A custom-made confocal microscope setup based on an Olympus IX 71 was used40,41 for the triple-FRET measurements with single -ATP synthases. Three different lasers were applied: two fiber coupled picosecond pulsed lasers with arbitrary repetition rates up to 80 MHz at 488 nm (PicoTa 490, Picoquant) and 635 nm (LDH-P-635B, Picoquant) were used, and the third, continuous wave laser at 532 nm (compass 315, Coherent) was switched by a fast acusto optical modulator (AOM type 335-192, Crystal Technologies). All three laser sources were externally triggered and synchronized by an arbitrary waveform generator (AWG 2041, Tektronix) in order to get the required duty cycle-optimized alternating laser excitation scheme as shown in Fig. 2. The three laser beams were combined to provide a common confocal excitation volume, as illustrated in Fig. 1, using two dichroic beam splitters (DCXR 488 and DC 540, AHF Tübingen). To determine and adjust the exact position of each of the confocal laser spots, a sample of polychromatic polystyrene beads (transfluospheres 488–635, Molecular Probes) embedded in a thin poly-vinyl-alcohol (PVA) film was used. After scanning an area of using a piezo-driven scan stage (P-517.2CD and PiFoc P-725.1CD, Physik Instrumente), the center of one polystyrene bead was determined with a fit algorithm using Gaussians to an accuracy of 20 nm. Subsequently, the alignment of the laser foci was improved. However, for the triple-FRET analysis, the three dimensional laser foci alignment was not very critical (in contrast to quantitative fluorescence cross correlation spectroscopy), because we probed the FRET changes for enzyme activity with 488 nm excitation qualitatively. If FRET is successfully detected within a photon burst, the FRET-based twisting of the rotor was analyzed quantitatively following 532 nm excitation. Start and end of a photon burst were defined by fluorescence intensity thresholds upon 532 nm excitation.