Single-Molecule Förster Resonance Energy Transfer (smFRET)
In 1948, Theodor Förster predicted that an excited donor fluorophore may transfer its energy onto a spectrally overlapping acceptor, given that the two dyes are in close proximity (2-10 nm). This energy transfer follows a strong distance dependence of 1/r6 which makes FRET suitable to probe the dynamics of ribozyme catalysis or to detect conformational changes in metabolite-sensing riboswitches. Interrogating biomolecular processes on the single-molecule level has two key advantages over ensemble approaches: (i) subensemble heterogeneities are unraveled that would otherwise be averaged out. (ii) kinetics can be obtained from equilibrium experiments without the need to synchronize the molecules. We have implemented three-color based smFRET in a total internal reflection fluorescence (TIRF) microscope that uses stroboscopic alternating laser excitation (sALEX) to monitor dozens of RNA molecules in parallel with time-resolutions down to 1 ms.
Time-correlated single-photon counting (TCSPC)
Fluorophore photophysics are known to affect FRET in various ways. Environment-sensitive probes like carbocyanines change their fluorescent lifetime, quantum yield and dynamic anisotropy according to the propensity of photo-induced cis-trans isomerization. Time-correlated single photon counting (TCSPC) measurements are routinely carried out in our lab to account for such photophysical effects that are generally referred to as RNA-induced fluorescent enhancement (RIFE).