NetNotes
Edited by Thomas E. Phillips University of Missouri
phillipst@missouri.edu
Selected postings from the Microscopy Listserver from September 1, 2016 to October 31, 2016. 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: site-specifi c protein labeling
Could anyone suggest what would the best feasible way to label my membrane bound protein of interest in cell (facing toward the cytosol) to study its orientation and/or interaction with a membrane using single molecule techniques like FRET/ FCCS/FLIM-FRET, etc. I am particu- larly interested in studying the orientation of a specific domain of the protein with respect to the membrane in the live cell. From literature, I have learnt of applying a specifi c ligand (bound to a fl uorophore) that would bind to the active site; but that might not work for my case as the protein domain is surface exposed and we don’t know of any such ligands binding to that surface. Also, is there any hope of using a small peptide directed to the protein domain (or, the amino acid stretch) bound to fluorophore for this purpose? Any help will be highly appreciated. Suparna
csuparna1@gmail.com Mon Oct 17
You are putting the bar very high. Considering cell membranes are dynamic and EM techniques require cells to be killed, don’t expect it to be an easy task. I would say the fi rst consideration is the distance between your protein domain of interest and the cell membrane. T ere is a chance that this distance is too short to be resolvable by classical fl uorescence microscopy techniques. T anks God Stefan Hell came on earth to save fl uorescence microscopists and now we have fl uorescence microscopes with very high resolution, however there is not always one nearby. You are talking about FRET, meaning you probably expect distances of several nm. In this case, I don’t see which technique would allow you to resolve this distance in live cells. Sure you’ll see a fl uores- cence due to FRET but you won’t be able to locate it with respect to the membrane, I am afraid. Please also consider the optical thickness of the plane observed in LM. I am not really up-to-date in confocal microscopy but I think you can at best get optical sections of 100nm, so you are limited in resolution in the Z axis as well. If distance is not such a big challenge than you can perhaps consider expressing your protein/ domain with a fluorescent tag, like GFP or RFP? Stephane Nizets
nizets2@yahoo.com Mon Oct 17
Specimen Preparation: LR White
How is LR White supplied and used? I am getting confused by the labels that say “catalyzed” and when to use the separate “accelerator.” Is the benzoyl peroxide accelerator only added for doing cold cures? What is the catalyst that is already mixed in? Does this aff ect the shelf life or heat cure? I thought I had this figured out but this morning a student brought me a vial of LR White that had polymerized overnight in the refrigerator. No accelerator added, straight from the stock bottle. Can’t fi gure that one out. I am now confused about this stuff and need some remedial education. Jonathan Krupp
jkrupp@deltacollege.edu Fri Oct 21
56
LR White, (London Resin White) is used mainly for post-embed immuno-electron microscopy. T e cut surface allows for epitopes to be exposed and labeled with antibodies without the need to etch or antigen retrieval techniques. Most times it contains the catalyst so polymerization can be done at 50ºC. Check with the manufacturer but it can also be UV polymerized (you may need to add a diff erent catalyst). Most samples are lightly fixed (no high concentrations of glutaraldehyde) not osmicated and dehydrated up to 70% ETOH (I usually go to 90% as the 70% is sometimes diffi cult to completely mix with the resin). Uranyl acetate and small amounts of tannic acid may be used in lieu of osmium. Sections are collected on Formvar grids because the resin is beam-unstable. Semi-thin sections can also be generated to try immunofl uorescence experiments. Michael Delannoy
mdelann1@jhmi.edu Mon Oct 24
TEM: focusing Zeiss EM109
Our facility has a secondhand Zeiss EM109 TEM, which we’ve recently had serviced. Part of this servicing included getting the coils adjusted mechanically, and it should be in peak condition. However, I have a lot of diffi culty getting things into clear focus. I’ve gotten (I think) quite experienced with aligning and stigmating it, but that doesn’t seem to fi x things — indeed, aſt er capturing a blurry image and realigning, the next image of the same sample often looks worse. I suspect that the stigmation is off , but I’m not sure; oſt en I can get better results by systematically capturing images at diff erent focus settings (though of course even with a digital camera this takes a long time, and doesn’t do the specimen any favors). Are there any tricks to this? One question that I have in particular concerns the projector stigmation. I set this using the caustic, which I try to get to look like a Mercedes-Benz symbol (a perfectly symmetrical three-pointed star in a circle). However, changing the low-mag focus (the taller inner knob that doesn’t click) changes the appearance of the caustic, such that at one focus setting (say, with the caustic spread out) the caustic is symmetrical, and at another (in this case when it’s smaller) it isn’t. Is there a “sweet spot” that I should have that focus set to (i.e., should I make the caustic as small as possible while still visible, or as large)? I should note that I have experimented a little with the stigmation while viewing specimens, and haven’t noticed any eff ect (unlike my experience with a Tecnai-12, on which I could correct the astigmatism just looking at the image). But I do suspect there is a stigmation problem somewhere in the scope, and just need to know how to fi x it. Any advice on the care and feeding of this microscope would be much appreciated. Aaron A. Heiss aheiss@amnh. org Wed Sep 14
It is very many years since I used a Zeiss 109, so my memory of its layout is long gone, but I may be able to help you? T e image in any TEM is created “in focus” by interaction between the objective
doi: 10.1017/S1551929516001061
www.microscopy-today.com • 2017 January
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