FIG. 1.

Overview of FCS and smFRET instrument and measurements. Left: Schematic of a typical confocal setup used in FCS or smFRET measurements. The laser is focused by a high NA objective lens to a diffraction-limited confocal volume. Emitted fluorescence is collected through the same objective, separated from the excitation light by a dichroic mirror and long-pass filter and finally focused onto the confocal pinhole. Center: FCS measures fluorescence intensity fluctuations over time of single-labeled molecules (at nM concentration) diffusing through the confocal volume. The signal is then processed using an autocorrelation algorithm to obtain the autocorrelation curves. Right: In smFRET measurements, each single donor–acceptor-labeled molecule produces burst of photons while it diffuses through the confocal volume in a timescale of a few milliseconds. The ET_eff values are then calculated for each photon burst and plotted as a histogram.