Basics of FRET
Fluorescence resonance energy transfer (FRET), also known as Förster resonance energy transfer, is a phenomenon whereby an “excited” donor fluorophore molecule transfers energy in a non-radiative process to a suitable acceptor fluorophore molecule. This transfer of energy is termed “non-radiative” because it occurs without the emission of a photon from the donor fluorophore. Instead, it involves the direct transfer of energy of a donor molecule in the excited state when its fluorescence emission spectrum overlaps with the excitation spectrum of an acceptor fluorophore. The resulting FRET signal from the acceptor is often referred to as “sensitized emission”.
FRET occurs between donor and acceptor molecules only after several conditions are met. First, the molecules must be sufficiently close – typically <10 nm, but more often a closer apposition is required to successfully demonstrate FRET. This distance dependence makes FRET a powerful tool, as such close apposition between molecules is often evidence of molecular interaction. A FRET signal is direct empirical evidence of this close physical relationship, independent of the resolution of the imaging system and independent of conventional colocalization techniques. The Förster distance is the distance at which the efficiency of energy transfer is 50% – typically between 4-7 nm, depending on the particular donor/acceptor pair, and is typically used to characterize the potential usefulness of a fluorophore pair. The efficiency of energy transfer increases exponentially as molecules become closer, and is exquisitely sensitive to the distance of separation.
Second, as mentioned above, the excitation spectrum of the acceptor must overlap with the emission spectrum of the donor. Greater spectral overlap generally increases the likelihood of FRET, but also complicates data analysis by increasing the risk of false-positive signals due to direct excitation of the acceptor.
Third, the donor and acceptor molecule dipoles must be oriented favorably in order for FRET to occur. In addition to proximity, the dipoles must be favorably aligned for efficient energy transfer. Most often this condition is met randomly with large populations of molecules and a standard value for dipole orientation is used in most FRET calculations.
These requirements make FRET particularly sensitive to spectral overlap and signal separation, placing strict demands on filter selection.