Benchmarking dynamic analysis methods in diffusion-based single-molecule FRET

Diffusion-based single-molecule Förster resonance energy transfer (smFRET) experiments detect bursts of fluorescence as individual labeled biomolecules transiently enter the observation volume, providing snapshots of their conformational dynamics. Several analysis methods exist for interpreting these measurements, each with different sensitivities to kinetic timescales, state separation, and dataset size, yet their relative performance has not been systematically characterized. As smFRET adoption grows beyond specialist laboratories, such a characterization is increasingly valuable. This study systematically benchmarks the commonly used dynamic analysis approaches using simulated datasets representative of typical experimental conditions, providing practical guidelines for analyzing conformational dynamics in diffusion-based smFRET.