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This lecture covers the practical aspects of using TrackMate for image analysis and data tracking, focusing on features, limits, and challenges. It explains the concept of tracking algorithms, the use of graphs to relate objects in time, and the process of segmenting data for tracking. The lecture also introduces TrackMate as an open platform for single-particle tracking, detailing its implementation, features, and support for 3D structures. It emphasizes the importance of spot detection, feature computation, and graph building in the tracking process, providing hands-on guidance for users. The presentation concludes with a discussion on detector settings, filtering processes, and the significance of manual curation in ensuring accurate tracking results.