Abstract
Single-cell resolution time-lapse microscopy of bacterial populations is a powerful tool for assessing cellular behavior and interaction dynamics. Realizing its full potential requires accurate image analysis: segmentation of individual cell objects, tracking of persistent cells from frame to frame, and connecting of mother cells to daughters during division events. Tracking is particularly challenging in densely packed populations or when cells move significantly; Leading software often struggles. To address this, we present TrackRefiner, a tool for refinement of bacillus cell tracking data. This package was specifically designed to refine the tracking outputs of CellProfiler. Benchmarks involve non-motile, rod-shaped bacteria; extension to motile species or other morphologies remains to be demonstrated. For timelapses with frequent imaging, TrackRefiner achieved-with one exception-over 98% detection accuracy and corrected 57-100% of tracking errors. TrackRefiner is published on PyPI and Anaconda . Source code, user manuals, and the benchmark dataset are available on Github and OSF .