Accurate measurements of cell morphology and behaviour are fundamentally important for understanding how disease, molecules and drugs affect cell function in vivo. Using muscle stem cell (muSC) responses to injury in zebrafish as our biological paradigm we established a ground truth for muSC behaviour. This revealed segmentation and tracking algorithms from commonly used programs are error-prone, leading us to develop a fast semi-automated image analysis pipeline that allows user defined parameters for segmentation and correction of cell tracking. Cell Tracking Profiler (CTP) is a package that runs two existing programs, HK Means and Phagosight within the Icy image analysis suite, to enable user-managed cell tracking from 3D time-lapsed datasets to provide measures of cell shape and movement. We demonstrate how CTP can be used to reveal changes to cell behaviour of muSCs in response to manipulation of the cell cytoskeleton by small molecule inhibitors. CTP and the associated tools we have developed for analysis of outputs thus provide a powerful framework for analysing complex cell behaviour in vivo from 4D datasets that are not amenable to straightforward analysis.

Analysis of cell shape and movement from 3D time-lapsed datasets is currently very challenging. We therefore designed Cell Tracking Profiler for analysing cell behaviour from complex datasets and demonstrate its effectiveness by analysing stem cell behaviour during muscle regeneration in zebrafish. AbstractAccurate measurements of cell morphology and behaviour are fundamentally important for understanding how disease, molecules and drugs affect cell function in vivo. Using muscle stem cell (muSC) responses to injury in zebrafish as our biological paradigm we have established a ground truth for muSC cell behaviour. This revealed that variability in segmentation and tracking algorithms from commonly used programs are errorprone, leading us to develop a fast semi-automated image analysis pipeline that allows user defined parameters for segmentation and correction of cell tracking. Cell Tracking Profiler (CTP) operates through the freely available Icy platform, and allows usermanaged cell tracking from 3D time-lapsed datasets to provide measures of cell shape and movement. Using dimensionality reduction methods, multiple correlation and regression analyses we identify myosin II-dependent parameters of muSC behaviour during regeneration. CTP and the associated statistical tools we have developed thus provide a powerful framework for analysing complex cell behaviour in vivo from 4D datasets.