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; 3) taking into account detector noise sources such as crosstalk, 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 manuscript that addresses all aforementioned issues in smFRET data analysis specialized for the case of pulsed illumination. Furthermore, we apply our method to both synthetic as well as experimental data acquired using Holliday junctions. Why It Matters In the first companion manuscript of this series, we developed new methods to analyze noisy smFRET data. These methods eliminate the requirement of a priori specifying the dimensionality of the physical model describing a molecular complex’s kinetics. Here, we apply these methods to experimentally obtained datasets with samples illuminated by laser pulses at regular time intervals. In particular, we study conformational dynamics of Holliday junctions.