Quantitative Stain Mapping in X‐Ray Virtual Histology

Abstract 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.