Holden, S.J. et al. Proc. Natl. Acad. Sci. USA 111, 4566–4571 (2014).

Following dynamic processes in living cells with photoactivated localization microscopy is extremely challenging. First, there is a limited pool of labeled molecules that is increasingly depleted during each localization step. Second, high irradiation intensities can cause phototoxicity during time-lapse imaging. Holden et al. overcome these challenges by performing automated photoactivated localization microscopy on different cells in synchronized populations of dividing bacteria. A μManager software module automatically adjusts the intensity of the activation laser to maintain appropriate densities of activated single molecules in an imaging system designed to minimize drift. An external phase-contrast system allows automated bacteria identification and orientation of molecule localizations with the outline of the cell. The methodology permitted nanoscale observation of the role of the protein FtsZ in bacterial cell division and the identification of previously unseen processes.