Analysis of photon trajectories from diffusing single molecules

In single-molecule free diffusion experiments, molecules spend most of the time outside a laser spot and generate bursts of photons when they diffuse through the focal spot. Only these bursts contain meaningful information and, therefore, are selected using physically reasonable criteria. The analysis of the bursts must take into account the precise way they were chosen. We present new methods that allow one to accurately determine the brightness and diffusivity of individual molecule species from the photon arrival times of selected bursts. We derive analytical expressions for the distribution of inter-photon times (with and without burst selection), the distribution of the number of photons in a burst, and the distribution of photons in a burst with recorded arrival times. The theory accurately treats the bias introduced due to the burst selection criteria. We use a Maximum Likelihood (ML) method to find the molecule's photon count rate and diffusion coefficient from three kinds of data, i.e., the bursts of photons with recorded arrival times (burstML), inter-photon times in bursts (iptML), and the numbers of photon counts in a burst (pcML). The performance of these new methods is tested on simulated photon trajectories and on an experimental system, the fluorophore Atto 488.