target computer.
Anyone interested may contact us for further details at:
I publish a monthly newsletter, Analytical Consumer, that reports on customer satisfaction with manufacturers of analytical equipment. We are currently doing a survey of TEM users, similar to an SEM study we published last July, in conjunction with Microscope News & Technology (who is also collaborating on this study). I am looking for labs with TEMs who would be willing to talk about their equipment.
I'm looking for:
What kind of TEM (manufacturer and model) do you use? How old is it (roughly)? Why did you buy from that particular manufacturer? Have you been satisfied with the equipment? Are you satisfied with the service? Do you get service from the equipment mfr, do it yourself, or have an outside service supplier? Do you have a service contract? What kind of samples do you typically use the TEM for? What kind of lab are you in? R&D, research, instrument facility, service lab, or ...? What kind of company is the lab?
If you answer these questions and want a copy of the survey, please include your address. If you would like to talk about it in person, give me a call at (508) 369-9079, or send me your phone number and I'll call you.
Thanks!
Jo Rita Jordan Editor and Publisher Analytical Consumer
jjordan-at-world.std.com or 76150.2171-at-compuserve.com
MICROSCOPY CONFERENCE (In conjunction with the 18th National Conference of the New Zealand Society for Electron Microscopy)
Dunedin, New Zealand 4th - 8th September 1995.
A Microscopy Conference will be held in Dunedin, 4th to 8th September, 1995. The conference will be held in conjunction with the 18th National Conference for the New Zealand Society for Electron Microscopy (NZSEM) however will cover all aspects of Microscopy with emphasis on the techniques of both Light Microscopy and Electron Microscopy.
The venue for the conference will be the Otago Medical School.
Workshop sessions will run on Monday and Tuesday (Sept 4th - 5th); the conference proper will run from Wednesday until midday Friday (Sept 6th - 8th).
Guest speakers include:
Professor John M Robinson, Ohio State University, U.S.A. Professor Robinson has published widely on the practical aspects of enzyme cytochemistry (in particular the recently developed cerium-based techniques) and immunocytochemistry. His application of these techniques utilises many forms of microscopy including conventional light microscopy, confocal microscopy, transmission electron microscopy and scanning electron microscopy.
Professor Anthony S-Y Leong, University of Adelaide, Australia. Professor Leong is well known for his work with microwave techniques, both at the light microscope and the electron microscope level. His application of microwave technology includes fixation and processing for L.M. and T.E.M., and the use of microwaves in immunocytochemistry.
Dr Brian Brooker, Institue of Food Research, England. Dr Brooker is a food scientist particularly interested in the structure and behaviour of oil-in-water emulsions and the influence of emulsifiers on their functions. He applies many microscopy techniques to study these difficult samples including conventional light microscopy, confocal microscopy, freeze fracture, X-ray microanalysis, cryofixation and electron microscopy.
Dr Brendon Griffin, Centre for Microscopy and Microanalysis, Perth, Australia. Dr Griffin's field of interest are the microscopy of rare minerals. He is experienced in microprobe analysis particularly EDS, environmental SEM and field emission SEM. He has a particular interest in EM education. Dr Griffin is currently president of the Australian Society for Electron Microscopy.
Trades displays will be a feature of the Conference. We are also planning a techniques forum with the invited guests forming the panel.
If you are interested in receiving more information about this conference you are invited to contact the organising committee at the address below;
Allan Mitchell Organising Committee, 1995 Microscopy Conference C/- Department of Anatomy and Structural Biology Otago Medical School P.O. Box 913 Tel; National 03 479 7301 International 64 3 479 7301 Dunedin Fax; National 03 479 7254 International 64 3 479 7254 New Zealand. email address; allan.mitchell-at-stonebow.otago.ac.nz
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
I publish a monthly newsletter, Analytical Consumer, that reports on customer satisfaction with manufacturers of analytical equipment. We are currently doing a survey of TEM users, similar to an SEM study we published last July, in conjunction with Microscope News & Technology (who is also collaborating on this study). I am looking for labs with TEMs who would be willing to talk about their equipment.
I'm looking for:
What kind of TEM (manufacturer and model) do you use? How old is it (roughly)? Why did you buy from that particular manufacturer? Have you been satisfied with the equipment? Are you satisfied with the service? Do you get service from the equipment mfr, do it yourself, or have an outside service supplier? Do you have a service contract? What kind of samples do you typically use the TEM for? What kind of lab are you in? R&D, research, instrument facility, service lab, or ...? What kind of company is the lab?
If you answer these questions and want a copy of the survey, please include your address. If you would like to talk about it in person, give me a call at (508) 369-9079, or send me your phone number and I'll call you.
Thanks!
Jo Rita Jordan Editor and Publisher Analytical Consumer
jjordan-at-world.std.com or 76150.2171-at-compuserve.com
I publish a monthly newsletter, Analytical Consumer, that reports on customer satisfaction with manufacturers of analytical equipment. We are currently doing a survey of TEM users, similar to an SEM study we published last July, in conjunction with Microscope News & Technology (who is also collaborating on this study). I am looking for labs with TEMs who would be willing to talk about their equipment.
I'm looking for:
What kind of TEM (manufacturer and model) do you use? How old is it (roughly)? Why did you buy from that particular manufacturer? Have you been satisfied with the equipment? Are you satisfied with the service? Do you get service from the equipment mfr, do it yourself, or have an outside service supplier? Do you have a service contract? What kind of samples do you typically use the TEM for? What kind of lab are you in? R&D, research, instrument facility, service lab, or ...? What kind of company is the lab?
If you answer these questions and want a copy of the survey, please include your address. If you would like to talk about it in person, give me a call at (508) 369-9079, or send me your phone number and I'll call you.
Thanks!
Jo Rita Jordan Editor and Publisher Analytical Consumer
jjordan-at-world.std.com or 76150.2171-at-compuserve.com
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