Single-Molecule Förster Resonance Energy Transfer (FRET)
smFRET is a powerful biophysical technique which can reveal spatial information about biomolecules in a way which is sensitive to both dynamic and static heterogeneity between subpopulations within the same sample. It can measure kinetics of conformational changes at equilibrium with no need for synchronisation, and can measure absolute distances for infering precise structural information.
FRET is the transfer of excitation energy from a short wavelength fluorophore to a long wavelength fluorophore (Förster 1948). The transfer has a strong distance dependence in the 3-10 nm range
and so is often exploited for studying biomolecules. This is typically used to study binary on/off processes at the ensemble level, such as cleaving, binding, opening etc.
Confocal Single-Molecule Detection and Alternating Laser Excitation (ALEX)
smFRET techniques typically achieve the high signal to noise ration needed to detect single molecules by using a reduced detection volume. This can be done using Total Internal Reflection Fluorescence Microscope (TIRFM) for immobilised molecules, or using a diffraction limited spot for freely diffusing molecules as in the smfBox. By alternating the excitation laser between donor and acceptor wavelengths, not only can FRET efficiency be measured (under donor excitation), but apparent dye stoichometry can also be accessed (Lee 2005). In this way, a low FRET efficiency molecule can easily be distinguished from a donor in the absence of an acceptor, which may arise from incomplete labelling or photobleaching.