Förster resonance energy transfer (FRET) is a photophysical process in which energy is transferred non-radiatively from an excited donor fluorophore to an acceptor chromophore through dipole-dipole coupling. The efficiency of FRET depends critically on the distance between donor and acceptor, making it a molecular ruler for measuring distances in the 1–10 nm range.

The efficiency of FRET decreases with the sixth power of the distance between donor and acceptor. The Förster distance, at which transfer efficiency is 50%, is typically 3–6 nm for common fluorophore pairs. FRET efficiency also depends on the spectral overlap between donor emission and acceptor absorption, the relative orientation of the transition dipoles, and the quantum yield of the donor.

The most common FRET pairs include CFP-YFP, GFP-mCherry, and Cy3-Cy5. In addition to organic dyes and fluorescent proteins, FRET can use quantum dots as donors and lanthanide chelates for time-resolved measurements (TR-FRET). Bioluminescence resonance energy transfer (BRET) uses a bioluminescent donor such as luciferase and requires no external excitation light.

FRET is detected by ratiometric measurement of donor and acceptor emission intensities, confocal microscopy-based fluorescence lifetime imaging (FLIM-FRET), or acceptor photobleaching. Intramolecular FRET sensors incorporate both fluorophores into a single molecule, where conformational changes alter FRET efficiency. Intermolecular FRET detects association between differently labeled molecules.

Applications include real-time monitoring of protein conformational changes, protease activity assays, protein-protein interaction detection in live cells, and nucleic acid hybridization assays. FRET-based biosensors report Ca2+ concentration, kinase activity, and membrane potential with high temporal resolution.