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Total internal reflection fluorescence (TIRF) microscopy allows the visualization of the dynamic membrane-associated actin-like MreB filaments in live bacterial cells with high temporal resolution. This chapter describes computerized analysis methods to quantitatively characterize the dynamics and morphological properties of MreB assemblies. These include how to (1) segment bacterial cells, (2) perform single-particle tracking (SPT) of MreB filamentous structures, (3) classify their dynamic modes using mean squared displacement (MSD) analysis, and (4) measure their dimensions and orientation.

MreB proteins are actin homologs present in nonspherical bacteria. They assemble into membrane-associated discrete filamentous structures that exhibit different dynamic behaviors along the bacterial sidewalls. Total internal reflection fluorescence (TIRF) microscopy, a sensitive method for studying molecular events at cell surfaces with high contrast and temporal resolution, is a method of choice to characterize the localization and dynamics of cortical MreB assemblies in vivo. This chapter describes the methods for visualizing fluorescently tagged MreB proteins in live Bacillus subtilis cells. We detail how to (1) grow B. subtilis strains for reproducible TIRF observations, (2) immobilize cells on agarose pads and (3) in CellASIC ® microfluidic plates, and (4) acquire TIRF images and time lapses.