Live-Cell Imaging and Single-Cell Tracking to Monitor Clonal Dynamics and Fate Transitions in Human and Murine Intestinal Organoids

The intestinal epithelium undergoes rapid self-renewal through stem cell division, cell differentiation, and migration. Understanding how individual cells commit to specific fates and how clonal dynamics emerge within this tissue requires methods that capture cellular behavior in real time and at single-cell resolution. Intestinal organoids recapitulate key features of epithelial self-organization and cell-type diversity, making them a powerful system for studying these processes in a controlled setting. Here we present a protocol for long-term confocal live-cell imaging (up to 72 h) and single-cell tracking in human and murine intestinal organoids, built around two complementary reporter strategies. First, we describe the generation of mosaic organoids by combining differentially labeled cell populations, enabling single-cell resolution of membrane-localized and cytoskeletal reporters that cannot otherwise be attributed to individual cells in a dense epithelium. Second, we use cell-type-specific fate reporters (MUC2 for goblet cells and DEFA5 for Paneth cells) to monitor secretory cell type transitions in real time. The protocol covers organoid culture, mosaic organoid formation, sample preparation with strategies to minimize phototoxicity, image acquisition over several days, and semi-automated single-cell tracking using OrganoidTracker, which reconstructs cell trajectories and lineages over time. While demonstrated in intestinal organoids, this framework is readily adaptable to other epithelial organoid systems, including gastric, pancreatic, and colonic models, broadening its utility for studies of epithelial biology, homeostasis, and disease.