Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Wednesday Speaker Abstracts

PHOTON-BY-PHOTON MAXIMUM LIKELIHOOD ANALYSIS OF FREELY DIFFUSING SINGLE MOLECULES Irina V Gopich 1 ; 1 National Institutes of Health, Laboratory of Chemical Physics, NIDDK, Bethesda, MD, USA Single-molecule FRET measurements of freely diffusing molecules provide powerful insights into biomolecular structure and dynamics through the analysis of photon bursts detected in confocal microscopy experiments. I discuss two photon-by-photon maximum likelihood methods for analyzing these bursts. The first method, colorML, estimates FRET efficiencies and conformational transition rates using a reduced likelihood function that avoids explicit modeling of translational diffusion. This approach is valid when all molecular states have equal brightness and diffusivity. ColorML is computationally efficient, applicable across a broad range of transition rates, and has been successfully used to study protein folding, binding, and transition path times. However, colorML can yield biased estimates when states differ in brightness or diffusivity, or when background noise is significant. Because bursts are selected based on a minimum photon count, brighter and more slowly diffusing species are preferentially detected, leading to overrepresentation of their populations in the analyzed data (burst-selection bias). The second method, burstML, addresses these limitations by explicitly modeling diffusion through the laser focus and incorporating burst-selection criteria directly into the likelihood function. BurstML can analyze a mixture of non-interconverting species, as well as dynamical systems, in which molecules undergo conformational changes while diffusing through the observation volume. In addition to FRET efficiencies, populations, and transition rates, it enables accurate estimation of state-specific brightness and diffusion times. The method is validated using simulated data and demonstrated on experimental protein systems, extending the applicability of photon-by-photon maximum likelihood analysis to systems where differences in brightness and diffusivity are unavoidable.

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