Three-dimensional high-content imaging of unstained soft tissue with subcellular resolution using a laboratory-based X-ray microscope

Three-dimensional analysis of biological tissue is crucial for linking tissue morphology to cellular function. While conventional histology remains the gold standard for visualising tissue architecture at subcellular resolution, it is limited by its destructive, two-dimensional nature and anisotropic resolution, hindering its use in capturing full three-dimensional representations of tissue volumes. Here, we demonstrate that phase-contrast X-ray microscopy enables non-destructive, three-dimensional virtual histology of unstained liver tissue with subcellular resolution. By automatically detecting cells’ nuclei over three-dimensional tissue volumes and quantifying relevant parameters (e.g. electron density, nuclear morphology), this instrument has the potential to provide novel capabilities in biomedicine linking volumetric tissue architecture with cell-scale quantification.