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Cammer, Michael-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/widefield-autofluorescence-in-unlabeled-cells-a-filter-mystery-tp7592150p7592165.html
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We would very much appreciate troubleshooting help with the question of autofluorescence increasing after exposure to UV light.
However, not using UV is not an option as the lab needs to use 4 probes for the experiments.
Cheers-
Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016
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From: Confocal Microscopy List <
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Sent: Tuesday, April 27, 2021 7:41:46 AM
To:
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Subject: Re: additional autofluorescence in unlabeled cells - a biochemical mystery?
[EXTERNAL]
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The solution to all of these problems may be to change the solution: use
PBS (pH 7.4) or 80% glycerol : 20% PBS (pH 7.4) instead of whatever is
being used now (and gently stir with pipet tip - not shaken, not
vortexed) the Glycerol : PBS). Old mounting media often oxidize the
various "antifade" abbreviations/acronyms (DABCO, PPD, etc). Sure, PBS
may permit more rapid photobleaching of some fluorophores; mitigated by
efficient operation of the microscope.
In 2021 there should be very few users who really need DAPI as their DNA
counterstain. Consider using Zombie NIR (BioLegend, and a much cooler
name than DAPI ... need appropriate emission path for 750nm). Perhaps
ThermoFisher can un-retire To-Pro-5. Eliminating DAPI also enables using
the wavelengths for Brilliant Violet 421 (BD Biosciences, also available
from BioLegend and in one flavor [streptavidin] Jackson ImmunoResearch),
or SuperBright 436 (ThermoFisher) or SuperNova v428 (Beckman Coulter).
And then multiplex with tandems. I also note that ThremoFisher recently
acquired Phitonex, so various NovaFluors now available from them (40plex
by flow using NovaFluors, Brilliants, more have been posted online by
Phitonex, most fluorescence microcsopy experiments are stuck at 3plex +
DAPI). See PubMed 33376221 for a recent 65plex microscopy paper with ~10
fluorophores per iteration (Radtke ... Germain 2020 PNAS ... not open
access - can always email Ron Germain for the PDF (emails on that page).
Happy 2021,
George
On 4/26/2021 9:31 PM, Cammer, Michael wrote:
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> When I first learned immunofluorescence, we added a small amount of glut to the PFA, and this caused autofluorescence. Therefore, we bleached the cells (I think after Saponin extraction) with NaBH4 (which is not compatible with Bodipy). After 4 washes, we would check the cells on a fluorescence microscope to assure that we had bleached them sufficiently. I believe other labs similarly use NaIO4 &/or glycine.
>
>
> This discussion is particularly apropos to us now because a lab asked for my help with what I first thought was a simple autofluorescence problem, but appears to be something more difficult. It also harkens to a discussion I saw elsewhere about imaging dapi after imaging green because the dapi may photoconvert.
>
>
> I was going to post a more detailed description of the problem, but the timing is right to post about it now.
>
>
> Rather than tell the story how we got to this point and discovered the problem, I will jump directly to the problem.
>
>
> Cell cultures that are fixed with fresh PFA in PBS and Triton extracted have a very faint background when excited with 488 nm light and similarly faint background with secondary ab or isotype labeling. The background is weak enough to not need further bleaching before the specific labeling. (So far, no problem.)
>
>
> The problem is that exposure to UV light (365 ex dapi filter with Hg lamp, 370 nm dapi filter with X-Cite LED, or 395nm or 405 nm LED or laser) causes the cells to emit brightly when subsequently excited at 488 nm. Practically this means that any imaging of dapi or Hoechst prevents subsequent imaging of green. This is dose dependent, so more exposure to UV means brighter green (no, we have not plotted a proper curve or looked for saturation, although we have a few data points). This is not due to dapi photoconverting; we ran controls of completely unlabeled cells mounted both in Prolong Diamond and glycerol (where the response appears to be stronger). They have seen this problem with a few cells types, so it's not something like a line mistakenly expressing a photoconvertible protein.
>
>
> Of course there are ways around this. Stop using dapi as a convenient way to find cells. Always take picture of green first with the last exposure being dapi. Switch nucleic acid labels to another color. But while these work when limiting the staining to three colors, this problem eliminates blue as a possible color. By widefield fluorescence, tiling is ruled out because the exposed circle is larger than the rectangular camera FOV.
>
>
> Have other people seen this problem? Any ideas?
>
>
> Thank you!!
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
>
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016
>
>
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> From: Confocal Microscopy List <
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> Sent: Monday, April 26, 2021 9:02 PM
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> Subject: Re: [EXTERNAL] widefield autofluorescence in unlabeled cells - a filter mystery?
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> Unlabeled cells do fluoresce in the yellow-green range. I have always thought it is because they have soluble molecules such as flavenoids in the cytoplasm. If the cells are permeabilized, for example, to use them in immunofluorescence procedures, there is much less of this background.
> Carol Heckman
> Bowling Green State University
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> Sent: Monday, April 26, 2021 12:47 PM
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> A colleague asked me to take a look at their widefield microscope. It is an inverted microscope with a 100W Hg source, excitation filter wheel, a couple of choices for dichroics in the microscope filter changer and a filter wheel in front of the camera. They are seeing unlabeled cells fluoresce green (FITC/GFP set) with an otherwise black background where there are no cells. The microscope is approximately 15 years old.
>
> My guess is that the excitation filters have failed (or are failing) after being on the receiving end of a 100W Hg lamp for all this time. Any other thoughts?
>
> Doug
>
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