Diffusion Measurements by Single-Molecule Spot-Size Analysis

We present a method to obtain diffusion coefficients from single-molecule images in video microscopy experiments. This method is based on the size of the single-molecule spot and delivers even for short trajectories a maximum number of data points and a higher statistical accuracy than does the common mean square displacement analysis. It is thus possible to obtain a diffusion coefficient from a single snapshot and to follow slow changes in single-molecule mobility in diffusion constant trajectories. Random walk simulations of single-molecule spots are used to validate the method.