With increasing interest in studying biological systems across spatial scales—from centimetres down to nanometres—histology continues to be the gold standard for tissue imaging at cellular resolution, providing an essential bridge between macroscopic and nanoscopic analysis. However, its inherently destructive and two-dimensional nature limits its ability to capture the full three-dimensional complexity of tissue architecture. Here we show that phase-contrast X-ray microscopy can enable three-dimensional virtual histology with subcellular resolution. This technique provides direct quantification of electron density without restrictive assumptions, allowing for direct characterisation of cellular nuclei in a standard laboratory setting. By combining high spatial resolution and soft tissue contrast, with automated segmentation of cell nuclei, we demonstrated virtual H&E staining using machine learning-based style transfer, yielding volumetric datasets compatible with existing histopathological analysis tools. Furthermore, by integrating electron density and the sensitivity to nanometric features of the dark field contrast channel, we achieve stain-free, high-content imaging capable of distinguishing nuclei and extracellular matrix.

JDF is a Rigaku employee and AEV was a Rigaku intern at the time this research was carried out; Rigaku has a potential interest in the commercial exploitation of the results presented in this article. OM and C-MT are Creatv Microtech employees; Creatv Microtech fabricated the X-ray masks used in this work and has a potential interest in their commercialization. MEn and AO are named inventors on patents held by UCL protecting some of the imaging technology discussed in this paper.

Research reported in this publication was supported by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Number R01EB028829. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Mask fabrication performed at the Center for Nanoscale Materials, a U.S. Department of Energy Office of Science User Facility, was supported by the U.S. DOE, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. AO was supported by the Royal Academy of Engineering under the Chairs in Emerging Technologies scheme (CiET1819/2/78) nd PDL via (CiET1819/10). YZ/PDL/CLW were supported in part by CZI grant DAF2022-316777.

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The IRB of Memorial Sloan Kettering Cancer Center( New York, USA) waived ethical approval for this work as it only used discarded pathology tissue

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