Upon imaging Acanthamoeba castellanii cells on a Leica SP8 confocal microscope, I get a moderate to strong level of signal from the nucleolus when trying to image a FISH probe with Atto550 as the fluorophore. The signal persists in preparations without the probe. I'm allowing signal from about 560 to 650 nm (I forget exactly, but if I had to guess). There are no other probes in these preps, the only other fluorophore I've introduced is DAPI that's included in the ProLong Diamond mounting medium I'm using. The cells were treated with colcemid but my understanding is that it doesn't fluoresce. Has anyone observed autofluorescence of the nucleolus before? One hypothesis I have is that the nucleolus is somehow causing refraction that makes it appear to have signal even though no fluorescence is occurring, but maybe this is unlikely.
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