Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes

Abstract: We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity minimum of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. A 22-fold reduction of photon detections over that required in popular centroid-localization is demonstrated. In superresolution microscopy, MINFLUX attained ~1 nm precision, resolving molecules only 6 nm apart. Tracking single fluorescent proteins by MINFLUX increased the temporal resolution and the localizations per trace by 100-fold, as demonstrated with diffusing 30S ribosomal subunits in living E. coli. Since conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond.