smFRET

Single-molecule FRET (smFRET) is a fluorescence spectroscopy technique that probes the conformational dynamics of biomolecules at the level of individual molecules. By labeling a molecule with a donor and an acceptor fluorophore, smFRET monitors distance changes between the labels in real time, revealing dynamic states that are often hidden in ensemble measurements.

The method relies on Förster resonance energy transfer, in which the efficiency E of energy transfer depends on the distance r between donor and acceptor as E = 1/(1+(r/R0)^6), where R0 is the Förster radius for the dye pair (typically 2–8 nm). In smFRET experiments, photons emitted by donor or acceptor are recorded as a function of time from individual molecules, which may diffuse through a confocal detection volume or be immobilized on a surface. From the photon stream one computes FRET efficiency trajectories and, often, inferred distance distributions or kinetic models.

Common experimental designs include confocal smFRET on freely diffusing molecules and surface-immobilized smFRET. Techniques such as alternating-laser excitation (ALEX) or pulsed interleaved excitation (PIE) are used to distinguish species that are donor-only, acceptor-only, or doubly labeled, and to correct for labeling or detection artifacts. Labeling strategies include covalent dye attachment to engineered sites on DNA, RNA, or proteins.

Data analysis yields information on conformational states, transition rates, and dwell times. FRET-efficiency histograms reveal state populations, while time traces and hidden Markov modeling resolve dynamic transitions between states. Applications span nucleic acid folding, riboswitch and protein conformational changes, enzyme mechanisms, and biomolecular interactions.

Limitations include photobleaching, dye mobility and orientation effects, spectral bleed-through, and the need for accurate calibration and corrections for background, direct excitation, and gamma factors.