FLIM - FRET

In my undergraduate research project, I learned about Förster resonance energy transfer. I was assigned to synthesize pyrene-based organic compounds and conducted fluorescence imaging. Fluorescence bleach started from small domain, and the hypothesis was that the oxidation product act as energy acceptor.

20 years fast-forward, I am conducting fluorescence lifetime imaging using two-photon microscopy. It seems like chloride indicator dye is serving as energy donor with tDtomato protein acting as accepter. This is not something I wanted to see, but the experiment is interesting. I still do not know if this is FRET effect or some biological difference in tDtomato positive cells. Truth will be in the experiment when done correctly and analyzed correctly.