How does FLIM FRET work?

FLIM FRET utilizes changes in the fluorescence lifetime of a sample to observe the interactions between donor and acceptor fluorescent molecules. In this technique, the donor fluorophore is excited by a laser pulse, and the emitted photons are collected and analyzed to determine their fluorescence lifetime. The fluorescence lifetime of a donor fluorophore is affected by the presence of nearby acceptor molecules, which can absorb energy from the donor through FRET. Comparing the donor lifetime in the presence and absence of acceptor molecules allows estimation of the distance between the donor and acceptor-labeled proteins. When the donor and acceptor fluorophores are close enough, energy transfer occurs, leading to a decrease in the donor's fluorescence lifetime. FLIM measures the fluorescence decay rate of a fluorophore from sub nanoseconds to hundreds of nanoseconds. Overall, it provides valuable information about intracellular protein-protein interactions and conformational changes in cellular proteins.