Open-top multisample dual-view light-sheet microscope for live imaging of large multicellular systems.

Multicellular systems grow over the course of weeks from single cells to tissues or even full organisms, making live imaging challenging. To bridge spatiotemporal scales, we present an open-top dual-view and dual-illumination light-sheet microscope dedicated to live imaging of large specimens at single-cell resolution. The configuration of objectives together with a customizable multiwell mounting system combines dual view with high-throughput multiposition imaging. We use this microscope to image a wide variety of samples and highlight its capabilities to gain quantitative single-cell information in large specimens such as mature intestinal organoids and gastruloids.

Multiscale light-sheet organoid imaging framework.

Organoids provide an accessible in vitro system to mimic the dynamics of tissue regeneration and development. However, long-term live-imaging of organoids remains challenging. Here we present an experimental and image-processing framework capable of turning long-term light-sheet imaging of intestinal organoids into digital organoids. The framework combines specific imaging optimization combined with data processing via deep learning techniques to segment single organoids, their lumen, cells and nuclei in 3D over long periods of time. By linking lineage trees with corresponding 3D segmentation meshes for each organoid, the extracted information is visualized using a web-based "Digital Organoid Viewer" tool allowing combined understanding of the multivariate and multiscale data. We also show backtracking of cells of interest, providing detailed information about their history within entire organoid contexts. Furthermore, we show cytokinesis failure of regenerative cells and that these cells never reside in the intestinal crypt, hinting at a tissue scale control on cellular fidelity.