Fig. 1: Three-color FRET.
From: Fast three-color single-molecule FRET using statistical inference
a When the donor (D) is excited by a laser (blue arrows), the excited energy decays to the ground state either through the radiative (rippled arrows) or non-radiative pathways (dashed arrows) with rate constant kD or the excited energy is transferred to A1 and A2 with transfer rates kET1 and kET2, respectively. The excited state energy of A1 can also be transferred to A2 with a transfer rate kET12. The two-color FRET efficiencies (E1, E2, and E12) are the efficiencies of the energy transfer between corresponding two dyes in the absence of a third dye. b Three-color alternating laser excitation or pulse-interleaved excitation (3c-ALEX/PIE) scheme. A molecule (light gray) containing all three fluorophores is excited by a donor excitation laser (Donor-ex) and an A1 excitation laser (yellow rippled arrow, A1-ex) alternately. The parameters are determined from the global analysis of the photon trajectories. The relaxation rate k and the population of the folded state pF are global fitting parameters. The fractions of acceptor 1 (ε1) and acceptor 2 (ε2) photon count rates in the folded (F) and unfolded (U) states are determined from the photon trajectories by D excitation. From the trajectories by A1 excitation, the fraction of A2 count rate, ε12, is determined. These acceptor fractions are converted to FRET efficiencies defined in (a) (see Methods). c The global analysis scheme of three- and two-color segments collected in a three-color CW donor excitation (3c-CW) folding experiment. From the two-color acceptor fractions, εDA1 and εDA2, two-color FRET efficiencies E12c and E22c are obtained, respectively. Using these values, two sets of three-color FRET efficiencies can be calculated and compared. d The global analysis scheme in a three-color CW donor excitation (3c-CW) binding experiment. The three-color and DA1 parts of the photon trajectories cannot be separately analyzed as in (c) because the unbound state is always two-color (DA1), whereas the bound state is either three-color or two-color (DA1) due to the binding to either A2-labeled (middle) or A2-unlabeled (left) binding partner. The three-color FRET efficiencies can be determined similarly using the acceptor fractions obtained from the maximum likelihood method.
