u-track 3D: measuring and interrogating dense particle dynamics in three dimensions

Particle tracking is a ubiquitous task in the study of dynamic molecular and cellular processes by live microscopy. Light-sheet microscopy has recently opened a path to acquiring complete cell volumes for investigation in 3-dimensions (3D). However, hypothesis formulation and quantitative analysis have remained difficult due to fundamental challenges in the visualization and the verification of large sets of 3D particle trajectories. Here we describe u-track 3D, a software package that addresses these two challenges with three algorithmic innovations. Building on the established framework of globally optimal particle association in space and time implemented in the u-track package and recent advances in gaining association robustness in the case of erratic motion, we first report a complete and versatile pipeline for particle tracking. We then present the concept of dynamic region of interest (dynROI), which allows an experimenter to interact with dynamic 3D processes in 2D views amenable to visual inspection. Third, we present an estimator of trackability, which provides for every trajectory a confidence score, thereby overcoming the challenges of visual validation of trajectories in dense particle fields. With these combined strategies, u-track 3D provides a framework for the unbiased study of molecular processes in complex volumetric sequences.