NetNotes


Edited by Thomas E. Phillips University of Missouri


phillipst@missouri.edu


Selected postings from the Microscopy Listserver from September 1, 2015 to October 31, 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: osmium


Could others please share their experiences with the premade


aqueous OsO 4 solutions? I have been making my own 4% solution, storing it in the fridge, and diluting a small volume just before use. However our EM lab isn’t very busy (3-4 preps every few months, although I am working on trying to get more users!) and I end up having to throw out a lot of the 20 ml solution that I make; membranes start looking kind of crappy and with the older solution even if the solution still looks relatively clear. I also see a lot of black specks in my unstained sections. I don’t know of anyone who uses the premade ampoules, so I don’t know if there are any signifi cant diff erences in image quality. T ey come in such small volumes, which would be really convenient for me! Blanca Carbajal Gonzalez bicarbaj@mtholyoke.edu Fri Sep 18 For the reasons you mention, we have been making use of the premade aqueous OsO 4 solutions for years. It works well for our own use and it is convenient when another user comes by asking for a few milliliters of OsO 4 . Occasionally we still make up a solution for odd ball recipes or large quantities. Louie Kerr lkerr@mbl.edu Fri Sep 18 I worked in biological microscopy for several years prior to a long career in an industrial polymer microscopy. In the bio lab, we invariably made our OsO 4 solutions from crystalline OsO 4 . In the polymer microscopy lab, we rarely used OsO 4 , oſt en in short bursts. Invariably, I was skeptical of the pre-made solutions because many of the vials went to waste as indicated by black precipitate on the inner surface of the vials and loss of amber color of the solution. T e issue, as you indicated, is quality versus price (through waste). To maintain high quality, I suggest you keep OsO 4 crystals on hand and make fresh batches as needed. T e price of the crystals is less than the prepared solution and you can be assured of the quality: Electron Microscopy Sciences sells 1 gram of OsO 4 crystals for $32 versus $53 for 10, 2 ml vials of 4% OsO 4 solution. When you make up a batch OsO 4 solution, break it up into several small vials and store each under an inert gas (nitrogen or argon) head to slow degradation of the oxide. When a vial begins to go bad, hopefully the unopened vials are still fresh and usable. Gary Brown microscopy.gmb@gmail. com Fri Sep 18


As a matter of curiosity, what is the consensus on the usable lifetime of a made up from crystal, 4% osmium in water stock solution, stored in a fridge? While on the subject of osmium stock solution storage, do people clean their stock solution storage bottle between making up their osmium stock solutions? Allan Mitchell allan. mitchell@stonebow.otago.ac.nz Tue Sep 22


I’ve not seen yet (I don’t think?) any mention of freezing the osmium samples. Is there a reason for this? In this facility, I make up fi ſt y 1 ml aliquots of 2% aqueous osmium, from 1 g osmium crystals, into clean 7 ml fl at bottom glass vials with polypropylene screw cap lids with foil inserts (to prevent splashes and vapor seepage), and store


52


them in the -20ºC freezer in double Tupperware boxes (one inside another - for extra safety) only taking out the required number of vials for each processing session. As they are such small quantities, they are safer to handle and defrost quickly. I have found that I can store the osmium in this way for years without noticing signifi cant artefact. I would also be interested to hear how people neutralize their osmium aſt er use. Nat Allcock nsa2@leicester.ac.uk Wed Sep 23 I always reduced osmium tetroxide and ruthenium tetroxide to their dioxides using a sodium bisulfi te solution (10 wt/vol %). Add an excess of the bisulfi te solution into the vial of the tetroxide and leave for a while, an hour or more. T is works well. Some labs reduced osmium tetroxide with unsaturated vegetable oil. Gary M Brown microscopy. gmb@gmail.com Wed Sep 23


Specimen Preparation: carbon coating


What would anyone out there recommend for a TEM grid carbon coater for ultraclean carbon deposition (dry/UHV system), with precision in the 1-5 nm thickness range and a real-time thickness monitor? Larry Scipioni les@zsgenetics.com T u Sep 17


For carbon coating, you may prefer to use a turbo-pumped system and a fast evaporator. You may have a try on two systems which I have experience with: - Cressington turbo coater 208 (carbon rod and e-beam guns are available) - Leica ACE series (carbon rod and carbon wire are available, to my knowledge; e-beam?) other machines from other companies will do the job as well, I assume. Quartz thickness monitor yes; but real-time thickness: you will have to wait for a few (5 to 10) seconds until the system settles (physics of heat transfer). Upon carbon evaporation, the quartz is getting heated as well and will give you some numbers which are not realistic. At the end, we fi nd this reproducible. Both machines work fi ne, in our hands, for light shadowing of bio-samples for STEM, TEM or SEM. try to fi nd a lab where you can have a test experiment with your samples, or convince the sales rep to give you a system for a week for several tries. As you do not explicitly state what you are going to do: “TEM grid carbon coater”: if you want to produce your own carbon supporting fi lm, you may have fi rst to shadow carbon onto mica, then fl oat this off (e.g., on a water surface), and then pick up the C-fi lm with (hydrophilized, glow-discharged) grids. Reinhard Rachel reinhard.rachel@biologie. uni-regensburg.de Fri Sep 18


Specimen Preparation:


bakeout chamber for cleaning STEM samples I’ve been trying to develop a procedure to clean our STEM samples prior to insertion into our ARM200CF microscope. In particular, we are trying to perform highly analytical work that involves long EELS / EDS mapping, so we would like to reduce hydrocarbon contamination on the surface of our samples. Since most of our specimens are oxide liſt outs, they are quite robust to heating and plasma cleaning. Typically


doi: 10.1017/S1551929515001108 www.microscopy-today.com • 2016 January


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