NetNotes
I have used even very old MEA stored at non-optimal conditions
for successful dSTORM measurements in the past. Some amount of absorbed water is usually tolerable in my opinion - although it might make it more difficult if you need to know the exact amount of MEA in your buffer. However, many dyes are somewhat robust in their behavior when it comes to the concentration of MEA. Additionally, while not ideal, a dSTORM measurement without an additional oxygen scavenger system present is also possible with some dyes (like AF647). While this might not always produce images for your next publication, they should be fine for demoing dSTORM, or checking sample preparation protocols. You might also have a look at this paper for a different approach to the issue of oxygen scavenger systems:
https://pubs.acs.org/ doi/abs/10.1021/ac400035k. Patrick Ten
patrick.then@
uni-jena.de
Although not suitable for live-cell STORM, your messages inspired
me to look back at some papers reporting the use of commercial Vectashield as a STORM imaging buffer. I’ve previously used it with good results as a more stable training/test sample. However, I see a more recent paper reported that Vectashield quenches AF647 (
https://www.nature .com/articles/s41598-020-63418-5). Simon, perhaps you can use sodium sulfite+thiol without glycerol for TIRF compatibility? Looks like that paper reported good results as well. Ben Hibbs
ben@kleinaustralia.com.au
I agree with what most people have already suggested: 1. MEA can
be aliquoted (solid or as 1M solution) and then frozen; 2. BME is another more toxic/stinky but more stable option; 3. MEA in sulfite is stable for longer than without sulfite; 4. Vectashield works well (I’m biased here), though the quenching is real, so it is a better option for tubulin samples than low abundance proteins. Diluting in 1/4 in glycerol rather than using it pure does help and I get good reconstructions (granted, on tubulin). Ko Olivier
niko.olivier@gmail.com
Stimulated Emission Depletion (STED) Microscopy Blowing Holes in Mounting Medium Confocal Listserver I’ve got a user who is using STED to look at neurons cultured on
poly-lysine-coated coverslips. Tey are fixed and mounted in ProLong Diamond. Some slides show holes or rings that develop during scanning, even with fairly low 775nm depletion laser power. I’ve imaged many ProLong-mounted slides with STED over the years with much higher power and never saw this before. Has anyone seen this, or have an idea what might be happening during mounting/curing to cause this? Te ProLong appears to be cured (at least to some degree) since the coverslips are stable. Tanks. Chris O’Connell
coconnell@uconn.edu
Hard to say without more detail about the samples, but I have
had rare samples where something similar has occurred. In particular, this was the case for malarial parasites and specific retinal samples. If your sample has any sort of chromophore or other highly absorptive component that is susceptible to absorbing the wavelength of the STED beam, then this can cause severe, localized damage to the samples. If you have any sort of plasmonic structures, this can also be an issue. If your STED has access to dynamic illumination schemes (DyMIN), then you can completely avoid this by automatically shutting off the STED beam when such extreme events are detected. Nicolai Urban
nicolai.urban@mpfi.org
To add to what Nicolai mentioned, and assuming you haven’t
explored this already, you might check to see if the user is flaming the coverslips versus acid washing during prep. Flaming may leave
2021 November •
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some carbon deposits that can absorb and create holes like you describe. Acid washing is generally preferable here. Jessica Shivas
jessica.m.shivas@
gmail.com
I agree with Nicolai that this happens (only) if the STED depletion
laser is absorbed by the sample. Tis can happen if the mounting medium has a slight hue in it, like Vectashield. We have also used ProLong and never observed this problem. Is it possible that your sample may have some staining residue present? If you use antibody staining, I wouldn’t think so, but with histological dyes this could possibly happen. If the samples are checked under bright field conditions, it might reveal if dyes or carbon particles are present. Steffen Dietzel
lists@dietzellab.de
Quality Control and Fitting of the Point Spread Function Confocal Listserver We image sub-resolution beads on our confocal microscopes and
perform a 3D gaussian fit to determine the center and full-width half maximum (FWHM) in x, y and z directions. We noticed that on our Nikon A1R HD microscope, the point spread function (PSF) is elliptical at 45 degrees when using a 60X oil, NA 1.4 lens. I was told this is because no quarter wave plate is inserted so the best brightness and contrast can be obtained. Tis raises three questions that I would like to ask to the community: 1. Do others with a Nikon A1R HD also notice this effect with high NA objectives? 2. Can someone explain why the brightness and contrast is better without the quarter waveplate? I do not have a physics background, and this is hard to follow for me, but I would like to understand the reasoning. 3. Does someone know a FIJI plugin or other free soſtware package that can do an elliptical (3D) fit to analyze the PSF for quality control so we can quantify the difference between the longest and shortest axes instead of comparing x and y? Herlinde De Keersmaecker
herlinde.dekeersmaecker@
ugent.be
We have Nikon confocal microscopes and also acquire PSFs.
Quite a long time ago we realized that the DIC slider (I assume that this is what you mean?) under the objective was making the PSF longer and a bit larger, but it was not at 45 degrees. Our policy since we realized this was to have no DIC slider by default. We keep them in an objective box near each microscope and instruct the users who require DIC how to insert all the DIC elements. Sylvie Le Guyader
sylvie.le.guyader@
ki.se
I would concur with Sylvie on the DIC prism. We have a couple of
spinning disk and widefield systems that show a significant 45 degree elliptical PSF in the lateral plane when a DIC prism is inserted. We also have a Nikon SoRa system which shows a very small ellipticity, also at 45 degrees, using the 100x objective. Te engineer suggested I tweak the correction collar to minimize this, which is something to try if you have one on your objective. As for soſtware which can fit the elliptical shape. If I understand correctly and you mean elliptical in the lateral plane, then PSFj can fit this shape. All the other PSF programs I am aware of only fit the x and y directions and won’t spot the elliptical problem. If you need full 3D fitting, then PSFj has an option to do this, although I have never used it myself. Claire Mitchell
camdu@warwick.ac.uk
From a paper (
https://doi.org/10.1117/1NPh.4.2.025002) a
while back, Dr. Micu and I discovered that most confocal systems are linearly polarized. I suspect this allows them to work with various DIC optics, and the Nikon microscopes in particular offer
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