Fig. 4: Stochastic simulations of smFRET efficiency.
From: Recovering true FRET efficiencies from smFRET investigations requires triplet state mitigation
a, Schematic of a nine-state photophysical model used for stochastic simulations of smFRET efficiency. For simplicity in representation, energetic relations between excited states are not maintained in the vertical dimension. b,c, Simulations of smFRET efficiency for Cy3–Cy5 (b) and LD555–LD655 (c) FRET pairs. Theoretical FRET efficiencies were calculated based on Förster theory without consideration of triplet states (Methods). Error bars in experimental FRET data represent s.d. in mean FRET efficiency values from five experimental repeats. The experimental data are crosstalk, direct excitation and γ-corrected. d,e, Simulations of population fractions of nine joint states at different excitation rates for Cy3–Cy5 (d) and LD555–LD655 (e) pairs. f, Power-dependent variations in the \(\zeta\)-correction parameter based on the nine-state photophysical model for Cy3–Cy5 and LD555–LD655 FRET pairs over a broad range of illumination intensities spanning those commonly used for TIRF and confocal imaging. g, Simulation of smFRET efficiency for the Cy3–Cy5 FRET pair across donor-acceptor triplet state lifetimes and excitation rates. h, Variations of \(\zeta\) for the Cy3–Cy5 FRET pair with varied donor and acceptor triplet state lifetimes and excitation rates. i, \(\zeta\)-corrected smFRET efficiencies for simulated Cy3–Cy5 FRET pairs at varied excitation rates.
