Fig. 5: The impact of non-Poissonian photon emission.

a Jablonski diagram of the dye pair AlexaFluor 488 and 594 based on Nettels et al. ⁵³ (left). Each state is denoted by the electronic states of donor (first symbol) and acceptor (second symbol). S and T stand for the singlet and triplet manifolds, respectively. Subscripts refer to electronical ground (0) and first excited (1) states. Red and green arrows indicate transitions that lead to the emission of acceptor and donor photons, respectively. Dark blue arrows are excitation transitions of the donor and light blue arrows indicate the direct excitation of the acceptor at the wavelength of the donor. Black arrows indicate energy transfer processes where singlet-singlet annihilation and singlet-triplet annihilation are indicated by the rates \({k}_{{SSA}}\) and \({k}_{{STA}}\), respectively (Supplementary Table 1). The classical Förster energy transfer rate is \({k}_{T}\). Gray arrows indicate singlet-triplet and triplet-singlet transitions. The coarse-grained (CG) model of the full photophysical scheme (right) has 4 states that interconvert at microsecond timescales. b FRET histogram of a particle with fixed donor-acceptor distance identical to the Förster distance simulated using the coarse-grained model in (a). c Average of all photon traces of the bursts in the simulation. The overlay was constructed by aligning the trajectories relative to the average arrival time of donor (green) and acceptor (red) photons. The mean arrival time was arbitrarily set to zero. Arrow indicates the mismatch between donor and acceptor signal. d Apparent FRET efficiency profile calculated from the data in (c). e Normalized correlation ratios for the data in (b–d). Inset: Zoom of the normalized correlation ratios. f FRET correlation function for the data in (b–d). Black line is a single-exponential fit. Arrow indicates the triplet-induced decay.