Single Photon smFRET. III. Application to Pulsed Illumination

Abstract

Förster resonance energy transfer (FRET) using pulsed illumination has been pivotal in probing complex single molecule dynamics within subcellular environments. However, there are still 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 overestimates, particularly uncertainty over state numbers; 3) taking into account all experimental details such as crosstalk and instrument response function contributing to uncertainty; in addition to 4) background. Here, we implement the Bayesian nonparametric framework described in the first companion paper that addresses all the 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 junction.

Single Photon smFRET. I. Theory and Conceptual Basis

“…First, inclusion of the IRF can be parallelized, potentially reducing the time-cost to just a calculation over a single data acquisition period. In our third companion paper [55], dealing with pulsed illumination, we improve computational cost by making the assumption that fluorophore relaxation occurs within the window between consecutive pulses, thereby reducing our second order formulation herein to a first order HMM, and allowing for faster computation of the likelihood in pulses where no photon is detected. In [55], we mitigate the computational cost of (3) by assuming physically motivated timescale separation.…”

“…24 and estimate it directly along with other parameters, as it is identical for all of the pulses. Later, in the third companion manuscript [55], we will use this approximation to provide a more computationally advantageous model.…”

“…in the third companion manuscript [64]. Using realistic values from the third companion manuscript [64], we set the excitation probability per pulse to be 0.005.…”

“…That is, the ambient photons and dark current are still modeled by an Exponential distribution though it is often further approximated as a Uniform distribution given that interpulse period if much shorter than the average background waiting time. The full formulation describing all background sources under pulsed illumination is provided in the third companion manuscript [64].…”

Fluorescence Lifetime: Beating the IRF and interpulse window

“…This posterior, however, assumes a nonstandard form and we cannot jointly sample all parameters. Therefore, we invoke a Gibbs sampling strategy for which we can sample individual parameters from the full conditional posteriors [53][54][55][56][57][58][59][60] . That is, the posterior of the parameter of interest conditioned on the remaining parameters.…”