TrackMate is an open-source Fiji plugin for automated, semi-automated, and manual tracking of single particles. It offers a versatile and modular solution that works out of the box for end users, with a simple and intuitive user interface. It is easily scriptable and adaptable, operating on 1D, 2D, 3D, or other single and multi-channel image variants. TrackMate provides visualization and analysis tools to assess tracking results. It is also easily customizable for specific tracking problems and allows developers to write their own detection, particle linking, visualization, or analysis algorithms within the TrackMate environment. This evolving framework enables researchers to quickly develop and optimize new algorithms based on existing TrackMate modules without writing new user interfaces. The current capabilities of TrackMate are demonstrated through three biological problems. First, it is used for Caenorhabditis-elegans lineage analysis to assess how light-induced damage during imaging impairs early development. TrackMate-based lineage analysis indicates the absence of a cell-specific light-sensitive mechanism. Second, it is used to investigate the recruitment of NEMO clusters in fibroblasts after stimulation by the cytokine IL-1, showing that photodamage can generate artifacts in the shape of TrackMate characterized movements that confuse motility analysis. Finally, it is validated for quantitative lifetime analysis of clathrin-mediated endocytosis in plant cells. TrackMate is a plugin within the Fiji ImageJ distribution for tracking, developed with several goals: usability, flexibility, and a data model suitable for a wide range of tracking applications. It is openly available, well documented, and includes several detection and tracking modules that allow combining manual and automated particle tracking approaches. TrackMate includes several visualization tools and features that facilitate data exchange with other tracking tools and analysis applications. It is designed for maximal flexibility, allowing users to tailor its capabilities through the addition of specific tracking, detection, visualization, or analysis modules. TrackMate uses a data model that makes it a useful tool for a wide range of tracking applications, ranging from single-particle tracking of subcellular organelles to cell lineage analysis. TrackMate has a modular design that allows using its processing core without relying on the GUI, in scripts or other software. This allows TrackMate to be used for batch analysis, potentially running on a remote cluster over many images at once. TrackMate is interoperable with other software, such as MATLAB and Icy, and can be extended and reused by external developers. TrackMate is designed for use with multidimensional light microscopy datasets from a wide range of modalities, including 2D brightfield, 3D TIRF imaging, and 4D laser scanning microscopy. It has been adapted for various biological tracking applications, including the analysis of phototoxic effects on cell division in C. elegans, the dynamics of NEMO punctate structures in response to cytokine stimulation, and the dynamics of clathrin at the plasma membrane of ArabTrackMate is an open-source Fiji plugin for automated, semi-automated, and manual tracking of single particles. It offers a versatile and modular solution that works out of the box for end users, with a simple and intuitive user interface. It is easily scriptable and adaptable, operating on 1D, 2D, 3D, or other single and multi-channel image variants. TrackMate provides visualization and analysis tools to assess tracking results. It is also easily customizable for specific tracking problems and allows developers to write their own detection, particle linking, visualization, or analysis algorithms within the TrackMate environment. This evolving framework enables researchers to quickly develop and optimize new algorithms based on existing TrackMate modules without writing new user interfaces. The current capabilities of TrackMate are demonstrated through three biological problems. First, it is used for Caenorhabditis-elegans lineage analysis to assess how light-induced damage during imaging impairs early development. TrackMate-based lineage analysis indicates the absence of a cell-specific light-sensitive mechanism. Second, it is used to investigate the recruitment of NEMO clusters in fibroblasts after stimulation by the cytokine IL-1, showing that photodamage can generate artifacts in the shape of TrackMate characterized movements that confuse motility analysis. Finally, it is validated for quantitative lifetime analysis of clathrin-mediated endocytosis in plant cells. TrackMate is a plugin within the Fiji ImageJ distribution for tracking, developed with several goals: usability, flexibility, and a data model suitable for a wide range of tracking applications. It is openly available, well documented, and includes several detection and tracking modules that allow combining manual and automated particle tracking approaches. TrackMate includes several visualization tools and features that facilitate data exchange with other tracking tools and analysis applications. It is designed for maximal flexibility, allowing users to tailor its capabilities through the addition of specific tracking, detection, visualization, or analysis modules. TrackMate uses a data model that makes it a useful tool for a wide range of tracking applications, ranging from single-particle tracking of subcellular organelles to cell lineage analysis. TrackMate has a modular design that allows using its processing core without relying on the GUI, in scripts or other software. This allows TrackMate to be used for batch analysis, potentially running on a remote cluster over many images at once. TrackMate is interoperable with other software, such as MATLAB and Icy, and can be extended and reused by external developers. TrackMate is designed for use with multidimensional light microscopy datasets from a wide range of modalities, including 2D brightfield, 3D TIRF imaging, and 4D laser scanning microscopy. It has been adapted for various biological tracking applications, including the analysis of phototoxic effects on cell division in C. elegans, the dynamics of NEMO punctate structures in response to cytokine stimulation, and the dynamics of clathrin at the plasma membrane of Arab