3D Tracking of Migrating Cells from Live Microscopy Time-Lapses

: With rapidly advancing microscopy techniques for live cell imaging, we are now able to image groups of migrating cells in many different in vivo contexts. However, as the resulting data sets become larger and more complex, following the behavior of these cells and extracting accurate quantitative data become increasingly challenging. Here we present a protocol for carrying out accurate automated tracking of cells moving over time in 3D, implemented as custom-built macro scripts for ImageJ. As opposed to many generic tracking workflows, the workflow we propose here accounts for the overall movement of the embryo, allows the selection of subgroups of cells, and includes a step for the complete assisted review of all 3D tracks. Furthermore, it is easy to add new custom track measurement to the code provided. Together, these present a reliable method for the precise tracking of cells, from which distinct subsets of cells can be selected from within a population.