Fluorescence resonance energy transfer (FRET) is a radiation-less energy transfer between two electronic dipoles occurring with a first-order rate constant inversely proportional to the sixth power of the distance between them2. It is a quantum mechanical phenomenon that occurs between two fluorophores (a fluorescence donor and a fluorescence acceptor) that are in molecular proximity of each other (< 80 Å apart), if the emission spectrum of the donor overlaps the excitation spectrum of the acceptor5: under these conditions, energy (E) is transferred non-radiatively from the donor to the acceptor with an efficiency defined by the equation below, where r is the distance between the two fluorophores and R0 (Förster distance) is the distance at which 50% energy transfer takes place (typically 20–60 Å).
R0 is dependent on the extent of spectral overlap between the donor and acceptor, the quantum yield of the donor and the relative orientation of the donor and acceptor. Excitation of a donor fluorophore in a FRET pair leads to quenching of the donor emission and to an increased, sensitized, acceptor emission. Intensity-based FRET detection methods include monitoring the donor intensity with or without acceptor photobleaching, the sensitized acceptor emission or the ratio between the donor and acceptor intensity5.