NetNotes
Edited by Bob Price University of South Carolina School of Medicine
Bob.Price@
uscmed.sc.edu
Q1
Selected postings are from recent discussion threads included
in the Microscopy (
http://www.microscopy.com), Confocal Micros- copy (
https://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy), and 3DEM (
https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem) listservers. Postings may have been edited to conserve space or for clarity. Complete listings and subscription information can be found at the above websites.
Mounting Media for STED Confocal Listserver For STED experiments we have always mounted our cells with
ProLong Gold. It is one of the media recommended in the Guide to STED Sample Preparation published by Leica and it works well in our hands. As it hardens, it is well known it squashes/shrinks the cells mainly in the Z dimension, thus affecting its shape. I have never read or heard that the squashing also has effects at the molecular level (i.e., changing molecular shape or distances between molecules), something that would have a negative impact in our STED observations, but do you think it could be the case? Is there any publication on this topic? And, is there any alternative mounting media to avoid this (hypothetical) artifact on the molecular structure of the sample? Xavier Sanjuan Samarra
xavier.sanjuan@
upf.edu
Te 3D squashing effect is something many of us have seen,
but I am another person who keeps intending to publish a thorough comparison of the effects and never has time! And whether it has an effect at the molecular distance level is a good question. You do not actually have to let the ProLong Gold harden. If you seal around the coverslips with quick-drying nail polish immediately aſter mounting, it doesn’t harden, and you retain more of the 3D information. Sure, you don’t end up with the higher refractive index of the cured mountant, but it’s not as though the completely cured ProLong Gold has a refractive index (RI) matching that of regular immersion oil anyway. We use non- hardened ProLong Gold/Diamond for 3D-SIM (using an OMX) and compensate for the RI mismatch using a different refractive index oil. I haven’t tried that with STED, and you need to be aware that the Cargille oils that we typically use for RI selection may induce some chromatic dispersion too, but you could give it a shot. Otherwise, unless you have adaptive optics on your system, you will have to see whether your results are better or worse without curing—i.e., balancing the negative effects of spherical aberrations against squashing effects! In the light sheet microscopy world, a lot of work has been done with RI matching, in particular using TDE-based mountants. And I now notice that Abberior has a mounting medium on their webpage called Abberior TDE which comes in different types—RIs to match immersion oil, silicone oil or glycerol. It says the TDE mountants are specifically designed for imaging thick specimens, which would imply to me that they minimize squashing artifacts, though I can’t see that it explicitly says that. I find the RI of uncured ProLong Gold to be pretty similar to that of Vectashield,
70 doi:10.1017/S1551929520001261
which is glycerol-based (this is just by empirical testing, i.e., which RI oil matches best on the OMX). So, I wonder whether the Abberior TDE glycerol version would work well with uncured ProLong Gold? Alison J. North
northa@mail.rockefeller.edu
Regarding curing vs non-curing media I cannot comment reliably
(I would love to hear if Abberior has done any comparisons for STED?) but back in my time at Oxford I know that there was a comparison done between non-curing mounting media in terms of inducing shrinkage and distortion artefacts in cells. I am not sure if that was ever published but the 90% glycerol mounting medium was shown to be the best. Personally, to avoid any potential issues with curing, I have used non-curing mounting media only. Currently I use SlowFade Diamond for STED which has performed really well. It is glycerol- based and all the usual STED fluorophores appear to work well. I have not tried SlowFade Glass yet as I presume this is intended for more deep-imaging samples. Jakub Chojnacki
jakubcho@gmail.com
Are the samples getting physically flatter as the mounting media
cure or are mismatches in refractive indexes (indices?) making depth appear different? Michael Cammer
michael.cammer@
nyulangone.org
Tanks, this is useful! So, are you using the SlowFade Diamond
with an oil-immersion objective, without significant spherical aberration artifacts? If so, then uncured ProLong media shouldn’t be a problem either—though it also depends on whether you are using an Abberior system—with AO—or a Leica system, right? As far as I understand, the one drawback of the Slowfade reagents is that the samples don’t last nearly as long—they should be imaged within a day or two—while I have kept ProLong mounted samples in the fridge for literally years without significant deterioration. Do you find that Slowfade samples go off quickly, or is it not as bad as I’ve been led to believe? Alison J. North
northa@mail.rockefeller.edu
Tis paper should answer all your questions on TDE including
shrinkage, dispersion, etc.
https://onlinelibrary.wiley.com/doi/ abs/10.1002/jemt.20396. Briefly, if you take a sample through a stepwise increase in TDE as described in the paper, the shrinkage is minimal. We saw some great improvements using TDE-based mounting medium over ProLong Gold for SIM (
https://hcbi.fas.harvard.edu/files/ hcbidoug/files/hcbi_recommended_mounting_media_0.pdf). Tis was before ProLong and SlowFade Glass were available. Te drawbacks to TDE are the longer amount of time required to prep the sample as it is stepped through different concentrations. Also, green and blue dyes photobleach almost immediately in high TDE concentrations (those needed to match oil immersion objectives), but are more stable when more water is present (glycerol/silicone objectives). Te Abberior folks have told me that the high RI ProLong/SlowFade Glass products shiſt the emission spectrum of red and far-red dyes making STED more difficult. We had some samples ready to test when we got shut down. Doug Richardson
ds.richardson@
gmail.com
www.microscopy-today.com • 2020 September
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