Tracking of individual freely diffusing fluorescent protein molecules in the bacterial cytoplasm

We combine stroboscopic laser excitation with stochastic photoactivation and super-resolution fluorescence imaging. This makes it possible to record hundreds of diffusion trajectories of small protein molecules in single bacterial cells with millisecond time resolution and sub-diffraction limited spatial precision. We conclude that the small protein mEos2 exhibits normal diffusion in the bacterial cytoplasm with a diffusion coefficient of 13.1 -+ 1.2 \mu m^2 s^(-1). This investigation lays the groundwork for studying single-molecule binding and dissociation events for a wide range of intracellular processes.