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

Single-molecule Förster resonance energy transfer (smFRET) provides nanometer-scale snapshots of biomolecular conformational dynamics, but extracting reliable dynamic information from diffusion-based measurements requires a careful choice of analysis method. This study benchmarks the widely used approaches, including qualitative indicators of dynamics and quantitative rate extraction via hidden Markov modeling, using simulated datasets spanning common experimental conditions and focusing on a two-state system. The assessment clarifies the timescales each method reports on, determines the number of bursts required for reliable inference, examines the differences in FRET efficiencies needed by each approach, and evaluates the accuracy of kinetic rate recovery. Together, these results provide practical guidelines for selecting analysis strategies and accurately interpreting conformational dynamics in diffusion-based smFRET. The accompanying datasets offer a resource for optimizing experimental design and developing new analytical methods.