My colleague and I did FISH using a FAM-labeled probe. We observed signals using FITC filters. However, when we tried to use the Cy3 filter, we could see intense and distinct signals not observed using the FAM filter.
This is very unsual. I have not encountered this in several years with FISH. I suspect contamination with another probe cocktail, as my colleague was doing FISH with another slide using Cy3-labeled probe. However, the signal patterns were totally different from the two slides.
I wonder if it is possible to get an intense signal from FAM labeled probes using Cy3 filter. Thank you!
Hi Nomar, use this site to look at overlapping em/ex of fluorophores.
https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html
If you input fluorescein and Cy3, you can see that there is some overlap that can lead to bleed-through. The stronger the fluorescein emission is, the more likely it is to bleed into the Cy3 channel.
Thank you Steingrimur. However, I just used Fluorescein alone. When we checked the signal, there is no signal using the FAM filter, only at the Cy3 filter. This is so unusual. I have not encountered this before.
Hi Nomar, I know that high concentrations of some fluorophores can show a red-shift. Maybe that's what's happening.
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