NetNotes


Edited by Thomas E. Phillips University of Missouri phillipst@missouri.edu


Selected postings from the Microscopy Listserver from September 1, 2018 to October 31, 2018. 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: thin film Hoping to get some advice on sample prep for using SEM to measure


CVD film thickness via cross-section. Film is sputtered Aluminum with nominal 1-micron thickness on a fluoropolymer substrate using approximately 20-40,000× magnification. Te aluminum film is too brittle for a razor cut and flakes off or doesn’t present a clean edge for measurement. We have tried with some success encapsulating with epoxy support layer before cutting. Also using liquid nitrogen for freeze fracture. Any suggestions how to prepare and mount sample would be much appreciated! Mike Toalson mike.toalson@elementpi.com Fri Oct 5 Typically for SEM, it is not the section we go for, but the block itself.


Te ultramicrotome would be used on the embedded block to “polish the surface”. Tis means taking as small as 60nm slices, gently on a small surface, say 1/2mm, using a diamond knife and sectioning out onto a water boat on the knife Possible if the aluminum is very, very thin. If it will separate from the block at all with a razor, then using an old used diamond is worth a try first. Usually, the trim by hand step with the feel of it under my fingers is how I can tell. Te flaking may be thickness dependent, does it “eat” the razor blade? If it eats the blade, it will harm the diamond. If that does not work, someone more experienced with grinding techniques may have some advice. Tis can be put into a holder that grabs the block, or some may epoxy to a stub. If any gluing is done, it needs to pump down in vacuum for 2-3 days before putting it to any SEM. Lou Ann Miller turtlelam@comcast.net Fri Oct 5 Two different ion beam polishing techniques should work for a


sample like this, assuming you have access to such equipment. Te first and easiest thing to do would be to cut a cross-section view using a dual beam (FIB). We’ve done samples like this before, and they turn out well, especially if you’re only interested in the Al layer thickness. Another way to prepare a decent cross-section is to use a broad beam ion polisher, though you may need to use a cryo-stage to keep the fluoropolymer substrate from charring and causing the Al layer to delaminate. Both the FIB and broad beam polisher would avoid any possibility of smearing and delamination caused by microtomy and conventional polishing. If you don’t have access to such equipment, then I’m sure you can locate a lab nearby that does. Christopher Winkler microwink@gmail.com Fri Oct 5


Microtomy: difficulty obtaining Tokuyasu ultrathin


sections of plant material I am having difficulty obtaining ultrathin sections of leaves and


flowers with the purpose of performing immunohistochemistry. My samples were fixed in 4% paraformaldehyde in TBS-T, infiltrated in an ascending grade of sucrose up to 2.3 M, then embedded on a sectioning pin surrounded by 2.3 M sucrose by dipping into liquid nitrogen. I am sectioning on an ultramicrotome equipped with a cryobox. I’m finding that sectioning at 1 µm thickness at –65°C has minimized the amount of sucrose flaking, and is providing me with the best sections for all conditions I have tried (from –60° to –90° at 5°C increments; 1.5 µm to 0.3 µm with 0.15 µm increments). However, I am still experiencing


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several issues: Te issues I’m having at 1 um sections; –65°C: – sections are wrinkling and curling off the knife edge – tissue sections do not adhere to slides aſter transferring out of cryo-box – tissue is largely fragmented upon imaging with Toluidine Blue staining and transmitted light, where epidermis, mesophyll, and epidermal outgrowths are floating all over the slide My major concerns relate to the sections not adhering to my slides aſter transfer, which will certainly wash off my slides during the immunolabeling work up, and the fragmentation of my tissue, which removes the spatial context needed for immunolabeling. I have tried several different types of slides including uncoated, pre-cleaned slides from Fisher, uncoated and non-cleaned slides from VWR, as well as PTFE coated slides from EMS, with no apparent difference in section adherence. If you have any tips, tricks or recommendations, please let me know, as I would be very grateful for any help on this technically challenging and patience-testing method. Sam Livingston samuel. livingston@botany.ubc.ca Wed Oct 17 You say you’ve tried several adhesives but have you tried chrom-


alum, which works well in many cases? It’s relatively easy to make. Tere are many different recipes, e.g., http://stainsfile.info/StainsFile/ prepare/adhesives/chromegelatin.htm. We resorted to this aſter poly- lysine-, agar-, and silane-coated slides—either purchased or prepared ourselves—proved unreliable with a particularly difficult tissue. I’m guessing the fragmentation of the tissue is because the cell walls are not sufficiently well-fixed/penetrated by the various solutions. Does happen with frozen plant tissues. For example, rapid-frozen/ freeze-substituted Arabidopsis roots have fantastically-preserved cell contents, but the cells tend to separate as you section the resin blocks. Tobias Baskin comments on this in one of his earlier papers, I think. Rosemary White rosemary.white@csiro.au Sun Oct 21


STEM Image Libraries: machine learning My colleagues and I would like to bring to your attention the


following development aimed at faster adoption of machine learning methods across electron microscopy community and enable ML/AI application in atomically resolved imaging. Modern machine learning is impossible without large volumes of labeled data. To enable faster adoption of machine learning methods in STEM, ORNL is working with Citrine Informatics to share an open library of images for the specific case of Si - vacancy complexes in graphene monolayers with plans to increase the amount of data in the library over time. Te initial library is available at (https://doi.org/10.25920/0xv3-8459) A paper that discusses the collection, analysis, and dissemination of this data is available at https://arxiv.org/abs/1809.04256 Te notebooks for the analysis workflow will be available at PyCroscopy (on GitHub) shortly and can be requested directly from Maxim Ziatdinov (ziatdinovma@ornl. gov <mailto:ziatdinovmax@gmail.com>) We hope that this initiative becomes adopted by the community. Please contact Malcolm Davidson (mdavidson@citrine.io), the leader of Citrine’s Open Data Initiative, for any questions about Citrine’s open data repository or ML platform. We’re putting the finishing touches on some tooling and will share a step- by-step guide to our workflow in the coming months if you’re interested in sharing your STEM data openly on Citrine’s platform.


doi:10.1017/S1551929518001220 www.microscopy-today.com • 2019 January


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