DOE OSTI · 3375070
Three-dimensional high-content imaging of unstained soft tissue with subcellular resolution using a laboratory-based X-ray microscope
Abstract
With increasing interest in studying biological systems across spatial scales—from centimeters down to nanometers—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 characterization 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 Hematoxylin and Eosin (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.
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Esposito, Michela [Univ. College London (United Kingdom)] (ORCID:0000000268271240), Astolfo, Alberto [Univ. College London (United Kingdom)], Zhou, Yang [Univ. College London (United Kingdom)] (ORCID:0000000154749605), Buchanan, Ian [Univ. College London (United Kingdom)] (ORCID:0000000213086451), Teplov, Alexei [Memorial Sloan Kettering Cancer Center, New York, NY (United States)] (ORCID:0000000348264518), Hutchinson, John Ciaran [Great Ormond Street Hospital for Children National Health Service Foundation Trust, London (United Kingdom)] (ORCID:0000000217082634), Endrizzi, Marco [Univ. College London (United Kingdom); The Francis Crick Institute, London (United Kingdom)] (ORCID:0000000278102301), Vinogradova, Alexandra Egido [Rigaku Americas, The Woodlands, TX (United States)], Makarova, Olga [Creatv MicroTech Inc., Chicago, IL (United States)], Divan, Ralu [Argonne National Laboratory (ANL), Lemont, IL (United States). Center for Nanoscale Materials (CNM)], Tang, Cha-Mei [Creatv MicroTech Inc., Chicago, IL (United States)], Yagi, Yukako [Memorial Sloan Kettering Cancer Center, New York, NY (United States)], Lee, Peter D. [Univ. College London (United Kingdom)] (ORCID:0000000238988881), Walsh, Claire L. [Univ. College London (United Kingdom)], Ferrara, Joseph D. [Rigaku Americas, The Woodlands, TX (United States)] (ORCID:000000018534642X), Olivo, Alessandro [Univ. College London (United Kingdom)] (ORCID:0000000271502951). 2026-03-17. Three-dimensional high-content imaging of unstained soft tissue with subcellular resolution using a laboratory-based X-ray microscope. https://doi.org/10.1073/pnas.2525239123
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