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Hi Doug,
Normally if the excitation filter is failing through burn-out, you'll see this as an increase in the overall noise reaching the camera. With that said, burn-out is usually very easy to spot visually when looking at the filter so if you haven't already, you may want to pull the filters out of the setup and check for obvious signs of degradation in the coating. They may be old filters (pre-hard-coated IBS days) which means it will be difficult to clean them, in which case they might need replacing.
As for seeing cells that aren't there, do the images you see coincide at all with the cells that you're trying to image? Or are they complete different?
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Peter Brunt
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-----Original Message-----
From: Confocal Microscopy List <[log in to unmask]> On Behalf Of Cromey, Douglas W - (dcromey)
Sent: Monday, April 26, 2021 12:48 PM
To: [log in to unmask]
Subject: 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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