TrackMate: An open and extensible platform for single-particle tracking. Highlights: TrackMate is a software tool for automated, and semi-automated particle tracking. TrackMate’s major development focus is on usability and extensibility. TrackMate leverages Fiji to help provides its functionality and extensibility. TrackMate is used for: C. elegans lineaging, NEMO assembly and clathrin dynamics. Challenging imaging problems can robustly be analyzed using semi-automatic methods.