Systematic Assessment of Freely-Diffusing Single-Molecule Fluorescence Detection using Brownian Motion Simulations

Single-molecule fluorescence detection (SMFD) experiments are useful in distinguishing between sub-populations of molecular species in measurements of heterogeneous samples. One of the experimental platforms for SMFD is based on a confocal microscope setup, where molecules in the solution randomly traverse an effective detection volume, formed by a tightly focused laser beam. The non-uniformity of the excitation profile and the random nature of Brownian motion, produce fluctuating fluorescence signals. For these signals to be distinguished from the background, single-molecule fluorescence burst analysis is frequently used. Yet, the relation between the results of burst analyses and the underlying spatial information of the diffusing molecules is still obscure and requires systematic assessment. In this work we performed three-dimensional Brownian motion simulations of SMFD, and tested the positions from which the molecules emitted photons that were detected and passed the burst analysis criteria for different values of burst analysis parameters. The results of this work verify which of the burst analysis parameters and experimental conditions influence both the position of molecules in space when fluorescence is detected and taken into account, and whether these bursts of photons arise purely from single molecules, or not entirely.