Quantitative analysis methods for free diffusion single-molecule FRET experiments

Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful technique for investigating the structure and dynamics of biomolecules. This review focuses on recent advances in quantitative methods to analyze freely diffusing molecules in smFRET. The methods include traditional approaches of analyzing FRET efficiency and advanced photon-by-photon techniques based on maximum likelihood estimation without binning photon sequences. More recently, methods explicitly accounting for molecular diffusion have been developed, addressing biases arising from variations in brightness and diffusivity among molecular states and species. Applications of these tools include studies of protein folding, DNA dynamics, and oligomerization processes of neurodegenerative proteins. These advancements expand the ability of free diffusion-based smFRET to elucidate the dynamic behavior of biomolecules on the timescales relevant to their biological processes.