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

Abstract

Bacteria exhibit surface motility modes that play pivotal roles in early-stage biofilm community development, such as type IV pili-driven “twitching” motility and flagellum-driven “spinning” and “swarming” motility. Appendage-driven motility is controlled by molecular motors, and 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. We treat each bacterial cell as a spherocylindrical lens and use finite element modeling to solve Maxwell’s equations and compute the diffracted light intensities associated with different angular orientations of the bacterium relative to the surface. By performing cross-correlation calculations between measured 2D microscopy images and a library of computed light intensities, we demonstrate that near-surface 3D movies of Pseudomonas aeruginosa translational and rotational motion are possible at high temporal resolution. Comparison between computational reconstructions and detailed hydrodynamic calculations reveals that P. aeruginosa act like low Reynolds number spinning tops with unstable orbits, driven by a flagellum motor with a torque output of ~2 pN μm. Interestingly, our analysis reveals that P. aeruginosa can undergo complex flagellum-driven dynamical behavior, including precession, nutation, and an unexpected taxonomy of surface motility mechanisms, including upright-spinning bacteria that diffuse laterally across the surface, and horizontal bacteria that follow helicoidal trajectories and exhibit superdiffusive movements parallel to the surface.

Phenotypic differences in reversible attachment behavior reveal distinctP. aeruginosasurface colonization strategies

“…Pseudomonas aeruginosa PAO1 and PA14 wild type (WT) strains were used in this study. For the flagellum localization data, PA14 WT and Δ pilA (deleting the major subunit of the TFP filament) (48) with FliC (the major subunit of the flagellum filament) modified to FliC(T394C) (49) were used. PAO1 was cultured as previously described (21, 23), and PA14 was cultured as previously described (8).…”

Pseudomonas aeruginosadetachment from surfaces via a self-made small molecule

“…Since MHQ affects the association of P. aeruginosa cells with surfaces, we further investigated its potential mechanism of action by using high-magnification imaging to examine the behavior of individual P. aeruginosa cells at the surface. P. aeruginosa cells can attach to the surface either by their pole (vertically) or by their side (horizontally) (19)(20)(21). Upon treatment with PBS, 76% (ntotal = 1347) of the cells attached to the surface vertically.…”

Engineering photonics solutions for COVID-19

“…From microscopy 1–3 to optical communications, 4–6 optical technologies permeate nearly every aspect of society. Therefore, when confronted with the challenges of a global pandemic, 7–12 it is not surprising that many solutions have been found in photonic devices and developed by optical engineers.…”