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

Abstract

3D cell cultures, in particular organoids, are emerging models to investigate healthy or diseased tissues. 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 nano-scale volume electron microscopy by performing 3D cell cultures in a single carrier amenable to all imaging steps. This allows to follow organoids growth, probe their morphology with fluorescent markers, identify areas of interest and analyze their 3D ultrastructure. We demonstrate this workflow on mouse and human 3D cultures, and use automated image segmentation to annotate and quantitatively analyze subcellular structures in patient-derived colorectal cancer organoids. Our analyses reveal local organization of diffraction-limited cell junctions in compact and polarized epithelia. The continuum resolution imaging pipeline is thus suited to foster basic and translational organoid research by simultaneously exploiting the advantages of light and electron microscopy. HighlightsO_LIEstablishment of 3D cell cultures in sample carriers directly amenable to high-pressure freezing (HPF) C_LIO_LI3D cell cultures in HPF carriers allow drug treatment and live-cell imaging C_LIO_LIMulti-scale imaging of 3D cultures from live-cell light microscopy to volume electron microscopy C_LIO_LIEstablishments of HPF conditions for mouse and patient-derived organoids C_LIO_LIDeep-learning automatic segmentation of ultrastructural detail and quantitative data-mining reveal different subcellular organization associated with epithelium polarity C_LI