I am using the acceptor photo bleach method with Venus and mTFP1. Recently, I have applied this sensor to a model and I was expecting to see an increase in FRET due to less tension, however they are not interacting as a FRET pair at all. When I photo bleach a ROI with the 514 laser line, the mTFP1 signal also decreases. This is indicating to me that they are not successfully transferring energy as they should, but I am not sure if that is the only reason for it. Also, I am seeing an increase in overall fluoresce compared to controls. This makes me think that maybe the fluorophores are creating other dipole bonds with nearby molecules, but again I am uncertain in my logic because I am still learning a lot about FRET.
Any insight would be greatly appreciated.
Hello Claira,
Optically, irradiation at 514 nm should not harm the mTFP1, so there should be another reason. I have not worked with this FRET pair, but can it happen that irradiation at 514 nm generates a lot of reactive oxygen species that then quench fluorescence of mTFP1? I guess, it also depends on what kind of sensor you have and where it is located inside the cell (assuming that your assay is cellular, not biochemical). Can you explain what you mean by the increase of overall fluorescence? At which length is this increase in fluorescence emission registered? It is not unusual to see increase in fluorescence of some fluorophores after a short pre-irradiation in case if in its initial state, the fluorophore is self-quenched (e.g., if it is located in high amounts in a small vesicular structure like endosome).
Regards, Darja
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