Bacteria display diverse motility patterns when traveling in complex three-dimensional (3D) environments. Unfortunately, 2D microscopy fails to detail critical features of 3D motile behavior. However, the infrequent application of 3D tracking techniques derives from challenges in usability and overall performance. To tackle this obstacle, I implement an easy and effective high-throughput 3D bacterial tracking method applicable in standard phase-contrast microscopy that is outlined in Taute, Tans, and Shimizu’s paper (2015). Bacteria (i.e., E. coli) can be localized at a micron-scale resolution by maximizing image cross-correlations amongst their diffraction patterns (phase rings) and a reference library. 

The diameter of each visible diffraction ring correlates to an object's z-distance relative to the focal plane. Image cross-correlations use a reference library to match a phase ring associated with a known z-distance with the ring observed amidst the swimming bacteria under the microscope. We can use this innovative technique to record swimming bacteria over a specified period, cross-correlate each phase-contrasted frame with our reference library, and simultaneously identify accurate z-distances of multiple bacteria during that time. Thus we can establish complex 3D trajectories, which we can then analyze for specific motility behavioral patterns of interest. 

Ultimately, this simple and practical approach will expose previously unidentified contributions of individual bacteria to the variability of motility patterns among a population. Despite working on this project for the previous ten weeks, necessary procedures have yet to be completed. Nevertheless, as we progress with this project and approach the finish line, the ease of application concerning this bacterial tracking method will render wide accessibility to 3D tracking and enable extensive opportunities to investigate bacterial motility in all kinds of complex 3D environments.