Quantitative single-molecule localization microscopy reports on protein numbers in signaling protein complexes

Knowledge of how proteins organize into functional complexes is essential to understand their biological function. Optical super-resolution techniques provide the spatial resolution necessary to visualize and to investigate individual protein complexes in the context of their cellular environment. Single-molecule localization microscopy (SMLM) builds on the detection of single fluorophore labels, which next to the generation of high-resolution images provides access to quantitative molecular information. We developed various tools for quantitative SMLM (qSMLM), an imaging method that both super-resolves individual protein clusters and reports on molecular numbers by analyzing the kinetics of single emitter blinking. This method is compatible with both fluorescent proteins and organic fluorophores. With qSMLM, we quantify protein copy numbers in single clusters, and we study how changes in the stoichiometry of protein complexes translates into function.