What are the advantages of FLIM FRET?

FLIM offers a distinct advantage by allowing the coupling of the donor with non-fluorescent acceptors. This eliminates any spectral crosstalk between the donor and acceptor, providing a clear measurement of the FRET efficiency. All fluorescent molecules exhibit a decay in fluorescence over nanosecond timescales, with the decay rate sensitive to environmental changes affecting fluorescence quenching. Additionally, FLIM widens the range of donor/acceptor pairs that researchers can utilize in their experiments. Also, FRET reduces donor fluorescence, and this reduction can be determined by examining the shortened fluorescence decay time of the donor when FRET is present.