Quantifying protein-protein interactions using confined photoconversion and single-particle tracking

Single particle tracking (SPT) has become a powerful tool to investigate the dynamics of single molecules in live cells. Typical SPT measurement require sparsely labeled molecules in order to identify and localize the individual molecules. This low density limits the rate at which diffusion-limited reactions can be observed. We are developing a novel technique that increases the rate of observable interactions by two orders of magnitude. We call this method SPT-REversible Saturable OpticaL Fluorescence Transitions (SPT-RESOLFT) because it uses a concept from the RESOLFT super-resolution method. The concept relies on saturated labeling and selectively imaging proteins of interest within roughly 100 nm spot. Fluorophores are activated using diffraction-limited Gaussian beam and deactivated outside of 100 nm by a Laguerre-Gauss donut beam of (p, l) = (0, 2) mode which corresponds to topological charge of 2. We experimentally demonstrate the concept of SPT-RESOLFT using EGFR-mEos4b, which form dimers with ∼1 s lifetimes, and with the gamma subunit of FcεRI, gamma-mEos4b, which forms stable dimers.