Dependence of the CCF on FRET. For the inclusion of FRET, we varied the quenching factors for fluorophore a from 0 ... 1 and for fluorophore b form 1 ... 2 (qa = 2-qb, qb = 1 ... 2), mimicking the quenching of the FRET donor and the enhancement of fluorescence of the FRET acceptor. We assumed identical detection volumes for both channels with axial radii of 260 nm, and axial radii of 1.3 μm, corresponding to observation volumes of 0.5 fl; the brightness of the two particles was set to 1500 counts/second in their respective channel, and 10% cross-talk from particle a into the detection channel B and 1% cross-talk of particle b into detection channel A; the diffusion times for the two particles were set to τDa = 3 ms, τDb = 0.3 ms.
