Single cell resolution 3D imaging and segmentation within intact live tissues

Epithelial cells form diverse structures, from squamous spherical organoids to densely packed pseudostratified folded tissues. Quantification of cellular properties in these contexts requires high-resolution deep imaging and computational techniques to achieve truthful three-dimensional (3D) structural features. Here, we describe a detailed step-by-step protocol for sample preparation, imaging and deep-learning-assisted cell segmentation to achieve accurate quantification of fluorescently labelled individual cells in 3D within a live tissue: the Drosophila wing disc. We share the "lessons learned" through troubleshooting 3D imaging, including considerations on the choice of microscopy modality and settings (objective, sample mounting) and available segmentation methods. In addition, we include a computational pipeline alongside custom code to assist replication of the protocol. While we focus on the segmentation of cell outlines from membrane labelling in the Drosophila wing disc, we believe it will be valuable for studying other tissues that demand complex analysis in 3D.