Single‐molecule fluorescence permits super‐resolution imaging, but traditional algorithms for localizing these isolated fluorescent emitters assume stationary point light sources. Proposed here are two fitting functions that achieve similar nanometer‐scale localization precision as the traditional symmetric Gaussian function, while allowing, and explicitly accounting for, directed motion. The precision of these methods is investigated through Fisher information analysis, simulation and experiments, and the new fitting functions are then used to measure, for the first time, the instantaneous velocity and direction of motion of live bacteria cells. These new methods increase the information content of single‐molecule images of fast‐moving molecules without sacrificing localization precision, thus permitting slower imaging speeds, and our new fitting functions promise to improve tracking algorithms by calculating velocity and direction during each image acquisition. Single molecules on the move: Two fitting functions are introduced for measuring in‐frame motion of isolated fluorescent emitters. These methods determine the instantaneous directionality and velocity of motion without sacrificing the localization precision. Theory, simulation, and experiments are used to validate the methods, and the fitting algorithms are applied to the motion of live bacteria cells (see picture)
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