A quantitative pipeline for whole-mount deep imaging and multiscale analysis of gastruloids

Whole-mount 3D imaging at the cellular scale is a powerful tool for exploring the complex processes of organoid morphogenesis, as it allows examining tissue architecture, cell types, and morphology simultaneously in 3D models. To address the challenges of whole-mount imaging in dense and diffusive tissues, we focused on gastruloids—3D embryonic organoids derived from mouse embryonic stem cells that replicate key aspects of in vivo organogenesis and present significant imaging challenges. We developed an integrated two-photon microscopy pipeline to image immunostained and cleared gastruloids in toto , achieving detailed visualization at both cellular and whole-mount scale. Our pipeline, implemented in Python with a Napari interface, includes image preprocessing to correct optical artifacts and segment individual cell nuclei. This enables comprehensive quantitative analysis of gene expression patterns, cellular events, and morphologies across multiple spatial scales using an interactive post-processing pipeline. This approach not only advances our understanding of gastruloids development but also lays the groundwork for applying these methods to other organoid and tumoroid systems, enhancing our ability to study a wide range of 3D biological models.