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 350 × 300 × 200 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.