For those still interested in the earlier question, here is additional information re: EM lab safety, courtesy of one of our labs OSHA safety inspectors/industrial hygenists.
-----------------------------
There is a (relatively, i.e. 1990) new OSHA regulation that applies specifically to laboratory use of hazardous chemicals: 29 CFR 1910.1450 "Occupational Exposure to Hazardous Chemicals in Laboratories." It's primary requirement involves developing and implementing a Chemical Hygiene Plan that sets forth procedures, equipment, personal protective equipment and work practices that are capable of protecting employees from health hazards associated with hazardous chemical use (chemicals that meet the definition of "health hazard" under OSHA Hazard Communication Standard). Employers who might fall under this application should get a copy of the regulation from their regional OSHA office; also available is a small pamphlet "OSHA 3119 - Exposure to Hazardous Chemicals in Laboratories" from the OSHA Publications Office (202) 523-9667. -- David
................................................................. David I. Jacobi (dj5-at-gtri.gatech.edu) Georgia Tech Research Institute/EOEML 022F O'Keefe Building, Atlanta, GA 30332-0837 Phone: 404/894-8089; FAX: 404/894-8275 Elizabeth (Eliesh) G. O'Neil Research Scientist I GTRI/EOEML Baker 271, 925 Dalney Street Atlanta, GA 30332-0827 (404) 853-0590 (office) (404) 894-6199 (FAX)
Maybe one of the manufacture-ers/distributers of slides & coverslips is watching: is there some film now being applied to slides & coverslips, or a change in cleaning methods that might leave such a film? Also: Mark Elliot: are you mounting with Histo-Mount? I found that that makes a real difference when using Hisot-Clear. Phil Oshel poshel-at-luc.edu
John C. Wheatley: Glad to see ASU worries about OSHA [& RCRA?]--you may be unique. For MSDS sheets--Sigma, Baxter, & I think Fisher--by law, everyone who sells chemicals, I think--provides free MSD sheets by call to 800 #'s in there catalogs. Most will fax if mail is too slow. Phil Oshel poshel-at-luc.edu
Phil P., Yes, I've found that 0.2 micron filtered seawater helps much with marine orgs. You may still need fome extra buffering for the pH. Karnovsky's or 4:1 formaldehyde:glut as per Dykstra works well as a fix for molluscs & protista--as a first try anyway. UNESCO published a book on the fixation & preservation of marine animals, esp. zooplankton. You might also try writng marine labs for theirs methods--many have written technique books--like U Alaska Fairbanks & MBL at Woods Hole. Phil Oshel poshel-at-luc.edu
Concerning type of glass used: I am doing cross sectional TEM on vacuum deposited thin films on glass slides, and noticed that several types of glass have a thin surface film structure different in composition than the bulk. For ex, some has a layer denuded of Na (sodaglass), corning glass has a layer denuded of Ba. Layer thicknesses measured about 50nm to 100nm.
Could it be that these layers are present in ordinary glass slides or cover glasses, and has different wetting characteristics ?
Dirk Knoesen Dept Atomic and Interface Physics, Utrecht Univ, Netherlands
Someone replied directly to me re: my slide water droplets comments--Maillot?--but the mssg got erased before I could read it. If this makes sense to whoever sent the note, could they resend it, please? Phil Oshel poshel-at-luc.edu
Yes, chemicals (senus lato.) are supplied with MSDS sheets. And I dutifully read them, catalog them, and inform all my users in the facility of their existance right in the middle of the chmical work lab. However my concern is the really limted amount of information provided by them. Particularly in regards to disopsal of small quantities used in University settings. The MSDS sheets tend to regard spills of tank car quantities not 2 ml lab quantiies, and state that you follow "State and local quidelines". Well state and local quidelines say nothing about 2ml of OsO4 or DAB.
Working with a full respirator and protective suit for 30ml of glutaraldehyde might be very safe, but is a little over kill, and prohibitively expensive, especially when down the hall there are 35 students hunched over several formalin infiltrated corpses in the gross anatomy lab. Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
JOURNAL OF MICROSCOPY, VOLUME 175, PART 3, SEPTEMBER 1994
This issue features five invited contributions, originally presented at the 6th European Congress for Stereology, Prague (September 1993).
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 176-185
Stereological analysis of the spatial Poisson-Voronoi tessellation
UTE HAHN & UDO LORZ, Freiberg University of Mining &, Technology, Institut fur Stochastik, Bernhard-von-Cotta-strasse 2, D-09596 Freiberg, Germany
SUMMARY
Stereological model tests and parameter estimators for the spatial Poisson-Voronoi tessellation are discussed. The tests aim to discriminate the Poisson-Voronoi tessellation from more regular or more irregular tessellations. The power of the model tests under some special parametric alternative hypotheses is investigated by simulation. Among the tests considered, the most powerful test is based on the variance of the section cell areas. Various stereological estimators for the model parameter of the spatial Poisson-Voronoi tessellation are compared with respect to their bias and variance by means of a Monte-Carlo study. Formulae are given for variance prediction. An estimator based on vertex counting is found to be best. Robustness is investigated by applying the estimators to Voronoi tessellations generated by other point process models.
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 186-194
Second order stereology for pores in translucent alumina studied by confocal scanning laser microscope
LARS KARLSSON & ANDERS LILJEBORG, Royal Institute of Technology, S-100 44 Stockholm, Sweden
SUMMARY
The three-dimensional (3-D) arrangement of pores in translucent alumina was investigated with a confocal scanning laser microscope (CSLM). By moving the focal plane of the CSLM down into the material, a stack of serial thin optical sections was obtained to produce a 3-D image of the pores. Computer-based image analysis was used to obtain the coordinates of the pore centroids. The distance distribution function G(r) and the second-order functions K(r), L(r), H(r) and g(r) were used to analyse the spatial point pattern of the pore centroids. Estimates of the preceding functions obtained from eight stacks of sections were compared with the corresponding functions for a 3-D stationary Poisson point process, which served as a reference model for complete spatial randomness. The analysis suggested that the pore centroids were arranged in an aggregated pattern within a range of about 10 micrometre.
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 195-204
Analysis of homogeneity of phase repartition in TiB2-Fe composites using variance and covariance analysis
J. M. MISSIAEN & J. M. CHAIX, Laboratoire de Thermodynamique et, Physico-Chimie Metallurgiques, CNRS URA 29 ENSEEG, BP 75 Domaine Universitaire, F-38402 Saint-Martin d'Heres Cedex, France
SUMMARY
The microstructure of sintered TiB2- Fe material has been studied by image analysis. The dispersion of phases, with particular attention to porosity, is investigated using chord lengths, covariance functions and variance of volume fractions. An extension of the usual point covariance analysis is made by sampling series of 10 contiguous fields, and measuring the covariance in the direction of field alignment. A model of multiscale structure is proposed to interpret the different transitions of the variogram as composition fluctuations at different scales. Boolean models are used to give a geometrical representation of these fluctuations: the dispersion of porosity can be seen either as 20-30-micrometre pore clusters or as a result of TiB2+Fe 30-50-micrometre clusters, probably formed during the elaboration process. Complementary information is obtained from the evolution of measured variance versus field size: a model is proposed to analyse the shape of the experimental curves. At small scales, 'short' integral ranges are determined, also obtained from the covariance. At very large scales (} 300 micrometre), composition fluctuations of about 2% are detected.
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 205-213
Modelling of vascular growth processes: a stochastic biophysical approach to embryonic angiogenesis
KONRAD SANDAU & HAYMO KURZ, Mathematik und Naturwissenschaften, Fachhochschule Darmstadt, Shofferstrasse 3, D-64295 Darmstadt, Germany
SUMMARY
In a computer simulation, growth of a capillary network is driven by a stochastic process on a planar hexagonal grid. Starting at a point source, the probabilities for the formation of new capillary elements depend on local biophysical knowledge. This knowledge is mainly derived from the flow theorem of Hagen- Poiseuille and the diameter exponent. The hexagonal grid is visualized as being supported by a cylinder or a sphere. An arterial tree results from the adaptive diameter augmentation, and is considered to have limited fractal properties. The dimension of its border, and the time course of growth and of blood pressure are compared with biological data from the chorioallantoic membrane (CAM) of incubated chicken eggs. The model is discussed with reference to the mechanosensitivity and cell-matrix interactions of endothelial cells, and CAM haemodynamics.
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 214-221
Shape processing and analysis using the calypter
ERIC PIRARD, Universite de Liege, Laboratoires de Geologie Appliquee, Avenue des Tilleuls 45, 4000 Liege, Belgium
SUMMARY
After presenting some preliminary criteria that should be respected by any automatic shape analysis technique, this paper focuses on the importance of the binary image encoding method. Most image analysers simply use a raster image to represent a binary object. If, occasionally, a vectorial description is available, it is merely chosen for its performances in data compression. Data compression and shape analysis have different goals and usual methods cannot satisfy both. The calypter is a new descriptor vectorizing the shape as a set of maximal inscribed discs. It is the most efficient means of accomplishing Euclidean mathematical morphology transformations and allows for further developments in binary shape processing. A simple adaptive contour filtering technique is presented. The calypter offers local and global perception of shape characteristics. It permits complete automation of the morphometric roundness charts used in many laboratories and also generates new shape parameters. A case study of three sand populations is presented to show the pertinence of a new 'equivalent roundness' parameter.
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 222-228.
Retention of vacuole contents of plant cells during fixation
Z. DONG, M. E. MCCULLY & M. J. CANNY, Department of Biology, Carleton University, 1125 Colonel Drive, Ottawa, Ontario, Canada K1S 5B6
SUMMARY
Changes in the semi-permeability of tonoplast during fixation were studied using beetroot tissue. Using vacuole betacyanin as the indicator, the permeability of the tonoplast was assessed by the leakage of this pigment as determined by changes in the optical density of the solution bathing the tissue. Cryo- analytical scanning electron microscopy was used to monitor the changes in ion concentration in cells during fixation. Fixatives were 3% glutaraldehyde or 4% formaldehyde in 0.025M phosphate buffer at room temperature or on ice. Results showed that glutaraldehyde, especially at low temperature, takes as long as 30h to disrupt the semi-permeability of the tonoplast of beetroot cells, while in formaldehyde on ice, beet cells lose their selective permeability in 15min. This study confirms that the semi-permeability of the tonoplast may not be lost until long after the cytoplasm has been fixed and suggests that this explains why cold fixation in 3% glutaraldehyde for about 12h has become the most reliable standard procedure for the successful preservation of vacuolated plant cells.
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 229-237
Central and peripheral nervous structures as seen with the confocal scanning laser microscope
P. CASTANO, A. MARCUCCI, A. MIANI Jr, M. MORINI, S. VERALDI & C. RUMIO, Institute of Human Anatomy, Via Mangiagalli 31, 20133 Milano, Italy
SUMMARY
Central neurons and peripheral nervous structures, e.g. cutaneous free endings, perifollicular nets, Meissners corpuscles and intramuscular fibres, were studied using various impregnation methods. The confocal scanning laser microscopes (CSLMs) used were equipped with different laser sources, in order to evaluate their limitations and advantages with these techniques and to contribute to a better understanding of the general morphology of the nervous system. When staining with silver sections with clouds of tiny silver granules which are beyond the resolution power of the conventional light microscope but which show a high reflectivity with the CSLM are obtained. Golgi-Cox mercuric impregnation, however, provides specimens which are precipitate- free, thus ensuring the reliability of information obtained. It does, however, have the disadvantage of being applicable only to the central nervous system. In all cases it is an advantage for the instrument to be fitted with different lasers (e.g. Ar and He-Ne), so as to optimize the images of samples impregnated with different methods. Notwithstanding the possibility that artefacts may distort the geometry of the sample and reduce the resolution, the images presented in this paper show that with careful selection of optical sectioning distances, the use of a suitable stack of sections and, if necessary, the aid of false electronic colours and of partial or complete rotation, it is possible to achieve a more precise interpretation of the morphology and organization of complex structures, such as those of the nervous system.
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 238-251
Optimizing the performance of confocal scanning laser microscopes over the full field of view
A. ENTWISTLE & M. NOBLE, The Ludwig Institute for Cancer Research, 91 Riding House Street, London, W1P 8BT
SUMMARY
To examine many of the imaging capabilities of confocal scanning laser microscopes rapidly and reliably over the whole field of view, three simple, easily prepared specimens are required: a mirror positioned on a carefully measured shallow gradient, a film of highly fluorescent material and a rectangular grid with a readily defined centre. Using these specimens the adjustment of any combination of confocal scanning laser visualization system and light microscope can be examined throughout the field of view. The effects of misalignment of the various subcomponents of a confocal scanning laser microscope on both the axial spread function of the plane and the shading pattern over the image field are described. Finally, where the design of the confocal optics permits, the three specimens can be used to facilitate the alignment of the various components to the optimal level achievable.
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 252-265
HREM image simulations for supported particle catalysts
MING-HUI YAO & DAVID J. SMITH, Centre for Solid State Science, Arizona State University, Tempe, Arizona 85287-1704, USA
SUMMARY
The imaging conditions for electron microscope studies of supported ultrafine particle catalysts have been investigated by multislice simulations. Images of Pt and ReO4 particles ranging from 0.4 to 2.3 nm in size were simulated in both plan view and profile view with a rutile (TiO2) support. It was shown that particle visibility varied greatly with the objective lens defocus. Optimum defocus was not favourable for supported particles in plan view since the ultrafine supported particles were the least visible at this defocus. Underfocusing, especially at defoci corresponding to half-spacing fringes in the TiO2 support, led to improved visibility and resolution of the supported particles. Although the structure and shape of supported ultrafine particles should be resolved better with a 400-kV high-resolution electron microscope, their detectability is poorer than with a 200-kV instrument. An ReO4 cluster should be detectable at 200kV on TiO2 supports up to 5nm in thickness, whereas it is only likely to be detectable at 400kV on supports up to 3nm in thickness. The simulations confirmed that optimum defocus is mst favourable for imaging supported particles in profile view. Atomic information for particles as small as a 13- atom Pt cuboctahedral cluster should be resolvable with a 400-kV instrument. The crystalline Ti monolayer observed on surfaces of Pt particles, which could explain the mechanism known as SMSI, was simulated as an example of profile imaging.
John Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, University of Michigan 2455 Hayward, Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 12:08
Date:9/2/94 URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL
Hi there, I am about to buy a video printer to replace the majority of our Polaroid prints on our SEM. I want something that will give me about Polaroid size (i.e. 4X5 inch ) but is cheaper. I think Sony and Mitsubishi make printers in this area. Printer needs to be 256 greys and needs to be connected to both an ESEM monitor and also a Mac monitor. Anyone have any suggestions as to the best product? Any to avoid? reply by email please and I will summarize to the net if need be. Please dont send messages saying I should be buying a Dye Sub printer, we have one, I want cheap student notebook type output.
John Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, University of Michigan 2455 Hayward, Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 12:14
Date:9/2/94 URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL
Sorry to ask a non microscopy question, but has anyone experience with PC and Mac to S-VHS converters? Actually it is microsocpy related, I want to video tape the Digital Instruments AFM in action (NO, I dont want to point a camcorder at the screen!). I need something that costs less than an arm and a leg and does the job nicely. Any experiences or comments? Email replies please. Thanks. john Mansfield
1.Who can provide me with information on lectin based labeling of carbohydrate polymers (e.g.EPS) for EM (and LM) studies? 2.Does someone have experience in thin film observations of carbohydrate polymers in TEM? 3.Does someone have suggestions (e.g. literature references) for chemical fixation of carbohydrate polymers (EPS) for TEM or SEM observations?
Regards,
Marcel Paques Unilever Research Laboratory The Netherlands Phone:(31)10-4605515 Email: Marcel.Paques-at-URLNL.Sprint.Com
We want to do the same thing as John, so if you would cc: to me as well I would appreciate it. Thanks-
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
On 2 Sep 1994, John Mansfield wrote:
} John Mansfield } North Campus Electron Microbeam Analysis Laboratory } 413 SRB, University of Michigan } 2455 Hayward, Ann Arbor MI 48109-2143 } Phone: (313)936-3352 FAX (313)936-3352 } jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: } 12:14 } } Date:9/2/94 } URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL } Subject: PC and Mac to S-VHS } } Sorry to ask a non microscopy question, but has anyone experience with PC and } Mac to S-VHS converters? Actually it is microsocpy related, I want to video } tape the Digital Instruments AFM in action (NO, I dont want to point a } camcorder at the screen!). I need something that costs less than an arm and } a leg and does the job nicely. Any experiences or comments? } Email replies please. } Thanks. } john Mansfield } }
Dear Scott, Two referrences for the information you need are: 1) Berger & Seltzer, in Studies in Penetration of Charged Particles in Matter, Pub. 1133, NAS-NRC (1964), available from NAS printing & publishing office, 2101 Constitution Ave, N.W., Washington DC -at-0148. 2) Berger & Seltzer, Stopping Powers and Ranges of Electrons and Positrons, NBSIR 82-2550 (1982) Office of Standard Referrence Data, National Bureau of Standards, Washington DC 20234. As you can see by the second address, these addresses may be outdated. What you need to do is to use the relationships 1/R = w1/R1+w2/R2+... and S = w1S1+w2S2... where the w's are weight fractions. These relationships are good to a few %. The proper units for the range & stopping power are g/cm**2 and MeV*cm**2/g. The earlier ref gives data for Kr & Ag and the later gives data for Kr and Mo. You will have to interpolate the data to get the values for Nb; N is listed in either ref. I can fax you the appropriate pages if you can't get the refs for your- self. I know we have been guarding our copies jealously--they've been here longer than I have. Who knows, NIST or NAS may even have more up-to-date pubs. If so, I'd be interested in obtaining them. Good luck. Yours, Bill Tivol
Regarding grating replicas... just remember Norbert's incredible efforts to make calibration possible with the light microscopes in the last century.\nina allen
We are looking for initial expressions of interest in the following position. I would be grateful if this could be advertised as widely as possible. Any at all interested should send a short CV to my e-mail address below, or fax (61)-9-380-1087, within the next few days.
Many thanks, Andy Johnson, Centre for Microscopy and Microanalysis, University of W.A.
TEACHING AND RESEARCH FELLOW A proposed joint position at The University of Western Australia between the Centre for Microscopy & Microanalysis, the Department of Physics and the Department of Chemistry with the possible involvement of the Department of Mechanical & Materials Engineering.
We are applying for a three year teaching and research position which will have good prospects of continuation for a person with a sound PhD in Physics, Chemistry or Engineering, some research experience involving transmission electron microscopy and electron energy loss spectrometry and an interest in teaching. We envisage the position will combine teaching and research of the latest electron energy loss imaging and diffraction techniques to Honours and Graduate Students with research in that area. The research equipment comprises a Philips EM430 TEM fitted with a Gatan Image Filter housed in the University's Centre for Microscopy & Microanalysis. The Centre also has a wide range of electron microscopes (3 SEMs, a FESEM, ESEM, JEOL 2000FXII TEM) and a microprobe. All are equipped with EDS and modern attachments. The staff of the Centre are actively engaged in teaching and research programs involving electron and confocal microscopy through which the Centre is in close collaboration with the academic departments of the University of W.A., other major universities in Perth and with industry. The fellow will be expected to be actively engaged in similar programs and in the courses of the participating Departments as well as their own research.
At present the University has called for preliminary submissions of the names of potential Fellows who, if successful, will be required to take up their positions by mid 1995.
Some of the important selection criteria are: * applicants will have completed a PhD or equivalent within the previous five years. * the quality of the applicants will be assessed by: their academic record, current & potential performance in research, potential performance in teaching.
This is a wonderful opportunity for a young person to advance their career in a broad academic environment.
Please send your CV, a short version is sufficient at this stage, without delay to Dr Andrew Johnson, Director, Centre for Microscopy and Microanalysis, University of W.A.. email: andy-at-earwax.pd.uwa.edu.au fax 61-9-380-1087
I recently purchase a Reichert Zetopan microscope and am trying to locate certain accessories for it. If anyone has any such items or know of individuals who might, please contact me with descriptions and prices or I will provide a list of desired items. Please contact me directly at:
e-mail: HOWEY-at-UWYO.EDU
OR
Richard L. Howey 703 So. 10 Laramie, WY 82070
Home Tel: (307) 742-3545 Office Tel: (307) 766-2200
You were asking about video printers for an ESEM. We have been using the Mitsubishi model p68 for several years now and are very satisfied. Each of our SEM's (3) has a printer attached which is switchable between the SEM screen and an EDS/MCA display. The Mitsubishi printer takes inputs as RGB (TTL or analog/PC or Mac) as well as regular composite video. We have set our printers up with a switch to toggle the inputs between video and RGB, allowing quick sequential images from either source. The switch connection, across the appropriate DIP switch on the rear of the printer, can be easily installed by a competent technician.
David Vowles Electron Microscopy Unit Australian National University PO Box 475 Canberra ACT 2601 Australia Tel:(61 6) 2493543 Fax:(61 6) 2494891 E-mail:vowles-at-rsbs-central.anu.edu.au
You were asking about video printers for an ESEM. We have been using the Mitsubishi model p68 for several years now and are very satisfied. Each of our SEM's (3) has a printer attached which is switchable between the SEM screen and an EDS/MCA display. The Mitsubishi printer takes inputs as RGB (TTL or analog/PC or Mac) as well as regular composite video. We have set our printers up with a switch to toggle the inputs between video and RGB, allowing quick sequential images from either source. The switch connection, across the appropriate DIP switch on the rear of the printer, can be easily installed by a competent technician.
David Vowles Electron Microscopy Unit Australian National University PO Box 475 Canberra ACT 2601 Australia Tel:(61 6) 2493543 Fax:(61 6) 2494891 E-mail:vowles-at-rsbs-central.anu.edu.au
I recently purchased a Reichert Zetopan microscope and am trying to locate certain accessories for it. If anyone has any such items or knows of individuals who might, please contact me with descriptions and prices or I will provide a list of desired items. Please contact me directly at:
e-mail: HOWEY-at-UWYO.EDU
OR
Richard L. Howey 703 So. 10 Laramie, WY 82070
Home Tel: (307) 742-3545 Office Tel: (307) 766-2200
We have a Siemens electronmicroscope no.302 here, still in good condition and with replacement electronic bulbs and numerous spare parts here.It was produced in 1959. It appears to be of scientific historic interest and might be offered to a technical museum . Morten M.Laane ,Prof.of biology , University of Oslo.
We have a Siemens electronmicroscope no.302 here, still in good condition and with replacement electronic bulbs and numerous spare parts here.It was produced in 1959. It appears to be of scientific historic interest and might be offered to a technical museum . Morten M.Laane ,Prof.of biology , University of Oslo.
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I've gotten a few messages recently asking why messages posted to the newsgroup donot appear to Microscopy and this is the reason why.
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Dear microscopists,
1.Who can provide me with information on lectin based labeling of carbohydrate polymers (e.g.EPS) for EM (and LM) studies? 2.Does someone have experience in thin film observations of carbohydrate polymers in TEM? 3.Does someone have suggestions (e.g. literature references) for chemical fixation of carbohydrate polymers (EPS) for TEM or SEM observations? 4.Does someone have suggestions (e.g. literature ref.) for specific staining procedures of carbohydrates for CSLM observation?
Regards,
Marcel Paques Unilever Research Laboratory The Netherlands Phone:(31)10-4605515 Fax:(31)10-4605671 Email: Marcel.Paques-at-URLNL.Sprint.Com
John- some of the best printers that we have looked at are from: Alden Electronics Inc. (508) 366-8851 40 Washington St. Westborough, MA 01581 they have several models which produce quality images for under $1.00/print. -Mike On 2 Sep 1994, John Mansfield wrote:
} John Mansfield } North Campus Electron Microbeam Analysis Laboratory } 413 SRB, University of Michigan } 2455 Hayward, Ann Arbor MI 48109-2143 } Phone: (313)936-3352 FAX (313)936-3352 } jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: } 12:08 } } Date:9/2/94 } URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL } Subject: Video Printers } } Hi there, I am about to buy a video printer to replace the majority of our } Polaroid prints on our SEM. I want something that will give me about } Polaroid size (i.e. 4X5 inch ) but is cheaper. I think Sony and Mitsubishi } make printers in this area. Printer needs to be 256 greys and needs to be } connected to both an ESEM monitor and also a Mac monitor. Anyone have any } suggestions as to the best product? Any to avoid? } reply by email please and I will summarize to the net if need be. } Please dont send messages saying I should be buying a Dye Sub printer, we } have one, I want cheap student notebook type output. } }
Nestor, General message to microscopy list received at this site, understood, and information filed for future reference. I double checked to make sure had proper address in address book. Jay
Nestor- apologize for cluttering your mail box with last message and this apology. I'm reading this via modem and it sometimes confuses text from one screen with the next screen. Your message was printed out please read and reply....after replying it reprinted text and this time said read and comply. I thought you were a terrible masochist requesting 1300 replies, but who am I to question the sysop. Jay
Anybody out in cyberspace have any experience with Unicryl resin? The ad says it is a hydrophilic LM & EM resin that stains readily with routine polychromatic stains like safranin. I currently use Epon but it requires etching with NaOH saturated ethanol to allow good safranin staining of mucin in goblet cells. if i could skip that step and still be able to use the same blocks for EM, it would be nice. any comments on this resin would be appreciated.
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)
Microscopy Research and Technique will be making its annual changes (rotation) to the Editorial Board for 1995. If you are interested in becoming an Editorial Board Member, please reply to my E-Mail address directly 75022.2723-at-Compuserve.com listing your name, affiliation, mailing address, phone number, Fax number, research interests, and most recent three publications.
Dear Alex, I can take you *back* a decade: Elementary Excitations in Solids by David Pines discusses electrons, phonons and plasmons from what appears to be a non-relativistic quantum-mechanical point of view. There are probably some referrences with QED as their starting point; however, I don't have any. For the main result you need, the stopping-power tabulations in Berger & Seltzer are probably your best beginning--see a previous post of mine. Yours, Bill Tivol
Dear Scott, for number of years I use average Z values calculated from D. Newbury's paper "Fundamentals of scanning electron microscopy for physicists: contrast mechanism" published in SEM 1977/I, IITRI, Chicago, pp 553-568. You can also find the particular formula in my paper in Scanning Vol.3, 4 1980 pp 288-291. To make it easy for you we wrote the programme calculating Z average. This is how you can get it: ftp to lab.csl.utas.edu.au change directory: cd pub/cameca get backsc.ftn quit Although programme is selfexplanatory you should try it on something easy at first such as SiO2----should give you value 10.69(!). Please contact me directly if you need more on the subject. Regards, Wis Jablonski OiC EM/XRay microanalyis at University of Tasmania, Australia Email W.Jablonski-at-csl.utas.edu.au
Dear Scott, For number of years I have been using average Z values calculated from formula by D. Newbury ( Fundamentals of scanning electron microscopy for physicists: contrast mechanism published in SEM 1977/I, IITRI, Chicago, pp 553-568). You can also find this formula with some practical applications in my paper in SCANNING Vol 3, 4 1980 pp 288-291. To make it easy for you we wrote the programme calculating Z average which includes W.This is how you can get it: ftp to lab.csl.utas.edu.au change directory: cd pub/cameca get backsc.ftn quit Although programme is self-explanatory you should try first on something easy such as Si and SiO2 ---average Z 14 and 10.69 respectively. Please contact me directly if you need more on the subject. Regards, Wis Jablonski OiC EM/XRay microanalysis at University of Tasmania, Australia. Email W.Jablonski-at-csl.utas.edu.au
How to avoid the almost systematic decomposition of inorganic fluoride compounds under the electron beam during a TEM experiment (JEOL 2010) ?
Any suggestion appreciated (inversely as the cost). Thanks in advance.
Armel Le Bail - Laboratoire des Fluorures, CNRS-URA-449, Universite du Maine, 72017 Le Mans Cedex, FRANCE - armel-at-ONE.univ-lemans.fr or lebail-at-naimn2.cnrs-imn.fr
How to avoid the almost systematic decomposition of inorganic fluoride compounds under the electron beam during a TEM experiment (JEOL 2010) ?
Any suggestion appreciated (inversely as the cost). Thanks in advance.
Armel Le Bail - Laboratoire des Fluorures, CNRS-URA-449, Universite du Maine, 72017 Le Mans Cedex, FRANCE - lebail-at-naimn2.cnrs-imn.fr or armel-at-ONE.univ-lemans.fr
There are other alternatives to Unicryl. LR White and LR Gold are both reasonably good EM/LM resins and are hydrophyllic - allowing fairly easy use of LM stains. JB-4 is an excellent LM resin with excellent LM staining properties, but can't be used for EM work. LR White and JB-4 both cost significantly less than Unicryl (i.e. %25 as much) and there doesn't seem to be additional benifits to the unicryl to justify the cost, specifically for general LM staining, as opposed to immunolabeling (for which I haven't heard of a good justification of the benifit vs. cost either).
You might consider one of these resins instead of Unicryl. But I am interested to here what other people have to say.
(Oh, yes I have no finacial ties with any of the above information or manufacturers)
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
For those of you out there who are tired of hand writing out tinney tiny little penciled labels for embedding with your tissues I have been running tests with HP Laserjet printed labels, with excellent results so far. It seems that the electrostatic deposition of the carbon black pigments (in the genuine HP tonner cartridges anyway) do not disolve in either Spurr's or Quetol 651 resin (I haven't tried any of the other yet) and do not seem to effect the blocks in any manner. You might want to give it a try.
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
Several people have asked about apparent missing messages. Due to the volume of mail this server is now handling I've had to restrict the number of attempts which mail tries to be delivered to inactive sites. Delivery is attempted twice for each address at ~6 hour intervals if it is not delivered on the second try then it the attempt to that particuliar address is stopped. I'm trying to find the best compromise in this procedure, since if we wait too long, then the mail queues get huge, but if we don't wait long enough, people who shut off their computers or whose nodes go off line do not receive mail. The 6 hour 2 attempt interval appeared reasonable for a start. I'd like to let it run for a bit longer, before changing it to another value.
Comments, suggestions will be appreciated, but do it off-line and to me direct....
} Date sent: Wed, 07 Sep 1994 10:01:13 +1100 } From: "Richard E. Edelmann" {REDELMAN-at-musom01.MU.WVNET.EDU} } Subject: Embedding Labels } To: Microscopy-at-aaem.amc.anl.gov } Organization: MU School of Medicine } Priority: normal
} } For those of you out there who are tired of hand writing out } tinney tiny little penciled labels for embedding with your tissues I } have been running tests with HP Laserjet printed labels, with } excellent results so far. It seems that the electrostatic deposition } of the carbon black pigments (in the genuine HP tonner cartridges } anyway) do not disolve in either Spurr's or Quetol 651 resin (I } haven't tried any of the other yet) and do not seem to effect the } blocks in any manner. You might want to give it a try. } } } Richard E. Edelmann } Electron Microscopy Facility Supervisor } Marshall University - School of Medicine } Huntington, West Virginia } A good description of this method can be found in J. A. Dant and R. E. Kingsley - Laser Printing Labels For TEM. Hints and Tips EMSA (MSA) Bulletin 21(1):67. In the past this lab laser printed large numbers of labels with an automatic numbering system. When using LR White there was always a collection of toner near the tissue but it never caused a problem in our application. In the present we no longer use laser printed labels because of problems encountered over the past two years. It is unclear if the problems we encountered are a result of changes made in toners to make them more environmentally friendly of the switch to micro-fine toner packs used in 600 dpi printers. For teaching labs - At least one student in each section will slip in a set of labels made on an ink jet printer or reduced using a copy machine each with disastrous consequences.
*************************************** David Garrett "DGARRETT-at-GAB.UNT.EDU" University of North Texas Dept. Biological Sciences (817)565-3964 Fax (817)565-4136 ***************************************
We've been using labels printed on an Apple LaserWriter for several years with good success in a variety of epoxies, paraffin and Historesin. If you can't get your font small enough, then reduce the document through the print dialog. Photocopied labels will also work.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
On Wed, 7 Sep 1994, Richard E. Edelmann wrote:
} } For those of you out there who are tired of hand writing out } tinney tiny little penciled labels for embedding with your tissues I } have been running tests with HP Laserjet printed labels, with } excellent results so far. It seems that the electrostatic deposition } of the carbon black pigments (in the genuine HP tonner cartridges } anyway) do not disolve in either Spurr's or Quetol 651 resin (I } haven't tried any of the other yet) and do not seem to effect the } blocks in any manner. You might want to give it a try. } } } Richard E. Edelmann } Electron Microscopy Facility Supervisor } Marshall University - School of Medicine } Huntington, West Virginia }
I have been doing the same with Epon-Araldite, also with very good results. I use Word for Windows, 6 point type, and print on a Hewlett-Packard Laserjet III.
---------------------------
On Wed, 7 Sep 1994, Richard E. Edelmann wrote:
} } For those of you out there who are tired of hand writing out } tinney tiny little penciled labels for embedding with your tissues I } have been running tests with HP Laserjet printed labels, with } excellent results so far. It seems that the electrostatic deposition } of the carbon black pigments (in the genuine HP tonner cartridges } anyway) do not disolve in either Spurr's or Quetol 651 resin (I } haven't tried any of the other yet) and do not seem to effect the } blocks in any manner. You might want to give it a try. } } } Richard E. Edelmann } Electron Microscopy Facility Supervisor } Marshall University - School of Medicine } Huntington, West Virginia }
I've been using computer generated embedding labels for years. Currently using an Apple Laserwriter printer with no problems. If your printer uses ink that bleeds in resin try soaking the labels in ethanol for 30-60min, dry them completely in warm oven and then embed. Anne
Anne Simpson Gomes
EM Unit, F09 "How's it going Eh?!!!"....... Univ of Sydney from NSW 2006 Australia The Compact Canuck!! Fax: (612) 552 1967
Since the topic has taken off, I thought I would add a bit. I have trouble with air bubbles if I push the label down into the well of Polybed. I have found that if\ I put the label on top and place it into the vacuum for a while then sink it, I don't have bubbles in my block.
A few weeks ago I was on the hunt for some histologic exotica, to which I received all the clues needed to track down the reagent. The quest continues: This time the subject is a little known beast named Safran du Gatinais!!!! for makin alcoholic Safran. What alchemy! Any ideas, where to get it from ???
All help deeply appreciated
Simon C. Watkins Director SBIC University of Pittsburgh Pittsburgh PA 412-648-3051
As you are probably aware, "Safran du Gatinais" is French for "Safranin from the Gatinais", which is a region near Paris. You may find that "Safranin O", a common stain available from various sources, might serve as a substitute? A bottle of Safranin O on my shelf, produced by Chroma-Gesellschaft (Stuttgart, Germany), was obtained many years ago from Roboz Surgical Instruments, 810 18th Street, N.W., Washington, D.C. 20006. I think it also appears currently in the Arthur Thomas catalog.
Kent A. Kent Christensen Department of Anatomy and Cell Biology Univ. of Michigan Medical School
----------------------------
On Thu, 8 Sep 1994, simon wrote:
} A few weeks ago I was on the hunt for some histologic exotica, to which I } received all the clues needed to track down the reagent. The quest } continues: This time the subject is a little known beast named Safran du } Gatinais!!!! for makin alcoholic Safran. What alchemy! Any ideas, where } to get it from ??? } } All help deeply appreciated } } Simon C. Watkins } Director SBIC } University of Pittsburgh } Pittsburgh PA } 412-648-3051 } } }
I would like to inquire if anyone has a double-tilt side entry specimen holder for the Philips EM300 TEM. There were two stages available for this instrument, one with smaller holders and (the one we have) that accepts the "normal size" specimen holders. We are using this for the current fall semester lab class and would like to borrow/purchase this type of holder.
Bob Roberts Arizona State University Center for Solid State Science PSB-234 Tempe, Arizona 85287-1704 (602) 965-4512
I would like to inquire if anyone has a double-tilt side entry specimen holder for the Philips EM300 TEM. There were two stages available for this instrument, one with smaller holders and (the one we have) that accepts the "normal size" specimen holders. We are using this for the current fall semester lab class and would like to borrow/purchase this type of holder.
Bob Roberts Arizona State University Center for Solid State Science PSB-234 Tempe, Arizona 85287-1704 (602) 965-4512
} Date sent: Thu, 8 Sep 94 16:48:37 EDT } From: Rajesh Patel {patelr-at-pilot.njin.net} } To: Microscopy-at-anlemc.msd.anl.gov } Subject: Microscopy Society of America
} } I am a member of MSA but it seems I never get any bullitens from them like } the quaterly bulliten and the list of members etc. } } Who do you contact? } The Business Office Microscopy Society of America P.O. Box MSA Woods Hole, MA 02543 Voice: (800)538-3672
I would like to inquire if anyone has a double-tilt side entry specimen holder for the Philips EM300 TEM. There were two side entry stages available for this instrument, one with smaller holders and the other (like we have) that accepts "normal size" specimen holders. We are using this for the current fall semester lab class and would like to borrow/purchase this type of holder as soon as possible. Thanks. Bob Roberts Arizona State University Center for Solid State Science PSB-234 Tempe, Arizona 85287-1704 (602) 965-4512
Greetings Microscopists, One of our users wants to do LM immunocytochem and standard EM on the same tissue in the same block. Because his antigen is relatively sparse, the ability to "gently" remove any resin would be a plus so that maximum staining could be attempted. Any number of resins come to mind; LR White most prominently. Does anyone else have suggestions that would be better for this purpose? Thanks in advance.
************************************************* _______________________ * * | | * * | _____ ______ | * Charles J. Butterick (Chuck) * |__| | | |__| * Electron Microscopy Center * | | * Department of Cell Biology and Biochemistry * ______| |______ * Texas Tech University Health Sciences Center * | __ __ | * Lubbock, Texas 79430 * |__| | | |__| * USA * | | * Phone 806 743-1633 voice * | | * 806 743-1219 fax * | | * Email emccjb-at-lubb.ttuhsc.edu * | | * * __| |__ * * |_______| *************************************************
I would like to inquire if anyone has a double-tilt side entry specimen holder for the Philips EM300 TEM. There were two stages available for this instrument, one with smaller holders and (the one we have) that accepts the "normal size" specimen holders. We are using this for the current fall semester lab class and would like to borrow/purchase this type of holder ASAP. Thanks.
Bob Roberts Arizona State University Center for Solid State Science PSB-234 Tempe, Arizona 85287-1704 (602) 965-4512
In my dealings with a "new" antigen to localize I always try numerous resins and fixation protocols until I am happy with the results. No one procedure will work all antigens. LR White is a good place to start. Good Luck.
Dear Chuck, you ask about doing LM and EM on the same specimens and blocks. This is by far the best strategy for immunocytochemistry because fixation protocols, antibody dilution and antigen distribution can be easily assesed at the LM level before progressing to electron microscopy. Resins are convenient to use because they are easy to section and store and the morphology is more easily understood by non-microscopists. They can all be used for immunocytochemistry at the light and EM levels but Epon, Araldite and Spurrs have many problems associated with them. Resins developed for immunocytochemistry include the London resins (LR White, LR Gold), the Lowicryls (K4M, K11M, HM20, HM 23) and, more recently, Unicryl. All of the latter can be polymerized by heat or by UV light and, within limitations, all can be used to infiltrate tissues at low temperature. All can be labeled with antibodies and protein A-gold for EM and all have been tested to give positive results (Schwarz,1994, Proceedings ICEM-13, Paris, France pp 225-226; see also Albrecht et al, 1990 Brain Res. 535:49-61; Schwarz et al 1993 Cell Tissue Res 273:417-425). One additional advantage, pointed out by Schwarz, is that antibodies do not penetrate resins, so only the surface is imaged and there is no out-of-focus signal to blur the image (similar to a confocal image). By using the same fixation and sectioning protocols and the same reagents for both LM and EM a labeling reaction is certain to occur for both. The amount of antigen to be detected may also affect the sectioning method to be used. For some antigens, it has been shown that higher labeling efficiencies are produced on cryosections. Frozen biological material, cryoprotected with sucrose, can be easily sectioned for both light and electron microscopy. As with resins, these sections can be labeled with fluorescent antibodies or colloidal gold for light microscopy (silver enhancement will make the gold visible at the LM level) and with colloidal gold for electron microscopy. Always remember, whichever method is used, the amount of signal at the EM level will depend on the amount of antigen present. Small numbers of antigen will result in small numbers of gold particles over the section.
Paul Webster Center for Cell Imaging Yale School of Medicine.
Laurie Marks asks about the appropriate form of the CTF for a tilted sample. Dear Laurie, If you think of a tilted sample as the limit of a sequence of samples consisting of flat segments at different heights, the CTF for a member of the sequence is the sum of the CTF's for each height (i.e. different values of defocus). The limit would be an integral. There may be complications due to the differences in magnification for different heights, but I can't figure out what they would be. Good Luck. Yours, Bill Tivol
Dear Chuck, you ask about doing LM and EM on the same specimens and blocks. This is by far the best strategy for immunocytochemistry because fixation protocols, antibody dilution and antigen distribution can be easily assesed at the LM level before progressing to electron microscopy. Resins are convenient to use because they are easy to section and store and the morphology is more easily understood by non-microscopists. They can all be used for immunocytochemistry at the light and EM levels but Epon, Araldite and Spurrs have many problems associated with them. Resins developed for immunocytochemistry include the London resins (LR White, LR Gold), the Lowicryls (K4M, K11M, HM20, HM 23) and, more recently, Unicryl. All of the latter can be polymerized by heat or by UV light and, within limitations, all can be used to infiltrate tissues at low temperature. All can be labeled with antibodies and protein A-gold for EM and all have been tested to give positive results (Schwarz,1994, Proceedings ICEM-13, Paris, France pp 225-226; see also Albrecht et al, 1990 Brain Res. 535:49-61; Schwarz et al 1993 Cell Tissue Res 273:417-425). One additional advantage, pointed out by Schwarz, is that antibodies do not penetrate resins, so only the surface is imaged and there is no out-of-focus signal to blur the image (similar to a confocal image). By using the same fixation and sectioning protocols and the same reagents for both LM and EM a labeling reaction is certain to occur for both. The amount of antigen to be detected may also affect the sectioning method to be used. For some antigens, it has been shown that higher labeling efficiencies are produced on cryosections. Frozen biological material, cryoprotected with sucrose, can be easily sectioned for both light and electron microscopy. As with resins, these sections can be labeled with fluorescent antibodies or colloidal gold for light microscopy (silver enhancement will make the gold visible at the LM level) and with colloidal gold for electron microscopy. Always remember, whichever method is used, the amount of signal at the EM level will depend on the amount of antigen present. Small numbers of antigen will result in small numbers of gold particles over the section.
Paul Webster Center for Cell Imaging Yale School of Medicine.
Laurie Marks asks about the appropriate form of the CTF for a tilted sample. Dear Laurie, If you think of a tilted sample as the limit of a sequence of samples consisting of flat segments at different heights, the CTF for a member of the sequence is the sum of the CTF's for each height (i.e. different values of defocus). The limit would be an integral. There may be complications due to the differences in magnification for different heights, but I can't figure out what they would be. Good Luck. Yours, Bill Tivol
Laurie Marks asks about the appropriate form of the CTF for a tilted sample. Dear Laurie, If you think of a tilted sample as the limit of a sequence of samples consisting of flat segments at different heights, the CTF for a member of the sequence is the sum of the CTF's for each height (i.e. different values of defocus). The limit would be an integral. There may be complications due to the differences in magnification for different heights, but I can't figure out what they would be. Good Luck. Yours, Bill Tivol
Looking for a fully automated batch processor for Kodak 4489 or similar TEM sheet film. Something that will handle at least 25 sheets of 3-1/4" x 4" film, preferably 50, from developing through drying. Benchtop unit preferred over freestanding. Anybody know of such equipment?
Larry Thomas Battelle, PNL Richland, WA 99352 tel: 509 376-3785 fax: 509 376-0418 le_thomas-at-pnl.gov
Laurie Marks asks about the appropriate form of the CTF for a tilted sample. Dear Laurie, If you think of a tilted sample as the limit of a sequence of samples consisting of flat segments at different heights, the CTF for a member of the sequence is the sum of the CTF's for each height (i.e. different values of defocus). The limit would be an integral. There may be complications due to the differences in magnification for different heights, but I can't figure out what they would be. Good Luck. Yours, Bill Tivol
We have had some internal disagreements about how thin a metal foil can be mechanically polished prior to jet polishing without inducing dislocations in the final thin section. I realize that this depends, to an extent, on the metal in question, but would like to get a feel for people's opinions/experience and a good reference that addresses this subject. Thank you, Garth B. Freeman, Sr. Research Scientist, Georgia Tech, Atlanta Georgia garth.freeman-at-gtri.gatech.edu
I recently purchased from an estate sale two boxes of chemical microscopy reagents of the type sold by Cargille and McCrone and taken from Chamot & Mason. On the inside cover of both boxes, on the "index" sheet, is printed the term "Shillaber Model." Does anyone know what that means? Who is or was Shillaber? Can someone provide me a reference? I'm just curious?
A postdoctoral position is available employing HREM techniques to surfaces under UHV conditions using a new multi-chamber combined UHV-HREM and surface science facility at Northwestern. Extensive electron microscopy experience is essential; hands on experience with any of MBE growth, XPS, Auger or high-level image analysis would help. Applications with the name of two referees should be sent to: Professor L. D. Marks Department of Materials Science and Engineering Northwestern University Evanston, IL 60208 ldm-at-apollo.numis.nwu.edu
{Pine.3.87.9409130834.A28895-0100000-at-crl6.crl.com} To: "Peter D. Barnett" {pbarnett-at-crl.com} Cc: microscopy-at-anlemc.msd.anl.gov Message-id: {01HH28NE9TZU94DWRU-at-minna.acc.iit.edu} MIME-version: 1.0 Content-type: TEXT/PLAIN; CHARSET=US-ASCII Content-transfer-encoding: 7BIT
Dear Peter,
That must be a reference to Charles Patten Shillaber, Fellow of the RMS and author of one of the best books on the practical use of photomicrography;"Photomicrography in Theory and Practice, John Wiley and Sons, Inc., 1944.
I am looking for an EPMA mineral standard, tugtupite (syn: beryllosodalite). Does anyone know where I can get it (either from requesting or purchasing) ?
Thanks in advance.
Long Liang Electron Microprobe and SEM Lab ARCO Exploration and Production Technology 2300 West Plano Parkway Plano, TX 75075 E-mail : LLIANG-at-is.Arco.COM
Message-Id: {MAILQUEUE-101.940914140050.416-at-eo.ine.philips.nl} To: ROBERT-at-CSSS.LA.ASU.EDU
DEAR MR. ROBERTS,
WE PICKED UP YOUR EMAIL REGARDING THE SEARCH ACTION FOR A PHILIPS EM300 DOUBLE-TILT SPECIMEN HOLDER AND WE ARE CHECKING VIA OUR CHANNELS IF ANYONE CAN HELP YOU.
AS WE ARE ALSO CHECKING IN SOME OTHER EUROPEAN COUNTRIES, IT CAN TAKE SOME TIME BEFORE WE HAVE A DEFINITE MESSAGE.
IF POSITIVE, WE WILL INFORM OUR SALES ORGANISATION PEI MAHWAH IN USA WHICH ACTIONS HAVE TO BE UNDERTAKEN TO SHIP THE HOLDER TO THE DESIRED LOCATION.
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INTERNATIONAL BUSINESS CENTRE PHILIPS ELECTRON OPTICS COMMERCIAL SUPPORT EINDHOVEN - THE NETHERLANDS BUILDING AAE-1 TELEPHONE + 31 40 766385 TELEFAX + 31 40 766786
9/14/94 Frozen tissue to epon?? 10:24 AM Hi, Does anyone have any ideas that this will or will not work; I have some unfixed adipose tissue that is in liquid nitrogen. I would like to look at the morphology of this tissue. I do not need it for labelling. What I was thinking of doing was leaving the tissue overnight in 70%GA/Methanol at -90, then gradually decreasing the temperature and the alcohol concentration, (do the reverse of low temperature embedding), chopping up the pieces a bit, then do the standard embedding . Would it be better to put it in Lowicryl (I don't think so)? What do you think? Thanks, Jeanne
} We would like to get some feedback on these two accelerators. In your } experience what is the shelf life of DMP-30? How much better is the storage } life if unopened? What quantity of BDMA is generally used as a replacement } for } DMP-30? How does it's shelf life compare? Any other rules of thumb? } As you might have guessed, we have had some intermittant problems with } soft embeddments, and suspect that the accelerator might be the culprit. } Thanks. } } Tim Bourett } DuPont Experimental Station } Wilmington, DE USA
Tim: I asked a similar question recently and a summary of the replies follows. I had used DMP30 for 18 years without problem. I generally keep my bottle at room temp in my hood. I never monitored shelf life but I am sure that most bottles lasted over a year. Recently I started having a problem with soft blocks. I switched to BDMA (2x DMP-30 concentration). The problem went away for our routine blocks but one of my grad students still had a weird problem with the interface of free plastic over some cell monolayers not polymerizing. This is with both BDMA and DMP-30. still haven;t solved that problem but I am still unsure if the problem was with the resins or the student. I haven't personally embedded any monolayers in epon since this problem started. I guess I will have to break down and do it myself to see where the problem is. For other tissues (bacteria, intestine) embedded in Epon, the BDMA works fine.
Summary:
On Tue, 12 Jul 1994, Tom Phillips wrote:
} I am tempted to try benzyl dimethyl amine (BDMA) in place of DMP-30 but don't } have the original reference. How much does one use? Anyone have the original } citation? I usually use: } 20 g EmBed812 + } 10 g DDSA + } 10 g NMA + } 0.6 g DMP-30.
I use them at the same ratio. The BDMA has a much longer shelf life.
Rick A. Harris Dept. of Molecular and Cellular Biology Microscopy Facility University of Calif., Davis
I have never tried BDMA, but Hayat gives several recipes for plastic where he shows X% DMP-30 OR 2X%BDMA. (Page 116 Hayat's third edition "Principles and Techniques of Electron Microscopy biological applications") He refered to Mollenhauer, H,H, 1969, Stain Technol., 39:111. Plastic Embedding Mixtures for EM.
Larry Hawkey Duke Neurobiology hawkey-at-neuro.duke.edu
To: tphillips (Tom Phillips)
We make rather large batches, about 233 grams at a time. We normally use 3.44 gm of DMP-30, and were told to use 4.3 gm BDMA. Hope this helps. ___________________________________________________________________ Randy Nessler rnessler-at-emiris.iaf.uiowa.edu The University of Iowa Central Electron Microscopy Research Facility
In a "Survey of Embedding Media For Electron Microscopy" by Audrey Glauert in the Journal of the Royal Microscopical Society 1962 Vol.LXXX pp 269-277 she suggests that DMP 30 and BDMA are interchangable, then follows several recipes (Kushida 1959, Finck 1960, Luft 1961) for Epon resin mixes all incorporating BDMA as the accelerant. The trend for some years has been towards using BDMA in preferance to DMP 30 - I'm not sure why but it is much less viscous so that alone makes it more convenient (Glauert mentions precipitates forming when DMP 30 is used in some resin mixes). I started using BDMA years ago and find it works every time. In the same Glauert reference and in her book "Fixation,dehydration and embedding of Biological Specimens" she suggests several epon 812/812 equivalent mixes, I've always found the medium hardness mixture excellent for most of our specimens.It is her recipe we use, more or less. I mix it thus: "mix A" =19ml 812 resin and 31ml DDSA;"mix B"= 26.5ml 812 resin and 23.5ml MNA.(weigh equivalent amounts if you prefer - I've never seen any noticable improvement with the more accurate weighing).
Mix parts A and B together when ready to use and at the same time add 15ul of BDMA per ml of your total resin/DDSA/MNA mixture. Hope this is of use. R Easingwood
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
We use BDMA in our PolyBed 812. We use 2X as much BDMA as we would use DMP -30. Supposedly BDMA is less viscous than DMP-30 and there fore allows for a quicker infiltration of specimens. It also has a slightly longer shelf life than DMP-30 (12 months as opposed to 9 months). We've had no problems since we switched. Hope this helps.
John Aghajanian Worcester Foundation for Experimental Biology
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)
Its been ages since I worked with BDMA so can't comment on that. but DMP-30 seems ok stored unopened at room temp, in a dark cupboard, for at least 2 years. Once opened, I dispose of the unused accelerator whenever a new bottle of the epoxy resin is opened. Tlhis seems to work and has not given us problem batches. A few years ago, I had many problems with soft blocks suddenly and consistantly appear from a vendor, who shall not be named. Switching vendors for my Spurr's resin eliminated the problems. The vendor blamed the problem on the quality of employees in their region. Never mind that 2 competitors in the same region seemed to have no problems in their quality.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
Greetings: I'm attempting to rejuvenate a carbon coater manufactured by a company called Mikros. Inspection of the guts showed a broken relay switch. I'd like to know if anyone knows who might be able to supply parts for this device. The part number I'm looking for is: 115 NO 120T. It is identified in the manual as a "delay relay" and is used in the automatic valving system. N.B. this is an old instrument, circa 1965, so I'm not holding my breath. However, it is exactly like the very reliable instrument I used as a grad student and I'm sure that with a bit of coaxing it could be a decent addition to the lab. Many thanks!
Dwight Beebe IRBV, Dept. de sciences biologiques beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
Many moons ago I also used a similar Mikros evaporator. The company is no longer in existance therefore you will need the services of a good electronics tech to find replacement parts. Good Luck
Why not freeze substitute. Your specimens will look great.
On 14 Sep 1994, Jeanne Barker wrote:
} 9/14/94 } Frozen tissue to epon?? 10:24 AM } Hi, } Does anyone have any ideas that this will or will not work; } I have some unfixed adipose tissue that is in liquid nitrogen. I would like to } look at the morphology of this tissue. I do not need it for labelling. What } I was thinking of doing was leaving the tissue overnight in 70%GA/Methanol at } -90, then gradually decreasing the temperature and the alcohol concentration, } (do the reverse of low temperature embedding), chopping up the pieces a bit, } then do the standard embedding . Would it be better to put it in Lowicryl (I } don't think so)? } What do you think? } Thanks, } Jeanne } } }
Help! I recently put some tissue into LR White for the first time, and I'm= having a terrible time with it in the beam!! Even on formvar/carbon,= 75KeV, my smallest condensor/objective apertures, the stuff's blowing up. = What's wrong???? Many thanks to all. Grace Kennedy UCSD
} 9/14/94 } Frozen tissue to epon?? 10:24 AM } Hi, } Does anyone have any ideas that this will or will not work; } I have some unfixed adipose tissue that is in liquid nitrogen. I would like to } look at the morphology of this tissue. I do not need it for labelling. What } I was thinking of doing was leaving the tissue overnight in 70%GA/Methanol at } -90, then gradually decreasing the temperature and the alcohol concentration, } (do the reverse of low temperature embedding), chopping up the pieces a bit, } then do the standard embedding . Would it be better to put it in Lowicryl (I } don't think so)? } What do you think? } Thanks, } Jeanne } First of all: How was your tissue frozen - just in liquid nitrogen or in a cryocoolant - ethane, propane or freons ? Good cryofixation is important step in preparastion and the final result - morphology will ,depend on it. I was using Epon and Lowicryl embeddings of the biological materials. In most cases water withdrawal was done by means of freeze-drying at low temperatures.
See: Wroblewski R and Wroblewski J. (1984) Freeze drying and freeze substitution combined with low temperature embedding. Histochemistry 81, 469-475.
Subject: Time:8:00 AM OFFICE MEMO Thanks. Date:9/15/94 I would like to thank everyone who gave me suggestions regarding my question about embedding tissue that has already been frozen in liquid nitrogen. I'm sure that now I will be able to get decent pictures, one way or another.... This listserver is great! Jeanne
At Fernbank Science Cnter in Atlanta, GA we are hoping to hold an independent study class for high school students in various areas of microscopy. We have chemists, bologists, physcists and electron microscopist on staff at Fernbank Science Center. We have on sight a Zeiss 960 SEM, Zeiss 962 SEM with EDX capabilities, monocular and bonocular (binocular) scopes with the capapbilities of changing them to polarizing (simple) scopes. I have had Polarizing Lgith (light) Microscopy training and we have a very activi professional microscopy group in the Atlanta area. We are looking for ideas for exploration in all areas of microscopy using the equipment above. We also a have a Zeis TEM. We hope to begin this course in the winter of 1995. We will have approximately 10 students for 2.5 hurs for 24 sessesions. If you have any ideas for proposals please let me know. I will be happy to share all information as it is developmed for students and teachers. . . . . . . . . . . . . . . . . . . . . . . . . . Staff Fernbank Science Center (phone)404-378-4311 gfs001-at-sol1.solinet.net
Dear Mary, I'd include some electron diffraction in addition to the more usual EM stuff, but that's my field, so maybe I'm biased. ED is a great example of the wave-particle duality for electrons, and since there is only one electron in the column at once (low dose conditions), the electron must interfere with it- self. (Thinking of the particle as a wave packet with narrow Gaussian distri- bution of momenta helps.) Besides, it's really neat to scan the grid in diff mode and to see the ED pattern suddenly appear. Clay minerals have some thin particles which give spectacular patterns. The high school students I've wor- ked with seemed to enjoy this. Good luck. Yours, Bill Tivol
X-Authentication-Warning: sunday.fhl.washington.edu: Host omnicron.fhl.washington.edu didn't use HELO protocol
Hello,
My name is Douglas Hansen. I have been listening in on the microscopy group for a little while. I am looking for several individuals or groups who would be interested in testing and evaluating a sub-micron dimension reference and calibration standard for SEM's. These reference standards are originals, or, if you will, master gratings, and not replicas. Our own evaluations of these reference standards indicate that they are easy to use and have well defined edges. The sample I would provide to those who volunteer will have a calibrated dimension of 0.293 microns. I will provide this sample free of charge and only ask in return that you provide feedback and agree that you will arrange to allow me to use your comments in advertising, etc. if I request it. If the test results are positive, these new standards will be available through most of the microscopy supply distributors within a few months. They are expected to cost about $250 to $300 dollars.
Please respond to me if you have interest in acting as a test site for these reference and calibration standards.
This is Douglas Hansen again. I have had a response to the standard test offer that surprised me greatly. I have had over 20 responses already. Therefore, I will have to withdraw this offer as of Friday Morning. Any e-mail which arrives in my account by 9:00 am Friday will be offered a test sample. All requests which arrive after this time will be sent a letter of regret. I am sorry, but I only have so many beta samples to pass out. However, I want to thank everyone for their interest. If the test results are as positive as our own testing has been, you will be able to purchase these standards through your favorite microscopy supplies distributor within 2 months.
Demo of SEM, AFM, Image analysis equipment by Leica At Greenville Hilton, (Heywood exit off 385 to downtown) Tues, Wed, Thurs, 9/20, 9/21, 9/22 For Further Info, contact 800-248-0665 ext 516 (Mike Webber)
ELECTRON MICROSCOPY FACILITY SUPERVISOR/INSTRUCTOR Miami University is seeking applicants for a full-time, nontenure-track, staff position of Electron Microscopy Facility Supervisor to begin as soon as possible. The successful applicant will hold an M.S. or Ph.D. degree in one of the biological sciences and will be responsible for the operation, maintenance, and supervision of an interdepartmental EM facility containing two TEMs, two SEMs, cryopreservation equipment, EDX spectrometer, five darkrooms, and an image analyzer. Duties include supervision of a full-time EM technician and teaching in microscopy courses. Collaboration on faculty research projects will be encouraged. Please submit curriculum vitae, transcripts of all course work, and three letters of recommendation to: David Pennock, Director, Electron Microscopy Facility, Miami University, Oxford, OH 45056. Application review will continue until the position is filled. Miami University is an Equal Opportunity/Affirmative Action Employer.
I would like some inputs from those already outputing images directly from TEMs. I have a Gatan 673 wide angle camera now outputting images to a SEIKOSHA VP1500 thermal printer from a Hitachi 7100 TEM. I would like to produce TIF or similar fomat images from the camera output, but I do not want to dupplicate the set up I now have in a different room, where a Targa plus 16/32 is already installed for running other softwares.
I need the least expensive video board that would allow this with a decent resolution. The board will be installed on a 486 COMPAQ, and hopefully an Ethernet card will allow me to get my mac to talk with with the PC.
Apology to all who have been trying to get that programme.I have been assured that it will work OK if you follow this: ftp lab.csl.utas.edu.au login name:anonymous password: {email name & address} ftp} cd pub/cameca ftp} dir backsc.ftn ftp} get backsc.ftn ftp} quit Hopefully this should fix it.
ASTIMEX Scientific Ltd. (University of Alberta, Canada) sells Mineral Mount code MINM25-53, diameter 25mm, 53 minerals of the good quality.One of them is tugtupite Na4BeAlSi4O12Cl with the composition as below: BeO 5.36% Na2O 26.50 Al2O3 10.90 SiO2 51.39 Cl 7.58 all in weight % , total 101.7 I purchased the mount about 5 years ago and I have been using it as the source of secondary stds but sometimes as primary stds when NBS stds are not at hand. Regards, Wis Jablonski Uni Tas
Message-Id: {MAILQUEUE-99.940919080550.928-at-eo.ine.philips.nl} To: microscopy-at-anlemc.msd.anl.gov
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The message could not be delivered to:
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Received: from relay.philips.nl by PHYSC1.BYU.EDU with PMDF#10201; Fri, 16 Sep 1994 09:42 MDT Received: from ine.philips.nl ([130.144.32.8]) by relay.philips.nl (8.6.9/8.6.9) with SMTP id RAA26461 for {hansen-at-physc1.byu.edu} ; Fri, 16 Sep 1994 17:45:50 +0200 Received: by ine.philips.nl; Fri, 16 Sep 94 17:43:30 +0200 Received: From EO_SW/WORKQUEUE by eogate.ine.philips.nl via Charon-4.0A-VROOM with IPX id 100.940916174437.384; 16 Sep 94 17:44:50 -0100
Not knowing what time zone you are in I do not know if this will reach you before your 9.00 deadline.However I am the product manager of the Philips Electron Optics SEM activity and as well project of the JESSI project 104 (similar to Sematech) that has as a goal a precise and accurate metrology sem.As such I would be pleased to participate in your test with particular referernce to low voltage metrology.Also I have recently been asked by the ISO organisation to set up a working group to define internationally acceptable standards and procedures for sem metrology.You may be interested in some involvement with this group Please let me hear from you re the test sample and in any case we should stay in contact.You should also contact M Postek at NIST and M Bennet at SEMATECH.Michael has been spending at lot of time on this topic,including a wel defined round robin test of sem's that you would find interesting and Marilyn B is (via TI) producing a mask metrology standard that will be available shortly.
Greetings, For use in vacuum evaporation, we are looking for a source of Tungsten wire that is 1mm diameter. The usual em supply house catalogs seem not to list it in this diameter in their catalogs. We did find some in the TAAB catalog, from England, but I am hoping to find a USA distributor. Thanks in advance for any help.
Try Alfa 800-343-0660 Their catalog # for 1.0 mm dia wire is 10411 Catalog lists $22.80/meter, $84.00/5 meters You may have tough time bending the wire to give any shape as it is usually very brittle.
To: MCI:CENTRAL cc: DRStadden:R_D:Armstrong Subj: Thermal Printers ------------------------------------------------------------------
I recently got word that our Codonics VP-3500 thermal printer, dedicated to an Amray 1820D SEM, was in need of a new print head. I had sent it cross-country to the only Seikosha repair facility, anticipating a $150 charge for cleaning the heads. (I was getting fine, but objectionable, white lines across the images, and couldn't get rid of them by running the cleaning sheet through the paper path, as per directions.) The cost of the new head is $1,350, + a $150 service charge. The 5-year-old unit was $6,700 new.
In terms of technological upgrades, what are my options for, say, under $7500? Of course, color capability is not required. Thanks for any thoughts on this matter.
Dave Stadden Armstrong World Industries, Inc. Lancaster, Pennsylvania
Tobias regarding a source of 1 mm tungsten wire...
A while back I received a brochure from
REFINING SYSTEMS INC. P.O. Box 72466 Las Vegas NV 89170 Tel (702) 368-0579
regarding metal wire, slugs, pellets, etc. I have no experience ordering from them or using their materials, but they did seem to have a range of products that might include the wire of a diameter you need.
please let me know if they have what you need and you liked their service, quality etc. Thanks.
good luck steve ---------------------------------------------------------- Dr. Steven Barlow EM Facility/Biology Dept. San Diego State University San Diego CA 92182-0057 phone: (619) 594-4523 fax: (619) 594-5676 email to sbarlow-at-sunstroke.sdsu.edu
Just a brief note about our ongoing experience that may be related to this thread. This summer I resumed doing light microscopy about a hiatus of about a year. We were consistently getting a water-like reaction when we transferred slides from 100% ethanol to xylene. We changed everything. I thought it was hydrated ethanol...we opened fresh 1 pt bottles of absolute ethanol. I thought my assistant was being sloppy...so I did the staining myself and it still happened. I thought the xylene dishes had been contaminated somehow...so I replaced them with fresh xylene. Our university has a chemical storehouse for frequently used chemicals like xylene. Either it has been a bad lot of xylene from one manufacturer or a generic problem with xylene from that manufacturer (I'd never used xylene with this company before...). The upshot is that we bought xylene from another manufacturer and we aren't having these problems. So I really think we may all be searching too hard sometimes for explanations for aberrations...I thought it was the slides, the coverslips, etc. long before I thought to believe it was bad xylene (another colleague of mine here had the same problem).
-- Nancy L Desmond, Ph.D. Department of Neurosurgery University of Virginia Health Sciences Center, Box 420 Charlottesville, VA 22908 804.924.5607 (voice) 804.982.3829 (fax)
Greetings: I'd first like to extend my thanks to the large number of people who responded to my request for help in locating a part for an old carbon coater I'm trying to revive. Everyone had excellent suggestions and provided addresses and phone numbers. And now...I'd like to find a protocol (that I'd heard of) that would allow me to use bacteria with attached phage for a TEM lab on negative staining. I have easy access to both E. coli and phage, but don't have a source for the technique. Aparently there is a way in which the phage can be induced to attach themselves to the bacteria without subsequent detachment. Anybody out there know about this? Please respond directly. I'll summerize and repost. Many thanks!
Dwight Beebe IRBV, Dept. de sciences biologiques beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
I require ideas re the cleaning of the following problematic SEM samples, please: Field-collected, museum samples of fly larvae, fixed in 70% eth-OH, very soft and delicate, even after fixation. Detritus and some slime appears to be fixed to the cuticle of some samples. I have tried the following: A weak Photo-flo solution with gentle brushing 100% picric acid etching 0.05% KOH Contrad soap (an alkaline anti-contamination soap)
Setae et al could be damaged by ultra sonic cleaner, so feel that chemical cleaning is the solution, but what???
Any ideas, please? John F. Putterill EM unit, Onderstepoort Veterinary Institute South Africa 0110
} I require ideas re the cleaning of the following problematic SEM } samples, please: } Field-collected, museum samples of fly larvae, fixed in 70% eth-OH, } very soft and delicate, even after fixation. Detritus and some slime } appears to be fixed to the cuticle of some samples. } I have tried the following: } A weak Photo-flo solution with gentle brushing } 100% picric acid etching } 0.05% KOH } Contrad soap (an alkaline anti-contamination soap) } } Setae et al could be damaged by ultra sonic cleaner, so feel that } chemical cleaning is the solution, but what??? } } Any ideas, please? } John F. Putterill } EM unit, Onderstepoort Veterinary Institute } South Africa 0110 } } John: There is a pretty good and simple apparatus you can make that will help clean your critters. It was in: Proc. Entomol. Soc. Wash. 93(1), 1991, pp. 204-205. If you need a copy of this article, send me a fax number and I will fax a copy to you or send me your address and I will mail a copy. Hope it works!
Phil voice: (410) 455-3582 fax: (410) 455-3875 E-mail: prutle1-at-gl.umbc.edu
A few months ago, I offered Field emission reference articles to anyone interested. I recently received a response requesting some literature. However, I lost the message in an unfortunate PC crash. would the person who requested the information please resend the request?
Thanks! Damon L. Heer
FEI Company 7451 N.E. Evergreen Parkway Hillsboro, OR 97124-5830
My apologies, I have committed the cardinal sin of sending a request that should have been sent to the listserv address to the list address. Again my humble apologies! John Mansfield.
I am currently preparing a manuscript and for sake of completeness would like to know if anybody is aware of work done using EELS to characterize minor or even trace metals in mineral samples. I've taken a look through the MSA proceedings for the last couple of years but haven't really spotted anything. Can anybody help, please?
Rich Leapman and Dale Newbury published a paper titled "Trace Elemental Analysis at Nanometer Spatial Resolution by Parallel-Detection Electron Energy Loss Spectrometry" last September, 1993, in Analytical Chemistry. Although they did not look at minerals, they did analyze NIST standard glass reference materials.
Hope this helps, John
} I am currently preparing a manuscript and for sake of } completeness would like to know if anybody is aware of work done } using EELS to characterize minor or even trace metals in mineral } samples. I've taken a look through the MSA proceedings for the } last couple of years but haven't really spotted anything. Can } anybody help, please? } } Thanks in advance. } } Greg McMahon } MTL/CANMET } Ottawa, Ontario } Canada
David Vowles Electron Microscopy Unit Australian National University PO Box 475 Canberra ACT 2601 Australia Tel:(616) 2493543 Fax:(616) 2494891 Email:Vowles-at-rsbs-central.anu.edu.au
John Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, University of Michigan 2455 Hayward, Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 9:53
Date:9/21/94 URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL
An number of you have seen me try and set my microscopy to digest mode. I accidentally sent the request to the list and not to the listserv-at-aaem.amc.anl.gov address. When I resent the message to the listserv address I got no reply or confirmation. I therefore assume that the version of the listserver software that Nestor is running does not support digest mode. Nestor is out of the country on business until October 6th and so I cannot ask him. If Russ Cook is monitoring this list maybe he can give some insight. So, for the time being I would not bother trying to set the digest mode. For those of you who don't know it, the digest mode sens you the messages in batches with all of the message titles at the head of the batch. That way you can scan the batch message header and see if there are messages that you need to read.
The best way of reading the mailing list is, in my humble opinion, to use the Usenet News group sci.techniques.microscopy. You only need to download/read the messages that are of interest to you. You can read news with either a local MAc or PC based news reader (e.g. NewsWatcher or Nuntius for the Mac) or from a Unix workstation (using rn, trn or xrn for example) you can read news via Gopher (links vary depending on your location) or via the WWW with NCSA Mosaic or some such other Web browser. The URL for sci.techniques.micrscopy is: news:sci.techniques.microscopy. OK? John Mansfield
John Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, University of Michigan 2455 Hayward, Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 10:14
Date:9/21/94 URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL
We have been trying out the World Wide Web and have set up some web pages that are designed to provide information on our microscopy laboratory and The Department of Materials Science and Engineering here at the University of Michigan. The pages are still under development but you can check them out at URL: http://www.engin.umich.edu/~jfmjfm/emal_info/emal_home_page URL: http://www.engin.umich.edu/~jfmjfm/mse_folder/mse_home_page URL: http://www.engin.umich.edu/~jfmjfm/mseandemalmain.html
Comments and Questions should be directed to me. Thanks John Mansfield
As John Mansfield noted, Nestor is out of town and cannot attend to the microscopy server. Unfortunately, he has not brought me in on the operation, so I cannot help any of you who have questions about its system management.
I would like to sign on to the microscopy e-mail conference group. I am an E.M. technician in neuroscience at U of Penn. Please send further instructions. Thanks. Sally
Applications are invited for a Microscopist in the Light Microscopy Laboratory of 3M's CRL - Analytical and Properties Research Laboratories. The Light Microscopy Laboratory supports the development, production, promotion and protection of all 3M products. This is a state of the art light microscopy laboratory that utilizes more than 30 different light microscopies to solve problems of importance to all the 3M divisions. Applicants should have broad experience in microscopy such as: bright field, dark field, differential interference contrast, phase contrast, polarized light, and interference microscopy; and sample preparation techniques such as metallography, microtomy, ion milling, and etching. Additionally, applicants should demonstrate expertise in one or more of the following areas: confocal microscopy; microspectroscopy (uv/vis, raman, color/appearance); forensic microscopy; image analysis and quantitative characterization of the general morphology of microscopic structures, particles, and defects; quantitative characterization of surface morphologies; and/or fiber and debris identification. This position requires an M.S. or Ph.D. in the physical sciences or engineering.
Job Description -
The successful candidate will apply their knowledge of light microscopy to the solution of problems submitted by 3M research and/or production facilities. This will be accomplished by working with our 3M requesters to identify appropriate microscopy techniques that provide useful answers, carryout the microscopical investigations, and effectively communicate the results. The candidate will be expected to actively pursue the development of new quantitative analytical microscopical techniques or methods. Also, the successful candidate will become a corporate problem-solving resource by sharing their expertise through the appropriate 3M forums.
For confidential consideration please submit resumes to (no phone calls): G. G. Kiperts 3M Center 224-1W-02 St. Paul, MN 55144-1000
Has anyone out there with a GATAN 622 camera attached to their TEM attempted to capture the video into a Macintosh AV? I am considering the purchase of a PowerMac, and would like to hear from anyone who has tried to capture video from the GATAN camera to determine if the AV option is a good investment.
F. Scott Miller Electron Microscope Lab smiller-at-umr.edu University of Missouri-Rolla voice: 314 341 4727 Rolla, MO 65401 fax: 314 341 6934
Sorry to bother you, but my e-mail address changed about a month ago. You may wish to update your directory; mail to the old address does not transfer. allen-at-aaem.amc.anl.gov Thanks and regards. Charlie
======================================== Charles W. Allen Electron Microscopy Center-HVEM-Tandem Facility MSD 212/E211 Argonne National Laboratory Argonne. IL 60439 USA
Sorry to bother you, but my e-mail address changed about a month ago. You may wish to update your directory; mail to the old address does not transfer. allen-at-aaem.amc.anl.gov Thanks and regards. Charlie
======================================== Charles W. Allen Electron Microscopy Center-HVEM-Tandem Facility MSD 212/E211 Argonne National Laboratory Argonne. IL 60439 USA
I am not familiar with the protocol you have in mind but try mixing bacteria and phage together, let sit a few, then fix with glut. This should prevent phages from falling off.
Has anyone out there with a GATAN 622 camera attached to their TEM attempted to capture the video into a Macintosh AV? I am considering the purchase of a PowerMac, and would like to hear from anyone who has tried to capture video from the GATAN camera to determine if the AV option is a good investment.
F. Scott Miller Electron Microscope Lab smiller-at-umr.edu University of Missouri-Rolla voice: 314 341 4727 Rolla, MO 65401 fax: 314 341 6934
Dear microscopists, could someone help me with information about a company and one of its products. How can I come into contact with Citifluor Ltd, London. Does somebody use its Citifluor solution? I It should prevent fluorescence stained objects (in my case aquatic bacteria on Nucleopore filter) from quick fading. Also other suggestions how I can protect DAPI- or AO-stained bacteria from a fast fading of fluorescence are appreciated.
Thanks
Guenter Jost Baltic Sea Research Institute Dept. Biological Oceanography Seestrasse 15 D-18119 Rostock-Warnemuende Tel.: 0381-5197227 Fax: 0381-5197440 jost-at-bio.io-warnemuende.d400.de
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Dear microscopists, could someone help me with information about a company and one of its products. How can I come into contact with Citifluor Ltd, London. Does somebody use its Citifluor solution? It should prevent fluorescence stained objects (in my case aquatic bacteria on Nucleopore filter) from quick fading. Also other suggestions how I can protect DAPI- or AO-stained bacteria from a fast fading of fluorescence are appreciated.
Thanks
Guenter Jost Baltic Sea Research Institute Dept. Biological Oceanography Seestrasse 15 D-18119 Rostock-Warnemuende Tel.: 0381-5197227 Fax: 0381-5197440 jost-at-bio.io-warnemuende.d400.de
Since a couple of months I'm now reading the interesting discussions on this server. In the studies of specimen with SAD there is a question that bothers me since a longer time: I'm trying to figure out the precision of the measurement of the lattice parameters in thin, ploycristalline Ni/Ti multilayers (grain size about 10nm) with SAD. The work was done on a Phillips EM430 and Hitachi HF2000 FEG microscope. Did anybody try to calculate whether there is a dependence of the position of the reflection in the diffraction pattern from the position of the grains in the selector aperture due to higher order lens errors? Does there exist literature about this problem?
What tissue will you be labeling? You realize that only the surface will be seen on SEM. BSE is probably the better imaging technique to employ using 20-30nm gold. Need a little more info.
I have used the CitiFluor antifadent for fluorescence and found it to work quite well. The product I used was AFT-10 tablets. One tablet dissolved in 1 ml of 10X PBS, brought up to 10 mls with glycerol (ie 90% glycerol/PBS final). This was to coverslip mounted brain sections using FITC and TRITC double immunofluorescence. The cost was app $110 USD plus tax and shipping for 10 tablets (about 2-3 years ago). Unfortunately, I have lost the FAX number for CitiFluor, but their address and phone follows. Good luck!
CitiFluor Limited The City University Northampton Square London EC1V OHB UK phone (Int): 441-253-4399 ext 3502
David Morilak Dept Pharmacology Univ Texas Health Science Center San Antonio
Subject: Time:10:18 AM OFFICE MEMO re} Kill my feed Date:9/23/94
Dear Erik, Perhaps you do not understand the organization of a list. The "your" which you refer to are other subscribers like yourself which have no control over the list administration. When you send junk mail to the list it is an attack on others like yourself. If you want to be removed from the list I suggest you contact the listserve manager Nestor Zaluzec (Zaluzec-at-aaem.amc.anl.gov) for instructions. Mike Mike_Schwartz-at-qm.yale.edu Fax 203-785-5263 Voice 203-785-4324
Michael Schwartz, Ph.D. Associate Professor Section of Neurobiology Yale University School of Medicine 333 Cedar St. New Haven, CT 06510
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I want this feed stopped - one of these messages will posted every day until the junk mail stops. This wastes my bandwidth as well as yours but since I have been paying for it for the last 3 months, how could it hurt to overload your systems with junk mail... since no-one will listen to me...
I just got a call from a person at an equipment remarketing company in New Mexico that will be auctioning some equipment from a large government lab, including a Coats and Welter field emission SEM (Model HPS 70). They are looking for good homes for this equipment and the auction takes place TOMORROW, Saturday, Sept. 24. If you want more information, please contact Edith Lewis directly by phone.
Edith Lewis Remarket, Inc. (505) 892-4983
Please don't reply to this list. Thanks.
John chandler-at-lamar.ColoState.EDU Fort Collins CO
Dear John, My Industrial Regional Buying Guide lists Inland Vacuum Industries, Inc 35 Howard Ave., Churchville NY 14428. (716) 293-3330. I don't know their mechanical pump oils, but I've been satisfied with their silicone DP oil. Good luck. Yours, Bill Tivol
Hi, We have been using 49 C melting point paraffin, which we purchased from BDH Chemicals, Poole, UK (Prod. #29839). Now, we are unable to find a source for this wax. Does anyone know of somewhere that we can buy this stuff? Thanks!
PS: Wtih respect to the "kill the feed" demand, I personally find the discussion of other techniques and aspects of EM/LM to be interesting and often directly useful in my research and teaching. I want to thank Nestor for his continued work and all the others who contribute to the discussions for making this a very productive source of information.
Dwight Beebe IRBV, Dept. de sciences biologiques beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
On my way out of the MSA/MAS meeting in New Orleans, I stopped by the Computer Workshop. I previewed a Macintosh program, Super SEM 1.1 by B.J. Griffin and A. van Riessen from the Center of MIcroscopy and Microanalysis at the University of Western Australia. Unfortunately a floppy disc that I put the ftp information on about this program and the software library was unrecognizable when I returned. Can anyone tell me how to obtain Super Sem?
Thanks, Lou Ross 101 Geological Sciences Bldg. University of Missouri Columbia, MO 65211 (314) 882-4777, FAX 882-5458 e-mail geosclmr-at-showme.missouri.edu
On my way out of the MSA/MAS meeting in New Orleans, I stopped by the Computer Workshop. I previewed a Macintosh program, Super SEM 1.1 by B.J. Griffin and A. van Riessen from the Center of MIcroscopy and Microanalysis at the University of Western Australia. Unfortunately a floppy disc that I put the ftp information on about this program and the software library was unrecognizable when I returned. Can anyone tell me how to obtain Super Sem in the US or do I need to contact the authors directly?
Thanks, Lou Ross 101 Geological Sciences Bldg. University of Missouri Columbia, MO 65211 (314) 882-4777, FAX 882-5458 e-mail: geosclmr-at-showme.missouri.edu
Get a life and stop crying. You sound like my 9-year old daughter when she hasn't had enough sleep!
OK, now for what you need. The following exerpt is from a recent administrative transmission from the listserver to all of us who actively subscribe to the list. The section on unsubscribing when you don't know your original subscription address is obviously relevant to you. If you still have trouble sorting it out after speaking to YOUR SysOp, contact Nestor Zaluzec (the list administrator) at Zaluzec-at-aaem.amc.anl.gov - and leave the rest of us alone!
FROM MISCROSCOPY LISTSERVER 9/5/94:
If you want to unsubscribe you must send a message to
LISTSERVER-at-AAEM.AMC.ANL.GOV donot send it to "Microscopy-at-..."
in the message put the following line.
UNSUBSCRIBE MICROSCOPY NAME-at-HOST (where NAME-at-HOST is your Email address)
and you MUST provide your original subscription address. If you try to unsubscribe from the listserver using an address which is not registered then the message will be ignored (and then forwarded to my Email).
Recently, I have been seeing a couple of individuals trying to unsubscribe mulitiple times. They are sending the message to the correct address BUT the Email addresses which they have been suppling as "their subscription addresses" are not registered on the system.
This most likely occurs, when an individual subscribes from one address then has their mail forwarded to a different computer. Unless the system receives your subscription address there is no way for it (or me if I have to do it manually) to find out who to delete. If you are not sure what address you subscribed with, then try checking the header lines of a recent Microscopy Email message. Usually in that header you will be able to see the route that has occured during your Email transit, particuliarly if your message is being forwarded locally. If you donot know what to do here, then check with you local System Manager.
David Morilak Dept Pharmacology Univ Texas Health Science Center San Antonio
This is Nestor, touching base from France. Up until today I have been out of contact with the listserver.
I have just seen your request however, I cannot unsubscribe you (or anyone else for that matter) unless you supply the address which you have originally subscribed to this server with. THERE IS NO RECORD OF ANY SUBSCRIBER WITH THE ADDRESS YOU HAVE SUPPLIED
SMTP%"anthrax!er298s1146-at-wimsey.com"
I WILL BE MORE THAN HAPPY TO HELP, BUT CANNOT DO IT WITHOUT YOUR PROVIDING THE CORRECT INFORMATION!!!!
To all subscribers, please if you forward your mail remember what your original subscription address is neither I nor the listserver system can read minds!! Especially across the Atlantic!
Reply to: RE} Measures of spatial distribution. Hi Dave, P.J. Clark, F.C. Evans (1954) Distance to nearest neighbor as a measure of spatial relationships in a population. Ecology 35:445-453. Re = 1/( 2 sqrt(N) ) = expected Mean distance between nearest neighbors. Ra = actual Mean distance. Q = Ra / Re Value if Q is: 1.0 for a random distribution. { { 1.0 for clumping or aggregation. } } 1.0 for a more regular or uniform spacing. Qmax: Maximum possible value for Q is 2 * sqrt( 2 / sqrt(3) ) - 2.1491 for a hexagonal (honeycomb) distribution. Notes: 1. See the original paper for derivation of statistics. 2. "Mixed" populations (for example, showing some clumping and some uniform spacing) can result in values of Q that do not tell you anything. In other words, some patterned distributions can result in Q = 1, which would niavely be interpreted as random. 3. Pages 263-295 of the following paper is where I came across the original reference and is my source for the above info: M.J. Potel, S.A. MacKay (1979) Preaggregative cell motion in Dictyostelium. J. Cell Sci. 36:281-309.
Enjoy,
George McNamara Universal Imaging Corporation --------------------------------------
To my image analyst comrades; I am looking for references to the measurements of dispersion. I am working with TEM images of dispersed inorganic materials in polymer blends - I've cooked up a nearest neighbor measure and the distribution thereof but I haven't been able to find any satisfactory (at least to my customer) theoretical workup on that method. Any thoughts on other distribution quality measures would be most welcome too. Thanks
Dave Calvert Eastman Chemical Co. Kingsport, TN Also found at calvert-at-emn.com
Does anyone know of any independents who can perform routine maintenance on a Hitachi H-7000 in the Upstate New York area? Tks in advance! Craig Lending SUNY Brockport, Biology clending-at-acspr1.acs.brockport.edu 716-395-5755
Dear Monica, Your message raises more questions than answers. What is the tissue you are working with? Are you seeking to label surface antigens, or internal ones? Access to the antigen is probably the biggest challenge. Some sort of fracturing may be required (particularly for internal antigens) coupled with some detergent extraction to allow access to the antigen.
In all likelihood you will need to use BEI to find your label. You may subsequently be able to use SEI once you've located the gold. And off hand, I can't think of a reference to immuno on whole tissues, you may wish to read Hodges GM, Southgate & Toulson (1987) Scanning Microscopy 1/1:301-318. Have fun! Cheers
Peter
Peter Vesk, E.M. Unit,F09 University of Sydney NSW 2006 Australia phone: 61 2 692 2351 (overseas) fax: 61 2 552 1967 peter-at-emu.su.oz.au
A member of the Royal Microscopical Society has a complete run of the Journal of Microscopy from 1973 to 1993 which he would like to donate to an Eastern European or Third World Institution or Library which would otherwise be unable to subscribe. This offer is not extended to individuals.
The recepient would have to pay the cost of packaging and delivery to their address.
Interested parties should contact the Royal Microscopical Society BEFORE 10 OCTOBER 1994. Please DO NOT USE EMAIL TO REPLY! Fax or post the name and address of the institution and the name of the person to contact regarding delivery arrangements to: THE ROYAL MICROSCOPICAL SOCIETY 37/38 ST CLEMENTS OXFORD OX4 1AJ UNITED KINGDOM FAX: +44 865 791237.
Please mark your fax/letter: For the attention of Gillian Wilson.
Does anyone out there know a source for barium permanganate? I want to use it as a post stain to enhance the contrast of plant cell walls. Any other suggestions to achieve the same end are appreciated. ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
} Does anyone out there know a source for barium permanganate? } I want to use it as a post stain to enhance the contrast of plant cell } walls. } Any other suggestions to achieve the same end are appreciated. } ********************************************************** } * Greg Erdos ** * } * Director, ICBR EMCL ** Phone 904-392-1295 * } * 218 Carr Hall ** FAX 904-392-8598 * } * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * } * Gainesville, FL 32611 ** * } **********************************************************
Pfaltz & Bauer 800-Call-1-PB carries barium permanganate (#B00203)- 50 g for $47.
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)
There is no place on an open scientific list such as this for rude exchange. Sorry for my rash response to "Kill my Feed!" - it wasn't intended for general distribution (also my mistake), but it was rude nonetheless. I hope at least the info provided will be useful...
David Morilak Dept Pharmacology Univ Texas Health Science Center San Antonio
Several people have posted mssgs to this list regarding used SEMs. Anyone with such info is asked to send it *especially for CHEAP or free scopes--budgets!* directly to:
Several mssgs were posted to this list earlier regarding used SEMs for sale. People with this info **especially for CHEAP or free SEMs--budgets, you know** are asked to send *directly* to:
Does anyone out there have any experience using the Cerius DLS-UI module (from Molecular Simulations). I basically want to introduce particular types of atoms on specific sites within a prototype unit cell and have the program calculate the resulting cell lattice parameters. However, I have not yet been able to get the program to do this and I was wondering if anyone else has. Thus far, responses from the manufacturer's support line have been rather ambiguous.
I have a light-element EDS detecter (ultra-thin window type) which doesn't perform well on light-element detection. For instance, low oxygen and carbon x-ray counts were detected during quartz and graphite analyses, respectively.
I think the problem is due to oil and/or dirt contaminations on the ultra-thin window. Does anyone have cleaned an ultra-thin window before? What kind of procedures I need to follow? Thanks in advance.
Long Liang Electron Microprobe and SEM Lab ARCO Exploration and Production Technology 2300 West Plano Parkway Plano, TX 75075 E-mail : LLIANG-at-is.Arco.COM
I've used both biological and non-biological and looking for specimen deterioration is one of the best methods. However, I will not allow anyone who has dried their samples with a chemical drying agent such as Hexamethyldisilazane (HMDS) or Pelldri (vendor name) to use my vacuum systems (SEM or Sputter coater). In my experience they contain vacuum volitile compounds which gunk up the vacuum system (as much as the EM supply companies may deny it).
In general well fixed, well dehydrated biological specimens do not present a problem for SEM's. But they may cause a problem for ultrahigh vacuum systems (Cold FEGs, and Auger) and special care needs be taken with insuring specimen dehydration. And I would be very cautious with a windowless EDS detector.
If you need absolute cleanliness for your non-biological work I would stay away from LTSEM stages (Low-Temperature or Cryo SEM of fully hydrated specimens) as they do dirty the vacuum system somewhat.
I haven't had any experience with silicone implants, I don't know if absolutely all the silcone is polymerized or bound up (or even how it is hardened). But possible resultant glass coated aperatures are not cool.
Lets hear what the rest of the peanut gallery out here has to say. Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
I've used both biological and non-biological and looking for specimen deterioration is one of the best methods. However, I will not allow anyone who has dried their samples with a chemical drying agent such as Hexamethyldisilazane (HMDS) or Pelldri (vendor name) to use my vacuum systems (SEM or Sputter coater). In my experience they contain vacuum volitile compounds which gunk up the vacuum system (as much as the EM supply companies may deny it).
In general well fixed, well dehydrated biological specimens do not present a problem for SEM's. But they may cause a problem for ultrahigh vacuum systems (Cold FEGs, and Auger) and special care needs be taken with insuring specimen dehydration. And I would be very cautious with a windowless EDS detector.
If you need absolute cleanliness for your non-biological work I would stay away from LTSEM stages (Low-Temperature or Cryo SEM of fully hydrated specimens) as they do dirty the vacuum system somewhat.
I haven't had any experience with silicone implants, I don't know if absolutely all the silcone is polymerized or bound up (or even how it is hardened). But possible resultant glass coated aperatures are not cool.
Lets hear what the rest of the peanut gallery out here has to say. Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
I have a Fisons(Kevex) Quantum Detector which has an ultrathin window. It seems to require cleaning about 3 or 4 times a year. My procedure is to use a plastic pipette to carefully flow ethanol down the detector tube so that it flows over the end of the tube with the window. Do not squirt the window itself with anything!! Use of a small magnifying loop to observe the window helps to determine when it is clean. I forgot to say, remove the collimator first. When you are finished an easy check of the window is a comparison of the peak heights of the copper K and Cu L lines for 20kv excitation. When clean I get a slightly higher L peak. Good luck and pray before doing the cleaning! Jim Kelly
Dear Greg, If you cannot find a supplier and you are willing to try some chemistry with potentially spectacular results, you can try to add Ba(OH)2 and KOH to H2MnO4. Actually, adding BaOH to K2MnO4 might just do the job. If you meant BaMnO4 rather than BaKMnO4, and the K would interfere, you can add concentrated H2SO4 to K2MnO4, carefully add Ba(OH)2, make sure the solution is cold, collect the precipitate--which should be a mixture of BaMnO4 and BaSO4--and acidify until the BaMnO4 dissolves. IF YOU DECIDE TO TRY THIS, BE AWARE THAT H2MnO4 IS **VERY** DANGEROUS. Paper or other organic material explodes on contact with H2MnO4. Let's hope the combination of BaOH and K2MnO4 is what you need. Good luck--really. Yours, Bill Tivol
Dear Robin, I'd be very worried about contamination of the instrument due to vol- atile organic compounds from biological specimens. We do essentially only bio- logical work, and our vacuums, etc. are not anywhere near what you need to have to be sure you get no surface contamination. Cryopreparative methods may give you a better chance--at least the volatiles can condense nearby--but the area the beam hits will still evolve organics. Perhaps replicas can be made of the original specimen--freeze-fracture, evaporate metal onto the specimen, then dissolve the original, leaving a metal shell to examine. I have not done this, but our lab did about 15-20 years ago. Good luck.
The most likely reason for low carbon and oxygen counts is icing of the detector (or window). Most systems are supplied with a de-icing control which warms up the detector while the bias voltage is turned off. This should be tried first before suspecting other contamination.
Richard Leapman National Institutes of Health Bethesda, MD
} Greetings to everyone, } } I have a light-element EDS detecter (ultra-thin window type) which } doesn't perform well on light-element detection. For instance, low } oxygen and carbon x-ray counts were detected during quartz and graphite } analyses, respectively. } } I think the problem is due to oil and/or dirt contaminations on the } ultra-thin window. Does anyone have cleaned an ultra-thin window } before? What kind of procedures I need to follow? Thanks in advance. } } Long Liang } Electron Microprobe and SEM Lab } ARCO Exploration and Production Technology } 2300 West Plano Parkway } Plano, TX 75075 } E-mail : LLIANG-at-is.Arco.COM
Greetings, Just a comment about fluorescent antifade compounds. In the past I have made side by side comparisons of several of these, came to a particular conclusion, and then found a colleague who had done similarly but came to a different conclusion. There are also some discrepancies in the various papers that have been published which make these comparisons. These differences seem to be based on differences in sample and/or fluorochrome. It appears that there is no "perfect" antifade, and that it may be worthwhile to try several for your given application.
In my lab, we use a product called Vectashield (from Vector labs; no affiliation; address available by request) that works great for our applications.
Dear Roy, Two indications that our EDS detector needs to be warmed up and cleaned are the FWHM of a standard peak (Mn K-alpha in our case) and differences in energy calibration when things get really bad. It's not too difficult to run a standard or to look at the FWHM's of a few peaks in the specimen. Comparison with standards taken when the system is known to be clean should tell you when you need to do something. I have to issue the caviat that I don't have light- element capability, and I am working with 1 MV electrons, so your experience may vary. Good luck. Yours, Bill Tivol
Received: from MAILQUEUE by MICROBIO1 (Mercury 1.11); Wed, 28 Sep 94 11:04:15 EST Return-path: {baskin-at-biosci.mbp.missouri.edu} Received: from emoryu1.cc.emory.edu by transporter.microbio.emory.edu (Mercury 1.11); Wed, 28 Sep 94 11:04:15 EST Received: from aaem.amc.anl.gov by emoryu1.cc.emory.edu (5.65/Emory_cc.4.0.1) via SMTP id AA02179 ; Wed, 28 Sep 94 11:02:21 -0400 Return-Path: baskin-at-biosci.mbp.missouri.edu Message-Id: {9409281502.AA02179-at-emoryu1.cc.emory.edu}
Greetings, Just a comment about fluorescent antifade compounds. In the past I have made side by side comparisons of several of these, came to a particular conclusion, and then found a colleague who had done similarly but came to a different conclusion. There are also some discrepancies in the various papers that have been published which make these comparisons. These differences seem to be based on differences in sample and/or fluorochrome. It appears that there is no "perfect" antifade, and that it may be worthwhile to try several for your given application.
In my lab, we use a product called Vectashield (from Vector labs; no affiliation; address available by request) that works great for our applications.
Our labs use Vectashield and / or Fluoromount-G from Southern Biotechnology { (available through Fisher).
The Vectashield works better for most less than ultrapure flourochromes, while the Fluoromount seems to give double the time to fade for the higher grade dyes. Specifically the dyes from Southern Biotech and Molecular probes are the better quality.
These evaluations are purely subjective as we have not done any time studies to varify what we have noticed during observations.
As an alternative, 4%Propyl galate (Sigma) can be prepared in glycerol and used as an alternative mounting medium. This works nearly as well as the Vectashield, IF you maintain a pH near 8.0. We routinely mix 4% PG/Glycerol with an equal amount of PBS for our routine medium. I should add this is for stains that will not be photographed. We save the more expensive material for this purpose. melsen-at-MICROBIO.emory.edu
Ref: Information about electronic enlargers/auto developers
Date posted 9-28-94, please respond within a few days only.
Sometimes ago I saw demonstrated an electronic pinpoint enlarger, but have not since then heard anything about it, nor have I seen it demonstrated at any meeting. I would appreciate some info. about such enlarger (a phone number if possible) and approximate cost. This I need for the preparation of a proposal. Also, what is your preference on automatic resin coated black and white paper developers. At present we use the Kodak machines but it will be replaced soon.
Thanks in advance for your help. ***** ************ ************** *************** *Cesar D. Fermin, Ph.D \||/ Fax (504) 587-7389 * *Tulane Medical School /||\ Answ. Mach.(504) 584-2618 * *Pathology/SL79 \||/ Secretary (504) 584-2436 * *New Orleans, La 70 112 /||\ Lab (504) 5841 * ***** ***************** ************************* ________________________________________________
Does anyone out there with thin window EDS detectors also do a lot of biological cryo SEM? If so are you beset by continual icing problems?
We will soon be replacing an old beryllium window detector with a new UTW (ultra thin window) detector, so this recent thread has been quite timely. We do a lot of cryo SEM on frozen hydrated plant materials and insects, usually for morphology, but also for EDS analysis.
I sometimes have to defrost surfaces with nitrogen gas introduced into the sample chamber, while keeping the sample fully frozen at about -160 C, just enough gas to warm up the surface frost to freeze-dry temperature, especially during Minnesota summers (we do 'poor-persons' cryo, no fancy cryo-transfer devices. Its slick, quick and easy, so not a problem, actually..........until now??)
Not only that, but the in-chamber cryo hook-up acts as a cryo pump, of course (I see slight improved high vacuum during a run) and when that hook-up warms up after shutdown, any trapped oil vapor and moisture is released in a concentrated dose into the chamber.
So am I in for a big headache with high rate of ice buildup on my thin window detector as a result of the cryo work that we do? As long as the sample remains totaly chilled at -160 C, there should not be much moisture released as a result of the e-beam scanning it, should there?
I wish I could park the detector snout in a "garage" when not being used for EDS, but seems like only true windowless detectors come with that option.
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
In message "Fermin, Cesar" writes: Also, what is your preference on automatic resin coated black and } white paper developers. At present we use the Kodak machines but it will be } replaced soon. }
In December, 1993, I saved a file of 23 responses to a query on B&W photo paper processors and have added 7 more since then(from mid-summer, 1994). I will send my file to Cesar and if anyone else wants a copy, e-mail your request to me privately and I will get one to you.
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
We are currently looking for software package(s) for visualization of stacks of digital images acquired with confocal/wide field microscopes. We know of one good commercial package, running on Silicon Graphics workstatins, but the price is a bit on the "steep" side. If anyone out there uses 3D visualization software (commercial or public domain), we would like to know which one it is, its price tag, the computer platform it runs on and how satisfied you have been.
Thank you in advance for your help. Massimo
_________________________________ Massimo Sassaroli Dept. of Physiology & Biophysics Box 1218 Mount Sinai School of Medicine 1 Gustave L. Levy Pl. New York, NY 10029-6574
Message-Id: {MAILQUEUE-101.940928151439.352-at-vanlab.paprican.ca} To: christoffersen-at-snmail.jsc.nasa.gov
Hi, I have been trying to decide on suitable standard samples for checking EDX detector performance myself and have a couple of suggestions to pass on:
- CaF2 should not produce an oxygen peak but will efficiently excite oxygen in ice which may be present on the crystal. (from Kevex's SuperQuantum brochure)
- CaCo3 (calcite) may be used to monitor the peak heights of carbon and oxygen relative to calcium, as well as to check the resolution of carbon and oxygen and check for the presence of silicon in the spectrum as an artifact produced by the detector.
I'd welcome any comments on the suitability of these two specimens for monitoring detector performance.
Have fun, Laurie
______________________________________________________________________ Laurie Frederick, A.SC.T. PAPRICAN Corrosion Control Group 3800 Wesbrook Mall The Pulp and Paper Research Vancouver, B.C. Institute of Canada Canada V6S 2L9
Two programs avail. through NIH are: 1) HVEM-3D (PC-based software) high voltage EM lab in Boulder, CO contact Jim Kremer {kremer-at-beagle.Colorado.Edu} he also has some great program for the SGI machine (see his abstract in the 1994 MSA proceedings) 2) SYNU (work station {SGI/SUN} software) microscopy and imaging resource in San Diego, CA contact: Mark Ellisman -at- UCSD or Simon Lee {simon-at-UCSD.edu}
both of these programs are free or close to it, and do everything the $k programs do.
-Mike On Wed, 28 Sep 1994 sassaroli-at-msvax.mssm.edu wrote:
} Hello fellow microscopists, } } We are currently looking for software package(s) for visualization of } stacks of digital images acquired with confocal/wide field microscopes. We } know of one good commercial package, running on Silicon Graphics } workstatins, but the price is a bit on the "steep" side. } If anyone out there uses 3D visualization software (commercial or public } domain), we would like to know which one it is, its price tag, the computer } platform it runs on and how satisfied you have been. } } Thank you in advance for your help. } Massimo } } _________________________________ } Massimo Sassaroli } Dept. of Physiology & Biophysics } Box 1218 } Mount Sinai School of Medicine } 1 Gustave L. Levy Pl. } New York, NY 10029-6574 } } sassaroli-at-msvax.mssm.edu } } } }
Hi I am searching help about the construction and use of the spindle stage. Is this device available from some manufacturer ? anybody can suggest me how constructing one by myself ? I know that exists a book on the subject (Bloss - The spindle stage, Cambridge Un. Press, 1981). Is this book still available ? Thanks
Regarding 3D Visualization software I received mail from a friend of mine, dealing with that subject. Below a copy of his message. Hope it will help you a bit furher,
A new release of the VolPack volume rendering library is now available by anonymous ftp (see instructions below).
VolPack is a software library written in C and intended for use in visualization applications. The library includes implementations of the volume rendering algorithm presented at SIGGRAPH '94 this past summer (see Philippe Lacroute and Marc Levoy, Fast Volume Rendering Using a Shear-Warp Factorization of the Viewing Transformation, Proc. SIGGRAPH '94, pp. 451-458). The algorithm is more than five times faster than any other direct volume rendering algorithm I know of. The library can render a 256 by 256 by 256 voxel volume in roughly one second on an SGI Indigo R4000, running entirely in software, and produces high-quality images.
Here is a brief list of features:
- Renders data sampled on a regular, three-dimensional grid. - Supports user-specified transfer functions for both opacity and color. - Provides a shading model with directional light sources, multiple material types with different reflective properties, and depth cueing. - Produces color (24 bits/pixel) or grayscale (8 bits/pixel) renderings. - Supports arbitrary affine view transformations. - Supports a flexible data format that allows an arbitrary C structure to be associated with each grid point. - Achieves very fast rendering times without specialized hardware.
The principal limitations of the algorithm are that performance is data-dependent (since some of the optimizations are based on coherence), and there are some constraints on resampling filter quality. For a technical discussion of the algorithm and the tradeoffs involved, please see the SIGGRAPH paper (also available by ftp).
A simple Tcl/Tk application based on VolPack is also available. A Tcl/Tk package with a complete set of Tcl bindings for the library routines will be available soon.
The complete VolPack distribution can be retrieved via the Web at URL:
http://www-graphics.stanford.edu/software/volpack
or by anonymous ftp from graphics.stanford.edu (36.22.0.39), directory pub/volpack:
ftp} cd pub/volpack ftp} get README ftp} binary ftp} get volpack.1.0b2.tar.Z ftp} bye
% zcat volpack.1.0b2.tar.Z | tar xvf -
Now look at the README file in the unpacked directory.
The current release is version 1.0beta2. It is a beta release. The distribution includes source code, a tutorial user's manual, man pages for all library routines, and some sample programs with one sample dataset. Other goodies are available at the ftp site.
Contact: Phil Lacroute (lacroute-at-weevil.stanford.edu)
-- Kees van der Wulp TNO - Nutrition & Food Research Institute INTERNET : vanderwulp-at-mbl.tno.nl Division of Toxicology VOICE : +31 15 843101 PO-Box 5815 FAX : +31 15 843989 2280 HV RIJSWIJK (NL) THE NETHERLANDS
Regarding your request for 3D vis. software I received a message from a friend of mine. Hope this will help you.
********************************************
A new release of the VolPack volume rendering library is now available by anonymous ftp (see instructions below).
VolPack is a software library written in C and intended for use in visualization applications. The library includes implementations of the volume rendering algorithm presented at SIGGRAPH '94 this past summer (see Philippe Lacroute and Marc Levoy, Fast Volume Rendering Using a Shear-Warp Factorization of the Viewing Transformation, Proc. SIGGRAPH '94, pp. 451-458). The algorithm is more than five times faster than any other direct volume rendering algorithm I know of. The library can render a 256 by 256 by 256 voxel volume in roughly one second on an SGI Indigo R4000, running entirely in software, and produces high-quality images.
Here is a brief list of features:
- Renders data sampled on a regular, three-dimensional grid. - Supports user-specified transfer functions for both opacity and color. - Provides a shading model with directional light sources, multiple material types with different reflective properties, and depth cueing. - Produces color (24 bits/pixel) or grayscale (8 bits/pixel) renderings. - Supports arbitrary affine view transformations. - Supports a flexible data format that allows an arbitrary C structure to be associated with each grid point. - Achieves very fast rendering times without specialized hardware.
The principal limitations of the algorithm are that performance is data-dependent (since some of the optimizations are based on coherence), and there are some constraints on resampling filter quality. For a technical discussion of the algorithm and the tradeoffs involved, please see the SIGGRAPH paper (also available by ftp).
A simple Tcl/Tk application based on VolPack is also available. A Tcl/Tk package with a complete set of Tcl bindings for the library routines will be available soon.
The complete VolPack distribution can be retrieved via the Web at URL:
http://www-graphics.stanford.edu/software/volpack
or by anonymous ftp from graphics.stanford.edu (36.22.0.39), directory pub/volpack:
ftp} cd pub/volpack ftp} get README ftp} binary ftp} get volpack.1.0b2.tar.Z ftp} bye
% zcat volpack.1.0b2.tar.Z | tar xvf -
Now look at the README file in the unpacked directory.
The current release is version 1.0beta2. It is a beta release. The distribution includes source code, a tutorial user's manual, man pages for all library routines, and some sample programs with one sample dataset. Other goodies are available at the ftp site.
Contact: Phil Lacroute (lacroute-at-weevil.stanford.edu)
From postmaster-at-compuserve.com Thu Sep 29 11:34:48 1994 Received: from frontier.tno.nl by mbimp1.mbl.tno.nl via SMTP (920330.SGI/920502.SGI) for imgp id AA10285; Thu, 29 Sep 94 11:34:48 +0100 Received: from arl-img-1.compuserve.com by frontier.tno.nl (4.1/1.53) id AA15203; Thu, 29 Sep 94 11:27:38 +0100 Received: from localhost by arl-img-1.compuserve.com (8.6.4/5.940406sam) id GAA25988; Thu, 29 Sep 1994 06:33:38 -0400
Re: ? EMDNRM - Mail Delivery Failure. No room in mailbox. 75022,2723 Re: 3D Visualization Software
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Dear Massimo,
Regarding 3D Visualization software I received mail from a friend of mine, dealing with that subject. Below a copy of his message. Hope it will help you a bit furher,
A new release of the VolPack volume rendering library is now available by anonymous ftp (see instructions below).
VolPack is a software library written in C and intended for use in visualization applications. The library includes implementations of the volume rendering algorithm presented at SIGGRAPH '94 this past summer (see Philippe Lacroute and Marc Levoy, Fast Volume Rendering Using a Shear-Warp Factorization of the Viewing Transformation, Proc. SIGGRAPH '94, pp. 451-458). The algorithm is more than five times faster than any other direct volume rendering algorithm I know of. The library can render a 256 by 256 by 256 voxel volume in roughly one second on an SGI Indigo R4000, running entirely in software, and produces high-quality images.
Here is a brief list of features:
- Renders data sampled on a regular, three-dimensional grid. - Supports user-specified transfer functions for both opacity and color. - Provides a shading model with directional light sources, multiple material types with different reflective properties, and depth cueing. - Produces color (24 bits/pixel) or grayscale (8 bits/pixel) renderings. - Supports arbitrary affine view transformations. - Supports a flexible data format that allows an arbitrary C structure to be associated with each grid point. - Achieves very fast rendering times without specialized hardware.
The principal limitations of the algorithm are that performance is data-dependent (since some of the optimizations are based on coherence), and there are some constraints on resampling filter quality. For a technical discussion of the algorithm and the tradeoffs involved, please see the SIGGRAPH paper (also available by ftp).
A simple Tcl/Tk application based on VolPack is also available. A Tcl/Tk package with a complete set of Tcl bindings for the library routines will be available soon.
The complete VolPack distribution can be retrieved via the Web at URL:
http://www-graphics.stanford.edu/software/volpack
or by anonymous ftp from graphics.stanford.edu (36.22.0.39), directory pub/volpack:
ftp} cd pub/volpack ftp} get README ftp} binary ftp} get volpack.1.0b2.tar.Z ftp} bye
% zcat volpack.1.0b2.tar.Z | tar xvf -
Now look at the README file in the unpacked directory.
The current release is version 1.0beta2. It is a beta release. The distribution includes source code, a tutorial user's manual, man pages for all library routines, and some sample programs with one sample dataset. Other goodies are available at the ftp site.
Contact: Phil Lacroute (lacroute-at-weevil.stanford.edu)
Laurie, My only commemnt about these 2 materials is that they both are quite beam sensitive. Using a focused beam (on the EPMA) or high magnification on the SEM will result in an evolved gas from the specimen. If you have a visible light optic system on your machine this process will be easily detectable. Hence, my suggestion would be to use beam currents no greater than 10 nA and relatively low magnifications (will require some experimentation), or a defocused beam of diameter } /=15um if you have that capability. In summary, "go easy on them."
} Hi, } I have been trying to decide on suitable standard samples for } checking EDX detector performance myself and have a couple of } suggestions to pass on: } } - CaF2 should not produce an oxygen peak but will efficiently excite } oxygen in ice which may be present on the crystal. (from Kevex's } SuperQuantum brochure) } } - CaCo3 (calcite) may be used to monitor the peak heights of carbon } and oxygen relative to calcium, as well as to check the resolution of } carbon and oxygen and check for the presence of silicon in the } spectrum as an artifact produced by the detector. } } I'd welcome any comments on the suitability of these two specimens } for monitoring detector performance. } } Have fun, } Laurie
Ed Vicenzi tel (609) 258-1464 office Princeton University tel (609) 258-1406 lab Princeton Materials Inst. fax (609) 258-6878 70 Prospect Ave. Princeton, N.J. 08540-5211 vicenzi-at-phoenix.princeton.edu
Does anyone have experience with high mag. SEM work using mica as a substrate? I am trying to image proteins (~50 nm X 10 nm X 3 nm) in a Hitachi S4500 and am having a great deal of difficulty. I have tried coating the samples with ~1 nm of Cr or Ir in a VCR ion beam coater. At higher accelerating voltages (~10 kV) the image is not stable with time. I think I may be destroying the mica at these voltages. At lower accelerating voltages (0.5 - 1 kV) the image is stable but the S/N is so low that I have difficulty focusing at mag's above 10k X.
Steve Eppell Facility Coordinator Center for Cardiovascular Biomaterials Case Western Reserve University sje-at-po.cwru.edu
There are basicly two types of spindle stages avilable: 1. detent spindle stage McCrone Accessories & Co. 850 Pasquinelli Dr. Westmont, Illinois 60559-1275 USA Phone: 708-887-7100 Fax: 708-887-7764 The price should be about $40-60 apiece. 2. Supper spindle stage Charles Supper X-ray Co. 15 Tech Circle Natick, Massachusetts 01760 USA Phone: 508-237-2995 Fax 508-655-3913 Catalog # 7058: Crystal spindle stage, $360. Supper spindle is a much better instrument, which allows you to rotate the crystal 360 degrees. However, you will need a x-ray goniometer head to mount the crystal. In the meantime, you will a lot more flexibility to orient the crystal using the two mutually perpendicular arcs on the goniometer head. The standard goniometer head made by Supper costs about $340 apiece. But you may find a cheaper one through other manufacturers. 3. F. D. Bloss' book "The Spindle Stage" is also available through McCrone at about $90. Its catalog number is 455. If you want to use double variation method, you will need a heating cell. Check with Prof. Micky Gunter at University of Idaho to see if he still makes it. His e-mail address is GUNTER-at-IDUI1.CSRV.UIDAHO.EDU. Spindle stage is a extremely powerful technique. Good luck!
Shu-Chun Su Hercules Inc. Research Center 8136-267 500 Hercules Road Wilmington, DE 19808-1599 Phone: 302-995-3498 Fax: 302-995-4135
On Thu, 29 Sep 1994, Giorgio Gasparotto wrote:
} Hi } I am searching help about the construction and use of the spindle stage. } Is this device available from some manufacturer ? anybody can } suggest me how constructing one by myself ? } I know that exists a book on the subject (Bloss - The spindle stage, } Cambridge Un. Press, 1981). Is this book still available ? } Thanks } }
My 2 cents: I've done a fair amount of bio SEM, using CPD, dessicator, freeze, Pel-Dri, & hexamethyldisilizane dried specimens. I've noted very little performance degradation. The major requirement is to make sure the specimens really *are* dry--don't look at them right afte mounting & drying. Yes, for ultra-high vacuum materials science WDX or EDX scopes used for quantitative work, outgassing from biological specimens can be a problem. But then materials science specimens outgass--how many polymers are completely polumerized? their components will also outgass. Besides: 1) most of the contamination from bio samples is acutally from the silver paint/paste--the solvents can take a long time to fully evaporate, eapecially when the mounted samples are in therequired dessicator 2) bio specimens aren't done shrinking until they're completely dry, which takes time! maybe LOTS of time...(I hope your users aren't measuring soft tissues in the SEM/ SEM images). This is my experience with SEMs running at 10-6 & 10-7. If you need 10-9 torr, you may have more problems. Basic cleanliness and back-filling with dry nitrogen (even in a low-humdity environment!) does more for keeping a scope clean. And using turbo-molecular pumps instead of diffusion pumps makes a *big* differnece. EDX detectors: The ice comments are very useful. I did run into a problem once with pump-oil contamination (from a diffusion pump)--the simple fix was a beam collimator over the detector. This not only took care of the oil, by keeping away from the detector (the collimator acts like a cold finger), byt got rid of a spurious Chromium peak. Phil Oshel poshel-at-luc.edu
In message Thu, 29 Sep 1994 09:34:55 -0400, sje-at-po.CWRU.Edu (Steven J. Eppell) writes:
} } Does anyone have experience with high mag. SEM work using mica as a } substrate? I am trying to image proteins (~50 nm X 10 nm X 3 nm) in a } Hitachi S4500 and am having a great deal of difficulty. I have tried } coating the samples with ~1 nm of Cr or Ir in a VCR ion beam coater. At } higher accelerating voltages (~10 kV) the image is not stable with time. } I think I may be destroying the mica at these voltages. At lower } accelerating voltages (0.5 - 1 kV) the image is stable but the S/N is so } low that I have difficulty focusing at mag's above 10k X. } } Steve Eppell } Facility Coordinator } Center for Cardiovascular Biomaterials } Case Western Reserve University } sje-at-po.cwru.edu } ============ Steve,
I have obtained relatively decent high mag images (X 150,000; upper detector, 7 kV) of Tobacco Mosiac Virus on freshly cleaved mica, coated with 2 nm of platinum (as measured by a quartz crystal thickness monitor; but who knows what the real film thickness on the mica and on the virus is!!), using our HITACHI 4500 FESEM. Rapid focusing to minimize "beam damage" before taking the picture seems to help. In general the specimen as well as the mica appear to deteriorate after they are subjected to just one or two slow scans (photo scan rate). I have found that obtaining high mag SEM images of biomolecules can be very challenging (to put it mildly)!
} Does anyone have experience with high mag. SEM work using mica as a } substrate? I am trying to image proteins (~50 nm X 10 nm X 3 nm) in a } Hitachi S4500 and am having a great deal of difficulty. I have tried } coating the samples with ~1 nm of Cr or Ir in a VCR ion beam coater. At } higher accelerating voltages (~10 kV) the image is not stable with time. I } think I may be destroying the mica at these voltages. At lower } accelerating voltages (0.5 - 1 kV) the image is stable but the S/N is so low } that I have difficulty focusing at mag's above 10k X. } } Steve Eppell } Facility Coordinator } Center for Cardiovascular Biomaterials } Case Western Reserve University } sje-at-po.cwru.edu
Steve, Looks like you have a problem of thin coating. The SE signal yielding is much higher at low voltage than high voltage.
Ya Chen ---------------------------------------------------------------------------- | Assistant Researcher/Cryo-SEM Coordinator | Integrated Microscopy Resource (IMR)-- | An NIH Biomedical Research Center | University of Wisconsin-Madison | 1675 Observatory Drive #167 \ / | Madison, WI 53706 \ / |------------------------------------------- \/ /--/ | TEL: 608-263-8481 / / / | TEL: 608-265-3083 / /-- { | FAX: 608-265-4076 | Email:YChen-at-macc.wisc.edu | Email:chen-at-calshp.cals.wisc.edu ----------------------------------------------------------------------------
======================================================================= Chris Frethem (612)624-4652 (voice) Cell Biology & Neuroanatomy (612)624-8118 (FAX) U of MN, Minneapolis e-mail: frethem-at-lenti.med.umn.edu
---------- Forwarded message ----------
Steve, Try mounting your proteins on carbon films or carbon coated Formvar membrane on TEM grids instead, and view at higher (10 kV - 30 kV) accelerating voltages. Your grid should be mounted in such a way that you do not generate secondary electrons from a surface below the support membrane; a brass or aluminum chip with a 2.5 mm hole drilled in it, for example. The grid can be simply held over the hole with carbon tape or paint. I'm not familiar with the S-4500 specimen holders so I can't advise how to mount this "holey chip", but make sure that the beam either passes on into vacuum "space" beyond the membrane or that you provide a surface that generates few secondary electrons under this chip. Simply gluing your holey chip to another thin brass or aluminum chip with a thin layer of carbon paint or using double-sided carbon tape should suffice. I have had little experience using mica as a substrate but I'm assuming the instability you're seeing over time at 10 kV is due more to charging than any other factor, especially considering the very thin Cr or Ir sputter coat. The stability you see at 0.5 to 1.0 kV is expected for the same reason. Eliminating the mica should reduce this problem. You will have to search for the ideal kV; too high a kV and your secondary yield may be very low as the beam will blast right through your proteins without creating much topographic contrast. Using Iridium instead of Cr will help at the higher kV's but of course the Ir has more granularity than the Cr, too. Search for the best compromises. We have a Hitachi S-900 and have dealt with some of the issues you bring up in your question, though we can't claim to have solved them all yet! Good luck, and I hope someone else out there can feed you (us) some info too. By the way, how are you preparing these proteins? Air drying from suspension? Crit. pt. drying? Are they fixed? Freeze dried? What are they?
Bye
======================================================================= Chris Frethem (612)624-4652 (voice) Cell Biology & Neuroanatomy (612)624-8118 (FAX) U of MN, Minneapolis e-mail: frethem-at-lenti.med.umn.edu
On Thu, 29 Sep 1994, Steven J. Eppell wrote:
} } Does anyone have experience with high mag. SEM work using mica as a } substrate? I am trying to image proteins (~50 nm X 10 nm X 3 nm) in a } Hitachi S4500 and am having a great deal of difficulty. I have tried } coating the samples with ~1 nm of Cr or Ir in a VCR ion beam coater. At } higher accelerating voltages (~10 kV) the image is not stable with time. I } think I may be destroying the mica at these voltages. At lower } accelerating voltages (0.5 - 1 kV) the image is stable but the S/N is so low } that I have difficulty focusing at mag's above 10k X. } } Steve Eppell } Facility Coordinator } Center for Cardiovascular Biomaterials } Case Western Reserve University } sje-at-po.cwru.edu }
South Bay Technology produces sample preparation equipment for TEM, SEM, metallography and crystallography - so if you want to do the samples on your own we can surely help. If, on the other hand, you prefer to use an outside service, I have the following suggestions of people to contact:
IBM Corp. Contact: Ron Anderson TEL: 914-892-2225 FAX: 914-892-2555
Philips Semiconductor Contact: David Su TEL: 408-991-4798 FAX: 408-991-4801
NREL Contact: Kim Jones TEL: 303-275-3734 FAX: 303-231-1030
I hope this helps! If I can be of any other assistance, please let me know.
Best regards-
David Henriks South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673
I am a biologist with limited experience with materials specimens....... a student wants to look at a magnetic sample and I would appreciate any suggestions as to the best way to handle this?
Because of their cell walls, yeast have proven to be difficult to fix and infiltrate successfully. There are a couple of options that I can recommend.
1. If you just need an overall view of ultrastructural organization, use glutaraldehyde followed by potassium permanganate fixation.
2. If you need to do deatiled ultrastructural work or immunolabeling, fix in glutaraldehyde, treat with sodium metaperoidate to make the wall more permeable, and then postfix in osmium. Byers and Goetsch, the first people to look at yeast by EM (as far as I am aware), use an enzyme treatment instead of periodate.
The methods that I use follow in detail. -------------------------------------------------------------------- KMnO4 Technique for Electron Microscopic Study of Yeast Membranes
Robin Wright University of Washington Department of Zoology, NJ-15 Seattle, Wa 98195
Fixation:
Grow cells to early log phase (1 OD/ml) in media. Make up fresh 5X fixative:
Stock Solution Amount Used Final Concentration
20X TBS 6.25 ml 5X 1 M CaCl2 0.125 ml 5mM 1 M MgCl2 0.125 ml 5mM 2.4 M Sorbitol 10.4 ml 1M Distilled H2O 3.1 ml 50% Glutaraldehyde 5 ml 10% Final Volume 25 ml (This is enough to fix 80 ml cells).
Pour 10 ml of 5X Fixative into 50 ml plastic tubes. Pour the 40 mls of cells into the fixative and mix immediately. Let sit at room temperature for 5 minutes. Spin cells down for 4 minutes speed four in the table top centrifuge. Pour off the media into the hood sink with lots of water (this may need to go into a special waste container nowadays). Gently resuspend the pellet in 12.5 ml 1X Fixative (diluted from leftover 5X stock). Let sit at room temperature for 2 minutes then refrigerate overnight 4oC).NOTE: You can get away with fixing many fewer cells with less fixative, or scale down this procedure to fix more samples.
KMnO4 Treatment: Make up 10 mls fresh 4% KMnO4 in water and filter with a 0.2 mm acrodisc. Wash cells fixed with glutaraldehyde four times with deionized water (20mls) using the table top centrifuge at speed 4 for 2-3 minutes. Resuspend in 4 ml water. Aliquot 2 ml into glass conical tube. (You can use the other 2 ml for immuno EM --see LR White protocol). Add 2 mls of 4% KMnO4 and let sit at room temperature for 5 minutes. Spin cells down at speed 4 for 2Ő. Pour off KMnO4. If the cells clump donŐt try to resuspend them completely (clumping is desirable). Add 6 ml 2% KMnO4 and mix with cells by gently resuspending with a pasteur pipet (only trying to get solution under the pellet ). Let sit at room temperature 1 hour. Rinse cells in water twice (or until all the KMnO4 purple color is gone) as before. Cover with 1% uranyl acetyate and let sit ON at 4oC.
Dehydration Series: Dehydrate as follows: (1) The cells are resuspended in 10 ml 50% ethanol and then spun in a conical, glass test tube, in a clinical centrifuge for 5 min. (The use of glass is important for maximizing clumping of the pellet into aggregates of convenient size.) (2) The supernatant is replaced with 1 ml of 70% ethanol. At this point, the cells will form a firm pellet that can be gently dislodged with a pasteur pipette and chipped into small, pepper-size fragments. An aliquot containing 10-20 small chunks is dispensed into glass, snap cap vials containing 70% ethanol. The remaining sample is stored at -20oC in Eppendorf tubes and is available for subsequent processing, if necessary. I have removed aliquots from samples stored in this manner after several months and noted no alteration in ultrastructure or immunoreactivity. (3) The glass vial is capped and placed on a rotating drum for 5 min. Dehydration is completed in subsequent incubations (5 min rotations) in 95% and 3 changes of 100% ethanol. For the absolute ethanol incubations, a new bottle of ethanol is opened to ensure absence of water. I have also used molecular sieves (a teaspoonful wrapped in several layers of lens tissue) to remove traces of water from absolute ethanol. (4) A convenient way to change solutions is to use a pasteur pipette to remove most of the liquid from the vial while it is tipped to allow the sample fragments to gather at one side of the vial. Then, place the end of the pipette flush with the bottom of the vial on the side opposite the sample. The vial is tipped to allow fluid to reach the pipette and the fluid is slowly aspirated. Sample fragments of appropriate size for processing will gather around the bore of the pipette, but will not enter, IF the pipette is properly oriented. This procedure allows solution changes to be relatively rapid and complete.
Resin: Ultra Low viscosity resin is toxic. Be very careful when you handle it. Use only in the hood and wear gloves. Mix resin in a tripour plastic beaker using a tongue depressor. Add in order: 25g vinyl cyclohexene dioxide 52.5g n-hexenyl succinic acid 1.5g DER736 0.2g DMAE Pour mixture into a dispenser bottle. If there is left over resin at the end of your experiment, store it at -70oC. Before you use resin frozen in this manner, allow it to come to room temperature , then uncap it.
Infiltration: (1) Patience is a virtue. The last 100% ethanol is replaced with a solution of 2 parts ethanol to 1 part resin. The vial is capped and returned to the rotator for 1 hr. (2) The resin mixture is replaced with 1:1 resin:ethanol mix and the capped vial rotated for at least 1 hour. (3) The resin is exchanged with a fresh 1:1 mix and the samples are rotated, UNCAPPED, overnight in a hood. The ethanol slowly evaporates, increasing resin concentration gradually. (4) The next day, the residual resin is replaced with fresh 100% resin and the samples are rotated for an hour. The resin is changed and the samples placed under vacuum (20 psi) for 15 min to degas, then returned to the rotator for 1 hour. (5) Small pellet fragments are removed from the vial, using a sharpened applicator stick. The fragment is gently "rolled" across a Kimwipe to remove residual resin and placed into 5 ml plastic embedding cups (Ted Pella, Inc.) containing resin. These cups are placed under vacuum for 1-2 hours.
Embedding: Preheat temperature block or oven to 60oC. Fill out labels with pencil and place into BEEM capsules (make 5 per sample ). Pour 2 drops of resin into BEEM capsules. Transfer a single, good size chunk of cells with an applicator stick (broken so that it has a sharp end) to the capsule. Use a dissecting microscope to help you center the cells and to get rid of the air bubbles (use an unbroken applicator stick). Fill the BEEM capsule to the line with resin. Allow the resin to polymerize in the 60oC temperature block. Section and stain with ReynoldŐs lead citrate. I usually dilute standard ReynoldŐ lead citrate 1:10 with 0.1M NaOH and stain for 1 min.
------------------------------------------------------------------ Processing Yeast Cells for Immunocytochemistry using LR White Resin Robin Wright Department of Zoology, NJ-15 University of Washington Seattle, WA 98195 206-685-3659
We have recently begun to routinely use LR White resin for all electron microscopic studies of Saccharomyces cerevisiae. This resin infiltrates whole cells nearly perfectly, with no special effort, and produces samples that have less lipid extraction than normally seen with epoxy resins. It is easy to section, stains well with uranyl acetate and lead citrate, and produces excellent results upon immunolabeling. In addition, LR White is premixed and much less toxic (non-carcinogenic) than typically used EM resins. The major drawback is that LR White sections are somewhat unstable in the electron beam and it takes a degree of patience to wait until all movement stops before exposing the film.
Use of van Tuinen & Reizman's technique (J. Histochem Cytochem 35:327-333, 1987) involving treatment of fixed cells with sodium meta-periodate was the critical factor in getting consistent results with LR White. This treatment apparently reduces sugar bonds in the wall to allow efficient infiltration of the resin. In addition, we found that the temperature of polymerization must be very carefully regulated and have resorted to using temp blocks (like those commonly used for restriction digestion) specially drilled to contain gelatin capsules. The entire technique is outlined below, drawing heavily on a paper I prepared for Methods in Cell Biology.
FIXATION Gross morphological alterations and changes in the presence and/or location of proteins can be induced by subjecting yeast to centrifugal forces (R. Preston, Carnegie-Mellon University, Pittsburgh, PA; personal communication; L. Pillus, University of California, Berkeley, CA; personal communication). To avoid such changes during fixation for immunofluorescence, the fixative can be added directly to the growing yeast culture (L. Pillus, personal communication). Encouraged by this observation, Chris Kaiser (University of California, Berkeley, CA) and I have tested its feasibility for electron microscopy with excellent results. Since the technique is considerably more rapid than others that require multiple washes prior to fixation, this direct fixation method has become our favored protocol.
(1) 1/10th volume of 10X prefixative solution (10% glutaraldehyde, 2% methanol-free formaldehyde, 0.4M potassium phosphate, pH 7) at room temperature is placed into a centrifuge bottle or tube. (It is good practice to dedicate reusable plastic and glassware for EM, to avoid any possibility of contamination of cultures, etc, with traces of fixatives).
(2) The culture (early log phase) is poured rapidly into the fixative and allowed to sit at room temperature for 5 min. During this time, the medium will turn quite dark (YPD) or yellow (YM).
(3) The cells are pelleted, resuspended in 1/10th volume of ice-cold, 1X prefixative (1% glutaraldehyde, 1% methanol-free formaldehyde in 0.04M potassium phosphate, pH 7), and allowed to complete fixation on ice for 30 min.
(4) Excess fixative is removed by 3 buffer washes (i.e. 0.04M potassium phosphate, pH 7), leaving the cell suspension in each change of buffer for at least 5 min.
(5) Note: Either cacodylate or phosphate buffer gives comparable results and the presence or absence of Ca++ and/or Mg++ (1mM each) does not make obvious differences in results.
PERIODATE TREATMENT
(1) The washed cell pellet is resuspended in 5ml freshly mixed 1% sodium metaperiodate (aqueous) and allowed to incubate at room temperature for 15 min. The sample may become clumpy at this point, an advantage for later steps.
(2) Cells are pelleted and washed once in phosphate buffer.
(3) The pellet is then resuspended in 50mM ammonium phosphate to block free aldehyde sites. No attempt is made to break the clumps of cells, just to ensure that the entire pellet is accessible to the reagent. After a 15 min incubation at room temperature, the cells are washed twice with distilled water.
DEHYDRATION
(1) The cells are resuspended in 10 ml 50% ethanol and then spun in a conical, glass test tube, in a clinical centrifuge for 5 min. (The use of glass is important for maximizing clumping of the pellet into aggregates of convenient size.)
(2) The supernatant is replaced with 1 ml of 70% ethanol. At this point, the cells will form a firm pellet that can be gently dislodged with a pasteur pipette and chipped into small, pepper-size fragments. An aliquot containing 10-20 small chunks is dispensed into glass, snap cap vials containing 70% ethanol. The remaining sample is stored at -20oC in Eppendorf tubes and is available for subsequent processing, if necessary. I have removed aliquots from samples stored in this manner after several months and noted no alteration in ultrastructure or immunoreactivity.
(3) The glass vial is capped and placed on a rotating drum for 5 min. Dehydration is completed in subsequent incubations (5 min rotations) in 95% and 3 changes of 100% ethanol. For the absolute ethanol incubations, a new bottle of ethanol is opened to ensure absence of water. I have also used molecular sieves (a teaspoonful wrapped in several layers of lens tissue) to remove traces of water from absolute ethanol.
(4) A convenient way to change solutions is to use a pasteur pipette to remove most of the liquid from the vial while it is tipped to allow the sample fragments to gather at one side of the vial. Then, place the end of the pipette flush with the bottom of the vial on the side opposite the sample. The vial is tipped to allow fluid to reach the pipette and the fluid is slowly aspirated. Sample fragments of appropriate size for processing will gather around the bore of the pipette, but will not enter, IF the pipette is properly oriented. This procedure allows solution changes to be relatively rapid and complete.
INFILTRATION
(1) Patience is a virtue. The last 100% ethanol is replaced with a solution of 2 parts ethanol to 1 part resin. The vial is capped and returned to the rotator for 1 hr.
(2) The resin mixture is replaced with 1:1 resin:ethanol mix and the capped vial rotated for at least 1 hour.
(3) The resin is exchanged with a fresh 1:1 mix and the samples are rotated, UNCAPPED, overnight in a hood. The ethanol slowly evaporates, increasing resin concentration gradually.
(4) The next day, the residual resin is replaced with fresh 100% resin and the samples are rotated for an hour. The resin is changed and the samples placed under vacuum (20 psi) for 15 min to degas, then returned to the rotator for 1 hour.
(5) Small pellet fragments are removed from the vial, using a sharpened applicator stick. The fragment is gently "rolled" across a Kimwipe to remove residual resin and placed into 5 ml plastic embedding cups (Ted Pella, Inc.) containing resin. These cups are placed under vacuum for 1-2 hours.
EMBEDDING
(1) The sample fragments are then removed, blotted on a Kimwipe, and transferred to labeled GELATIN capsules filled with resin (one fragment per capsule). The longer, narrower portion of the capsule is filled with resin and the wider, shorter portion used as a cap after the sample is introduced. The capsules should have been dried overnight in a 60oC oven.
(2) The sample is allowed to sink to the bottom, positioned in the center of the capsule, and then placed under vacuum for 15 min.
(3) Polymerization is accomplished in a tempblock that has been drilled to contain gelatin capsules. The block is equilibrated at 45 -50oC and polymerization continues for 2 days. A thick pad of aluminum foil is placed on top of the block to maintain heat.
(4) Lois Banta and Scott Emr recommend a 3-day polymerization of LR White at 4oC using UV irradiation, but I have no direct experience with this method.
Grid Preparation and Sectioning
My first immunocytochemistry attempt was a resounding disaster, since the sections floated off all 20 grids during the first wash. That experience underscored the necessity of using a technique that ensures the sections will stay in place during the entire procedure. A formvar-coating technique of Bonnie Chojnacki (Carnegie-Mellon University, Pittsburgh, PA) has proven wonderfully effective for this purpose (see below). Nickel grids (200 - 300 mesh) are used, since they are less "reactive" than the standard copper ones. However, nickel grids readily become magnetized and it will save a great deal of frustration to have non-magnetic forceps on hand for handling them. The "tennis racket" style grids with handles (Ted Pella, Inc.) are highly recommended for the ease of handling they afford.
The hydrophilic nature of LR White requires that the water level in the boat be kept very low to prevent water from leaping onto the blockface as it passes the knife edge. Other special handling is not necessary. For serial sections, Fahrenbach's method (trimming the block to have wide leading and trailing edges and using diluted rubber cement to coat these edges) works amazingly well(Fahrenbach, 1984).
a. Grid Preparation
(1) Nickel grids are placed into a glass vial containing 100% ethanol and sonicated for 1 min. They are then rinsed several times in ethanol, dumped onto filter paper in a glass petri dish, and dried in a 50-60oC oven.
(2) The washed grids are placed in a glass petri dish containing a dilute formvar solution (1 ml 2% formvar in ethylene dichloride into 25 ml 24:1 ethylene dichloride:chloroform). A pasteur pipette can be used to wash the grids into one edge of the dish.
(3) The grids are individually removed with forceps and placed onto clean filter paper to dry. Only the bars are coated with formvar, leaving the entire grid space open for view of the section. "Sticky-bar" grids prepared in this manner are usable as soon as they have dried and are effective indefinitely.
b. Sectioning and Section Mounting
(1) It is wise to prepare a sufficient number of grids for several immunolabeling experiments at one time, taking into consideration all the controls and parameters to be tested. Each grid should contain as many sections as possible. Silicon mats with divided sections (available from any EM supplier) are very convenient for storing grids securely.
(2) LR White sections should not be exposed to chloroform vapors. The hydrophilic nature of the resin spreads the sections to eliminate compression while the section floats on the water surface.
(3) Sections are manipulated into a group using an eyelash glued to a sharpened applicator stick (clear nail polish works well). The dull side of a coated grid is carefully lowered over a group of sections floating in the boat and pressed into the the water surface, without breaking surface tension. The grid is removed and inverted onto a Kimwipe to blot excess moisture. After air drying, the grid is placed onto a silicon mat for storage.
(4) In view of the many grids required to do a complete immunolabeling experiment, I have incorporated Alice Taylor's (University of California, Berkeley, CA) "assembly-line" method. Sections are allowed to accumulate in the boat until a sufficient number have been cut. During the later stages of cutting, grids are loaded into 10 forceps, each of which has been fitted with a narrow piece of tygon tubing. The tubing is pushed down toward the forcep points, holding the forcep closed and keeping the grid in place until needed. The loaded forceps are propped up, ready for use. By having 10 forceps available with grids, the time for loading the sections onto the grids is reduced considerably.
c. Securing the sections
(1) If the sections are to be used immediately, it is essential to secure the sections onto the grid. The dish containing the grids loaded with sections is placed into a 50- 60oC oven for 1 - 2 min. This treatment does not affect immunolabeling or ultrastructure and ensures that the sections do not leave the grid, even under harsh conditions.
(2) Air-drying the grids for 1 - 2 days is also usually effective, but I have lost sections when the heating step is omitted. d. Storing Sections Polymerized resins are surprisingly fluid (Aldrich and Mollenhauer, 1986). Movement of embedded material occurs both in blocks and on sections. For optimal resolution, the sections should probably be used within a few days. In practice, I have detected no noticeable changes in immunolabeling or resolution after 3 months, but the possibility of alterations should be kept in mind.
NOTE: LR White-embedded samples are amenable to immunofluorescence as well as to immuno-gold labeling. Sections should be lifted from the boat using a bacteriological loop. The loop with the water film and sections is then touched to a glass slide that has been coated with a very thin layer of 1% gelatin. (The slide should be dry.) Allow the water to dry from around the sections and then fix the sections in place with 2% formaldehyde in PBS (10 min is sufficient, or you can store the slides in this solution until ready to use.) Rinse in PBS several times and then process as you would for immunofluorescence of whole cells. Use of Coplin jars for washes is recommended.
IMMUNOLABELING
Generation of reagent antibodies of high specificity is of utmost importance for accurate immunolocalization at the electron microscope level. While this requirement cannot be overstated, it is beyond the scope of this article to review the techniques for preparation of the antibody probes. De Mey (1983) is an appropriate initial source for this information. The antiserum should be affinity-purified. While theoretically feasible, immunoadsorption to remove undesired antibodies does not produce a serum with the required specificity for immunocytochemistry. Attempts to utilize preadsorbed sera in collaboration with Johanna Reneke and Jeremy Thorner (University of California, Berkeley,CA) and with Alex Franzusoff and Randy Schekman (University of California, Berkeley, CA) have convinced us of the necessity of affinity purification. Even a miniscule percentage of "contaminating" antibodies left after immunoadsorption can produce severe problems in interpretation. Background staining of the cell wall, vacuole, and nucleus are especially problematic. The time involved in preparation of cells for immunolabeling merits use of the best possible antibody. A prudent investigator would attempt immunofluorescent localization before immunocytochemistry, since it is easier, quicker, and might provide sufficient data without resorting to the more challenging, time-consuming steps of immunocytochemical studies. In addition, immunofluorescence will provide a standard against which immunocytochemical data can be interpreted. LR White sections can be used for immunofluorescent studies. In fact, use of sections provides a much greater intensity of staining than normally seen with whole cell preparations, perhaps due to greater accessibility of the antibody to the antigen. The easiest way to use LR White section for light microscopy is to mount them on solid nickle grids. The sections should be lifted very carefully from below to eliminate folds and "corrugations" in the sections. Obviously, the use of solid grids precludes use of normal phase microscopy, but the sections are so thin (1/50th the width of a yeast cell) that normal light microscopy (including Nomarski) are useless anyway. Preparation of colloidal gold reagents conjugated to Protein A is quite simple (see Roth, 1982 and 1983; Smit and Todd, 1986). Immunoglobulin-congugated gold reagents purchased from commercial sources (Janssen, Piscataway, NJ) give less background in our hands, however. Whether this difference is due to better conjugation techniques or to inherent increases in specificity of immunoglobulin as compared to Protein A is not clear. We generally use Goat-anti-rabbit immunoglobulin-conjugated gold (GARG), purchased from Janssen. We have noticed some problems with clumping and non-specific staining with 15nm GARG particles from this supplier (see results) but reagents with smaller gold particle size were satisfactory.
Controls: Convincing Yourself and Colleagues that the Results are Real For control experiments, the same rules apply to all immunochemical techniques. Labeling of sections in the absence of primary antibody will control for immunoreactivity of the secondary antibody alone. Using preimmune serum in the primary incubations will provide evidence that the labeling observed is dependent on the immune response induced after the antigen is introduced into the animal. In the case of affinity-purification, the pass-through from the affinity column contains those antibodies in the animal that do not react with the antigen and provides a similar control as the preimmune serum and is analogous to controls based on immunoadsorption. In addition to controls based on varying the antibody probe, yeast offers a wealth of possibilities to test whether or not the observations are valid. For example, strains that either overproduce or that lack the particular antigen can be immunolabeled and the patterns compared to that of the wild-type strain. Controls based on reproducibility merit mentioning: conclusions should be based on multiple experiments, including observation of duplicates from a single experiment, labeling of sections from different blocks of a single fixation, and labeling of sections from blocks of cells fixed on another day and/or with a different fixation procedure.
Experimental Procedures Set up of Incubation Chamber:
a. It is very helpful to set up the incubation chamber with labels at the start of the experiment. The chamber consists of a box with a tight-fitting lid, of sufficient size to contain all the grids and all the solution droplets. A padding of paper towels or sponge-cloths (very thin sponges about 6" X 6", from the grocery store) is placed into the bottom of the dish and thoroughly wetted to maintain humidity throughout the labelling steps. The surface should be fairly flat. Paper towels are folded, pressed onto the sides of the dish, and moistened.
b. Onto the wet pad, a length of Parafilm is positioned. Small, colored adhesive-dots ("sticky dots") labeled with the identity of the grid (i.e. strain, fixation variation, etc.) are placed on the left side, at approximately 1 inch intervals down the length of the Parafilm strip. Across the top of the Parafilm, 4 sticky dots are also positioned. The first and third represent the position where droplets of blocker will be placed. The second is the position of the primary antibody (1o) and the fourth is the position of the gold-conjugated secondary antiserum (2o). It is good practice to do duplicates of each incubation, so that a backup is available if technical problems occur.
Reagents: PBST (140mM NaCl, 3mM KCl, 8mM Na2HPO4, 1.5mM KH2PO4, and 0.05% TWEEN-20) is used throughout the immunolabelling protocol, for all washes and as the vehicle for the blocker. Blocker is PBST containing 2% ovalbumin. Glass distilled water of high purity (as for EM) is used and solutions are filtered through a 0.22m filter (Acrodisc) before use. Blocker solution can be prepared, filtered, aliquoted, and stored frozen at -20oC. If the blocking solution has been frozen, it refiltered before use. Both 1o and 2o are diluted into blocker. The dilution factor for the 1o must be empirically determined, using a dilution series. The gold-conjugated 2o should be adjusted to A525 = 0.3 for 15nm particles and to A525 = 0.13 for 5-10nm particles (from Daniela Brada).
Blocking: 20 ul droplets of 2% ovalbumin in PBST are positioned in the appropriate position on the parafilm sheet. The appropriate grid is submerged into the solution and allowed to incubate for 15 min at room temperature. Submerging is preferable to floating, since it will allow labeling of exposed antigen on both sides of the section.
Incubation in Primary Antiserum:
a. After blocking, the grid is removed from the blocker, touched to a Kimwipe to removed excess fluid. For this and subsequent blottings, the forceps tip and grid should be held sideways on the tissue surface, so that fluid between the forceps tips is also removed. This step should be done rapidly, not allowing the sections to dry.
b. The grid is then submerged in a 20ml droplet of diluted 1o antiserum. Length of incubation is probably a matter of convenience. The results presented here were from 2 hour incubations at room temperature. Overnight incubations at room temperature or 4oC have also been successful and may allow a lower concentration of 1o to be used.
Washes:
a. Washes are performed in the wells of porcelain or glass spot plates. The wells are marked and filled with PBST, and the spot plate is placed an orbital shaker.
b. After removal and blotting of excess fluid from the grid as described above, the grid is submerged in the appropriate well. The shaker is adjusted so that the solution is moving as rapidly as possible without spilling out of the well.
c. After 5 min, the grid is removed, blotted, and transferred to the next well. A total of 3 (5 min) washes are performed and the grid is blotted and transferred to a second droplet of blocker.
Incubation in Secondary (Gold-conjugated) Antiserum:
a. After a 15 min incubation in blocker, the grid is blotted and transferred to the diluted 2o. The grid is incubated for 1 hour at room temperature and then washed as above (III.B.5).
b. After the final PBST wash, the grid is washed in distilled water by dipping 10 times with rapid up and down motion in a 5ml beaker of water. This wash removes salts that would crystallize on the section, obscuring the view in the electron microscope. The grid is blotted on a Kimwipe, transferred to a labeled silicon mat, and allowed to air-dry.
OBSERVATION I generally look at the grids prior to staining with uranyl acetate or lead citrate, just to get an overall picture of the labeling pattern. When I am ready to sit down and take pictures, the grids are stained in 2% aqueous uranyl acetate for 1-5 min and in Reynold's lead citrate for 30 sec.
If you encounter any unexpected problems, or if these instructions are unclear, please contact me. I would also appreciate hearing whether or not this technique was successful in your studies and to be updated if you find variations that are important.
If you are going for standard high quality morphology then use 2% glutaraldehyde in 0.1M cacodylate buffer w/ added 0.005M CaCl2. The are growing in some sort of culture medium, so concentrate a sample of them in a tube with a contrifuge then reduce the volume of culture medium such that you can comfortably add an (approx.) equal volume of fixative to the tube. Then resuspend the yeast (gently) in the fix and allow to fix for about 1 hour (I would guess). Centrifuge again and draw off the fix, then wash with rinsing buffer. I've used this method for years on all types of suspended single cell preparations with good results. Good luck and let me know how it goes.
Dan
On Thu, 29 Sep 1994 BARBARA.HARTMAN-at-1773.220.SCHERING-PLOUGH.sprint.com wrote:
} } GREETINGS, } } WHAT IS THE BEST FIXATIVE FOR YEAST? I AM USED TO DEALING WITH ANIMAL } TISSUES SUCH AS LIVER AND KIDNEY SO THIS IS A NEW ONE FOR ME. } } THANKS } } } } BARBARA HARTMAN } SCHERING PLOUGH RESEARCH } FAX 201-579-4211 } PHONE 201-579-4343 } } }
Another substrate you might want to try is silicon wafer, especially if you have a friendly lab doing work on semiconductors nearby. We've found it useful for some imaging of TMV to test coatings. It conducts nicely and yields low background. Otherwise I would agree with the use of TEM grids supporting a carbon coated film as suggested by Chris Frethem.
As regards coatings Cr has a low signal yield due to its low atomic number, so reducing the background is important particularly at high kV. Platinum coatings of around 2nm will give a nice strong signal at the expense of a little resolution. With the size you give for your protein you may find the platinum grains mask detail.
The degrading image will be a combination of radiation damage, contamination and charging, cooling your specimen will help greatly (particularly for the former two) if you have the capability. I wish you luck and patience! Cheers
Peter
Peter Vesk, E.M. Unit,F09 University of Sydney NSW 2006 Australia phone: 61 2 692 2351 (overseas) fax: 61 2 552 1967 peter-at-emu.su.oz.au
JOURNAL OF MICROSCOPY, VOLUME 176 PART 1, OCTOBER 1994 ******************************************************
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 1-7.
Review: The measurement of intracellular antigens and DNA by multiparametric flow cytometry
R S Camplejohn, Division of Oncology, St Thomas' Hospital Medical School, Lambeth Palace Road, London, SE1 7EH
SUMMARY
The aim of this paper is to provide a strategy for measuring intracellular antigens combined with DNA content in cells or nuclei. A series of protocols are included which enable the majority of such antigens to be labelled and further information is provided for cases in which the standard methods prove to be inadequate. The basic principles of cell permeabilization/fixation are described, thus explaining how methods can be divided into three basic categories: (a) alcohol fixation with or without detergent pretreatment; (b) paraformaldehyde fixation followed by permeabilization with alcohol or detergents; (c) permeabilization of unfixed cells. The preparation of nuclear suspensions from paraffin-embedded material is described and the possibilities and problems of staining such suspensions for nuclear antigens are discussed. Examples of results obtained with the detailed protocols are given for staining with antibodies directed against proliferating cell nuclear antigen (PCNA), Ki-67 antigen and KiS1 antigen. Details of published studies of a variety of intracellular antigens are given in two tables. The power of multiparametric flow cytometry in the study of cell proliferation, differentiation and response of cells to damage is underlined.
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 8-16.
Rapid estimation of bacterial antibiotic susceptibility with flow cytometry
D J Mason, R Allman, J M Stark & D Lloyd, Division of Microbiology, St Thomas' Campus, Lambeth Palace Road, London, SE1 7EH
SUMMARY
Bacterial antibiotic susceptibility was rapidly estimated for Escherichia coli and Staphylococcus spp. by flow cytometry. This was achieved by measuring the uptake of a negatively charged membrane potential sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol and observing changes in low-angle light scatter (excitation light scattered by up to 15 degrees). Estimations of ampicillin, gentamicin and ciprofloxacin susceptibilities were possible within 2-5 h from a plate culture, depending on the species and antibiotic used. This includes the time necessary to establish steady-state growth in liquid culture.
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 17-22.
Rapid assay for pathogenic salmonella organisms by immunofluorescence flow cytometry
A C Pinder & Rosemary G McClelland, Department of Food Biophysics, Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich, NR4 7UA
SUMMARY
Multi-parameter flow cytometry was investigated for the rapid detection of specific serotypes of salmonellas (S. typhimurium and S. montevideo) labelled with fluorescent monoclonal antibodies, both in pure culture and in a typical food matrix (full-fat milk). In all cases, the method was accurate to levels below 10 000 target cells per ml for a total assay time of about 30 min. After 6h non-selective enrichment in the presence of a 10 000-fold excess of competing micro-organisms (Escherichia coli) the corresponding detection limit was about 20 cells/ml. These results suggest that flow cytometry has a significant potential for the detection of pathogenic micro-organisms in the food industry.
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 23-33.
Scanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging
The fluorescence photobleaching method has been widely used to study molecular transport in single living cells and other microsystems while confocal microscopy has opened new avenues to high-resolution, three-dimensional imaging. A new technique, scanning microphotolysis (Scamp), combines the potential of photobleaching, beam scanning and confocal imaging. A confocal scanning laser microscope was equipped with a sufficiently powerful laser and a novel device, the 'Scamper'. This consisted essentially of a filter changer, an acousto-optical modulator (AOM) and a computer. The computer was programmed to activate the AOM during scanning according to a freely defined image mask. As a result, almost any desired pattern could be bleached ('written') into fluorescent samples at high definition and then imaged ('read') at non-bleaching conditions, employing full confocal resolution. Furthermore, molecular transport could be followed by imaging the dissipation of bleach patterns. Experiments with living cells concerning dynamic processes in cytoskeletal filaments and the lateral mobility of membrane lipids suggest a wide range of potential biological applications. Thus, Scamp offers new possibilities for the optical manipulation and analysis of both technical and biological microsystems.
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 34-44.
Optical microscopical evaluation of pyrite oxidation of heap leached samples from a Pittsburgh seam coal
Gino A Irdi & Harold B Booher, United States Department of Energy, Pittsburgh Energy Technology Center, PO Box 10940, Pittsburgh, Pennsylvania 15236, USA
SUMMARY
The US Department of Energy and the US Bureau of Monies conducted experiments to determine the feasibility of reducing the pyritic sulphur content of a conventionally cleaned Pittsburgh seam coal by heap leaching. Two identical heaps, one indoor and one outdoor, were constructed, and sprayed with recycled rainwater/leachate for approximately 200 days. Lump samples were selected prior to initiation of heap leaching activities and from both heaps after 5- and 11-month intervals for microscopic (petrographic) examination. These examinations revealed no significant differences in pyritic sulphur removal between indoor and outdoor heaps. Samples from the starting coal were also selected and examined to see how different pyrite morphologies behaved during artificial weathering. Dendritic and framboidal pyrite forms appeared to be the more reactive forms.
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 45-53.
A comparison of the analysis of the Fresnel contrast in high and low resolution images for the characterization of the rigid body displacements at a grain boundary
S H Stobbs, D L Medlin, M J Mills & W M Stobbs, University of Cambridge, Newnham College, Cambridge, CB3 9DF
SUMMARY
The Fresnel contrast in high-resolution images of the Sigma=3{112} aluminium twin boundary is quantitatively assessed to determine whether the rigid body displacements might be measured. It is demonstrated that the effects of the shears at the boundary preclude this possibility. By comparison, simulations of low-resolution images of the same boundary show that the Fresnel fringe contrast in this type of image is negligibly affected by the shear displacements as well as being of considerably higher contrast. The reasons why it is thus only the low-resolution images that can provide high-resolution data on the displacements are discussed.
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 54-62.
Fractal characterization by frequency analysis. Part III: Effect of noise
Manuel Pancorbo, Eloy Anguiano & Miguel Aguilar, Instituto de Ciencia de Materiales, Consejo Superior de Investigaciones, Cientificas C-3-301, Universidad Autonoma de Madrid, Cantoblanco 28049 Madrid, Spain
SUMMARY
The effect of noise in the fractal characterization by frequency analysis of surface images obtained by scanning tunnelling microscopy (STM), atomic force microscopy (AFM) or profilometry has been studied. The origin of noise and its relationship to the signal is discussed. A procedure to simulate noisy images is presented. From the study it is concluded that the method usually used to characterize noise in STM is not valid and it is shown that fractal characterization of surfaces when noise is present by traditional frequency analysis methods is not possible. A new method to perform both the noise characterization and the fractal characterization of surfaces when noise is present is proposed.
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 63-74.
Direct imaging in a water layer of human chromosome fibres composed of nucleosomes and their higher order structures by laser plasma X-ray contact microscopy
Yasuhito Kinjo, Kunio Shinohara, Atsushi Ito, Hisako Nakano, Makoto Watanabe, Yasuhiro Horiike, Yukiko Kikuchi, Martin C Richardson & Kazuo A Tanaka, Radiation Biology Division, Tokyo Metropolitan Isotope Research, Centre, Setagaya, Tokyo 158, Japan
SUMMARY
X-ray contact microscopy with a 300-ps-duration laser-plasma X- ray source has been used to image hydrated human chromosomes. Clearly imaged are individual nucleosomes and their high-order particles (superbeads), elementary chromatin fibrils c. 30nm in diameter and their higher-order fibres of various sizes up to c. 120nm in diameter. The results demonstrate that X-ray microscopy is now capable of opening a new path of investigation into the detailed structures of hydrated chromosome fibres in their natural state.
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 75-82.
Relocation accuracy on HOME computerized microscopes
James H Tucker, Richard Dye, Joanne Sprey, Christopher Sowter, Evelyn Gray & Gerard Brugal, MRC Clinical and Population, Cytogenetics Unit, Western General Hospital, Edinburgh, EH4 2XU
SUMMARY
A major practical advantage of the HOME (highly optimized microscope environment) computerized microscope is the facility for relocating cells or other microscopic objects. Features can be marked directly on the microscope image using a mouse-driven cursor, and an interactive finder can then be used to relocate the marked features. Tests on a prototype HOME microscope have shown that positions can be relocated with an accuracy of standard deviation (SD) of less than 7 micrometre. The marked features could also be relocated on a second HOME microscope, although with a somewhat reduced accuracy (standard deviations of less than 17 micrometre). The system provides a very user- friendly environment for tasks requiring relocation of microscopic objects.
I have just removed a message of over 500 lines concerning Yeast Fixation. Can I make an appeal to people not to send so much detail straight to the server -- this type of information should go over more private channels directly between interested parties. Yeast fixation may be an important topic, but not everyone is interested is all the details.
Just a small note: In 1986 the lab I was working in ran some experiments on KMnO4 fixation of fungi (yeasts are fungi too) and we found that KMnO4 did not "Kill/Fix" the fungi we were working with. We were growing the fungi in the presence of up to 12% KMnO4! Albiet the growth morphology was very abnormal but the fungi were still growing.
In Robin's protocol for Yeast cell fixation the glut is responsible for the killing/fixation, the KMnO4 is primarily staining. 1 hour in 2-4% glut for SEM is o.k., but for TEM I would recommend 1-2% for 10-20 min max followed by 1% OsO4 2-6 hours. HIghly recommend the use of Na cacodylate buffer for fungal fixation, having tried others, and in reviewing the literature the general concensus is that Na Cacodylate is the best buffer for fungal specimens. (This comes from 12 years of fungal ultrastructural work).
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
I realize that this is probably not good equite to post a test here on this listserver, but I have not recieved any mail since the first of September. I just wanted to know if it is working. Thanks for allowing me the use of the listserver. Clarissa
On tues I called Pfaltz and Bauer to check on the current price for BaMnO4, which they gave me. By Wed when I faxed them my order, they had discontinued the item. On monday, which I hope is not too late, I will try ICN. ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
Does anyone out there have any experience using the Cerius DLS-UI module (from Molecular Simulations). I basically want to introduce particular types of atoms on specific sites within a prototype unit cell and have the program calculate the resulting cell lattice parameters. However, I have not yet been able to get the program to do this and I was wondering if anyone else has. Thus far, responses from the manufacturer's support line have been rather ambiguous.
B. G. Demczyk Univ. of Michigan
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