Decoding the Pattern of Photon Colors in Single-Molecule FRET

Conformational dynamics of a single molecule can be studied using Forster resonance energy transfer (FRET) by recording a sequence of photons emitted by a donor and an acceptor dye attached to the molecule. We describe a simple and robust method to estimate the rates of transitions between different conformational states and the FRET efficiencies associated with these states. For a photon trajectory with measured interphoton times, the pattern of colors is decoded by maximizing the appropriate likelihood function, This approach can be used to analyze bursts of photons from diffusing molecules as well as photon trajectories generated by immobilized molecules. The procedure is illustrated using simulated photon trajectories corresponding to two-state and three-state molecules. The method works even when the photon colors appear to be scrambled because of high background noise, the photophysical properties of the conformers are similar, or the conformational and photon count rates are comparable. The consistency of the model with the data can be checked by recoloring the photon trajectories and comparing the predicted and observed FRET efficiency histograms.