FRET is a mechanism of energy transfer between donor and acceptor fluorophores. Energy transfer occurs when two fluorophores with overlapping donor emission and acceptor excitation spectra are in close proximity (<10 nm). Upon exciting the donor molecule, there is non-radiative energy transfer to the acceptor. Therefore, fluorescence from the acceptor is only observed when the fluorophores are in close proximity. The efficiency of energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, meaning that FRET is very sensitive to small distance changes (Eq.1).

Whereby, R is that distance between donor and acceptor fluorophores and R0 is the Förster distance. This is the distance where the efficiency of energy transfer is 50 %.