SpFRET MFD experiments

For multi-parameter fluorescence detection (MFD) measurements, sample preparation was performed as described for spFRET TIRF experiments. The complexes were diluted to <50 pM to minimize the probability of having two or more molecules in the confocal observation volume at a time and measured for a period of 2 h. The experiments were performed on a homebuilt confocal microscope described in Mapa et al. (51). Pulsed interleaved excitation was achieved using picosecond pulsed interleaved lasers (PicoQuant, Berlin) with the wavelengths 532 and 640 nm. A laser power of 100 μW for each laser, measured at the back aperture of the objective, was used.