http://confocal-microscopy-list.275.s1.nabble.com/widefield-autofluorescence-in-unlabeled-cells-a-filter-mystery-tp7592150p7592175.html
Dominika Żurek‐Biesiada Sylwia Kędracka‐Krok Jurek W. Dobrucki
also causes autofluorescence to name a few. Spectral imaging and unmixing
Just my two cents. Good luck!
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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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> 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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> 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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