High-Speed “4D” Computational Microscopy of Bacterial Surface Motility*

Bacterial surface appendage-driven motility modes play key roles in early-stage biofilm community development. Examples include type IV pili-driven “twitching” and flagellum-driven “spinning” and “swarming” motilities controlled by molecular motors. Analysis of surface motility behavior is complicated by its inherently 3D nature, the speed of which is too fast for confocal microscopy to capture. Here, we combine electromagnetic field computation and statistical image analysis to generate 3D movies close to a surface at 5 ms time resolution using conventional inverted microscopes. Application of this technique on Pseudomonas aeruginosa, accompanied with hydrodynamic calculations, revealed that these tiny organisms act like spinning tops in the low Reynolds number regime. They undergo complex flagellum-driven dynamical behavior, including precession and nutation. We also observe an unexpected taxonomy of surface motility mechanisms, like horizontal bacteria that follow helicoidal trajectories and exhibit superdiffusive movements parallel to the surface. We further apply this technique to discern the effects on motility by each set of flagellum stators, MotAB and MotCD, in P. aeruginosa.