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).

E = R06/(R06 + r6)

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.

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