NetNotes
the same order inside as we see at the surface. I don’t think it would make a crystal if it was not similarly ordered inside, but I guess one has to look. So they would like to cut sections to see an arbitrary surface. I embedded some crystals in hard epoxy and tried sectioning with a diamond knife; the crystal shatters and the section separates where the crystal was, leaving small shattered fragments clinging to the edges of the resin. I have to admit that I did not use my good diamond knife, but an older one that produces a few knife marks and is certainly not optimally sharp any longer—because I didn’t know if it would damage the knife. Te resin sectioned fine. I tried various cutting speeds, and thicknesses from 50 nm to 90 nm, all with the same outcome. Te questions: Is it possible to section crystals in this way? Would this likely damage a “standard” biological diamond knife? Would FE-SEM of a cleaved crystal surface be a better approach? Is there another approach to take? Dale Callaham
dac@research.umass.edu Tue May 4 No. You can’t prepare Si specimens with a microtome. I wrote a
short note on the subject in Microscopy Today, 14, 6, November 2006, pg 58, titled “Just Say NO to Microtoming Silicon!” that explains why you will wind up with a pile of Si dust and possibly damage your knife. You can download an MT Archive PDF at http://www.
microscopy-today.com/index.jsf;jsessionid=15B61AFA7C53C9E9CA EAE496FEF4BA5E. Polishing and etching your mounted specimens will work, but
inasmuch as your particles will be randomly oriented, you can’t count on seeing the “nice hexagonal packing” in all of the particles. Te crystal lattice is present in all of them—it just won’t be visible unless you find particles oriented in one of the principal crystallographic orientations. Te orientation for a hexagonal surface, etc. Ron Anderson
randerson20@tampabay.rr.com Tue May 4 Te small fragments that are attached to the sections might have
the answer for you. Kind of “micro-cleaving” approach. Look at them in your TEM and evaluate the microstructure, near the sharp edges are the best locations to find thin (Jerzy Gazda
jerzy.gazda@
ceriumlabs.com Tue May 4 I definitely second this idea. However be careful that most
oſten the grid is turned upside down into the microscope and if the particles deposited on it do not firmly attach, they will contaminate the microscope. So take care to shake the grid a bit to get rid of the loosely attached particles before inserting the specimen holder. Stephane Nizets
nizets2@yahoo.com Wed May 5
Specimen Preparation: LR White vs Gold Can anyone give me a comparison of LR White (LRW) and LR
Gold (LRG)? Tom Bargar
tbargar@unmc.edu Mon May 24 It depends on your application. LRG is UV polymerized; LRW
is generally polymerized with heat. LRW works with osmicated tissue, LRG doesn’t. It is a lot easier to get nice thin sections of LRG compared to UV polymerized Lowicryls. I like LRG for EM immuno- cytochemistry but if you want to do immuno-staining of 0.5 µm sections, nothing compares to BMMA. Tomas E. Phillips phillipst@
missouri.edu Mon May 24 I used LRW and polymerized it with heat also. It came with a
little packet of white powdered accelerator, but I didn’t use it. It did polymerize with heat, I believe a 60°C oven. I have never used LRG because it was more expensive. Barbara L. Plowman bplowman@
pacific.edu Tue May 25
58
Specimen Preparation: UV and low temperature resin embedding I am trying to polymerize LR White at –20°C (no success aſter
24 hours), in a Leica AFS unit. Te UV system consists of 10 UV LEDs that are ~10 cm above the floor of the chamber. I can get the resin polymerizes in a 60°C oven in ~12 hours. If the problem is the LR White, can anyone advise me as to which resins work well with low temperature with UV polymerization? Charlene Wilke c-wilke@
northwestern.edu Tu May 6 Is it possible that you somehow have confused LR White with
LR Gold? LR Gold is the one people used to polymerize at –20°C (and get endless ribbons of sections out of it). Or Lowicryls, of course. Lowicryls on Leica AFS = tons of literature. Vlad Speransky
vladislav_speransky@nih.gov Tu May 6 I am no LR White specialist, but I remember some past discus-
sions about it. First of all you must make sure to avoid contact with oxygen in the air to allow polymerization. Second, your must make sure that UV light can penetrate your sample. Stephane Nizets
nizets2@yahoo.com Fri May 7
Specimen Preparation: freeze substitution I have been performing high pressure freezing (HPF) and freeze-
substitution (FS) on cell culture with vaccinia. Tried a number of FS cocktails in an attempt to determine the best ultrastructure freeze media. When I use 1% and 2% OsO4 + 0.1% uranyl acetate in acetone, the mature virions are electron dense and I couldn’t resolve fine detail. I recently tried 0.5% OsO4 + 0.1% uranyl acetate in acetone with the anticipation that the mature virions would be less dense. Surprisingly, the virions had reversed contrast. Te FS schedule was –90°C 3 days and gradually warmed to 21°C, acetone washed 4X and resin infiltrated. One suggestion was to use 1% OsO4 + 0.1% uranyl acetate in acetone and acetone wash at –80°C instead of leaving in OsO4 for the duration. Can anyone explain why concentration change of the OsO4 resulted in negative contrast? Other cocktails used were 0.5% glutaraldehyde +0.1% tannic acid in acetone, 2% uranyl acetate/methanol in acetone. Both resulted in patches of cytoplasm loss. Karen Kelley
vau@ufl.edu Tue Jun 1 In an earlier life I did a ton of freeze substitution although never
of viruses. My experience is that the osmication reaction is sensitive to subtle triggers at –80°C. Sometimes aſter FS in acetone, rinsing the now light brown tissues in 100% ethanol several times and then storing in ethanol overnight led to an intense osmication reaction with the tissue and ethanol solution going pitch black. I don’t have a solution for you but you could try FS in tetrahydrofuran. We got startling different views with this solvent compared to acetone or ethanol. It might be worth a try. Tom Phillips
phillipst@missouri.edu Tue Jun 1
Specimen Preparation: osmium + ferrocyanide I embedded routinely processed mouse brain in EmBed 812, no
problems. Te following week I embedded similar tissue processed the same except that I used an osmium ferrocyanide mix for osmication instead of plain buffered osmium. All other steps were identical. Te Os-ferrocyanide brain is impossible to cut, acts like it was not infiltrated. Pure plastic cuts fine. Is it possible that Os-ferrocyanide reacts with something in the EmBed 812?? Geoff McAuliffe mcauliff@
umdnj.edu Tu May 13 I have done 1000+ blocks of osmium + ferrocyanide fixed tissues
into Epon-type resins without problem. I haven’t used this comboni- tation in a while so I can remember whether I used it once the switch to Dpon 812 substitutes occurred but think I probably did. I doubt that is the problem. Tom Phillips
phillipst@missouri.edu Tu May 13
www.microscopy-today.com • 2010 September
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