Fluorescence Resonance Energy Transfer (FRET) is a powerful technique for characterizing distance-dependent interactions on a molecular scale. It is one of the few tools available that is able to measure intermolecular and intramolecular distance interactions both in-vivo and in-vitro.
FRET involves the excitation of a donor fluorophore by incident light within its absorption spectrum. This radiative absorption elevates the donor fluorophore to a higher-energy excited state that would normally decay (return to the ground state) radiatively with a characteristic emission spectrum. If, however, another fluorophore molecule (the acceptor) exists in proximity to the donor with its energy state characterized by an absorption spectrum that overlaps the emission spectrum of the donor, then the possibility of non-radiative energy transfer between donor and acceptor exists. As an example, Figure 1 shows the overlap of the cyan fluorescent protein (CFP) emission spectrum and the yellow fluorescent protein (YFP) absorption spectrum; this pair supports a strong FRET interaction.
Figure 1: CFP (donor) and YFP (acceptor) absorption and emission spectra. Overlap between CFP emission and YFP absorption (shaded region) leads to efficient FRET interaction.
The radiationless energy transfer described above is mediated by dipole-dipole interactions (Van der Waals forces) between the donor and acceptor fluorophore molecules that vary as the inverse 6th power of distance between the two molecules. The rate of energy transfer from donor to acceptor, kF, is approximately
kF ~ kD(r0/r)6
where kD is the radiative decay rate of the donor fluorophore, or inverse of the fluorescence emission lifetime in the absence of the acceptor fluorophore (typically 1 - 50 ns), r is the distance between the two molecules, and r0 is the "Förster distance" that characterizes the 50% efficiency point of the energy transfer. FRET is suited to measuring changes in distance on the order of the Förster distance, which is typically 20 to 90 Å. This length scale is far below the Rayleigh-criterion resolution limit of an optical microscope (typically 2500 Å for visible light at high numerical aperture), thus illustrating the power of FRET for measuring extremely small distance interactions.
