Processing TIRF Microscopy Images to Characterize the Dynamics and Morphology of Bacterial Actin-Like Assemblies

• DOI: 10.1007/978-1-0716-0219-5_9
• Publication Date: 2019-12-26T12:02:34Z
• Authors: Cyrille Billaudeau; Arnaud Chastanet; Rut Carballido-López
• Keywords: 0301 basic medicine; 0303 health sciences; [SPI.OPTI] Engineering Sciences [physics]/Optics / Photonic; Single-particle tracking (SPT); Bacteria; 2D anisotropic Gaussian fitting; 500; 530; Time-Lapse Imaging; Actins; Single Molecule Imaging; Image analysis; 03 medical and health sciences; [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology; Bacterial Proteins; Microscopy, Fluorescence; Kymograph; [SPI.OPTI]Engineering Sciences [physics]/Optics / Photonic; Automated dynamic classification; Mean squared displacement (MSD); MreB; [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology

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.