Light and electron microscopy continuum-resolution of 3D cell cultures

3D cell cultures, in particular organoids, are emerging models in the investigation of healthy or diseased tis-sues. Understanding the complex cellular sociology in organoids requires integration of imaging modalities across spatial and temporal scales. We present a multi-scale imaging approach that traverses millimeter -scale live-cell light microscopy to nanometer-scale volume electron microscopy by performing 3D cell cul-tures in a single carrier that is amenable to all imaging steps. This allows for following organoids' growth, probing their morphology with fluorescent markers, identifying areas of interest, and analyzing their 3D ultra -structure. We demonstrate this workflow on mouse and human 3D cultures and use automated image seg-mentation to annotate and quantitatively analyze subcellular structures in patient-derived colorectal cancer organoids. Our analyses identify local organization of diffraction-limited cell junctions in compact and polar-ized epithelia. The continuum-resolution imaging pipeline is thus suited to fostering basic and translational organoid research by simultaneously exploiting the advantages of light and electron microscopy.