Supplementary Figure 3: Schematics of a typical confocal setup with alternating laser excitation and pulsed interleaved excitation.
From: Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study
Schematics of a typical confocal setup with alternating laser excitation / pulsed interleaved excitation and color-sensitive detection. The most important elements are specified: Objective (O), dichroic mirror (DM), pinhole (P), spectral filter (F), avalanche photo diode (APD) and electronic micro- or picosecond synchronization of laser pulses and single photon counting (Sync). Elements used for the correction factors in Table 2 (main text) were: F34-641 Laser clean-up filter z 640/10 (right after Laser 640 nm); DM1: F43-537 laser beam splitter z 532 RDC ; DM2: F53-534 Dual Line beam splitter z 532/633; DM3: F33-647 laser- laser beam splitter 640 DCXR; FG: F37-582 Brightline HC 582/75; FR: F47-700 ET Bandpass 700/75; Objective: Cfi plan apo VC 60xWI, NA1.2; Detectors: MPD Picoquant (green), tau-SPAD, Picoquant (red); Pinholes: 100 µm; ; Laser power at sample: ≈ 100 µW; Beam diameter ≈ 2 mm; Diffusion time of Atto550 and Atto647N around 0.42 ms and 0.50 ms, respectively. For details on all used setups and analysis software, see Supplementary Note 8.
