This protocol describes imaging and computational tools to collect and analyze live imaging data of embryonic cell migration. Our five-step protocol requires a few weeks to move through embryo preparation and four-dimensional (4D) live imaging using multiphoton microscopy, to 3D cell tracking using image processing, registration of tracking data and their quantitative analysis using
computational tools. It uses commercially available equipment and requires expertise in microscopy and programming that is
appropriate for a biology laboratory. Custom-made scripts are provided, as well as sample datasets to permit readers without
experimental data to carry out the analysis. The protocol has offered new insights into the genetic control of cell migration during
Drosophila gastrulation. With simple modifications, this systematic analysis could be applied to any developing system to define cell
positions in accordance with the body plan, to decompose complex 3D movements and to quantify the collective nature of cell
migration
