Live-cell imaging is a highly used technique to study cell migration and dynamics over time. Although many computational tools have been developed during the past years to automatically detect and track cells, they are optimized to detect cell nuclei with similar shapes and/or cells not clustering together. These existing tools are challenged when tracking fluorescently labelled membranes of cells due to cell’s irregular shape, variability in size and dynamic movement across Z planes making it difficult to detect and track them. Here we introduce a detailed analysis pipeline to perform segmentation with accurate shape information, combined with BTrackmate, a customized codebase of popular ImageJ/Fiji software Trackmate, to perform cell tracking inside the tissue of interest. We developed VollSeg, a new segmentation method able to detect membrane-labelled cells with low signal-to-noise ratio and dense packing. Finally, we also created an interface in Napari, an Euler angle based viewer, to visualize the tracks along a chosen view making it possible to follow a cell along the plane of motion. Importantly, we provide a detailed protocol to implement this pipeline in a new dataset, together with the required Jupyter notebooks. Our codes are open source available at Github2021.

Keywords:3D segmentationcell trackingdeep learningirregular shaped cellsfluorescent microscopy.