A postdoctorial position is available for work on the structure of fibers of sickle cell hemoglobin (HbS) and the red cell cytoskeleton.
RED CELL CYTOSKELETON Red cells are able to elastically deform. The extraordinary importance of this property can be appreciated from recalling that, although the human red cell is a biconcave disk eight microns in diameter, it can easily pass through capillaries half this size. This remarkable feat is accomplished by the red cell transiently changing its shape during passage after which its well- known biconcave form spontaneously and nearly instantly recovers. We are studying the structural basis of this property using cryoelectron microscopy.
McGough, A. and Josephs, R. (1990) On the structure of erythrocyte spectrin in partially expanded skeletons. Proc. Notl. Acad. Sci. USA 87: 5208-5212.
Li Li and Robert Josephs. Cryo-Electron Microscopy of Human Erythrocyte Membrane Skeletons. Proceedings of the 51st annual meeting of the Microscopy Society of America San Francisco Press, San Francisco. p. 116 (1993).
SICKLE CELL HEMOGLOBIN Sickle cell anemia is caused by the intracellular polymerization of sickle cell hemoglobin to form rod-like fibers. A knowledge of the fiber structure could be used for the design of an agent that could block fiber formation. We have combined the structure of hemoglobin (known from X-ray crystallography) and the molecular coordinates of the fiber (determined from electron microscopy) in order to synthesize a model which shows the intermolecular contacts of the fiber. This approach has allowed us to determine the contacts which form between molecules in the fiber. We are now studying how various mutations (some obtained by site directed mutagenesis) affect fiber structure. Such studies are expected to account for the structural and chemical properties of fibers in terms of intermolecular interactions.
Watowich, S., Gross L., and Josephs, R. (1989) Intermolecular Contacts within Sickle Hemoglobin Fibers. J. Mol. Biol. 209: 821-828.
M. R. Lewis, L. J. Gross, and R. Josephs. Variable Pitch in Frozen Hydrated Sickle Hemoglobin Fibers: An Image Analysis Model Study. Ultramicroscopy, 56 303-317 (1994).
Michael R. Lewis, Leon J. Gross, and Robert Josephs. Cryo- Electron Microscopy ofDeoxy-Sickle Hemoglobin Fibers. Microscopy Research and Technique 27 459 - 467 (1994).
Interested individuals please contact Robert Josephs The University of Chicago 920 East 58th Street Chicago, IL 60637 E-Mail Bob-at-befvax.Uchicago.Edu
Dr. P.Mummery from materials at Oxford , UK Dr. G. Smith from Shell Research Labs , Chester, UK
Thank you jjm Professor John J. Millar Head, Department of Applied Physics Electron Microscope Unit RMIT, GPO Box 2476V, MELBOURNE AUSTRALIA 3001 +613 660 2602 fax 613 660 3837 email jjmill-at-rmit.edu.au
This message is in MIME format. The first part should be readable text, while the remaining parts are likely unreadable without MIME-aware tools. Send mail to mime-at-docserver.cac.washington.edu for more info.
The following contains the compiled information that participants sent me regarding a Quantitative Image Analysis (Q-IA) mailing list.
It is divided into 4 sections. 1. ID#: Name and email address 2. Questions and comments made by participants 3. Q-IA that participants are currently ABLE to perform. 4. Computer and Software used
Immediately afterwards is my suggestion of where a (temporary?) Q-IA mailing list should be--and my reasons for choosing this site.
SECTION 1: NAME & EMAIL ADDRESS --------------------------------
ID# Name e-mail 1 ? smejkag-at-ccsmtp.ccf.org 2 Alan Hall hall-at-scientia.up.ac.za 3 Alex Almasan almasaa-at-athene.hh.ri.ccf.org 4 Alistair Young a.young-at-auckland.ac.nz 5 Andreas Becker becker-at-ps1515.chemie.uni-marburg.de 6 Bo Johansen boj-at-bot.ku.dk 7 Brain Wade bwade-at-ux1.cso.uiuc.edu 8 Carolyn Smith clsmith-at-codon.nih.gov 9 Chang Seng chang-at-newton.umsl.edu 10 Cheng-Lun Na na-at-utsw.swmed.edu 11 Craig Daly cdaly-at-biomed.gla.ac.uk 12 David A. Jans daj224-at-huxley.anu.edu.au 13 David boyle david.boyle-at-gensia.com 14 David Morilak morilak-at-uthscsa.edu 15 Dominique Bataille Bataille-at-citi2.fr 16 Don-Carlos Abrams don-at-rpms.ac.uk 17 Donna Wagahoff DWAGAHOF-at-wpsmtp.siumed.edu 18 Doug Chaytor doug.chaytor-at-dal.ca 19 Douglas L. Rosene drosene-at-bu.edu 20 Edgar Riverdal edgarr-at-labmed.uio.no 21 Eric Kokko KOKKO-at-EM.AGR.CA 22 Feona Hansen-Smith hansensm-at-oakland.edu 23 Gary Krichau gkrichau-at-unlgrad1.unl.edu 24 Gert van Cappellen vanCappellen-at-endov.fgg.eur.nl 25 Glenn Holm karuzis-at-wccf.mit.edu 26 Gordon Robertson robertson-g-at-vanlab.paprican.ca 27 Hakan Hall hakanh-at-psyk.ks.se 28 Jakob Walter jhwpatho-at-zedat.fu-berlin.de 29 James Crandall jcrandall-at-shriver.org 30 Jeff Reece reece-at-niehs.nih.gov 31 Jeffrey Kopp jbkopp-at-nih.gov 32 John F Smiley JSmiley-at-nwu.edu 33 Judy Trogadis judy-at-camtwh.eric.on.ca 34 Kate Whitley k.whitley-at-ucl.ac.uk 35 Lauran Oomen lauran-at-nki.nl 36 Marc Brande brande-at-SDSC.EDU 37 Maxx Abraham abraham-at-atax.eng.uab.edu 38 Michael Binks reba999-at-ucl.ac.uk 39 Michael J. Herron herro001-at-maroon.tc.umn.edu 40 Michael Rudisill mrudisil-at-hsc.usc.edu 41 Mike Esteman mikee-at-lilly.com 42 Norm Granholm Norman.Granholm-at-UC.Edu 43 Paul C. Dolber dolber-at-cs.duke.edu 44 Paul Goodwin pgood-at-fred.fhcrc.org 45 Petra Nederlof nederlof-at-genmic.biochem.mpg.de 46 R. Thomas Zoeller tzoeller-at-bio.umass.edu 47 R. Wade Schuette schuette-at-eecs.umich.edu 48 Richard Thrift RichardT-at-Depotech.com 49 Rosemary White r.g.white-at-sci.monash.edu.au 50 Stephen Helfer s.helfer-at-rbge.org.uk 51 Steve Rogers srogers-at-delphi.beckman.uiuc.edu 52 Steven Bagley steven.bagley-at-man.ac.uk 53 Ted Gaten gat-at-leicester.ac.uk 54 Vlasta P Spongr spongr-at-acsu.buffalo.edu 55 Ze'ev Silverman szeev-at-bgumail.bgu.ac.il
ID# Name question 1 ? 2 Alan Hall ? how quantify RNA from material embedded in Quetol with oso4? 3 Alex Almasan 4 Alistair Young ?what's good embedding compoud for confocal } 100um? 5 Andreas Becker quantitative PCR techniques 6 Bo Johansen 7 Brain Wade interest: quantify chlorophyll 8 Carolyn Smith 9 Chang Seng Semper6 plus is programmable IA software 10 Cheng-Lun Na ? Any EM autoradiography analysis software for Mac? 11 Craig Daly 3D of blood vessels; receptor distribution 12 David A. Jans interest: quantify flourescent labelled nuclear protein 13 David boyle Quantified radiolabelled tissue 14 David Morilak 15 Dominique Bataille ? how quantify stained antigen? 16 Don-Carlos Abrams 17 Donna Wagahoff 18 Doug Chaytor 19 Douglas L. Rosene ?how create standards for absolute IHC quantification? 20 Edgar Riverdal ? how quanify flourescent staining? 21 Eric Kokko 22 Feona Hansen-Smith 23 Gary Krichau 24 Gert van Cappellen ?how quantify IHC and in-situ hybridization? 25 Glenn Holm autoradiograms; cell counting and classification 26 Gordon Robertson 27 Hakan Hall autoradiograms; receptor distribution and densities 28 Jakob Walter ?macro for count labelled cells? ?est. MW with NIH? 29 James Crandall 30 Jeff Reece interest: [Ca++] in live cells 31 Jeffrey Kopp ? what microscope and CCD camera to buy? 32 John F Smiley quanitify IHC, 2 approaches: area stained; line intercept 33 Judy Trogadis recently published quantitative flourescence of receptors 34 Kate Whitley ? how quantify flourescent staining? 35 Lauran Oomen FISH analysis; enzyme levels 36 Marc Brande 37 Maxx Abraham Goal: automated flourescence immunoassay system 38 Michael Binks 39 Michael J. Herron 40 Michael Rudisill ? how quantify flourescence? 41 Mike Esteman 42 Norm Granholm ?advice on software? 43 Paul C. Dolber ? comments on background subtraction and calibration? 44 Paul Goodwin 45 Petra Nederlof ? how quantify flourescence? 46 R. Thomas Zoeller ? is computer enhancement of a signal legitimate? 47 R. Wade Schuette 48 Richard Thrift Interest: quantify hydrogen ions 49 Rosemary White ? how best quantify WB? 50 Stephen Helfer cell (fungal/plant) measurments 51 Steve Rogers 52 Steven Bagley 53 Ted Gaten ? how quantify dot blots? 54 Vlasta P Spongr 55 Ze'ev Silverman ?how quantify silver grains over cells?
SECTION 4: COMPUTER & SOFTWARE USED --------------------------------------
ID# computer software 1 Mac NIH 10 Mac NIH 12 Mac NIH 15 Mac NIH 18 Mac NIH 20 Mac NIH 23 Mac NIH 27 Mac NIH 28 Mac NIH 31 Mac NIH 32 Mac NIH 38 Mac NIH 39 Mac NIH 41 Mac NIH, IPLab, Prism; Image Pro Plus 43 Mac NIH 46 Mac NIH; BioQuant 51 Mac NIH; Photoshop, Analyze 53 Mac NIH 55 Mac NIH 5 Mac, PC NIH 6 Mac, PC NIH, Image Pro Plus, Image-1 7 Mac, PC NIH, Inspector 8 Mac, PC NIH; Image 1 13 Mac, PC NIH; V150 19 Mac, PC NIH; Inquiry, Bioquant 24 Mac, PC ImageQuant; Zeiss CLSM 34 Mac, PC NIH, Photoshop; Foster Findlay's PCImage 40 Mac, PC NIH, Photoshop, Zeiss CLSM, MathLAB 11 Mac, PC, SGI NIH, MetaMorph, Imaris & MicroVision 30 Mac, PC, SGI NIH, MetaFluor, Zeiss CLSM, VoxBlast, AVS 35 Mac, PC, SGI NIH, SCIL-Image, Photoshop 49 Mac, PC, SGI Own 52 Mac, PC, SGI NIH, PC Image, Semper,AVS 9 Mac, PC, SGI, Sun Semper6 Plus 37 Mac, PC, SGI, Sun NIH; Matlab, PV-Wave, Image Analyst 44 Mac, PC, SGI, Sun NIH, OPTIMAS, ImageQuant, ISee, Prism 16 Mac, PC, Sun Kontron, ContextVision +own 4 Mac, SGI NIH, Voxelview, Photoshop, 3DVIEWNIX 36 Mac, SGI NIH 45 Mac, SGI NIH +others 47 Mac, Sun NIH, Khoros, MATLAB, IMagist-II 17 PC Bioquant and MCID 22 PC Optimas 25 PC Biocom (French commerical software) 29 PC Eutectics NTS; IBAS 48 PC Optimas 4.0 50 PC DataCell OPTIMAS 54 PC Photoshop 33 PC, SGI, NextStep BioRad confocal software, ICAR (3D) 21 PC, Unix Visilog, PCI2 26 IBAS 2 3 14 42
--------------------
From SECTION 4, most if not all participants with Macintosh computers are using NIH Image software (some in combination with Photoshop), while participants using PC or Unix (SGI or SUN) stations do not seem to share a common software package. It seems apparent that most participants (42/55) are familiar with NIH Image. [Admittedly, these results may be biased since I could not find a mailing list for SGI or SUN users doing imaging. If you know of one, please let me know.]
For this reason, I suggest that we use the NIH Image mailing list as ONE centre for Q-IA discussion. If a PC or UNIX mailing list performing image analysis exists, I would certainly join in.
To subscribe to the NIH Image mailing list:
send an email message to
listserv-at-soils.umn.edu
with a message containing
"subscribe nih-image FIRSTNAME LASTNAME"
Obviously, there are many experienced and knowledgable people that have been successful in certain areas of quantitative image analysis as evidenced by this impromptu survey. I hope your questions can be answered now that you know where (and who) to ask.
By the way, may I suggest that if you are asking a question regarding quantitative image analysis, that you put "Q-IA: ..." as part of the subject. And, finally, I am trying (if time permits) to create a Q-IA Web Page. If you have any suggestions as to what I should put on it, feel free to email me directly.
Looking forward to reading your comments.
Clint Young Department of Psychiatry University of British Columbia
The Belgian Society for Microscopy is rapidly expanding. In order to reestablish contacts with fellow countrymen and -women abroad, we kindly ask all Belgians who are doing research in the field of any microscopy technique (EM, AFM, STM, NFOM, AM, IRM, NMRM, .....) to leave their coordinates with us so we can inform you of the activities of our society. In Septemeber 1995 a leaflet including a return slip for membership will be mailed to everyone in our files. I hope te hear from you soon,
Nick Schryvers co-secretary BVM-SBM RUCA, University of Antwerp Groenenborgerlaan 171 B-2020 Antwerp Belgium tel: 32-3-2180247 fax: 32-3-2180257 e-mail: schryver-at-ruca.ua.ac.be
EPMA=Jeol 733; 15Kv; probe curr 200x10E-07A. While doing spot (2um) analysis on a metal alloy Fe, Ti, C and O it ocurred that the electron beam did not hit the target beneath the optical cross of the binoculars. It hit a point about -70um on the x-axis, i.e. to the left of the cross. Double CHECKING the probe's alighnment on ZrO2 fluorescent standard confirmed nothing wrong there, and the spot precisely beneath the cross. Focussing on a fine crack of the brass sampleholder showed a drop in Cu-counts just when the crack was beneath the cross; spot still beneath the optical cross.
The sample was earthed with, maybe a bit excessive amount, of SILVER DAG to the sample holder. Watching simultaniously two ratemeters monitoring Cu and Ag, it was evident that the SILVER DAG DEFLECTS THE BEAM AWAY FROM THE SILVER MASS. Checking with a different spot af silver, one can deduct that the amount of deflection relates directly to the amount of silver. The larger the silver mass the greater is the deflection. The silver dag in question is not from my usual supplier, and not my brand of colloidal silver I am normaly using.
CAN ANYONE PLEASE HELP EXPLAINING THIS PHENOMENON. Thanks. Leon.
} \\\\\\\\\\\\\\\\\\\\\} WWW: http://www.puk.ac.za} \\\\\\\\\\\\\\\\\\\\\\} } Leon Smuts-Electronmicroprobe-University of Potchefstroom-South Africa} } ///////////////////} e-MAIL: plbls-at-puknet.puk.ac.za} ///////////////////}
Dear Microscopists, } } } From what source might we be able to obtain some crystalline graphite, in } order to prepare a thin graphite support film, as described in an article in } _Micron_, 1977, vol. 8:41-46 by Sumio Iijima? } } We've tried Ted Pella, SPI, Ernest Fullam, and Union Carbide. No luck. } } Any leads would be appreciated, } } Yolande Berta } School of Materials Science and Engineering } Georgia Tech } yolande.berta-at-mse.gatech.edu } }
You may try:
Sigri Great Lakes Carbon Corp., Specialty Graphite
Bay City, MI 48708 USA 800-248-8218 _Call Toll Free. 517-895-7740 _FAX
To all the microscopy list:
One great place to search on the Internet is to register to the Thomas Register of American Manufacturers at http://www.thomasregister.com/
After registration, it's free to use the "Supplier Finder" to search for products or for company names Georges Veilleux Researcher INRS-Energie et materiaux C.P. 1020 Varennes, Qc Canada J3X 1S2
The University of Chicago is looking for a new home for the following SEM: a JEOL JSM-35U, equipped with a two-element annular solid state backscattered electron detector, a beam current stabilizer, a liquid nitrogen baffle, a liquid nitrogen cold finger and a Kevex 7077 (PDP 11-23) x-ray microanalysis system with a horizontal-entry (motor-driven) Si-Li detector with a Be window. The SEM vacuum system has been upgraded with two Edwards E2M8 direct drive rotary pumps and an Edward Diffstak diffusion pump. The SEM and EDS system are in good condition and the photo CRT was replaced two years ago. The University expects some cost recovery to aid in acquisition of a new SEM-EDS system. If interested, please contact directly (i.e., do not send a message to the Microscopy Listserver!):
Andrew M. Davis Enrico Fermi Institute University of Chicago 5640 South Ellis Avenue Chicago, IL 60637 312-702-8164 312-702-9505 fax a-davis-at-uchicago.edu
The Department of Plant Sciences, University of Cambridge has been awarded a BBSRC Case studenship starting in October 1995 which will be co-directed with Dalgety plc Food Technology Centre in Cambridge. Dalgety plc is a major international company wide wide interests in food and agriculture. Thge studentship will centre on the analytical microscopy and biochemistry of the structure and cellular location of soluble and non-soluble non-starch polysaccharides in cereals and pulses in relation to their role in the processing of food stuffs. Candidates holding or expecting a first or upper second degree in a relevant subject should apply as soon as possible to Patrick Echlin Director Multi-Imaging Centre, Department of Plant Sciences, University of Cambridge, Downing Street Cambridge CB2 3EA UK. Tel; +44-1223-333946. Fax; +44-1223-333953.Send CV and names of two academic referees. Successful candidates will receive an additional payment of #2250 per annum and will be elible to participate in Dalgety's internal management training programme. Closing date for applications is July 20 1995, and interiews are scheduled for July 24/25. The Multi-Imaging Centre has unparalleled facilities for coherent light and high energy beam microscopy and in situ analysis. If you are a biochemist, a food scientist of a plant scientist, we can teach you the analytical microscopy.
Patrick Echlin Director, Multi-Imaging Centre School of Biological Sciences University of Cambridge
Does anyone know if there is any EM autoradiography analysis software available for Macintosh? Any comment is welcomed.
Cheng-Lun Na Cell Biology and Neuroscience UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75235-9039 PH: (214) 648-3597 (214) 648-2032 E-Mail: na-at-utsw.swmed.edu
Cheng-Lun Na Cell Biology and Neuroscience UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75235-9039 PH: (214) 648-3597 (214) 648-2032 E-Mail: na-at-utsw.swmed.edu
I am in the initial stages of gathering information on TEM and SEM negative scanning/digitization and the subsequent printing of these images. I'd appreciate receiving your thoughts on how best to approach these topics, who manufactures such scanners, which are best for EM negs, which software should be considered, should I be considering a souped up laser printer or some other type (dye sublimation, etc.).
Any input regarding these topics would be quite helpful and greatly appreciated. Thanks in advance.
John
___________________________________________________ | | | John G. Aghajanian, Ph.D. | | Worcester Foundation for Experimental Biology | | 222 Maple Avenue | | Shrewsbury, MA 01545-2737 | | | | Tel: 508 842-8921 ext. 147, 161 | | Fax: 598 842-9632 | | JOHNA-at-sci.wfeb.edu | | | |_________________________________________________|
In response to Frank Wubbolts' question regarding transfer of the frame store image from a JEOL JSM-5400 to a PC:
Yes, this can be done, assuming the 5000 series frame store is similar to the 6000 series SEM. We have a 6300F equipped with such a system made by JEOL USA. They make two systems. ARC (for image archiving) saves the image as a TIFF file which can be saved to a floppy, optical disk, or hard drive. The Vision system also does archiving, plus it allows control of the microscope via an external PC.
You might first check with your JEOL representative in the Netherlands to see if a comparable system is marketed there. If not, try contacting Steve Hamilton at JEOL USA in Peabody, MA at 508 535-5900.
Good luck! Jim Stets Air Products and Chemicals, Inc. Allentown, PA stetsjr-at-apci.com
Dear colleagues I am having a great deal of difficulty locating water soluble PVA mol wt 10,000 as used by Tokuyasu (1989). The suppliers approached carry PVA only in excess of 14,000 mol wt, or products which have a purity between 10 and 30K mol wt. Can anyone help with advise?
Thank you.
James Wesley-Smith EM Unit University of Natal Durban, South Africa James Wesley-Smith Electron Microscope Unit George Campbell Building University of Natal Durban, South Africa
Dear John, You wrote about finding the best equipment for scanning & printing. I'd like to say that the best equipment can depend on what kind of nega- tives you need to scan. I do electron crystallography, so I need accurate quantitation of diffraction patterns. These patterns have a lot of light area with occasional spots of high OD. In order to get what I need, the scanner has to be one with a small light source so that the area surrounding the dark spot doesn't transmit any light to the detection system. We have a Perkin-Elmer 1010A, which works quite well for this, but there are other brands with which I have had no experience. Linear diode arrays and CCD cameras are not good for quantitating ED patterns, but they are much fas- ter for scanning ordinary EM images. Yours, Bill Tivol
We have had very good results permeabilizing cells for immunochemistry using Brij 58 for 1-5 minutes prior to fixation and immunochemistry. About 1% of cells are totally destroyed, about 25% don't permeabilize, but the remaining cells label nicely with fluorescent antibodies and even 15 nm Gold conjugated antibodies. See Schliwa et al PNAS, USA 78:4329-33, 1981 for details on Brij. Hope this helps-
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 * **************************************************************
On Fri, 7 Jul 1995, Felicity Lawrence wrote:
} Hi Everybody! } } I'm a fairly new confocalist (we have recently bought a Leica TCS 4d with } inverted microscope). A researcher here wants to localise internalised } structures in BHK cells infected with dengue virus. He initially fixed the } cells with cold acetone for 1 minute but this caused dissolution, and hence } total loss of structure, of the membrane. To minimise damage to the } membrane, he tried fixing in ethanol. Unfortunately, the membrane remained } intact, so much so that the antibody-FITC conjugate couldn't penetrate. He } is now talking about slam freezing his cells, sectioning them in a } cryoultramicrotome, followed by labelling. This seems tedious to say the least. } } Can someone recommend a fixation agent/schedule to give both adequate } morphology and adequate access also? } } Many thanks, } } Felicity Lawrence } Analytical Electron Microscopy Facility, } QUT, Brisbane. Australia. }
We've been using Panasonic 500 MB and 1 GB optical disks (actually magneto-optical, or so I've been told) for over one year with excellent results. We ordered our JEOL VISION package with the Panasonic Drive installed. This is used in a multi-user lab. So far several dozen users have purchased the 500 MB disks and have had excellent results. Nobody seems to want 1000 images on one disk, so the 500 MB version is the most popular. They are read-write many times. The disks have come down in price recently with the 500 MB version selling for $89.79 from Media Source, telephone 800-241-8857. Stanley L. Flegler Center for Electron Optics Michigan State University flegler-at-pilot.msu.edu
I need to demonstrate that routine TEM prep. methods, ie. GA fixation, OsO4 postfix, ethanol dehydration, and spurrs embedding, can be employed to preserve and demonstrate the presence of synthetic polymers: polymer gels, polyacrylamide gels, polyhema, and the like.
I am wondering if any of you microscopists out in hyperspace are doing anything like this and would share your methods, experiences, references, and so on.
Thanks in advance for any help.
John
___________________________________________________ | | | John G. Aghajanian, Ph.D. | | Worcester Foundation for Experimental Biology | | 222 Maple Avenue | | Shrewsbury, MA 01545-2737 | | | | Tel: 508 842-8921 ext. 147, 161 | | Fax: 598 842-9632 | | JOHNA-at-sci.wfeb.edu | | | |_________________________________________________|
Does anyone out there know if the National Society for Histotechnology has a bulletin board similar to this one, and how to access it? Thanks-Reply directly. Grace Kennedy
There's an ad for a TEM technician at NIH (Bethesda, MD) on page 1963 of the latest issue of Science. Check it out if interested.
Bob
Robert R. Wise, PhD Director, UWO Electron Microscope Facility Department of Biology University of Wisconsin Oshkosh Oshkosh, WI 54901 (414) 424-3404 (414) 424-1101 fax wise-at-vaxa.cis.uwosh.edu
Someone just brought in a (rather sketchy) protocol for freeze-substitution of mammalian tissue, utilizing tetrahydrofuran (THF) or diethyl ether, followed by araldite embedment. She will be cryofixing tissue from a marine invertebrate, probably by plunging into liquid propane. Our main question (for now) is, Do we just start adding Araldite to the solvent, using a "normal" schedule? Will the Araldite mix well with either solvent? Do any of you forsee any problems?!
We are going this route to perform some EDX analysis. Any hints and tips about any part of the proposed procedure will be greatly appreciated!
Thanks in advance! Aloha, Tina Weatherby Carvalho Biological EM Facility University of Hawaii
Subject: Time: 11:51 AM OFFICE MEMO Removable storage Date: 7/8/95
A posting came through on the listserver the other day that I managed to discard, but it involved a question of removable storage, I think using magneto-optical technology. I thought it would be useful to alert members of the microscopy community, esp. those overseas (or who have been hibernating for the last several months), to the hottest new removable storage drive system currently available, made by Iomega. The Iomega Zip drive costs $200 and uses 100 Mbyte 3.5" disks which cost $15 each in a 10-pack, or $20 singly. These drives are shipping now (but the high demand means that you will undoubtedly find them backordered). Coming in Sept. is the Iomega Jaz drive for $500, which will offer a 1.3 gigabyte disk, based on Winchester technology (fast), for $100 each. For many microscopy labs, the capabilities offered by these devices would fill a niche for image storage, transfer, portability, convenience, etc. at very affordable prices. The Zip disks may become an industry standard, replacing floppy diskettes, in the forseeable future. A number of outside users of our laboratory are going to be taking their large image files home using this technology--it seems like the way to go for microscopists. BTW--To qualify this post, I also own stock in the company, and if you are smart, you'll buy some too!! (Larry's hot tip of the day!)
Hi, Missed the original post, but just saw Jay Jerome's note regarding methods of cell permeabilization. A second reference to such a procedure for plant material is: Meiners, S. et al. Planta 1991 184:443-447. The material used to successfully permeabilize root cells was saponin, a plant gylcoside, available from Aldrich (cat #S130-2). Saponin, which is rather nasty to work with, apparently is effective by itself, that is, it is not necessary to use pectinase or cellulase in addition to the saponin. The paper reports the incorporation of both FITC-conjugated dextrans as well as FITC-conjugated antibodies.
Good luck!
Dwight Beebe Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca Dept. de sciences biologiques Voice:514-872-4563 Universite de Montreal FAX:514-872-8496 4101, rue Sherbrooke est Montreal, PQ H1X 2B2 Canada
I would not go investing my money in this "storage fad"! Like all the Winchesters, Bernoulli, Syquests and Flopticals... it s magentic media and will be obsolete in the next year. Most of the forementioned are already! The only one with a true advantage is Syquest. For 2 reasons its reliability and speed. (If we all agree that 14ms is fast). In any event, when rewritable optical can obtain such competitive speeds as current Hard Drives and room temperature blue lasers can pack over 2 Gigs on a $10 piece of media then we have something to invest in. However ZIP's are just OK (and that is if you can get one; they are back-ordered everwhere probably until the next temporary "fad" hits the mail-order section of your favorite vendor). BTW- I don't have stock in any of them nor advocate their use, but I have used a 105MG Syquest for 4 years and never had a problem with a cartridge and its actually faster than my stock Hard Drive that came with the Macintosh. ZIP drives are slower than Magneto Opticals... and that is really slow! Sincerely, Larry Kordon Nikon, Inc.
I'm struck by the number of "subscribe" and "unsubscribe" messages which are broadcast to all subscribers. The "subscribe" ones I can understand to a degree, people starting to subscribe don't necessarily know much about it, but WHY, oh WHY, do we get so many "unsubscribe" messages.
For crying out loud:
TO UNSUBSCRIBE, send an email message to:
LISTSERVER-at-AAEM.AMC.ANL.GOV
message should be:
UNSUBSCRIBE MICROSCOPY YOURID-at-YOURADDRESS
DON'T SEND IT TO MICROSCOPY-at-AAEM.AMC.ANL.GOV, OR IT GETS BROADCAST TO EVERYONE ON THE MAILING LIST
It's pretty simple isn't it?
Ritchie Sims Geology Department University of Auckland Auckland, New Zealand
Check out our new Home Page at Michigan with link to much microscopy info. URL=http://www.engin.umich.edu/microscopy/ OK?
Jfm
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, John.F.Mansfield-at-umich.edu or jfmjfm-at-umich.edu they all reach me! URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html
} } Check out our new Home Page at Michigan with link to much microscopy info. } } URL=http://www.engin.umich.edu/microscopy/ } } OK? } } } } Jfm } } } } } I get a "not found" message if I try to use the URL: } } http://www.engin.umich.edu/microscopy/ } Whoops it should have been: } URL=http://www.engin.umich.edu/microscopy I'll try and fix it so both versions work. Until then the URL should be: } URL=http://www.engin.umich.edu/microscopy
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, John.F.Mansfield-at-umich.edu or jfmjfm-at-umich.edu they all reach me! URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html
A postdoctoral position is available immediately involving research in the microstructural characterization of hard magnetic materials, magnetic thin films, and nanoparticles. The research involves extensive Transmission Electron Microscopy studies on a wide variety of materials using JEOL JEM 2000 FX and JEOL JEM 100C TEM's, and an AMRAY 20 SEM. Candidates must have experience in conventional and high resolution TEM, Lorentz microscopy, energy dispersive X-ray analysis, and X-ray diffraction. Appointment is for one year. Send a resume and name, address, phone, and FAX of 3 references by August 1, 1995 to: Professor George C. Hadjipanayis, Dept. of Physics and Astronomy, University of Delaware, Newark, DE 19716-2570, USA, FAX 302-831-1637.
The UNIVERSITY OF DELAWARE is an Equal Opportunity Employer which encourages applications from qualified minority groups and women.
Although I have many years of experience in TEM, I have never done any electron diffraction studies. Could anyone who has experience in that field point me to a comprehensive text on electron diffraction: how and why it works, procedures, interpretation of patterns etc.? It looks like I will be going to try some steps in that direction.
Thanks for your time and help!
Herman Meekes Biological Sciences ______________ ______________ University of Missouri ---__ \ / __--- 109 Tucker Hall ------__\---/__------ Columbia, MO. 65211, USA \( )/ Tel: 314-882-0171 V Fax: 314-882-0123 / \ e-mail: hmeekes-at-biosci.mbp.missouri.edu /___\
} } Dear list readers! } } Although I have many years of experience in TEM, I have never done any } electron diffraction studies. Could anyone who has experience in that field } point me to a comprehensive text on electron diffraction: how and why it } works, procedures, interpretation of patterns etc.? It looks like I will be } going to try some steps in that direction. } } Thanks for your time and help! } Dear Herman,
This list is not complete, but it will hold you for a while:
Diffraction Physics by Cowley Electron Diffraction Techniques by Cowley Electron Diffraction by Pinsker Structure Analysis by Electron Diffraction by Vainshtein Electron Microscopy of Thin Crystals by Hirsch, Howie, Nicolson, Pashley & Whelan
For soimeone just starting out in electron diffraction, I would recommend first trying one or all of the following: Chapter 15, "Electron Diffraction" by Alderson & Halliday in the book TECHNIQUES FOR ELECTRON MICROSCOPY, Ed: D. H. Kay, F. A. Davis Publishers, 2nd. Ed. 1965 Chapter 2, "Electron Diffra tion in the Electron Microscope" in the book PRACTICAL ELECTRON MICROSCOPY IN MATERIALS SCIENCE, by J. W. Edington. Originally published by Van Nostrand, 1976 and now out of print, but possibly available from TechBooks, 301-871-1663. Both of the above give a gentle introduction to ED, with good illustrations and worked examples, etc. You may also find the following helpful, after you've read the above: INTERPRETATION OF ELECTRON DIFFRACTION PATTERNS, by Andrews, Dyson & Kewon, Plenum Press, i967 All of these are rather 'old' books and may be hard to find. You probably can't buy any of them, but may find them in a large scientific library. I don't know of any recent book that treats the subject in an easy way that would be useful to a beginner. If you have questions, don't hesitate to contact me (bigelow-at-umich.edu), W. C. Bigelow
For someone just starting out in electron diffraction, I would recommend first trying the following: Chapter 15, "Electron Diffraction" by Alderson & Halliday in the book TECHNIQUES FOR ELECTRON MICROSCOPY, Ed: D. H. Kay, F. A. Davis Publishers, 2nd. Ed. 1965 Chapter 2, "Electron Diffraction in the Electron Microscope" in the book PRACTICAL ELECTRON MICROSCOPY IN MATERIALS SCIENCE, by J. W. Edington. Originally published by Van Nostrand, 1976 and now out of print, but possibly available from TechBooks, 301-871-1663. Both of the above give a gentle introduction to ED, with good illustrations and worked examples, etc. You may also find the following helpful, after you've read the above: INTERPRETATION OF ELECTRON DIFFRACTION PATTERNS, by Andrews, Dyson & Kewon, Plenum Press, i967 All of these are rather 'old' books and may be hard to find. You probably can't buy any of them, but may find them in a large scientific library. I don't know of any recent book that treats the subject in an easy way that would be useful to a beginner. If you have questions, don't hesitate to contact me (bigelow-at-umich.edu), W. C. Bigelow
I had some years of TEM experience in (mostly) biological studies, and was similarly confronted by the need to learn something of electron diffraction techniques. I concur with W.C. BigelowUs opinion about the Edington text. Last I heard, it was about $32.00, paper bound still from Van Nostrand Reinhold, NY (ISBN 0-961-2934-0-3). Another gentle introduction is in the Glauert series Vol 1 part II, An Introduction to Electron Diffraction by B.E.P.Beeston. Still another one that may help is G. Thomas and M. Goringe, Transmission Electron Microscopy of Materials , Wiley-Interscience. 1979. ISBN 0-471-12244-0. Hope this helps.
} To: Microscopy-at-aaem.amc.anl.gov } Subject: ZIP Drives? } } I would not go investing my money in this "storage fad"! Like all the } Winchesters, Bernoulli, Syquests and Flopticals... it s magentic media and } will be obsolete in the next year. Most of the forementioned are already! The } only one with a true advantage is Syquest. For 2 reasons its reliability and } speed. (If we all agree that 14ms is fast).
Yes, that's true, BUT ... As I heard, there are problems with reliability of the data storage. Problems were found on 270MB mechanics (SCSI or AT), but not on 105MB. The main problem is dust.If any dust penetrates into disk , the disk media can be (will be) scratched. In Bernoulli system, there is no such high danger. But the main problem here is price for the mechanics as well as for the data media.
Does anybody know something more about IOMEGA Jaz drive???? The relation between capacity and the price seems to be fine!
} 105MG Syquest for 4 years and never had a problem with a cartridge and its
David Pejchl
======================================================================== Institute of Scientific Instruments | E-mail: david-at-ISIBrno.Cz Academy of Sciences of the Czech Republic | pejchl-at-Sci.MUni.Cz Kralovopolska 147, 612 64 Brno | Phone: +42 5 41321246 Czechland | Fax: +42 5 41211168 ========================================================================
X-Sender: smgxstg-at-pop-server.bcc.ac.uk X-Mailer: Windows Eudora Version 1.4.4 Mime-Version: 1.0 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable
I am trying to find a reliable way of getting 50 micrometre Vibratome sections of paraformaldehyde/glutaraldehyde perfusion fixed brain tissue to embed absolutely flat. I need to resection them to 1 micrometre and then section the semithins to ultrathins. The tissue is HRP/DAB labelled and I am trying to find and section individual cells to carry out image analysis.
The vibratome sections are fairly large in area (up to 20 x 10mm) and there are a lot of them (50 to 100).
At the moment I fix them in Osmium Tetroxide as flat as possible on a glass surface, but they always buckle slightly. After dehydration and infiltration each section is mounted in resin on a PTFE coated slide under a silicon coated coverslip.
This is where the first problem arises. If the coverslip is pressed hard enough to flatten the section, it breaks up due to the stiffening effect of the Osmium and the buckled nature of the section.
The smaller sections that do survive are frequently still too buckled and uneven to use the 50 or 100 times objective mag on the LM. This makes focusing and photography very difficult.
I have tried Osmium fixing the sections between a slide and coverslip, but if they are pressed hard enough to keep them flat, the fix does not penetrate to the centre of the section.
Although I achieve some results with my 'buckled' sections, the whole process would be more reliable and reproducible if the large 50=B5m sections were flat in the first place.
The only methods I have seen all seem to be on much smaller sections, but I do need to have a lot of large sections.=20
I'm sure someone else must be doing this sort of thing and that there is a relatively simple way of achieving this goal.
Does anybody have a method or a reference to a well described method?=20
REGARDING Cryosectioning of unfixed cells Good morning,
A colleague of mine is trying to cut 5 micron sections of unfixed Cos cells. This is the first time that she is working with unfixed cells, and she is having a little problem. The cells were embedded in agar, then frozen in OCT. The cells look terrible. There is not enough time, or enough of the cells to play around with the different variables, so I am asking if anyone has any suggestions that I can pass on to her, or any references that discuss protocols that might help her.
} [snip] The cells were embedded in agar, then frozen in OCT. } The cells look terrible. There is not enough time, or enough of the cells to } play around with the different variables, so I am asking if anyone has any } suggestions that I can pass on to her, or any references that discuss } protocols that might help her. } Dear Jeanne, My guess is that the freezing is not fast enough to prevent ice crystal formation. High-pressure freezing might be good enough to preserve several- micron-thick specimens, but neither plunge-freezing nor slam freezing will preserve whole cells, especially cells embedded in agar. If your colleague does not have access to a high-pressure freezer, perhaps a call to Balzers will help. Good luck. Yours, Bill Tivol
Applications are invited for an Electron Microscopist in the Department of Materials Science and Engineering, at the University of Washington, specializing in maintaining and training of scanning and transmission electron microscopes and use of related techniques in materials and/or biological sciences at the Electron Microscopy Consortium. The microscopist is expected to (i) perform day-to-day alignment, operation, service, and maintanence of the instruments, (ii) train basic uses of the instrument, (iii) and prepare SEM and TEM samples and (iv) perform analysis of material in conjunction with researchers using these instruments. A strong background in the operation of SEMs and TEMs is essential and additional experience with STEM, EDS, EELS techniques and microscopy software is desirable. Duties also include supervision of thin film and bulk sample preparation by ultramicrotoming and ion-beam milling of inorganic and biological samples. Materials projects involve, small particles, metals, ceramics, composites, including polymers and biological hard tissues, geological and beam-sensitive materials. A strong background in a field of materials science, biology, chemistry, solid-state physics, or geology is also important.
Applicant should send a complete package, including (i) a letter of interest with a statement of research and career objectives, (ii) a resume with publications and a list of instrumental expertise, (iii) names of three references, by August 31, 1995 to:
Prof. Mehmet Sarikaya Materials Science and Engineering Box 352120 University of Washington Seattle, WA 981920-2120
We have recently gone digital and now everyone is asking what is the best way to compose and label pictures etc. We use Macs and Photoshop to get our images, now the question is what is the best way to add labels etc. and prepare them for printing.
I know this will generate a lot of personal preferences. That's OK, I have an open mind and just want to know whats going on in this area. Freehand, Illustrator, Quark, Pagemaker, all will do the job, but we have learned that sometimes certain applications are better for specific jobs. I would like to take advantage of your collective experiences to learn more and help the users in my lab.
Jonathan Krupp Microscopy and Imaging Lab University of California Santa Cruz, CA 95064 (408) 459-2477 jmkrupp-at-cats.ucsc.edu
Can anyone please give me information and mixing instructions on: Biodur E20 Red Biodur E20 (hardner) Biodur Thinners E For use of making a cast of fine bloodvessels, or any other suggestions?
thanks
H. van der Merwe University of Pretoria Faculty of Veterinary Science Department of Anatomy Onderstepoort 0 1 1 0 SOUTH AFRICA
Jeanne Barker asks about sectioning frozen, unfixed cells; "The cells were embedded in agar, then frozen in OCT. The cells look terrible. There is not enough time, or enough of the cells to play around with the different variables, so I am asking if anyone has any suggestions that I can pass on to her, or any references that discuss protocols that might help her." Bill Tivol is correct in pointing ou the concequences of freezing damage, a variable that is usually ignored by histologists cutting sections for light microscopy. However, I am not sure that the researchers involved in this project would have the time, skill or patience to start preparing their cells by high pressure freezing (BTW Muller lab URL is http://www.em.biol.ethz.ch/em-lab/emhome.html).
It would be much simpler to fix the cells and then cryoprotect them before freezing. Liquid nitrogen can then be used for freezing without having to worry about vitrification. Simply immersing the fixed cells into a sucrose solution (our histologists use 10%) is usually sufficient for cryostat sectioning.
For really thin sections and improved resolution we routinely prepare sections (300 - 500 nm thick) of formaldehyde fixed material that has been cryoprotected in 2.1 M sucrose (the Tokuyasu method). The sections are cut at -60*C in a cryochamber of an ultramicrotome with a dry knife and then transferred onto glass slides, but this might be too involved for routine work.
Be aware that the sucrose will make the block softer so lower cutting temperatures may have to be used.
Paul Webster Center for Cell Imaging Yale School of Medicine
Thanks to all who responded to my request regarding information on sapphire knives. I thought I would provide a synopsis of what I have found out. My original question was one of mostly curiosity. Perhaps others are curious as well.
At least two Japanese companies (one named Sakura, the other ?) have been making sapphire knives for some time. They are both still in business although they do not seem to currently have a distributor in the US. The last catalog to list sapphire ultramicrotome knives for sale in the US was the 1988 BioRad Microscopy Accessories catalog (6th edition). Their "Crystome" sapphire knife for ultramicrotomy listed for $1495 (5mm cutting edge, synthetic sapphire).
The BioRad inventory has been sold several times in the last 3 years and most of it resides with Structure Probe Inc and Energy Beam Sciences. Both EBS and SPI continue to support the BioRad catalog such that an order placed with BioRad is forwarded to and processed by SPI or EBS (depending on the product). Steve Slap of Energy Beam Sciences says that although EBS does not maintain a stock of sapphire knives, a quick fax by EBS to the suppliers in Japan is all that is needed to order one. Also, SPI has several sapphire knives in stock in the US, according to Chuck Garber. EBS markets a sapphire Vibratome knife, but it is not designed for ultramicrotomy.
No one who responded to my original query had actually used a sapphire knife for ultramicrotomy. One person knew someone who had conducted tests with a sapphire ultramicrotome knife on a tough sample years ago and found that it dulled significantly faster than diamond. This latter person thought that sapphire might be adequate for ultrathin sectioninng of soft (biological) tissues but not harder samples.
The distinct advantage of sapphire over diamond is that knife widths of } 5mm are quite possible (because the material is synthetically manufactured), making them attractive to histologists. For instance, the sapphire Vibratome knife from EBS is } 25 mm wide and is far superior in sectioning performance to glass histology knives. Diamond knives in that size range are, quite obviously, unavailable (unless you want to buy the Hope Diamond and have it made into a histology knife--NOT).
Apparently a US market for sapphire ultramicrotome knives never developed. Probably (according to Chuck Garber) because domestic (US) suppliers of diamond knives solved some early problems with quality and availability and also because the Yen became so strong against the dollar that the price difference between sapphire (produced in Japan) and diamond (produced in the US) shrank. I do not know what the situation is in other parts of the world. Are there any microscopists in Japan who are on the net and could provide some information? Because they are still available in Japan, I am guessing there must be a market for them there (?).
Bob
Robert R. Wise, PhD Director, UWO Electron Microscope Facility Department of Biology University of Wisconsin Oshkosh Oshkosh, WI 54901 (414) 424-3404 (414) 424-1101 fax wise-at-vaxa.cis.uwosh.edu
In response to Colin Veitch's post re: downloading/uploading TEM settings to a PC:
} Is anyone out there using a Mac for a similar purpose and if so could they } give us some advice on the code required. We are reluctant to use a PC as } that would mean bringing in yet another computer into the TEM room as there } are already 3 in there.
...I can offer this:
We are currently using a Mac (Quadra) running 'SoftPC with Windows' in place of an IBM PC for saving alignments on a Philips CM30. Communication is over an RS232 line. The situation sounds similar to yours. Since we already had the IBM software and the Mac software was not yet available, it was a quick fix that we are still using (about a year later). One thing to note is that the IBM software runs slower in emulation on a Mac, but saving and loading settings is still done rather quickly (several seconds). We plan to eventually switch to the Mac-based remote control software.
-Dennis ____________________________________________________ Dennis C. Winkler National Institute of Standards and Technology (NIST) CSTL - Surface and Microanalysis Science Division Microanalysis Research Group (301) 975-2184 dcw-at-enh.nist.gov (Disclaimer: These are my opinions and not the official position of NIST.)
X400-Received: by mta bottom.ctd.anl.gov in /PRMD=ANL/ADMD= /C=US/; Relayed; Thu, 13 Jul 1995 10:41:46 -0500 X400-Received: by /PRMD=ESnet/ADMD= /C=US/; Relayed; Thu, 13 Jul 1995 10:41:45 -0500 X400-Received: by /PRMD=uk.ac/ADMD= /C=gb/; Relayed; Thu, 13 Jul 1995 10:41:38 -0500 X400-Received: by /PRMD=UK.AC/ADMD= /C=GB/; Relayed; Thu, 13 Jul 1995 11:40:46 -0500 X400-Received: by /PRMD=UK.AC/ADMD= /C=GB/; Relayed; Thu, 13 Jul 1995 10:47:10 -0500 X400-Received: by /PRMD=UK.AC/ADMD= /C=GB/; converted (ia5 text (2)); Relayed; Thu, 13 Jul 1995 11:40:54 -0500
Does anyone have any ideas on interfacing a Link AN 10000 with imaging programmes to a PC? I gather that Link once made a device but since producing PC based systems, this is no longer available. Does anyone else produce a nsimilar item? Please keep any replies SIMPLE - I am a fully paid up member of "computer illiterates" Secondly, anyone have any experience of the "Crystal" imaging system once available as an accessory for SEM's?
Many Thanks
Richard
Richard Pring Long Ashton Research Station Long Ashton Bristol, UK. BS18 9AF
A friend tried out a sapphire ultra knife when they first appeared. Epon embedded tissue dulled it quite quickly. And lower cost, high quality diamond knives appeared and became the obvious choice to purchase
Several years ago a sales rep. from one of Leica's predecessor companies (can't remember exactly what the company was called then) loaned us a sapphire vibratome blade to try out. It was about 4 cm long and 1 cm wide, shaped like a single edge injector blade, but transparent crystal. There wasn't much improvement over disposable razor blades for fixed tissue, but it was wonderful for soft things like unfixed embryos sectioned for organ culture.
Glen MacDonald Hearing Development Laboratories University of Washington Seattle, WA 98195-6515 (206)543-8360 glenmac-at-u.washington.edu
I have a client that would like to have pictures taken of 40 to 50 micron microbes used to degrade hydrocarbons in soil (patented process). He is interested in both optical and EM. If you can provided this service, please send me following information. PLEASE check your send address. It should be spignole-at-ix.netcom.com or spb-at-wwa.com.
OPTICAL:
Type of optical microscope:
What techniques would you use ie phase contrast or Hoffman modulated contrast? Other?
Under what conditions can you grow the cultures?
Methods of fixation available:
Type of output: polaroid 35 mm digital format
Charges:
SEM
Type of instrument:
How would you prepare the sample?
Variable pressure or E-SEM
CryoSEM
Type of output polaroid or digital Charges:
Thanks:
Susanne Pignolet Brandom Ph.D. MicroWorld Resources and News 708-548-6522
We developed the sapphire blade for the Vibratome at the end of 1990 as a joint project with a company with ultramicrotomy knife experience and with the assistance of Dr. Madhu Kalia, Dept. of Pharmacology, Thomas Jefferson University. Technical Products International, manufacturer of the Vibratome, offers sapphire knives for the Vibratome through its worldwide distributors (there are 5 Vibratome distributors in the USA, including my company, Energy Beam Sciences). You can contact Ralph Willen at TPI toll-free at 800-729-4421 for more information. Using these sapphire blades, our customers have done routine serial sectioning of mildly fixed tissue with a Vibratome 1000 of thicknesses from 5-10 microns, revealing subcellular features never before seen in Vibratome sections (with microscopic resolution similar to that obtained with 1 micron plastic sections). With a Vibratome 2000, sections as thin as 2 microns have been obtained. Note:sapphire knives can be resharpened indefinitely. Steven Slap, Energy Beam Sciences
This request is being posted on the "microscopy" and "nih-image" listservers
Folks:
If anyone has experience with image analyzers from the Pias Co., particularly the model PIAS-2, please contact me privately (e-mail, phone, whatever).
Thanks in advance.
Bill ========================== Bill Heeschen/Analyt. Sci.- Mat. Char. ----- 1897-F Building / The Dow Chemical Co. --- --- Midland, MI 48667 U.S.A. --- D O W --- phone: (517)636-4005 fax: (517)636-5453 --- --- Email: waheeschen-at-dow.com ----- ========================== R
} ...I can offer this: } } We are currently using a Mac (Quadra) running 'SoftPC with Windows' in place } of an IBM PC for saving alignments on a Philips CM30. Communication is over } an RS232 line. The situation sounds similar to yours. Since we already had } the IBM software and the Mac software was not yet available, it was a quick } fix that we are still using (about a year later). One thing to note is that } the IBM software runs slower in emulation on a Mac, but saving and loading } settings is still done rather quickly (several seconds). We plan to } eventually switch to the Mac-based remote control software.
Please explain what you mean by "the Mac-based remote control software." Does such a product currently exist????
Best Regards, John
John R. Minter, Ph. D. Phone: (716) 722-3407 Eastman Kodak Company FAX: (716) 477-3029 Analytical Technology Division email: minter-at- kodak.com Rochester, NY 14562-3712
The opening for an electron microscopist in the Materials Science department at the Univ. of Washington, announced here on July 12, is a staff position NOT a faculty position. A Ph.D. is not required.
The job includes some opportunities for collaborative research on funded projects.
} Please explain what you mean by "the Mac-based remote control software." } Does such a product currently exist????
} Best Regards, } John R. Minter, Ph. D.
When I looked into it a year ago, Philips gave me a part number (PW6444/00). We will soon be getting a Philips CM300 and, as we understand it, we will be getting the Mac-based remote control software.
Hope this helps, Dennis ____________________________________________________ Dennis C. Winkler Surface and Microanalysis Science Division National Institute of Standards and Technology (NIST) (301) 975-2184 dcw-at-enh.nist.gov (Disclaimer: These are my opinions and not the official position of NIST.)
We also had difficulty getting low molecular weight Polyvinyl alcohol (PVA). The suppliers tech people offered differing testing procedures changed the molecular weights seen, dramatically. Some calling around, showed the concern, among people doing cryoultramicrotomy, was that the viscosity remained low, with good soluability in water, rather than a strict question of molecular weight. 30,000-70,000, molecular weight, (4-6 cps) PVA was purchased from Sigma, (Catalog # P-8136) that is "cold water soluable" This has worked well in our lab for cryoultramicrotomy. I'd be interested in what others success has been with various PVA's. later dlb
We are interested in setting up some sort of file server for image retrieval purposes. Images would be put into the server from a variety of microscopes (light, SEM, TEM). Our current LAN consists of several PC's, Macs and a SUN Sparc 5 workstation that is used on our Voyager III EDX system. What would be needed to set up a fast, large capacity server? Suggestions or names of others to contact would be appreciated. Thank you.
############################################################################# John J. Bozzola, Director Center for Electron Microscopy Southern Illinois University Carbondale, IL 62901-4402 U.S.A. Phone: 618-453-3730 Fax: 618-453-2665 Email: bozzola-at-siu.edu Web: http://www.siu.edu/departments/shops/cem.html #############################################################################
An alternative method to improve the morphology is to immerse the tissue in freon in a container immersed in liquid nitrogen. The freezing is faster because the freon doesn't form the thin isolating layer of gas around the specimen as occurs when the tissue is frozen in liquid nitrogen. ============================================================================= Francisco Javier Hernandez Blazquez | Universidade de Sao Paulo - Faculdade de | e-mail fjhblazq-at-usp.br Zootecnia e Engenharia de Alimentos | Voice: 55 195 616122 r. 265 Departamento de Ciencias Basicas/Histologia| r. 278 Av. Duque de Caxias Norte, 225 CP 23 | Fax: 55 195 611689 CEP 13630-000 Pirassununga (Sao Paulo) | 55 11 8694831 BRAZIL | ==============================================================================
} I would not believe that there are still some people on this } earth using Freon or similar compounds, if I had not read } this e-mail. Freon should not be used any more, at all. } Liquid Propane or Ethane do the same job. } BUT: even immersing in Freon, Propan, or Ethan is not good enough } to freeze a sample with a thickness of more than a few microns without } damaging it. Only high pressure freezing will do it (up to a thickness } of 200 to 500 micron; see papers by M.Mueller et al. Zuerich). } If you can't do this, you will have to fix your tissue chemically in } advance and possibly cryo-protect it before freezing. This MAY } POSSIBLY give you a proper ultrastructure, depending on your } tissue or cells, if you are lucky. } Without this, your sample will be damaged during freezing and } substitution. --- Best regards, } } - - Reinhard Rachel {Reinhard.Rachel-at-biologie.uni-regensburg.de} } | )| ) Universitaet Regensburg, Lehrstuhl fuer Mikrobiologie } | \| \ D - 93040 Regensburg (Tel.: xx49-941-943-4534) } }
X-Sender: pop00362-at-popserver.uni-konstanz.de X-Mailer: Windows Eudora Version 1.4.3 Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" To: Microscopy-at-aaem.amc.anl.gov
Dear list-participants,
does anyone know how to contact Eico Engineering (Japan) the easiest way from Germany; is there a German or European dealer or address?
I am interested in their MF-2 quick freezing device equipped with a magnetron; does anyone have any experiences and information about performance?
I would like to know the email address of EDAX EDS service in USA, so that I can contact them. I have a DX-4 EDS here, and have some problems. If I cannot get the address, then I will show these problems to the public for help.
Therefore, if any one who knows the address, please tell me.
Many thanks.
Zhen Quan Liu
EM laboratory Peking University Beijing 100871 China
I would like to know the email address of EDAX EDS service in USA, so that I can contact them. I have a DX-4 EDS here, and have some problems. If I cannot get the address, then I will show these problems to the public for help.
Therefore, if any one knows the address, please tell me.
Many thanks.
Zhen Quan Liu
EM laboratory Peking University Beijing 100871 China
I would like to know the email address of EDAX EDS service in USA, so that I can contact them. I have a DX-4 EDS here, and have some problems. If I cannot get the address, then I will show these problems to the public for help.
Therefore, if any one who knows the address, please tell me.
I am learning using NUPOP software for email service, if you get this message twice, I am sorry about that.
Many thanks.
Zhen Quan Liu
EM laboratory Peking University Beijing 100871 China
Our group is considering upgrading an ACAS 570 non confocal microscope system to the Meridian Ultima. We are primarily concerned about capability to do confocal and photoactivatable caged compound studies. Before we plunk down the cost of upgrading we would like to hear from any users of the Ultima about the pros and cons of the system, particularly with regard to these types of uses.
I would also appreciate it if anyone could supply me with the address of the confocal listgroup. Thanks!
Dave Zawieja Assistant Professor Dept. of Medical Physiology Texas A&M Univ.- Health Science Center College Station TX 77843 Phone:(409) 845-7465 FAX:(409) 847-8635 dcz-at-tamu.edu
You wrote: } } I would like to know the email address of EDAX EDS service in USA, } so that I can contact them. I have a DX-4 EDS here, and have some } problems. If I cannot get the address, then I will show these } problems to the public for help. } } Therefore, if any one knows the address, please tell me. } } Many thanks. } } Zhen Quan Liu } } EM laboratory } Peking University } Beijing 100871 } China } } FAX ( 86 10 ) 256 1615 } } Email zqliu-at-pku.edu.cn } Let me first say that I am sorry that you are having problems and that this must be a frustrating situation considering your location.
With regard to showing your problems to the public, please be adviced that this is a published forum and therefore libel laws do apply. In addition, by threatining to show problems to the public, your actions could be interpreted to indicate malecious intent.
Second, in the same light that vendors should not use this forum as a commercial vechicle, should customers use it as a mechanism for telling the world about problems?
Is this another moral and legal issue for this listserver to consider?
Another approach might include asking if there are individuals that are using the same equipment that could help through email. Then communicate the problems to only those that are able to help.
Susanne Pignolet Brandom, Ph.D MicroWorld Resources and News
_________________________________ Massimo Sassaroli, D.Sc. Dept. of Physiology & Biophysics Box 1218 Mount Sinai School of Medicine 1 Gustave L. Levy Pl. New York, NY 10029-6574
"Dr. Andrew P. Somlyo" {aps2n-at-elvis.med.virginia.edu} offered this very "illuminating" reply to what, at first sight, is a very thoughtful message by Reinhard Rachel. Does Dr. Somlyo suggest that the adverse effects of Freon on the atmosphere demonstrated by a great number of scientific studies are "nonsense"? Or that the remainder of Reinhard's message is "nonsense". As far as I am concerned, this is yet another blatant example of bandwidth abuse. I read most messages coming down this pipeline, even though many of them are outside my field of interest. If I do not have anything constructive to say, I usually keep my mouth shut and my fingers off the keyboard and the reply button. Perhaps Dr. Somlyo could also learn to be more sensitive and selective in his future replies!!
Sorry, but this sort of things get to me sometimes.
Sincerely,
Massimo Sassaroli
} This one-liner was the reply of Dr. Andrew P. Somlyo } } NONSENSE } } On Sat, 15 Jul 1995, Reinhard Rachel t4534 wrote: } } } I would not believe that there are still some people on this } } earth using Freon or similar compounds, if I had not read } } this e-mail. Freon should not be used any more, at all. } } Liquid Propane or Ethane do the same job. } } BUT: even immersing in Freon, Propan, or Ethan is not good enough } } to freeze a sample with a thickness of more than a few microns without } } damaging it. Only high pressure freezing will do it (up to a thickness } } of 200 to 500 micron; see papers by M.Mueller et al. Zuerich). } } If you can't do this, you will have to fix your tissue chemically in } } advance and possibly cryo-protect it before freezing. This MAY } } POSSIBLY give you a proper ultrastructure, depending on your } } tissue or cells, if you are lucky. } } Without this, your sample will be damaged during freezing and } } substitution. --- Best regards, } } } } - - Reinhard Rachel {Reinhard.Rachel-at-biologie.uni-regensburg.de} } } | )| ) Universitaet Regensburg, Lehrstuhl fuer Mikrobiologie } } | \| \ D - 93040 Regensburg (Tel.: xx49-941-943-4534) } } } }
_________________________________ Massimo Sassaroli, D.Sc. Dept. of Physiology & Biophysics Box 1218 Mount Sinai School of Medicine 1 Gustave L. Levy Pl. New York, NY 10029-6574
} You wrote: } Let me first say that I am sorry that you are having problems and that } this must be a frustrating situation considering your location. } } With regard to showing your problems to the public, please be adviced } that this is a published forum and therefore libel laws do apply. In } addition, by threatining to show problems to the public, your actions } could be interpreted to indicate malecious intent. } } Second, in the same light that vendors should not use this forum as a } commercial vechicle, should customers use it as a mechanism for telling } the world about problems? } } Is this another moral and legal issue for this listserver to consider? } } Another approach might include asking if there are individuals that are } using the same equipment that could help through email. Then } communicate the problems to only those that are able to help. } } Susanne Pignolet Brandom, Ph.D } MicroWorld Resources and News }
Ahem. One of the purposes of the listerver *is* to get folk talking together to help each other with problems. If a company is so paranoid about its products that they try to censor their customers on the net, then they certainly deserve any bad press they get from it.
Having a problem with a product and letting folk know about it is not libel by any stretch of the imagination. Asking for help with a solution is a reasonable thing to do, and carries no negative ethical connotations.
On the other hand, the idea of trying to gag customers *does* lead to some interesting ethical questions, I think.
} With regard to showing your problems to the public, please be adviced } that this is a published forum and therefore libel laws do apply. In } addition, by threatining to show problems to the public, your actions } could be interpreted to indicate malecious intent. } } Second, in the same light that vendors should not use this forum as a } commercial vechicle, should customers use it as a mechanism for telling } the world about problems? } } Is this another moral and legal issue for this listserver to consider?
=========================
I thought that the purpose of the listserver was to share information about techniques and products. I can tell you from personal experience that it is very important to share such information - ESPECIALLY regarding equipment and products. As long as one presents the facts and allows the other side to respond (which, of course, we do) then ethical vendors should have nothing to fear. I know that I would have probably selected a different vendor for a recent purchase that we made had I more information.
On the other hand, one seldom writes letters to praise a job well done or a good product. Maybe we need some good press too.
Peace.
############################################################################# John J. Bozzola, Director Center for Electron Microscopy Southern Illinois University Carbondale, IL 62901-4402 U.S.A. Phone: 618-453-3730 Fax: 618-453-2665 Email: bozzola-at-siu.edu Web: http://www.siu.edu/departments/shops/cem.html #############################################################################
The Merck Index lists two groups or asbestos: serpentines (such as chrysotile) and amphiboles (anthophyllite, actinolite, tremolite, crocidolite). Are all equally harmful or do their toxicity vary? In particular, how does tremolite rank for toxicity?
Bob
Robert R. Wise, PhD Director, UWO Electron Microscope Facility Department of Biology University of Wisconsin Oshkosh Oshkosh, WI 54901 (414) 424-3404 (414) 424-1101 fax wise-at-vaxa.cis.uwosh.edu
The Merck Index lists two groups or asbestos: serpentines (such as chrysotile) and amphiboles (anthophyllite, actinolite, tremolite, crocidolite). Are all equally harmful or do their toxicity vary? In particular, how does tremolite rank for toxicity?
Bob
Robert R. Wise, PhD Director, UWO Electron Microscope Facility Department of Biology University of Wisconsin Oshkosh Oshkosh, WI 54901 (414) 424-3404 (414) 424-1101 fax wise-at-vaxa.cis.uwosh.edu
Not to over react, but the original posting on this subject appeared (at least to me) as a request for information about how to contact EDAX by Email.
The rest I would attribute to a language problem as the originator of the message was from China. I do not think that the message was sent as a "threat" but rather a poorly worded statement saying that
"If I can't get hold of EDAX then I will ask for help in the public forum. "
Let's not go overboard, I think that Zhen Quan Liu was simply asking for information.
To help Zhen Quan Liu I have forwarded the Email message to EDAX by FAX and asked them to contact him. At present EDAX does not have a "general" EMAIL address but I understand that many of their service reps have individual accounts.
It's been a long time since I did asbestos quantitation but my gut feeling about the undesirable physiological effects of asbestos fibers in the alveoli of the lungs could be due to ends of the fibers puncturing the cells and allowing other toxins to enter the cells by capillary action on the fiber surface. As I recall, some forms of the fibers have a central channel or groove that could facilitate this effect.
Doug Davis EML Berkeley 26 Giannini Hall Berkeley CA 94720
On Mon, 17 Jul 1995 wise-at-vaxa.cis.uwosh.edu wrote:
} To all, } } The Merck Index lists two groups or asbestos: serpentines (such as } chrysotile) and amphiboles (anthophyllite, actinolite, tremolite, } crocidolite). Are all equally harmful or do their toxicity vary? In } particular, how does tremolite rank for toxicity? }
Here's an excerpt from "Pathology of Occupational Lung Disease" by Churg&Green:
begin excerpt
Fiber type, fiber size, and fiber aspect (length to width) ratio all appear to play a role in disease. There is fairly clearcut evidence that amphiboles are more dangerous than chrysotile in the genesis of human mesotheliomas; the importance of fiber type in regard to asbestosis and lung cancer is unclear.
Some of the earliest animal studies of asbestosis suggested that long fibers produced more disease than short ones, and this observation has been repeatedly demonstrated for both nonneoplastic lesions and mesothelioma. The effects of short fibers.. is disputed. One point of view suggests that short fibers have no or minimal effects; the other suggests that, although the effects of individual short fibers may be less than individual long fibers, larger numbers of short fibers may be just as important as smaller numbers of long fibers...
... The effect of aspect ratio is similarly uncertain...
I am sorry if I misinterpreted the posting from Zhen Quan Liu.
My intention was not to threaten or to intimatdate, but to caution. I realize that this was a mistake and an over reaction on my part. I hope that my actions will be excused.
This forum provides a great mechanism for communicating with other microscopists and again I am very sorry for causing a disruption.
Susanne Pignolet Brandom
All acts of stupidity are my own as are my opinions
Is it possible to identify accumulations of pollutants, heavy metals etc. in bone and hair material by x-ray microanalysis i.e. are the elements ever present in sufficient concentration to detect variations? If anyone has any experience of this, please would they comment and send me any references that they think may be useful. Thank you.
Is it possible to identify accumulations of pollutants, heavy metals etc in bone and hair material by x-ray microanalysis, i.e. are the elements ever present in sufficient concentrations to detect variations? If anyone has any experience of this, please would they comment and send me any references that they think might be useful. Thank you.
**************************************
From:
Julie Sheffield-Parker, Oxford Instruments PTY Ltd., P. O. Box 7, Pennant Hills, NSW 2120, Sydney, AUSTRALIA.
} Date: Tue, 18 Jul 1995 16:01:37 +1000 } X-Sender: oisydney-at-ozemail.com.au } To: Microscopy-at-aaem.amc.anl.gov } From: oisydney-at-ozemail.com.au (Julie Sheffield-Parker) } Subject: Microanalysis of hair and bone samples. } } Is it possible to identify accumulations of pollutants, heavy metals etc. in } bone and hair material by x-ray microanalysis i.e. are the elements ever } present in sufficient concentration to detect variations? If anyone has any } experience of this, please would they comment and send me any references } that they think may be useful. Thank you. } } } } ************************************************* } From:- } } Julie Sheffield-Parker, } Oxford Instruments Pty. Ltd., } P. O. Box 7, } Pennant Hills, } NSW 2120, } Sydney, AUSTRALIA } } Tel: ++ 61 2 484 6108 } Fax: ++ 61 2 484 1667 } E-Mail: oisydney-at-ozemail.com.au } } *************************************************
Hi Julie,
I would suggest to try to carefully incinerate the hair and/or bone samples and then analyze the ash residue. This way you will increase the the concentration of contaminants to a measurable level. The incineration can be performed at about 900 deg C in an ordinary laboratory furnace with sample put into a platinum crucible covered with a platinum lid. By precisely weighing the sample before and after incineration you can even quantify. I actually did this sort of analysis with foodstuff as well as spices and obtained good and reliable results.
Another possibility would be X-ray microfluorescence done in the SEM with a special although simple attachment put onto the EDS detector. The X-ray fluorescence will decrease significantly the detection limit although quantitation is more difficult. Contact Dr. I. Pozsgai who is an expert in this matter. He works for Research Institute for Technical Physics, Budapest, Hungary. His e-mail address is as follows:
pozsgai-at-falcon.mufi.hu
Good luck, and contact me if you succeed!
Kris Kristof KOVACS University of Veszprem, Central Laboratory P.O.Box 158, Veszprem, HUNGARY H-8201 Phone: +36-(88)-421-684 Fax: +36-(88)-426-016
} } Is it possible to identify accumulations of pollutants, heavy metals etc. in } } bone and hair material by x-ray microanalysis i.e. are the elements ever } } present in sufficient concentration to detect variations? If anyone has any } } experience of this, please would they comment and send me any references } } that they think may be useful. Thank you. } } } } Julie Sheffield-Parker, } } I would suggest to try to carefully incinerate the hair and/or bone samples } and then analyze the ash residue. This way you will increase the the } concentration of contaminants to a measurable level. The incineration can be } performed at about 900 deg C in an ordinary laboratory furnace with sample } put into a platinum crucible covered with a platinum lid. By precisely } weighing the sample before and after incineration you can even quantify. I } actually did this sort of analysis with foodstuff as well as spices and } obtained good and reliable results. } } Another possibility would be X-ray microfluorescence done in the SEM with a } special although simple attachment put onto the EDS detector. The X-ray } fluorescence will decrease significantly the detection limit although } quantitation is more difficult. Contact Dr. I. Pozsgai who is an expert in } this matter. He works for Research Institute for Technical Physics, } Budapest, Hungary. His e-mail address is as follows: } } pozsgai-at-falcon.mufi.hu } } Good luck, and contact me if you succeed! } } Kristof KOVACS } Dear Julie, The advantage of x-ray microanalysis is that the elements of interest can be localized to within a fraction of a micrometer (if the probe size is small enough). Both sensitivity and quantitation are better with bulk methods (atomic absorption, neutron activation, etc.). If you need to see the pattern along a hair strand, or localize an element in bone, however, XMA is a good technique. As to your original question, some pollutents may be present in sufficient concentrations, but the sensitivity of XMA is only about a large fraction of 1%, or a few millimolar. You will have to figure out whether what you are interested in is that concentrated. If you are going to incinerate the samples, do not use XMA--you have already lost the position information. Good luck. Yours, Bill Tivol
No one in the "EM prep lab" business has ever attempted anything like this before so please be patient as we develop it in a way that will be both interesting as well as informative to anyone accessing the information that is now "up" on our site.
This project represents our first steps toward the all-electronic SPI Supplies "SourceBook" of products for the EM laboratory. We believe that use of the new electronic media eventually will permit significant reductions in the normally high marketing and distribution costs associated with just about anything going into the EM laboratory, leading to lower prices for our customers.
Any comments, suggestions, or other input you think might help us make an even better "site" should be directed to "webmaster" at {SpiSupp-at-aol.com} .
Chuck
====================================================== Charles A. Garber, Ph. D. Ph: 1-(610)-436-5400 President 1-(800)-2424-SPI SPI SUPPLIES FAX: 1-(610)-436-5755 PO BOX 656 e-mail: GVKM07A-at-prodigy.com West Chester, PA 19381-0656 USA Customer Service: SpiSupp-at-aol.com
-- [ From: Charles A. Garber, Ph. D. * EMC.Ver #2.10P ] --
In response to the question of Julie Sheffield-Parker on July 18 about the analysis of hair and bone samples for heavy metals, "pollutants", etc., because of the always possible presence of metallo-organics, which would be volatilized away at the temperatures of operation of the typical muffle furnace, removal of the organics via "room temperature ashing" is preferred since one eliminates the possibility of loss of heavy metals.
Note: I am disclosing my own (possible) financial interest in this reply, since our firm manufactures such plasma etchers (e.g. the SPI Plasma Prep II). Now for the painful part: Other firms manufacturing "room temperature" ashing equipment would include a) Denton Vacuum, Inc. and b) Fisons VG Microtech.
Chuck
====================================================== Charles A. Garber, Ph. D. Ph: 1-(610)-436-5400 President 1-(800)-2424-SPI SPI SUPPLIES FAX: 1-(610)-436-5755 PO BOX 656 e-mail: GVKM07A-at-prodigy.com West Chester, PA 19381-0656 USA Customer Service: SpiSupp-at-aol.com
Could you use bulk methods on subsections of a hair strand or on collections of hair strands?
For example, if you have the follicles, you could chop up all the strands of hair and collect all the segments "follicle to 1 cm", "1 cm to 2 cm", etc. Then use the more sensitive methods to characterize the exposure to pollutants over time.
You could get more finely spaced data by chopping the hairs up into smaller segments, but that would reduce the total amount in any one batch.
} } } Is it possible to identify accumulations of pollutants, heavy metals etc. in } } } bone and hair material by x-ray microanalysis i.e. are the elements ever } } } present in sufficient concentration to detect variations? If anyone has any } } } experience of this, please would they comment and send me any references } } } that they think may be useful. Thank you. } } } } } } Julie Sheffield-Parker, } } } } I would suggest to try to carefully incinerate the hair and/or bone samples } } and then analyze the ash residue. This way you will increase the the } } concentration of contaminants to a measurable level. ...
} } Kristof KOVACS } } } Dear Julie, } The advantage of x-ray microanalysis is that the elements of interest } can be localized to within a fraction of a micrometer (if the probe size is } small enough). Both sensitivity and quantitation are better with bulk methods } (atomic absorption, neutron activation, etc.). } Bill Tivol }
We are having a problem locating a vendor for Freon 113. We have recently depleted our stock which we obtained from Bio-Rad who no longer carries this product. We have contacted Ted Pella, Inc.; Polaron and Energy Beam Sciences and none carry Freon 113. Is this form of Freon no longer available in the U.S.? If anyone knows of a vendor who still supplies Freon 113, please e-mail pertinent info. If this product is no longer available, what possible alternatives can be used in the final stage of dehydration after the ethanol drying series and before critical point drying?
Thanks in advance for any help--Gerald Harrison U. of P. S.E.M. Lab jerry-at-biochem.dental.upenn.edu
We use liquid CO2 quite successfully for CPD. Be sure it is very dry. We have gas company heat dry tanks before filling and use a filter on the line. With these precautions we get excellent results.
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 * **************************************************************
On Tue, 18 Jul 1995 jerry-at-biochem.dental.upenn.edu wrote:
} Dear fellow subscribers to Microscopy ListServer, } } We are having a problem locating a vendor for Freon 113. We have } recently depleted our stock which we obtained from Bio-Rad who no longer } carries this product. We have contacted Ted Pella, Inc.; Polaron and Energy } Beam Sciences and none carry Freon 113. Is this form of Freon no longer } available in the U.S.? } If anyone knows of a vendor who still supplies Freon 113, please } e-mail pertinent info. If this product is no longer available, what } possible alternatives can be used in the final stage of dehydration after } the ethanol drying series and before critical point drying? } } Thanks in advance for any help--Gerald Harrison } U. of P. S.E.M. Lab } jerry-at-biochem.dental.upenn.edu } }
Thanks for all the helpful advise. E.M.S. at (215)646-1566 still has Freon 113 at $110.00 for a case of four (450 ml). However, since several suggestions were to go straight from 100% EtOH to very dry CO2, we will also try this.
Aloha! A colleague here in Honolulu will be moving to Omaha in December, and will be looking for a job in EM or histology. She asks if there is a local microscopy society there that she can hook up with-? She figures she can call hospitals, universities, etc. cold (she called me cold and we found her a nice EM job), but she would like to get in touch with other like-minded microscopists.
Mahalo in advance, Tina
Tina Weatherby Carvalho Biological EM Facility University of Hawaii tina-at-ahi.pbrc.hawaii.edu
Mr-Received: by mta PETVAX.MUAS; Relayed; Wed, 19 Jul 1995 10:38:22 -0400 Mr-Received: by mta PETVAX; Relayed; Wed, 19 Jul 1995 10:38:24 -0400 Mr-Received: by mta SRVR01; Relayed; Wed, 19 Jul 1995 10:40:33 -0400 Disclose-Recipients: prohibited
I would appreciate colleagues comments regarding what they have found to be an optimal etching time for sodium ethoxide on resin sections prior to LM immunogold labelling. Thanks in advance.
I just discovered that our Denton Vacuum evaporator has been running on silicone diff pump oil. My question is does anyone have any good recomenndations for cleaning out ALL the silicone oil so we can replace it with Santovac 5?
Tina- Nebraska is part of the Central States Microscopy Society. The Secretary to contact is Sue Jaconis, 11444 Lackland Road, St. Louis, MO 63146. In my 1994 membership list, I see 3 members from Omaha, two at St. Joseph Hospital and one at Creighton University. I'll send you these names if you'd like. Steven Slap
For those of you that have access to the WWW, there is a complete listing of all LAS (Local Affiliate Societies) associated with MSA. Contact names & addresses are provided. Just scroll through the list.
The URL is: http://www.amc.anl.gov
then look for information on the Microscopy Society of America and further down the hot list will be the LAS information.
I saw nothing listed in the Omaha area, but there was a listing for Societies in Iowa, Oklahoma, and "Mountain States".
I would like to say thanks to all those people taking care of my previous email, and most of them have provided the address and FAX number of EDAX in USA and the person's name whom I might contact with.
Email is a very convenient way for communication and discussion in the field of EM. It is new to most Chinese people in China, even in universities. Also it is quicker and cheaper. The cost of one page of FAX in Beijing is equal to 1/6 of my one month wage. That is why I prefer email.
What I wanted to discuss with EDAX is pure scientific and technical. It is obvious that my poor English did confuse some people, I do apologize here for that. Anyway, I shall send EDAX a fax, telling them my technical and scientific problems.
Due to the problem of my local computer, I cannot get the email addresses of those people who wrote to me, so that I cannot answer or say thanks to them individually. They are:
Don Grimes, Yi Feng, Babara Hartman, Miami University, Susanne Brandom, Sid Patel, X. G. Ning, Z. P. Wang, Sender from Australia
I hope this email will make things clear.
If you think this mail is suitable, please transfer it to "microscopy", otherwise please return to me.
Many thanks
Zhen Quan Liu
Peking University Beijing, China Email zqliu-at-pku.edu.cn
One of my colleagues would needs a TEM of a negatively stained phage prep. Can anyone point me to a recent reference? TIA.
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)
The Department of Ophthalmology at UMDNJ, New Jersey Medical School in Newark has two technical positions open: ELECTRON MICROSCOPIST: This position requires a bachelor's degree plus a minimum of 3 years relevant experience. We are seeking a highly skilled and motivated individual to perform research on the eye who can perform all aspects of transmission and scanning electron microscopy. Animal work will include euthanasia and tissue harvesting. Individual should have excellent darkroom skills. Knowledge of MacIntosh, image analysis, ICC and ISH highly desirable. RESEARCH TEACHING SPECIALIST: This position requires a bachelor's degree plus a minimum of 1 year relevant experience. We are seeking a histologist with experience embedding and sectioning plastic embedded tissue. Animal work is required. Knowledge of PC and Mac computers and darkroom highly desirable.
If interested, please send your resume to Ilene Sugino UMDNJ-Ophthalmology DOC 6th Floor 90 Bergen Street Newark, NJ 07103 or fax me at 201 982-7762
I thought this response from Andrew Somlyo was worth sharing...........amongst other things, it does point out the need for care when using ethane or propane. re Regensburg's message: 1. It ignores an extensive literature dealing with depth of ice- crystal-free freezing as a function of coolant, plunge-velocity, etc. and metal mirror properties, while operating at atmospheric pressure (e.g.: Bald J. Microscopy 140: Pt.1 17-40, 1985 and refs. therein) . 2. Worse, the message ignores extensive experimental evidence showing ice-crystal-free (even vitreous) freezing at atmospheric pressure, albeit to a limited specimen depth. ( For muscle, a more "difficult" specimen then, say, liver,see e.g.: Somlyo A.V. et al: J. Cell Biol. 74:828-857, 1977; ibid 90:577-594, 1981; Padron et al; J. Microscopy 151 Pt.2 81-102,1988; Sosa et al; Biophys. J. 67:283-292, 1994). 3. Advocacy of explosive coolants without an accompanying warning of its potential dangers ( e.g.: Ryan and Liddicoat J. Microscopy 147: 337-340, 1987), true, is not nonsense, but worse. Electrical discharge dur ing operation of a stimulator can cause far more damage than the minute amount of Freon released into the atmosphere. 4. The uninformed use of the term "damage" can be misleading. As also noted by Peter Ingram, high pressure can cause more "damage" to sub- cellular composition than freezing at atmospheric pressure. 5. I want to protect the environment, but, as an American, I also believe in the separation of church and state. Therefore, until someone provides
quantitative evidence to show that the amounts of Freon released by vast hordes of cryo-electronmicroscopists have a SIGNIFICANT effect compared to, for example, bovine eructations, I consider the LEGAL use of Freon to be a matter of individual choice, not to be subject to the dictates of , often uninformed ( see also the Shell-Atlantic fiasco) devotees of the Green religion.
I investigated the matter of how to remove silicone oils from vacuum apparatus rather thoroughly while writing the section on Diffusion Pump Oils in my book on "Vacuum Methods in Electron Microscopy". Here is what I was able to come up with (p. 185): "Silicone oils themselves are also very difficult to remove from the parts of an electron microscope because they are so viscous and insoluble. Some instrument manufacturers have refused to issue service warranties on instruments if silicone oils are used in the diffusion p[umps. The cleaning procedure recommended by the Dow Corning Corporation (I called them and discussed the matter personally with one of their engineers) consists simply of wiping away as much of the oil as possible with a dry cloth or tissue, followed by repeated wiping with cloth pads moistened with toluene, xylene, trichloroethylene, or perchloroethylene." In addition to the above solvents, kerosene is sometimes recommended. Repeated rinsing with one or mopre of these solvents may ultimately get your system free enough of the old oil for your purposes. As mentioned on pp. 188-190 of my book, You may want to check with the manufacturer of the pump before changing the type of oil you use in it, to be sure Santovac will work in it satisfactorily. If it was designed for use with DC-704 silicone fluid, it's heater may not put out enough heat to give a suitable rate of boiling with Santovac. If it uses DC-705, this will probably not be a problem. P.S. you can order my book from Portland Press, Ashgate Publishers, Old Post Road, Brookfield, VT 05036-9704 (Ph: 800-535-9544) - it contains loads of just this kind of practical information.
} I would appreciate colleagues comments regarding what they have found to be an } optimal etching time for sodium ethoxide on resin sections prior to LM } immunogold labelling. Thanks in advance. } } Walt Bobrowski } Parke-Davis Research } Ann Arbor MI
For immunoEM you only need to *oxidize* the thin section using hydrogen peroxide or periodic acid. We routinely use 10% hydrogen peroxide for 10 min. This step is of course only necessary after embedding in epoxy resins. It is more easy and efficient to embedd in Lowicryls or Unicryl for immunoEM. *Etching* is performed if you want to stain the semithin epoxy resin sections. We use sodium ethoxide + toluene (2:1) for 3-5 min. If you need more information, feel free to contact me.
================================================================== 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 ==================================================================
I normally make up a saturated solution of ethanolic sodium hydroxide several days before I need to use it. It is ready when the solution has turned dark brown in colour and I have found that it's etching properties get stronger with time. Slides holding semithin sections (0.5 micron) are immersed in etching solution for 1 hour before being rinsed well in ethanol. Care should be taken to ensure that the slides are completely covered in ethanolic sodium hydroxide to prevent the formation of crystals.
I apologize my earlier posting was poorly worded. I was looking for a protocol so I could negatively stain a preparation of bacteriophage prepared by one of my colleagues. Thanks to those who offered TEM's as well as pointers to methods.
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)
I just remembered that, while writing my book on Vacuum Methods, I also ran across a detergent that is advertised as being suitable for removing silicone and other oils (p. 72). This is a product known as RBS-35 manufactured by the Pierce Chemical Co., P.O. Box 117, Rockford, IL 61105 (Ph: 815-968-0747). This stuff may be intended primarily for use on glassware, and so may require treatment in a boiling solution, I just don't recall; however, you might contact the manufacturer and see if they can suggest a method of using it on a DP. Incidentally, there will be silicone oil all through the pumping line and the hose attached to the DP, as well as inside the evaporator, and so these parts will also have to be cleaned if you want to get the system reasonably free of silicones. Best of luck! W. C. Bigelow (bigelow-at-umich.edu)
A student here is interested in looking at proteins with EM. How does one go about this? How does one stain proteins with Au? If anyone can point us toward a reference or offer advice, it would be greatly appreciated. I apologize for such a basic inquiry, but I'm only a materials scientist, and mostly a metallurgist at that.
Thanks --------------------------------------------------------- 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 Fax: 801/581-4816 | UUUUU U | | U U --------------------------------------------------------- UUUUU Of making many books there is no end, and much study wearies the body. -Eccl 12:12
The Integrated Microscopy Resource at the University of Wisconsin-Madison is pleased to provide the "Directory of Microscopists on the Net." It's available on the World Wide Web at
A fill-in form allows you to submit your name, organization/institution, email address, and research interests. A search engine allows keyword search, or the whole list can be viewed.
Having just started, the directory is fairly small. We could use your help in building up the directory and making it a useful resource that facilitates communication and collaboration.
Sincerely, Stephan Spencer Integrated Microscopy Resource University of Wisconsin-Madison Web: http://www.bocklabs.wisc.edu/imr/imr.html email: sspencer-at-rhino.bocklabs.wisc.edu
Please be aware that changing from DC704 to Santovac 5 results in a lower pressure in the stack, you will suffer some loss in pumping speed. More important is that you lower the changeover point from roughing to backing.
With DC 704 the pressure is about 300mTorr so you can changeover at 150-200mTorr with little consequence. The pressure with Santovac 5 was related to me to be about 150mTorr so you must go to 50-100mTorr of roughing.
Call me if you have any questions, as I recall you have an auto system and the resetting of the auto sensors must be done or problems will result.
} To all, } } A student here is interested in looking at proteins with EM. How does one } go about this? How does one stain proteins with Au? If anyone can point us } toward a reference or offer advice, it would be greatly appreciated. I } apologize for such a basic inquiry, but I'm only a materials scientist, and } mostly a metallurgist at that. } } Thanks } --------------------------------------------------------- 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 Fax: 801/581-4816 | UUUUU U } | | U U } --------------------------------------------------------- UUUUU } Of making many books there is no end, and much study wearies the body. } -Eccl 12:12
Hello Jeff, A simple way to look at peptide preparations is by negative staining with 2% uranyl acetate (UAc). Just put a drop of your suspension on formvar-coated copper grids and contrast with the uranyl acetate. If you want to discover a partic= ular peptide you can step to immunoEM. With specific antibodies or antisera against the peptide you will be able to reveal it in TEM. Dilute the protein suspension with a phosphate buffer + 0.1% (w/w) BSA. Place a small drop on formvar-coated nickel grids for about 1 min. Dry and let the grid float for 1 h on a drop of antiserum diluted in PBS-BSA. Wash many times for 5 min on drops of PBS and place them then for 1 h on a drop of protein A-Au (5 or10 nm) or protein G-Au diluted 1:10 in PBS-BSA. Wash and contrast with 2% UAc as above. Good luck. Sverker
=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D= =$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D= =$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D Sverker Enestrom M.D., Ph.D. Department of Pathology University of Linkoping, Sweden Phone: +46 13 22 15 20 =46ax: +46 13 13 22 57 =$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D= =$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D= =$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D=$D
Please find enclosed Second Announcement of Summer School on Morphological Image and Signal Processing being held in Zakopane, Poland, Sept. 27-30. This message has been sent to several list, so I am sorry if it happen to reach you more than once.
Summer School on Morphological Image and Signal Processing Zakopane, Poland, September 27-20, 1995
Programmme Committee
J. Chojcan (Gliwice, Poland) E. Dougherty (Rochester, USA) M. Gabbouj (Tampere, Finland) J. Goutsias (Baltimore, USA) S. Marshall (Glasgow, UK) W. Mokrzycki (Warsaw, Poland) I. Pitas (Thessaloniki, Greece) J. Serra (Fontainebleau, France) F. Vajda (Budapest, Hungary)
Organising Committee
H. Heijmans (Amsterdam, The Netherlands) - Scientific Director J. Smieja (Gliwice, Poland) Local Arrangements V. Starovoitov (Minsk, Belarus) K. Wojciechowski (Gliwice, Poland) - General Director
Invited Lecturers
J. Astola (Tampere, Finland) H. Heijmans (Amsterdam, The Netherlands) P. Jonker (Delft, The Netherlands) M. Schmitt (Fontainebleau, France) L. Vincent (Peabody, USA)
Venue
Zakopane is the most famous and attractive place in the Polish Tatra Mountains. It is located near the Slovakian border within a short distance from Krakow and Katowice.
Accommodation
Participants are awaited in the "Siwarna" rest house, Zakopane -Koscielisko, 32 Sywarne St. The accommodation is arranged in single, double and three- bedded rooms and is available from Tuesday, September 26th (with supper on that day as the first meal) to Saturday, September 30th (supper on that day included). Full board is provided (three meals daily). For extra payment the rest house offers various recreation facilities (swimming pool, sauna, massage, solarium, well equipped gym-hall, tennis court).
Registration fee
The registration fee of 150 ECU includes participation fee, full board and accommodation costs, social events and school proceedings. It should be paid before September 1st, in USD (equivalent of 150 ECU on the day of payment) to the following account
Bank Slaski I/O Gliwice 312679-0020016301
with annotation "Summer School Gl 1330"
The registration fee can be also paid after arrival, in cash. In that case it will be increased to 170 ECU.
A small number of grants is available for young researchers from Eastern European countries.
Registration
The registration desk in the lobby of "Siwarna" will be open on Tuesday, September 26th, from 16:00 to 22:00 and on Wednesday, September 27th, from 8:00 to 14:00.
Social Events
Banquet Mountain trip/ Sightseeing tour (one day)
The days of social events will not be established until the beginning of the Summer School, since mountain trip depends on weather conditions.
Transport
There are regular train links with Zakopane from Warsaw, Wroclaw, Krakow and Katowice (in which airports are located). The timetables are available upon request under address given below. There are also regular bus links between Zakopane and each of these cities. To reach "Siwarna" from the bus or railway station you may take one of a number of minibuses (price about 1 zloty), a taxi or a bus to Koscielisko Valley. In the last case leave at Krzeptowki II stop and continue on foot (about 2 km).
Currency
Two currencies are valid in Poland: new zloty and old zloty. One new zloty equals ten thousand old zloties All prices are usually given in new zloty or both new and old zloties. USD exchange rate is about two and a half new zloties for one USD. Money can be exchanged in exchanged offices (marked "Kantor") and banks.
Weather
Although in September weather in Zakopane is usually nice, since it is situated in high mountains, participants should be prepared for any kind of weather, including strong wind and rain.
hi everyone-- i'm an undergrad just learning about this stuff, doing tem of the cruciate ligaments of the knee. i'm having some trouble obtaining fresh specimens in the right age range for my study and was wondering how important it was to use never-frozen specimens, and use fresh-frozen instead. i've heard that it's not the best thing to do but i can't seem to find documentation on it. my goal is to measure collagen fiber diameters, so if these would remain unchanged by the freezing, this would be the best route for me.
any help would be greatly appreciated. if possible, please send replies to me directly because i just subscribed to the server and am not sure if i am on the mailing list yet. thanks in advance...
tracy vogrin musculoskeletal research center pittsburgh, pa
Does anyone have experience using freeze substitution media other than the standard acetone or alcohols? I am trying to immuno-localize a substance which these readily extract. I have had a suggestion for n-butanol but I have no information on the compatibility of this (or other media) with substitution equipment or with various epoxies. Any advice would be appreciated.
From Hans Ris, hris-at-facstaff.wisc.edu, Integrated Microscopy Resource, University of Wisconsin, Madison, WI. USA. Walt Bobrowski and Sverker Enestrom commented recently on epon extraction for LM and Immunogold labelling using sodium ethoxide. Sodium or Potassium ethoxide or methoxide have been used in the past to remove epon from blocks or sections for LM cytochemistry or SEM imaging. Experience has shown that these agents are inefficient and highly destructive for cell structures. In 1990 Iwadare et al, Stain Technol.65,205-209, published an improved and less destructive protocol for LM cytochemistry and immunolabelling (crown ether complex). Dr Marek Malecki and I have explored this new technique for high resolution SEM imaging of thick epon sections after epon removal. The results have been published in the Journal of Structural Biology 111, 148-157(1993). This new technique preserves nanometer structures in the nuclear pore complex and insect flight muscle. After extraction of the epon immunolabelling of actin, myosin and alpha-actinin was successful. The extraction solution is commercially available. Source and details of procedures are described in the J.Struct.Biol. 111,148 (1993) paper by Ris and Malecki.
A large volume of messages relating to specimen preparation issues and concerns are exchanged on this listserver on a steady basis. It is obvious that any information on related training, service, or product resources are much needed and appreciated by the subscribers.
I would like to inform those of you with materials science applications that AMC Group offers advanced hands-on specimen preparation workshops semiannually on a regular basis. The upcoming workshops for Fall `95 have been scheduled as follows:
SEM/TEM SITE-SPECIFIC CROSS-SECTIONING 1. Wedge Polishing for TEM, Dallas, TX (Oct. 25-27) 2. Focused Ion-Beam (FIB) Milling, Hillsboro, OR (Nov. 1-3) 3. Microcleaving, Sunnyvale, CA (Nov. 6)
LM/SPM/SEM/TEM MATERIALS ULTRAMICROTOMY ***All to be held in Phoenix, AZ 1. UM for Surface Preparation (Oct. 16-17) 2. UM for Thin Section Preparation (Oct. 16-18) 3. Advanced UM (Oct. 19-20)
To receive a copy of the workshops brochure (including the registration form) please contact us directly. Thank you.
Rene E. Nicholas Business Manager AMC Group (602) 949-4203 FAX: (602) 947-7615 E-mail: AMCGroup2-at-aol.com
Repeated attempts to unsubscribe have been futile! Please help! I sent UNSUBSCRIBE messages each with one version of my e-mail address ddd-at-tx.technion.ac.il or ddd-at-techunix.technion.ac.il to the listserver LISTSERVER-at-AAEM.AMC.ANL.GOV but alas to no avail. Sorry to bother all subscribers, but maybe its time to renew instructions to list users.
7/24/95 Wicking off of PVA 7:21 AM Dear Microscopists, Has anyone developed a method of wicking off PVA from grids in a consistent manor, or found an alternative way of getting the excess PVA off the grids? I have too much inconsistency. There must be a better way... Thank-you, Jeanne
} Date sent: Fri, 21 Jul 1995 12:07:09 -0700 } To: Microscopy-at-AAEM.AMC.ANL.GOV } From: kim_rensing-at-mindlink.bc.ca (Kim and Cathy Rensing) } Subject: Freeze Substitution media
} Does anyone have experience using freeze substitution media other than the } standard acetone or alcohols? I am trying to immuno-localize a substance } which these readily extract. I have had a suggestion for n-butanol but I } have no information on the compatibility of this (or other media) with } substitution equipment or with various epoxies. Any advice would be } appreciated. } } Thanks, Kim Rensing } } Dear Kim Try the freeze-substitution medium of Kaeser (1989). This medium relies on the ability of dimethoxy- propane to dissociate into methanol and acetone in the presence of water. The medium is made up of:
6 ml DMP (acidified with three drops of 0.1 N HCl in 25 ml DMP) 3 ml acetone 2.25 ml methanol 0.75 ml 10% uranyl acetate in methanol 0.6 ml 25% aqueous glutaraldehyde
The addition of 0.1 g of osmium is your call, as is the percentage of glutaraldehyde recommended. The reference to this information is Journal of Microscopy, Vol. 154, Part 3, pp 273-278 (1989).
Good luck.
James Wesley-Smith Electron Microscope Unit George Campbell Building University of Natal Durban, South Africa
The CS-Auto has been superceded by a new piece of equipment. Many labs. with better funding than my own and greater need of freeze-substitution have purchased the new equipment. Is it therefore reasonable to try and get hold of a second hand CS Auto? I used one once on loan and it was excellent (apart from the large quantities of low viscosity resin that we spilt).
I hear a rumour that many laboratories have CS-Auto freeze substitution equipment that has been made redundant by purchase of the more modern system. If any one is interested in selling one to me for minimal cost, please get in touch. Obviously a UK based instrument would be easier to collect!
Best wishes, Paul
Dr Paul V. Hatton Lecturer in Biomaterials School of Clinical Dentistry University of Sheffield Claremont Crescent SHEFFIELD S10 2TA
Tel. (0114) 271 7938 Fax. (0114) 2665326 or 2797050
Message-Id: {v01510102ac39363c0410-at-[146.139.72.78]} Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" To: microscopy-at-aaem.amc.anl.gov
} Dear All, } } IS there a standard document describing the EMSA/MAS format } for EDX spectra? Is it on the network? } Many thanks } Trevor Sewell
There is a document on the net which describes the MSA/MAS format, but a more complete description of the format can be found in the EMSA Bulletin 21:2, pages 35-41, November, 1991. I looked at the on-line document this AM and it needs some corrections. If you follow its instructions in upper-case text, you should be OK. I will attempt to get a fully correct version put into place later this week. The document can be obtained by anonymous ftp from:
Russell E. Cook Electron Microscopy Center for Materials Research Argonne National Laboratory 9700 South Cass Avenue MSD-212 Argonne, IL 60439 (708)252-7194 FAX: (708)252-4798
While writing a response for you, I hit some key that not only killed my current document but my email listing as well.... Sorry I can't address you by name.
I process cryo electron microscopy almost daily. PVA/Ua staining is a matter of routine in our lab and we rarely have a problem.
First we make wire loops out of 30 AWG wire (from an EM supplier) and stuff the ends into micropipet tips. These can be used over and over again after cleaning with H2O.
Then we use a stock of 2% PVA(aq) and 3% UA(aq) (foil covered) kept at 4C.
When ready we mix 900ul of PVA with 100ul of Ua in a syringe and push it through a syringe filter onto ice chilled parafilm for staining (approx. 4 min) Invert and float the grids to stain.
We pick the grids up with the wire loops and (here is the important step) CENTER THE GRIDS WITH A FEW TAPS OF THE FINGER AND BOLT ALL OF THE STAIN FROM THE CORNERS WITH FILTER PAPER (we cut small pie shaped wedges out of the paper). That's blot not bolt.
Then stand the pipet tip up in a 96 well tissue culture plate. Or whatever.
Anyway, this method works very well and if you wish to ask me any questions, feel free. I would be honored to help.
Jaime A. Dant Washington University School of Medicine Molecular Microbiology St. Louis, MO 63110 jaime-at-borcim.wustl.edu (314) 362-4987
LM folk: We are labelling the muscles in small animals with BODIPY/ phalloidin. It looks like we are going to need freeze-sub to get good structural preservation (the animals contract violently in fixatives, and don't anesthetize). Freeze- sub appears to imply glutaraldehyde, which gives intense background staining (not surprising). Does someone out there have a favorite recipe for using Borohydride to reduce background in Glutaraldehyde-fixed material for fluorescence microscopy? TIA Julian Smith III Biology Winthrop University
We are trying to electropolish pure Pd and Pd/Ga alloys, but are having difficulty with getting a smooth surface. So far (following the method of Witcomb) we have been using 70% glacial acetic acid, 4% perchloric acid, 18% glycerol, 8% butyl cellosolve.
This stuff is *really* viscous at low temperature! We have found that we don't get polishing at the 40 volts reported by Witcomb, but have had to go higher (even to 80 volts!) During the polishing the electrolyte temperature climbs from 3-4C up to 15C due to lack of heat transfer to the ice bath.
Does anyone have any suggestions on improving our polishing procedure or on keeping our electrolyte temperature down?
Thanks in advance, Henk
Henk Colijn colijn.1-at-osu.edu OSU Campus Electron Optics Facility ------------------------------------------------------------------- An optimist believes that we live in the best of all possible worlds. A pessimist fears this is true.
Does anybody know of any good books for the processing of marine organisms and tissues? There seems to be plenty of references for mammalian tissues, viruses, bacteria, etc., but I am having problems finding a good reference for doing TEM/SEM on salt and fresh water samples. I would also like to know if there is an ultrastructural atlas available for these types of samples. If you know the publisher and ISBN number that would be a great help also.
Do not reply to me, READ the entire posting before replying.
ADJUNCT LECTURER POSITION
THE UNIVERSITY OF MICHIGAN
The Department of Materials Science and Engineering at The University of Michigan, Ann Arbor, is seeking candidates for a non-tenure track lecturer. Responsibilities include: assisting or leading in the development of new undergraduate teaching materials and methods that place a strong emphasis on multimedia presentation techniques; the development of self-paced, interactive learning modules; participation in the development and teaching of new undergraduate laboratory modules that place a strong emphasis on processing and physical and mechanical property characterization. The department currently enrolls approximately 140 undergraduate students and is an integrated materials program encompassing metals, ceramics, polymers and EMO materials. Applicants must have a Ph.D. in materials science and a demonstrated interest in undergraduate education. Minimum one year appointment, renewable to three years based on funding and performance. Send resume and list of references to:
Professor Albert F. Yee, Chairman Department of Materials Science and Engineering The University of Michigan 2300 Hayward Street Ann Arbor, MI 48109-2136 or afyee-at-engin.umich.edu
An Equal Opportunity Affirmative Action Employer
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, John.F.Mansfield-at-umich.edu or jfmjfm-at-umich.edu they all reach me! URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html
Thanks to all who responded to my (rather vague) question regarding asbestos. I got a lot of good leads to follow up.
Bob
Robert R. Wise, PhD Director, UWO Electron Microscope Facility Department of Biology University of Wisconsin Oshkosh Oshkosh, WI 54901 (414) 424-3404 (414) 424-1101 fax wise-at-vaxa.cis.uwosh.edu
We are setting up to do TEM of caddis fly larvae. Does anyone have a good protocol (or reference therefor) for TEM processing of insects? Will fixatives get through the intact exoskeleton? Is there any way to dissect them (to promote penetration of fixative) without ending up with a bunch of squished insect goo?
Bob
Robert R. Wise, PhD Director, UWO Electron Microscope Facility Department of Biology University of Wisconsin Oshkosh Oshkosh, WI 54901 (414) 424-3404 (414) 424-1101 fax wise-at-vaxa.cis.uwosh.edu
Hello... We have a new Chair for Microbiology who is interested in obtaining a used Sorval MT-2B Ultramicrotome for one of his people who trained on this instrument and desires one. Please e-mail such availability, price and information to gtcole-at-opus.mco.edu or call Dr. Garry Cole (419)381-5423. Thanks. Appreciate your help. Beverly Giammara (419)381-4996
Microwave-accelerated chemical fixation methods have been used with success to fix insects for TEM:
1. Lindley VA: A new procedure for handling impervious biological specimens. Microsc Res Tech 1992, 21:355-360
2. Smid HM, Schooneveld H, Meerloo T: Microwave fixation of water-cooled insect tissues for immunohistochemistry. Histochem J 1990, 22:313-320
3. contact me directly for detailed info.
In message writes: } We are setting up to do TEM of caddis fly larvae. Does anyone have a good } protocol (or reference therefor) for TEM processing of insects? Will } fixatives get through the intact exoskeleton? Is there any way to dissect } them (to promote penetration of fixative) without ending up with a bunch of } squished insect goo? } } Bob } } } Robert R. Wise, PhD } Director, UWO Electron Microscope Facility } Department of Biology } University of Wisconsin Oshkosh } Oshkosh, WI 54901 } (414) 424-3404 } (414) 424-1101 fax } wise-at-vaxa.cis.uwosh.edu }
Gary R. Login, D.M.D., D.M.Sc. Assistant Professor of Oral Pathology Beth Israel Hospital Department of Pathology 330 Brookline Avenue Boston, Massachusetts, 02215
Is anyone aware of a stain or cytochemical reaction to detect the presence of iodine in tissue? -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- Greg Erdos Phone: 904-392-1295 -at- -at- Scientific Director, -at- -at- ICBR Electron Microscopy Core Lab -at- -at- 218 Carr Hall Fax 904-846-0251 -at- -at- University of Florida E-mail: gwe-at-biotech.ufl.edu -at- -at- Gainesville, FL 32611 -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-
A colleague of mine without access to the microscopy listserver asked me to send out the following request. Any information on forthcoming courses, workshops or meetings (1995/1996) on microwave techniques in the UK, Europe, Australia or USA would be welcome. The emphasis should be on electron microscopy - TEM of tissue (human or animal) preferably in the field of pathology. Thank you, Hercules Els EM Unit, Fac Vet Sci, Univ of Pretoria Onderstepoort hjels-at-OP1.up.ac.za
} Hi, } } I would like to "smoothen" a surface of a polystyrene bulk sample. } } The surface roughness should be in the range of nanometer (or better?). } } I think normal polishing methods do not apply here since they are } } to coarse. } } Does anyone have an idea of a good method that could help? } } Thanks very much in advance for your help. } } jandt-at-msc.cornell.edu }
Quite an opportunity (unfortunately at someone's expense) is before you:
A company experiencing funding difficulties is selling a number of very high end Zeiss, Wild and Nikon scopes for both Cell and Animal work, as well as much other equipment involved with bio and recombinant studies.
Please address your Attention and inquiries to:
LESLIE FLINT or CAROLE KURAHARA GenPharm International 297 North Bernardo Ave Mountain View, CA 94043 415-964-7024 415-964-3537 (fax)
If you are serious abous some of their high end equipment, you may be able to persuade them to delay the sale deadline which is this Friday, or to conduct an auction (perhaps on the net ?)
Regards to all,
Ed Monberg LMDC 3101 Whipple Road Union City, CA 94587-1216
Dear Dr. Cole, Beverly Giammara posted a message on the microscopy listserver that you are looking for a used Sorval MT-2B ultramicrotome. I recommend that you contact Bill McGee at Microtome Service Co. at (315)451-1404. Bill is a former Sorval microtomy specialist who refurbishes and services microtomes. I confirmed with Bill that he has an MT-2B in stock. Best regards, Steven Slap
Hello out there fellow Hitachi H-7000 owners. We have an intermittent problem which you might have encountered and may know an answer to.
Intermittently- particularly if the microscope has been (pumping but) unused, operating the photo lever to take a film will cause the HV to cut out. Not from wet film, since it happens with film pumped for long periods in the microscope. Can't observe a rise in pressure when operating shutter so can't confirm a leak on the shutter shaft O ring.
I would have thought any leak into the camera area would be powerfully buffered by the pumping capacity up the column and wouldn't affect gun vacuum enough to cause a cut out.
Any relevant experiences will be gratefully received.
Mel Dickson, m.dickson-at-unsw.edu.au The University of NSW, Sydney, NSW Australia
I'm looking for a method to localize ornithine transcarbamylase HISTOCHEMICALLY, or failing that if anyone knows of a good antibody.... etc
Thanks. ------------------------------------------------------------------ Simon C. Watkins Ph.D Director; Structural Biology Imaging Center Scaife 840 University of Pittsburgh Pittsburgh PA 15261 Tel:412-648-3051 Fax:412-648-1441 -------------------------------------------------------------------
just a thought or two.... Assuming a clear non HIPS sample (crystal PS), the best way to polish the surface is with a hot, highly polished metal surface. The result would be similar to injection molding. There is also a kit available from Sporty's Pilot Shop for polishing acrylic windshields on airplanes. I think it is called "Micromesh". It might work with polystyrene. It is a slow process because you must avoid heat build up.
TREA-at-chevron.com ----------
To: MICROSC1--INTERNET MICROSCOPY
} Hi, } } I would like to "smoothen" a surface of a polystyrene bulk sample. } } The surface roughness should be in the range of nanometer (or better?). } } I think normal polishing methods do not apply here since they are } } to coarse. } } Does anyone have an idea of a good method that could help? } } Thanks very much in advance for your help. } } jandt-at-msc.cornell.edu }
Integrated Microscopy Resource (IMR) a Biomedical Research Resource
at:
The Wisconsin Center 702 Langdon Street Madison, WI 53706
#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#* Presentations will focus on biological problems for which a combination of microscopies [i.e. integrated microscopy] has been used. The speakers will demonstrate by example the power, potential and limitations of various microscopical techniques. The techniques which will be discussed include: DIC, Confocal, 2-Photon Excitation Imaging, SEM, TEM, Cryo-specimen Preparation, and AFM. #*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*#*
SCHEDULE
FRIDAY (EVENING), SEPTEMBER 29, 1995 6:00 - 7:55 OPENING RECEPTION 8:00 - 8:45 Ken Jacobson, Univ. N. Carolina-Chapel Hill 8:50 - 9:35 John Sedat, Univ. California-San Francisco 9:40 - 10:25 Edward Salmon, Univ. N. Carolina-Chapel Hill
SATURDAY, SEPTEMBER 30, 1995 8:30 - 9:15 D. Lansing Taylor, Carnegie-Mellon University 9:20 - 10:05 Jeff Lichtman, Washington University 10:10 - 10:40 COFFEE BREAK 10:45 - 11:30 John White, Univ. Wisconsin-Madison 11:35 - 12:20 Steven Smith, Stanford University 12:25 - 1:25 LUNCH (on your own) 1:30 - 2:15 Gary Borisy, Univ. Wisconsin-Madison 2:20 - 3:05 Ralph Albrecht, Univ. Wisconsin-Madison 3:10 - 3:55 Paul Bridgeman, Washington University-St. Louis
5:00 - 6:00 EXHIBITOR'S SOCIAL 6:00 - 8:00 BUFFET DINNER
SUNDAY (MORINING), OCTOBER 1, 1995 9:00 - 9:45 Kent McDonald, Univ. California-Berkley 9:50 - 10:35 Stanley Erlandsen, Univ. Minnesota 10:40 - 11:10 COFFEE BREAK 11:15 - 12:00 Hans Ris, Univ. Wisconsin-Madison 12:05 - 12:50 Eric Henderson, Iowa State University
FEES: General Registration $100.00 (Late Fee: $130.00) (Includes: Opening Reception, Social and Buffet Dinner, Coffee Breaks and Materials)
Local Registration $ 80.00 (Late Fee: $110.00) (Includes: Opening Reception, Social, Coffee Breaks and
Materials)
FOR ADDITIONAL INFORMATION CONSULT OUR WEB SITE
http://www.bocklabs.wisc.edu/imr/imr.html
TO RECEIVE A BROCHURE AND REGISTRATION FORM
WRITE: IMR Univ. Wisconsin-Madison 1675 Observatory Drive Madison, WI 53706
Following the symposium, the IMR will be conducting a 2-day workshop. We will be presenting lectures and provide "hands-on" experience for the following techniques: * 2-photon excitation imaging * 4D DIC imaging * Cryo-SEM * High pressure freezing * Reversible embeddment for SEM and TEM
Workshop attendence will be limited to 25 participants. A letter of application is required. Once accepted a fee of $150.00 will be due.
I run an old JEOL JXA-5A probe, which long ago lost its WDS detectors, and we now use only EDS.
The maximum beam deflection available in the scanning mode is about 200 x 200 microns (-at- 15kV), which is acheived by driving the deflection coils (X and Y) with about 0.3 volts. Since the coils are about 6 ohms, this means 50 mA, and 0.015 watts. I would like to be able to scan a larger area, even if just for the collection of secondary or backscattered electron images and not for point analyses.
It seems to me that the reason why JEOL limited the deflection to such small values was probably so that the beam stayed in the focal area of the original WD spectrometers. With the EDS setup I feel that decent analyses could be made at considerably larger deflections, although this doesn't really matter, my main concern is to be able to get BSE or SE images of larger areas ie smaller magnifications. The thing that stops me from just going ahead and building new deflection coil drivers with more output is my strong desire not to burn out the deflection coils, as on this 25-year-old instrument they would not be replaceable, and I would end up with no imaging facility at all, and a heap of disgruntled users!
I asked my local JEOL branch, in Sydney, Australia, for any information about the maximum permissible currents through the deflection coils, but after some time I was informed that such information was no longer available for an instrument of such age. I don't really want to pull the column apart to inspect the coils, as I'm scared of the possible realignment problems on reassembly.
My feeling is that 0.015W is not very much power for what is presumably a coil wound of copper wire, and that 0.5W could be ok for any "reasonable" coil. 6 ohms suggests to me either reasonably thick wire or a very few turns of fine wire. The former could presumably stand more current (how much? 100mA? 250mA?), the latter not.
Does anyone out there have any information, experience, or thoughts which could be useful?
thanks
Ritchie SimsRitchie Sims Department of Geology University of Auckland Auckland, New Zealand
You need booster amps for these coils. The amps need to have "hard" current= outputs which put out the appropriate current regardless of the voltage lev= el.
Collar a friendly EE who knows about Op-Amps, especially ones which can= dissipate, say, 5 watts and respond at the speeds of interest.
Depending on the inductance of the coils and the supplies you choose, (start with =B115VDC) and a monolithic amp such as NSC OP10, and a voltage to drive it, it should be fine.
Regards,
Ed Monberg {em-at-mediacity.com}
510-429-1060 Fax 429-1065 LMDC, (Laser Motion Development Co.) 3101 Whipple Road Union City, CA 94587-1216
Greg Erdos writes: } } Is anyone aware of a stain or cytochemical reaction to detect the presence } of iodine in tissue? } Dear Greg, Starch reacts with iodine, but not I-, and the reaction is very sensitive. Use something to oxidise the I- to I (if necessary or desir- able), and add starch solution. I used this method to detect peptides by perparing chloropeptides, adding KI to get iodopeptides, then adding starch. The method showed the presence of peptide when ninhydrin showed nothing. Good luck. Yours, Bill Tivol
X400-Received: by /PRMD=SCHERING-PLOUGH/ADMD=TELEMAIL/C=US/; Relayed; Fri, 28 Jul 1995 13:53:38 -0400 X400-Received: by /PRMD=SCHERING-PLOUGH/ADMD=TELEMAIL/C=US/; Relayed; Fri, 28 Jul 1995 13:53:38 -0400 X400-Received: by /PRMD=SCHERING-PLOUGH/ADMD=TELEMAIL/C=US/; Relayed; Fri, 28 Jul 1995 13:53:38 -0400
A colleague is interested in the phone number and location of a company called 'Caltag' that sells antibodies. Any information will be greatly appreciated.
Thank you!
Barbara Hartman E-Mail: Barbara.Hartman-at-Schering-Plough.sprint.com Phone: 201-579-4343 Fax: 201-579-4211
Dear microscopy-netters! Does anybody of you have any PC sharewares (free programs) for Image Processing and Morfology Analysis? I have so much structural images from SEM for analysis... Please send me programs or any helpful information! Thanks Siencerely Igor lapsker at Lapsker-at-barley.cteh.ac.il
I have been asked to process a range of starfish tissues which include the external layers and gonad tissue. I propose to fix the soft tissues in 2% glutaraldehyde made up in filtered seawater, buffer wash in 0.2m cacodylate buffer plus 2% NaCl (approx 1000 mosmols) then fix in osmium tetroxide in 0.2M buffer.
Question: Do I need to use 0.2M buffer to wash the tissue before OsO4 fixation or can I use the seawater with the OsO4? My concern is whether the seawater and OsO4 react. My main problem is as follows: I assume I have to decalcify the external layers. Can someone please send me a method for decalcifying tissues from marine specimens? My standard mammalian decalcifying fluid is as follows: 41.3 disodium EDTA, 4.4g NaOH and make up to 1000mls with distilled water. To use the fluid I made up as follows: 1.75 % glutaraldehyde in decalcifying fluid. Could I make up the decalcifying fluid in seawater instead of distilled water or will the EDTA react with seawater?
Thanks in anticipation.
Allan Mitchell
Richard Easingwood South Campus Electron Microscope Unit Otago Medical School PO Box 913 Dunedin NEW ZEALAND
Igor: The best software that fits the description of free image analysis software for manipulation and morphology analysis is NIH Image (from the US National Institutes of Health). It will run on virtually any Apple Macintosh computer you have available to you. NIH Image contains a powerful scripting language and the PASCAL source code is available free of charge if you are interested in some serious coding. If you are unable to find a Macintosh, you can buy a Mac emulator for your DOS/Windows computer which has been successfully tested with the more-recent versions of NIH Image.
NIH Image is available by anonymous FTP at: zippy.nimh.nih.gov
There is a mail list for discussion of NIH Image (and general digital imaging issues, as well). To subscribe, send the message: subscribe nih-image YourNameHere to the list server: listproc-at-soils.umn.edu
For a fee of $100 you can get the NIH Image software and manual (I think) mailed to you from: NTIS 5285 Port Royal Road Springfield, VA 22161 USA
The Macintosh emulation software which runs under DOS and Windows 3.1 is called Executor and is available for $99. Contact them by e-mail at: questions-at-ardi.com
Good Luck,
Bill ========================== Bill Heeschen/Analyt. Sci.- Mat. Char. ----- 1897-F Building / The Dow Chemical Co. --- --- Midland, MI 48667 U.S.A. --- D O W --- phone: (517)636-4005 fax: (517)636-5453 --- --- Email: waheeschen-at-dow.com ----- ========================== R
Thank you to those who replied to my query, this is a marvellous facility.
I took up my courage and screwdrivers, gained access to the deflection coils, and was able to measure the coil wire gauge, which is, in case anybody ever needs to know, about 0.15mm diameter ie about the same as 3 Amp fuse wire. So I made a similar test coil on a plastic former, which softened the former at 0.8 A but seems indefinitely content at 0.4 A. This gives me sufficient courage to up the drive to the coils by a factor of 5X the present max of 50mA ie 250mA.
thanks again
Ritchie SimsRitchie Sims Department of Geology University of Auckland Auckland, New Zealand
Dear microscopy-netters! Does anybody of you have any PC sharewares (free programs) for Image Processing and Morfology Analysis? I have so much structural images from SEM for analysis... Please send me programs or any helpful information! Thanks Siencerely Igor lapsker at Lapsker-at-barley.cteh.ac.il
We are working in a marine biology laboratory studying echinoderms. For electron microscopy, we fix routinely our samples with the following protocol (all in final concentrations): - fixation: 3 hours in glutaraldehyde 3%, cacodylate buffer 0.1M, NaCl 1,55%. pH=7.8, osmolarity: 1030 mOsm. - buffer washes: 3 x 10 minutes in cacodylate buffer (0.2M), NaCl 1.84%. pH=7.8, 1030 mOsm. -postfixation: 30' in OsO4 1%, cacodylate buffer 0.1M, NaCl 2.3%.` pH=7.8, 1030 mOsm. - three buffer washes before deshydratation.
According to our experience in the subject, this protocol seems to be one of the best to fix the tegument and other tissues in echinoderms.
If you need to decalcify your samples, there are two methods: -simple embedding method: after postfixation and the three buffer washes, one bath of EDTA 10% (pH 7.8) during 24 hours. This method is easy but the ultrastructure is poorly preserved (diffuse appearance). -double embedding method: here, you decalcify the samples after the embedding. As the EDTA needs to reach the calcified parts, you must abrade the resin. It is very important that each skeletal element (spines, plates...) be at the surface of the abraded resin otherwise the decalcification will be incomplete. Then one bath of EDTA 10% pH 7.8 in a vacuum jar during 48 hours, followed by a wash of bidistilled water (24 hours, under stirring). Dry your samples on filter paper before desiccation by P2O5 in a vacuum jar during 24 hours. Then impregnation with the resin (Spurr) in the vacuum jar during three hours (with P205 inside the jar) before final embedding. This method is described in Holland & Grimmer 1981. Cell Tissue Res. 214: 207-217. Altough longer, this method gives a better ultrastructure than the first method.
Good luck and fun,
Laurent Ameye & David Gillan e-mail : lameye-at-ulb.ac.be
Marine Biology Laboratory (CP 160/15) Free University of Brussels (ULB) 50 Av F.D. Roosevelt B-1050 Brussels, Belgium. Fax: ++/32/2/6502796 Tel: ++/32/2/6502970
We are working in a marine biology laboratory studying echinoderms. For electron microscopy, we fix routinely our samples with the following protocol (all in final concentrations): - fixation: 3 hours in glutaraldehyde 3%, cacodylate buffer 0.1M, NaCl 1,55%. pH=7.8, osmolarity: 1030 mOsm. - buffer washes: 3 x 10 minutes in cacodylate buffer (0.2M), NaCl 1.84%. pH=7.8, 1030 mOsm. -postfixation: 30' in OsO4 1%, cacodylate buffer 0.1M, NaCl 2.3%.` pH=7.8, 1030 mOsm. - three buffer washes before deshydratation.
According to our experience in the subject, this protocol seems to be one of the best to fix the tegument and other tissues in echinoderms.
If you need to decalcify your samples, there are two methods: -simple embedding method: after postfixation and the three buffer washes, one bath of EDTA 10% (pH 7.8) during 24 hours. This method is easy but the ultrastructure is poorly preserved (diffuse appearance). -double embedding method: here, you decalcify the samples after the embedding. As the EDTA needs to reach the calcified parts, you must abrade the resin. It is very important that each skeletal element (spines, plates...) be at the surface of the abraded resin otherwise the decalcification will be incomplete. Then one bath of EDTA 10% pH 7.8 in a vacuum jar during 48 hours, followed by a wash of bidistilled water (24 hours, under stirring). Dry your samples on filter paper before desiccation by P2O5 in a vacuum jar during 24 hours. Then impregnation with the resin (Spurr) in the vacuum jar during three hours (with P205 inside the jar) before final embedding. This method is described in Holland & Grimmer 1981. Cell Tissue Res. 214: 207-217. Altough longer, this method gives a better ultrastructure than the first method.
Good luck and fun,
Laurent Ameye & David Gillan e-mail : lameye-at-ulb.ac.be
Marine Biology Laboratory (CP 160/15) Free University of Brussels (ULB) 50 Av F.D. Roosevelt B-1050 Brussels, Belgium. Fax: ++/32/2/6502796 Tel: ++/32/2/6502970
I'm looking for a source of the plastic sheet material made of ACLAR (TM?). A source of slides and/or coverslips made of the stuff would be ideal, but I will go for anything that might be used in lieu thereof. Also, if anyone has experience using this material for TEM embedding, I would be interested in their technique and opinion. Thanks in advance for any help you can give me.
Dan
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % % % Daniel Possin Work: 206/ 543-7489 % % Dept. of Ophthalmology RJ-10 FAX: 206/ 543-4414 % % University of Washington Home: 206/ 778-1714 % % Seattle WA 98195 USA Email: oemlab-at-u.washington.edu % % % % "The chinese expression 'cheung meng ba sui, gong hey fat choy' is % % equilvalent to Vulcan expression 'live long and prosper'. It's a % % small universe and getting smaller everyday". % % % %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
CAN ANYONE RECOMMEND AN INEXPENSIVE FRAME GRABBER THAT CAN EASILY INTERFACE WITH AN ANALOG JEOL 840-I , PC PREFERABLE . I HAVE A 486 WITH 16 MEG RAM THATS READY FOR IT BUT COULD ALSO GET HOLD OF A MAC. I ALSO HAVE ALL THE IMAGE ANALYSIS SOFTWARE. MY ONLY PRESENT WAY TO DIGITIZE SEM IS BY SCANNING MY 4X5 NEGS, BUT WOULD LIKE TO SKIP THE PHOTO STEPS. ATTEMPTING TO QUANTITATE IMMUNO GOLD SIGNALS IN BSE MODE (SLOW SCAN)
MicroscopyListserver Archive Email Extraction Software Version NJZ07060908