High-throughput 3D tracking of bacteria on a standard phase contrast microscope

Bacteria employ diverse motility patterns in traversing complex three-dimensional (3D)
natural habitats. 2D microscopy misses crucial features of 3D behaviour, but the applicability
of existing 3D tracking techniques is constrained by their performance or ease of use. Here
we present a simple, broadly applicable, high-throughput 3D bacterial tracking method for
use in standard phase contrast microscopy. Bacteria are localized at micron-scale resolution
over a range of 350x300x200 μm by maximizing image cross-correlations between their
observed diffraction patterns and a reference library. We demonstrate the applicability of our
technique to a range of bacterial species and exploit its high throughput to expose hidden
contributions of bacterial individuality to population-level variability in motile behaviour. The
simplicity of this powerful new tool for bacterial motility research renders 3D tracking
accessible to a wider community and paves the way for investigations of bacterial motility in
complex 3D environments.