This document constitutes the TrackMate documentation. It was initially part of the supplemental information attached to the TrackMate paper (Tinevez et al., 2017). It is divided in four parts, that group sections by interest.

• The first part contains three tutorials, aimed at end-users. They are meant to guide users with the TrackMate plugin as a single-particle tracking Fiji plugin. They cover the three applications of the plugin: – automated single-particle tracking; – manual curation and correction of tracking results; – manual and semi-automatic tracking.

• The second part contains technical information. It documents the use of some Track- Mate components and gives their exact definition, accuracy and performance. This part is meant to guide the user in choosing the right algorithm for their application.

• The third part is aimed at advanced users, that want to analyze the tracking results in another so ware package, or use a scripting language to configure and run TrackMate programmatically.

• The last part is made of seven tutorials aimed at Java developers and document how to extend TrackMate with custom modules. The seven tutorials document a specific Track- Mate module (detection, particle-linking, etc) but are best read first in order, as they introduce developers to the annotation mechanism used for automatic plugin discovery.