3D single-molecule tracking using one-and two-photon excitation microscopy

Three dimensional single-molecule tracking (3D-SMT) has revolutionized the way we study fundamental cellular processes. By analyzing the spatial trajectories of individual molecules (e. g. a receptor or a signaling molecule) in 3D space, one can discern the internalization or transport dynamics of these molecules, study the heterogeneity of subcellular structures, and elucidate the complex spatiotemporal regulation mechanisms. Sub-diffraction localization precision, sub-millisecond temporal resolution and tens-of-seconds observation period are the benchmarks of current 3D-SMT techniques. We have recently built two molecular tracking systems in our labs. The first system is a previously reported confocal tracking system, which we denote as the 1P-1E-4D (one-photon excitation, one excitation beam, and four fiber-coupled detectors) system. The second system is a whole new design that is based on two-photon excitation, which we denote as the 2P-4E-1D (two-photon excitation, four excitation beams, and only one detector) system. Here we compare these two systems based on Monte Carlo simulation of tracking a diffusing fluorescent molecule. Through our simulation, we have characterized the limitation of individual systems and optimized the system parameters such as magnification, z-plane separation, and feedback gains.