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

Posted by Jonkman, James on
URL: http://confocal-microscopy-list.275.s1.nabble.com/widefield-autofluorescence-in-unlabeled-cells-a-filter-mystery-tp7592150p7592152.html

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Hi, Doug.  Are these your own samples or your colleague's samples?  I had someone with a similar problem on one of my microscopes a few months ago.  Their no-primary control cells showed pretty strong fluorescence, and so they brought some cells that had no labeling whatsoever and they still fluoresced.  Eventually we got talking about what kind of cells these were, and it turns out they were some kind of stem cells, which got me thinking that maybe they had previously done cell sorting.  Indeed, they had used fluorophores for cell sorting and for some reason they didn't realize that the fluorescence would persist after fixing for microscopy.  I had the advantage of knowing that that my microscope filters and everything was not the problem, but it still took several sessions before this truth came out.  Maybe your case is completely unrelated but I thought I would just throw this out there!  Good luck with your trouble-shooting,
James

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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cromey, Douglas W - (dcromey)
Sent: Monday, April 26, 2021 12:48 PM
To: [hidden email]
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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