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Abstract

Single molecule tracking is an indispensable tool in life sciences due to its unmatched potential to observe and investigate functional dynamics of (macro)molecular machineries in living cells in a minimally invasive manner. The limiting factor for the spatio-temporal resolution of such studies is typically the finite photon emission rate of fluorescent single molecules. This work assesses to which extent the remarkable localization efficiency of MINFLUX nanoscopy allows for a higher spatio-temporal resolution than conventional super-resolution tracking approaches to reveal previously unresolvable subcellular dynamics. To this end, the phase scanner, an innovative scanning device was designed and built. It allows to interferometrically generate and scan various point spread functions by electrooptically controlling the phase shift between mutually coherent beamlets arranged in the back focal plane of a microscope objective. MINFLUX localizations were recorded that outperform the spatio-temporal resolution of an idealized camera-based localization by more than a factor of six. The unprecedented performance of the presented MINFLUX microscope was demonstrated by tracking Kinesin-1 motor proteins for the first time under physiological conditions in a single molecule tracking study. With these results, MINFLUX emerges as the most powerful technique when the highest spatio-temporal resolution is required in single molecule tracking studies.