We use cookies to give you the best online experience and to improve our service. By using our website you agree to our use of cookies in accordance with our privacy policy.

Research

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).