Single-photon smFRET: II. Application to continuous illumination.

Here we adapt the Bayesian nonparametrics (BNP) framework presented in the first companion article to analyze kinetics from single-photon, single-molecule Förster resonance energy transfer (smFRET) traces generated under continuous illumination. Using our sampler, BNP-FRET, we learn the escape rates and the number of system states given a photon trace. We benchmark our method by analyzing a range of synthetic and experimental data.

Single-photon smFRET. I: Theory and conceptual basis.

We present a unified conceptual framework and the associated software package for single-molecule Förster resonance energy transfer (smFRET) analysis from single-photon arrivals leveraging Bayesian nonparametrics, BNP-FRET. This unified framework addresses the following key physical complexities of a single-photon smFRET experiment, including: 1) fluorophore photophysics; 2) continuous time kinetics of the labeled system with large timescale separations between photophysical phenomena such as excited photophysical state lifetimes and events such as transition between system states; 3) unavoidable detector artefacts; 4) background emissions; 5) unknown number of system states; and 6) both continuous and pulsed illumination. These physical features necessarily demand a novel framework that extends beyond existing tools.

Single-photon smFRET. III. Application to pulsed illumination.

Förster resonance energy transfer (FRET) using pulsed illumination has been pivotal in leveraging lifetime information in FRET analysis. However, there remain major challenges in quantitative single-photon, single-molecule FRET (smFRET) data analysis under pulsed illumination including 1) simultaneously deducing kinetics and number of system states; 2) providing uncertainties over estimates, particularly uncertainty over the number of system states; and 3) taking into account detector noise sources such as cross talk and the instrument response function contributing to uncertainty; in addition to 4) other experimental noise sources such as background. Here, we implement the Bayesian nonparametric framework described in the first companion article that addresses all aforementioned issues in smFRET data analysis specialized for the case of pulsed illumination.