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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 <[log in to unmask]> on behalf of Carol Heckman <[log in to unmask]>
Sent: Monday, April 26, 2021 9:02 PM
To: [log in to unmask]
Subject: Re: [EXTERNAL] widefield autofluorescence in unlabeled cells - a filter mystery?
[EXTERNAL]
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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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From: Confocal Microscopy List <[log in to unmask]> on behalf of Cromey, Douglas W - (dcromey) <[log in to unmask]>
Sent: Monday, April 26, 2021 12:47 PM
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Subject: [EXTERNAL] widefield autofluorescence in unlabeled cells - a filter mystery?
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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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