I have a question from a user about a strange phenomenon we haven’t seen and wondered if others have seen this. The user labeled a retinal section with CF405M WGA (408/452nm), anti-iFlag 488, AlexaFluor647-phalloidin and the tissue is expressing mCherry. Has anyone seen mCherry convert to green emission after 405nm excitation? (a relatively high level of 405nm laser power was used)
Thanks for any feedback,
Lisa Cameron
Duke LMCF
(example images of before and after 405nm exposure available, but are not mine, so did not post initially)
So you have 4 different fluorophores in there and you’re seeing an increase in the green channel intensity after irradiation with 405?
What makes you conclude it was the mCherry converting? Have you tried it with a sample that only had mCherry? (Or only one other fluor). Seems like a case in which some single-labeled controls could help you narrow down the range of possible explanations.
I apologize for leaving out info.
The labeling pattern of the mCherry channel image is seen in the green emission channel after exposure of the sample to 405nm illumination, but not before.
Agreed about the separate fluor labels. I will work with the user more, just wondering if anyone had seen this phenomenon before. Thanks.
well, I think if there’s one thing we can all agree on, it’s that fluorophores are complicated
People have reported red to green photoconversion in orange/red proteins. Dave Piston published on that phenomenon 10 years ago. Note however: they explicitly mention in that article that they tried mCherry and mApple and saw no photoconversion with any wavelengths between 405 and 633nm.
but without seeing the raw intensity values in your image, it’s hard to surmise what might be happening in your case.
If you are imaging immediately before and after 405 excitation (and I know very little about photoconversion, so I may be way off base here ), would you expect to see similar decreases in intensity in the red channel when compared to increases in the green? Obviously there would be some bleaching, the QE wouldn’t be the same, etc, but it might be interesting to plot the R-G pixel values against each other in a variety of ways (before vs after), and see if you get a decently strong R value.
Thanks for the information.
I communicated with the user who asked me about this more and he tested fluors alone and found that with mCherry alone this apparent conversion did not happen - it only happened when the CF405M WGA label was used in combination.
