Can anyone explain why my EGFP expression increased (but Ai9 RFP decreased -like normal) after imaging with a confocal microscope?

24 May 2024 2 8K Report

I have been trying to image a receptor in the brain for weeks now. It is an EGFP-cre G-coupled protein receptor. The model is a iCre based EGFP KI mouse model crossed with an Ai9 reporter line. I have not been able to get anything really for this receptor with the EGFP, but we do know the mouse model is correct.

I decided to return to some of my old slides to look at them again and saw the GFP expression was much brighter in the areas I had already taken images of, but the RFP was photobleached as expected. I noticed this and had no idea so I decided to test a dumb theory that somehow the laser confocal increased the expression of the receptor/GFP or that it just needed more time exposed? So I took a new completely unused slide and took a whole slide image at the start of the imaging, then I chose some areas and imaged those areas up close further, after I did this a few times, I went back and took another whole slide image and saw what I have seen before that is confusing me. I have zero idea and everyone that I have asked has zero ideas.

Any ideas or thoughts or answers would be greatly appreciated!!

Alireza Naderi Sohi

Dear Emma

Thanks for sharing this astonishing observation.

I think one reason behind this event could be the change in EGFP folding or EGFP microenvironment created by laser with a high intensity which may finally cause an increase in fluorescence.

From another point of view - although I am not an expert in this field, I think due to some reasons, in the first imaging, some of the excited electrons did not return to the lower states (levels) at the time of emission, and in the second imaging, when some electrons went to the excited state again, those previous excited electrons, together with new ones, have returned to the lower states. therefore you have observed a greater intensity of emission. In fact, the laser radiation in the second imaging has caused a kind of stimulated emission that you can search about.

Sam Siljee

This is really baffling me too!

I know that some lasers are temperature dependant.

Could it be that the laser intensity is changing as the lasers warm up?

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