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Förster resonance energy transfer is a nonradiative transfer of energy from an excited state of a FRET donor to a nearby FRET acceptor, through coupling of their electric dipoles. The transfer step does not emit a photon that the FRET acceptor subsequently absorbs. It competes with the FRET donor's own fluorescence and other routes out of its excited state. A fluorescent FRET acceptor can then emit fluorescence; an absorbing but nonfluorescent FRET acceptor can still receive the transferred energy.^[ref]

The striking use of FRET is to turn a change in molecular proximity into an optical readout. Proteins carrying a FRET donor and a FRET acceptor may change the readout when they associate, as in the chemotaxis FRET assay; two labels on one protein may report a change in its shape. Interpretation depends on the FRET spectral overlap, FRET dipole orientation factor and labeling geometry, as well as separation. A change in FRET is therefore evidence about proximity under specified conditions, rather than a direct measurement of an arbitrary distance.^[ref]

For a single pair with fixed Förster distance $R_0$ and label separation $r$, the FRET efficiency isAt $r=R_0$, half the FRET donor excitations transfer energy. At $r=R_0/2$, the model gives $E=64/65\approx 98.46\%$; at $r=2R_0$, it gives $E=1/65\approx 1.54\%$. The sixth power makes FRET especially useful near the Förster distance, with little sensitivity to further separation once transfer is already negligible. These are model calculations, not measurements of a particular pair.

A FRET acceptor receives the excited state energy. Its absorption must support FRET spectral overlap with the FRET donor. FRET sensitized emission requires a fluorescent FRET acceptor; measuring a shortened FRET donor lifetime does not.

A FRET donor supplies the initial excited state energy. Its fluorescence provides both an emission channel and a reference FRET donor lifetime when no FRET acceptor is present. Calling a label a FRET donor describes its role in a chosen pair, rather than a permanent classification of the molecule.

FRET efficiency is the probability that an excitation of the FRET donor ends in transfer to the FRET acceptor. Write $k_D=k_{\mathrm{r}}+k_{\mathrm{nr}}$ for the total decay rate without a FRET acceptor, including radiative fluorescence and other decay, and $k_T$ for the transfer rate. For constant competing rates,The integral counts transfer events among all excitations in a population with exponential decay. Substitution gives the separation law above. This short rate argument assumes one FRET donor, one FRET acceptor, weak coupling, an available FRET acceptor, and fixed geometry during the decay; it is not a model for arbitrary interacting labels.^[ref]

In FRET photobleaching controls, selectively destroy the FRET acceptor's ability to absorb and test whether FRET donor fluorescence increases. Under unchanged excitation and detection, complete loss of transfer, and an intact FRET donor,This is a destructive comparison. Monitor acceptor loss, donor-only samples exposed to the bleaching illumination, and an unbleached reference region or time trace. Incomplete bleaching, donor damage, movement or label replenishment violate the comparison. During ordinary recordings, gradual unequal bleaching can instead produce an apparent change of FRET without a biological change.^[ref]

In Receptor sensitivity in bacterial chemotaxis by Victor Sourjik and others, Victor Sourjik and Howard C. Berg used a cyan fluorescent tag on CheZ as FRET donor and a yellow tag on CheY as FRET acceptor. Escherichia coli biological cells adhered to a polylysine-coated coverslip in a continuously perfused chamber. A fluorescence microscope excited them with a $442$-nanometre laser; split emission bands fed two photon-counting detectors. The yellow/cyan emission ratio fell when attractant was added and rose when it was removed.^[ref]

Saturating attractant increased cyan emission by an amount matching selective bleaching of the yellow tag: removing the FRET acceptor supported relief of FRET donor transfer. Strains carrying the tagged pair but lacking other chemotaxis machinery showed no stimulus-dependent emission ratio change. Weak illumination reduced bleaching drift.^[ref]

The inference is a change in the population of nearby tagged CheY/CheZ pairs associated with CheY phosphorylation. It neither measures each pair's separation nor directly observes motor motion. The readout also averages many biological cells, and perfusion takes finite time. The pathway interpretation belongs to the chemotaxis FRET assay; the general measurement lesson is that molecular association can alter FRET while the geometry of an individual associated pair stays the same.

FRET sensitized emission is FRET acceptor fluorescence caused by transfer from an excited FRET donor. Excite predominantly the FRET donor and collect both emission bands. Reduced FRET donor emission and increased FRET acceptor emission are useful together, but an acceptor-to-donor emission ratio is not automatically the FRET efficiency: brightness, label abundance and channel collection also enter.^[ref]