Theory and Analysis of Single-Molecule FRET Experiments

Inter-dye distances and conformational dynamics can be studied using single-molecule FRET measurements. We consider two approaches to analyze sequences of photons with recorded photon colors and arrival times. The first approach is based on FRET efficiency histograms obtained from binned photon sequences. The experimental histograms are compared with the theoretical histograms obtained using the joint distribution of acceptor and donor photons or the Gaussian approximation. In the second approach, a photon sequence is analyzed without binning. The parameters of a model describing conformational dynamics are found by maximizing the appropriate likelihood function. The first approach is simpler, while the second one is more accurate, especially when the population of species is small and transition rates are fast. The likelihood-based analysis as well as the recoloring method has the advantage that diffusion of molecules through the laser focus can be rigorously handled.