NetNotes


Edited by Thomas E. Phillips University of Missouri


phillipst@missouri.edu


Selected postings from the Microscopy Listserver from May 1, 2015 to June 30, 2015. Complete listings and subscription information can be obtained at http://www.microscopy.com . Postings may have been edited to conserve space or for clarity.


Specimen Preparation: glycogen


For several years, we have lost the ability to visualize glycogen by thin section TEM. As I recall, this occurred when we changed bottles of DBP in our Epon-substitute/Araldite mixture. Has anyone else had the glycogen “disappear”, and come up with a solution? Cynthia Goldsmith cgoldsmith@cdc.gov Tue May 12


Glycogen can become coalesced into amorphous unstained regions in the cytoplasm under certain fixing/staining conditions. I have found this in certain cell types (i.e., muscle, lung tissue) if you use non-reduced osmium followed by un-buffered uranyl acetate (aqueous) en bloc staining. Generally if you use non-reduced osmium, I would en bloc with buffered (0.1 M maleate, pH 6.2) 2% uranyl acetate. If you do use reduced osmium (K 4 Fe(CN) 6 ), then you can continue with aqueous uranyl acetate. A remedy for material already in plastic is to triple stain the sections: 1% filtered tannic acid (10 min) followed by 2% aqueous uranyl acetate, then lead citrate. This will fill in the amorphous regions and stain the glycogen there. I have done this with lung tissue. Michael Delannoy mdelann1@ jhmi.edu Wed May 13


Specimen Preparation: mitochondrial contrast


We have a few mammalian skeletal muscle samples that are already embedded in Spurr’s resin. They were conventionally fixed with glutaraldehyde and OsO 4 , and en bloc stained with uranyl acetate. The mitochondria are poorly contrasted within the muscle fibers using uranyl acetate (50% ethanolic solution) and lead citrate post-staining. Section thickness is 70 nm. Are there any modified post-staining techniques that can preferentially enhance contrast of the mitochondria vs the surrounding muscle fibers? David Lowry dlowry@asu.edu Tue May 26


You can try triple staining with 1% filtered and aqueous tannic


acid (10 min) before the uranyl acetate and lead. The grids must be Formvar coated as the copper will react with the tannic acid. Michael Delannoy mdelann1@jhmi.edu Wed May 27


Specimen Preparation: non-radioactive uranyl acetate


I just read a post on uranyl acetate and wanted to ask you a question regarding a particular uranyl acetate sold by EMS as non-radioactive. Does this work the same as radioactive ones? Has anybody used this non-radioactive uranyl acetate? Does it have any kind of limitations? Their description is at this website: http://www. emsdiasum.com/microscopy/products/chemicals/tannic.aspx#22400 Fumiya Watanabe fxwatanabe@ualr.edu Thu May 21 Uranyl acetate is always radioactive. There are uranium isotopes whose decay rates are quite low, and in some states, the total radioac- tivity is deemed low enough so that uranyl acetate can be discarded


50


down the sink. I expect that this is what EMS has in mind. Bill Tivol wtivol@sbcglobal.net Fri May 22


As mentioned by Bill, uranyl acetate is at best depleted, but never non-radioactive. What you came across is actually a non-radioactive uranyl acetate substitute, which consist of “Two lanthanide salts, samarium and gadolinium triacetate”. Check the paper quoted in the data sheet. As a general note, the staining properties of uranyl acetate vary substantially between manufacturers and thus between countries (as nobody wants to ship the stuff unless necessary). Testing is the only way to find out. Chris Buser cbuser125@gmail.com Fri May 22


Image Analysis: particle sizing


I had an animated discussion with a colleague material scientist. The question is: is it possible to measure the size of microparticles (some aluminum-containing mineral) by embedding them, preparing a fine flat surface, coating and analyzing in REM with W gun in BSE mode. My colleague says it is no problem, really. I say I see 2 objections (really): - the first one is that the interaction volume is much bigger in BSE mode than in SE mode (Mr. Chapman’s rule #3), decreasing the lateral resolution, which is already a problem for particle sizing in REM - the second one is more theoretical: by sectioning microparticles, we get all sorts of cross- sections. By measuring the diameter of the cross-sections we don’t get the measure of the mean diameter of the particles but a much more spread size distribution which is not centered on the actual diameter of the particles. Stephane Nizets nizets2@yahoo.com Tue Jun 2


You are right. The problem of size distribution caused by serial sectioning can be overcame by a) Assuming all of the particles are the same size and are randomly distributed in the embedding medium - dangerous assumptions at best or b) Serial sectioning the block and imaging the block face - this requires an ultramicrotome within the SEM (= REM) chamber, but such things exist now: Leighton, S.B. 1981. SEM images of block faces, cut by a miniature microtome within the SEM - a technical note. Scan Electron Microsc 1981, (Pt 2): 73–6. Wanner, A.A., et al., 2015. “Challenges of microtome-based serial block-face scanning electron microscopy in neuroscience.” J Microscopy doi: 10.1111/jmi.12244 or c) Micro-CT within the SEM or by itself. This still leaves all of the issues in your first objection and the basic “where’s the edge?” question. Even in secondary imaging this is an issue. How important depends on the particle size - if they’re micrometers in size the error won’t be too big relative to the particle; if they’re nanometers in size, the error can be relatively big. Either way, why not use light or TEM microscopy to size the particles? These would be more accurate. Phil Oshel oshel1pe@cmich.edu Tue Jun 2


Interaction volume in BSE is bigger than in SE but is not so big if accelerating voltage is low enough. This volume can be small enough, at low voltage, if the mean atomic number of particles is not too poor. Many BSE detectors are now efficient under 5kV; if you


doi: 10.1017/S1551929515000735 www.microscopy-today.com • 2015 September


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