Automated Optimization of Bacterial Tracking Pipelines With TrackMate 8.

Quantitative analysis of bacterial dynamics in time-lapse microscopy requires robust tracking pipelines, yet selecting and optimizing algorithms for specific experiments remains challenging. Indeed, microbiologists are confronted with numerous algorithms that must be carefully chosen and parameterized to achieve optimal tracking for their experiments. We present an automated methodology to determine optimal tracking configurations for microbiological applications. It is based on TrackMate 8, a novel version of the TrackMate Fiji plugin extended with microbiology-specific tools. Our approach systematically evaluates algorithm-parameter combinations optimizing biologically relevant metrics (e.g., cell-cycle accuracy, bacterial morphology) and includes: (1) integration of deep-learning algorithms (Omnipose, YOLO, Trackastra) adequate for bacterial images in TrackMate; (2) a TrackMate-Helper extension for parameter optimization; and (3) a tracking and segmentation editor for tracking ground-truth generation. We demonstrate the effectiveness of the methodology on two use cases showing its adaptability to diverse experimental conditions. This methodology enables microbiologists with a widely applicable, automated framework to optimize tracking pipelines, facilitating quantitative analysis in bacterial imaging. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Impact of the respiratory chain in the growth and morphology of Escherichia coli Basic Protocol 2: The impact of mutations on the motility of Helicobacter pylori.