Abstract Three dimensional (3D) geometrical models are not only a powerful tool for quantitatively characterizing complex tissues but also useful for probing structure-function relationships in a tissue. However, these models are generally incomplete due to experimental limitations in acquiring multiple (&gt;4) fluorescent channels simultaneously. Indeed, predictive geometrical and functional models of the liver have been restricted to few tissue and cellular components, excluding important cellular populations such as hepatic stellate cells (HSCs) and Kupffer cells (KCs). Here, we performed deep-tissue immunostaining, multiphoton microscopy, deeplearning techniques, and 3D image processing to computationally expand the number of simultaneously reconstructed tissue structures. We then generated a spatio-temporal singlecell atlas of hepatic architecture (Hep3D), including all main tissue and cellular components at different stages of post-natal development in mice. We used Hep3D to quantitatively study 1) hepatic morphodynamics from early post-natal development to adulthood, and 2) the structural role of KCs in the murine liver homeostasis. In addition to a complete description of bile canaliculi and sinusoidal network remodeling, our analysis uncovered unexpected spatiotemporal patterns of non-parenchymal cells and hepatocytes differing in size, number of nuclei, and DNA content. Surprisingly, we found that the specific depletion of KCs alters the number and morphology of the HSCs. These findings reveal novel characteristics of liver heterogeneity and have important implications for both the structural organization of liver tissue and its function. Our next-gen 3D single-cell atlas is a powerful tool to understand liver tissue architecture, under both physiological and pathological conditions.

Hep3D: A 3D single-cell digital atlas of the liver to study spatio-temporal tissue architecture