Camera-based single-molecule FRET detection with improved time resolution.

The achievable time resolution of camera-based single-molecule detection is often limited by the frame rate of the camera. Especially in experiments utilizing single-molecule Förster resonance energy transfer (smFRET) to probe conformational dynamics of biomolecules, increasing the frame rate by either pixel-binning or cropping the field of view decreases the number of molecules that can be monitored simultaneously. Here, we present a generalised excitation scheme termed stroboscopic alternating-laser excitation (sALEX) that significantly improves the time resolution without sacrificing highly parallelised detection in total internal reflection fluorescence (TIRF) microscopy. In addition, we adapt a technique known from diffusion-based confocal microscopy to analyse the complex shape of FRET efficiency histograms. We apply both sALEX and dynamic probability distribution analysis (dPDA) to resolve conformational dynamics of interconverting DNA hairpins in the millisecond time range.