NetNotes


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


Selected postings from the Microscopy Listserver from November 1, 2018 to December 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: fluorescent beads surviving histological treatment I am doing a difficult search and Googling leads me to direc-


tions I don’t want. I hope you can help me with this. I want to use fluorescent microparticles as positive controls for an injection in skin samples. Te skin samples are injected, and then they are fixed, dehydrated, and embedded in paraffin (using Histoclear instead of xylol), using classical histological methodology. I want to be able to detect the fluorescence in paraffin sections with- out any further treatment. I don’t want to detect the beads by immune detection or any labeling method on the paraffin sec- tions. Optimally, the beads are 3-5Â µm in size and have a fluo- rescence spectrum similar to TRITC. Alternatively, I am looking for metallic beads (like gold or silver) with a diameter of several micrometers (unusual), which I can probably detect in bright field. A colored solution is no alternative; I want to detect par- ticles. Stephane Nizets nizets2@yahoo.com Tue Dec 18 What are your microparticles composed of? Make sure


that they are not soluble in alcohols or Histoclear before your process your tissues. If you have any doubts, I would suggest freezing the samples and cutting cryosections. Tissues are fixed, cryo-protected with sucrose, and then frozen, so there are no solvents involved. I can send you an excellent protocol that gives very good histology (off-list, since Nestor’s filters will block attachments). Please let me know if you would like to try that. Lee Cohen-Gould lcgould@med.cornell.edu Tue Dec 18 You could use gold nanoparticles, for instance a BSA stabi-


lized gold tracer, of any size and use silver enhancement on the section to enlarge their size for bright field observation. Very easy. Feel free to contact me off-list. I will be happy to help. Jan Leunissen leunissen@aurion.nl Tue Dec 18 Have you considered using ceramic microbeads instead of


the metallic ones? I’ve never used them, but they should come in the size range you desire. Christian Feldhaus christian. feldhaus@tuebingen.mpg.de Tu Dec 20


Ultramicrotomy: alternatives to water I have a user cutting thin sections of a polymer that is sol-


uble in water, methanol, ethanol, and other organic solvents. Any thoughts about alternative liquids? Chris Gilpin gilpin@ purdue.edu Wed Nov 28 How about fully fluorinated liquids, such as Galden by


Inland Vacuum? Another one of chemically inert variants, such as Perfluorodecalin maybe? Valery Ray vray@partbeamsystech. com Wed Nov 28 Not so sure these would be compatible with Diamond


knives and their mounting cements. Best to ask Helmut Gnaegi (helmut.gnaegi@diatome.ch) of Diatome for ideas how to do this. Al Coritz acoritz@emsdiasum.com Wed Nov 28


46 doi:10.1017/S1551929518001426


TEM: screen lifter problem My Philips CM12 TEM has a tendency to shut off if I liſt


the focus screen too fast (too hard?) to expose the bottom mount camera. Tis is relatively new to me, as I was using a side mount camera and never had to liſt the plate. Is this normal? If it’s not, other than being slow and delicate, what is the cure? Frank Karl frank_karl@ardl.com Wed Dec 5 Disassemble feed-through shaſt assembly, clean it, and


replace O-rings. Viewing windows O-rings may need replace- ment. Ditto large screen assembly. Most O-rings around pro- jection chamber are Buna-N rubber. Install Viton O-rings instead. Tese last forever, more or less. Vitaly Feingold vitaly@ sia-cam.com Wed Dec 5 I have had a CM12 since 1990 (oh yes, I remember it very


well—the boxes were delivered on Dec 4th, 1990, two days before St. Nicklaus day), and it is still in use. It had quite a few peculiarities, but never heard of this one. No, this is not nor- mal. We have a bottom-mount camera in operation (TVIPS) since 1998, daily, heavily used—no problems with any of the phosphor screens, and I keep my fingers crossed that we will never have a problem like this. Reinhard Rachel reinhard. rachel@biologie.uni-regensburg.de Wed Dec 5 It is really strange as Reinhard wrote. We have been running


CM12 since 1985 up to 2014 when HT tank has gone. We had some problems with leaks in the viewing chamber (O-ring of dif- fraction beam stopper assembly and sealing of 35 mm camera) during those years. But those leaks never led to the microscope complete switch-off. Usually HT was switched off and IGP pump, too. However, we observed that aſter strong discharge the CM12 was sometimes switched off, and this also restarted the EDAX system connected to the CM12. What tells you the plate exposure meter when you change the intensity on the main screen? Is the exposure time changing? I hope that you solve the problem soon. Oldrich Benada benada@biomed.cas.cz Wed Dec 5 It is a little unclear what you mean by the Microscope “shuts


off.” Is it just the HT, the vacuum system, or all the electronics? Assuming that it isn’t all the electronics, it sounds like there is a vacuum leak on the screen liſt lever. If you are operating, the loss of vacuum will shut off the emitter and the HT and (quite likely) the IGPs as well. Te vacuum page will then show the IGP shut off and V6 open so that the diffusion pump is pumping on the column and gun. Te fix would be to disassemble the screen liſt feedthrough and replace the O-rings. Henk Colijn colijn.1@osu.edu Wed Dec 5


EDS: spectral imaging My lab switched to TermoFisher Pathfinder soſtware from


an older NSS. Still on the “uphill” part of the learning curve. My question concerns accessing the EDS map images in Spectral


www.microscopy-today.com • 2019 March


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