Re: widefield autofluorescence in unlabeled cells - a filter mystery?

Posted by Zdenek Svindrych-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/widefield-autofluorescence-in-unlabeled-cells-a-filter-mystery-tp7592150p7592155.html

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Hi Doug,
picture = 1000 words. Post a (link to a) picture.
It could be autofluorescence. Do the unlabeled cells look green on other
widefield fluorescence microscopes?
It could be transmission. Block all light between the sample and the
condenser. Does that help? Even a room light on the ceiling could work as
darkfield illumination. Btw, what's the objective lens?
It does not look like filter bleed through. That usually looks like (quite
non-uniform) signal independent of the cells.
Are the cells live or fixed? In PBS or in index-matching mounting medium?
Best, zdenek

On Mon, Apr 26, 2021 at 12:50 PM Cromey, Douglas W - (dcromey) <
[hidden email]> wrote:

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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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--
--
Zdenek Svindrych, Ph.D.
Research Scientist - Microscopy Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth