Quantitative Stain Mapping in X‐Ray Virtual Histology

Virtual histology using X-ray micro-computed tomography offers 3D tissue visualization, yet lacks the specificity of conventional histology, where targeted stains selectively highlight features like cell nuclei. While X-ray-compatible stains have now been developed, an unsolved challenge is the quantitative separation of their signal from the underlying tissue signal, which is essential for tissue-specific imaging. This work presents the first method enabling 3D stain mapping on a histologically relevant scale. Applied to murine kidneys stained with different hematein-lead complexes, the approach extracts molar contrast agent distributions alongside high-contrast morphology at the micrometer scale and is successfully validated against K-edge subtraction imaging. Moreover, the dual optical-X-ray properties of the stain enable direct spatial correspondence between X-ray-derived concentration maps and conventional optical histology of the same specimen, establishing a bridge between virtual and traditional histology. In summary, the proposed methodology provides tissue-specific virtual histology with objective, quantitative metrics across millimeter to centimeter-sized 3D volumes, opening pathways for immunospecific labeling and automated analysis of disease progression without physical sectioning.