Evaluating non-destructive X-ray-based 3D histology using contrast-enhanced micro-CT to visualise and quantify...

Visualising and quantifying vascular 3D microstructure are essential for linking it to mechanics and disease. This study evaluated Phospho-Tungstic Acid (PTA) and 1:2 Hafnium-substituted Wells Dawson Polyoxometalate (1:2 Hf-POM) as Contrast-Enhancing Staining Agents (CESAs) for X-ray-based 3D Histology (X3DH) using Contrast-Enhanced Micro-focus Computed Tomography (CECT), as alternatives to classical 2D histology for visualising and quantifying collagen and elastin. Native and enzymatically digested porcine carotid artery segments, with varying elastin and collagen content, were CESA-stained and μCT-imaged to generate X3DH data, used to calculate Elastin-Stained Volume Fraction (E-SVF). Equivalent CESA-stained and unstained sections underwent Verhoeff's (VH), Picrosirius Red (PR), and Haematoxylin-Eosin (H&E) histology to measure Elastin-Stained Area Fraction (E-SAF), Collagen-SAF (C-SAF), and cell density. X3DH results were compared with histology to evaluate correspondence and staining effects. Both CESAs produced strong signal colocalisation with elastic lamellae and weaker staining in the remaining ECM. E-SVF for both CESAs correlated strongly with E-SAF, but E-SVF values were about two times lower than E-SAF (r²_PTA=0.86, p < 0.0001, slope_PTA=0.42 | r²_1:2Hf-POM=0.86, p < 0.0001, slope_1:2Hf-POM=0.49). This highlights CECT as a non-destructive, accurate method for quantifying vascular microstructure with fewer artefacts than classical 2D histology. VH and PR staining were visually and quantitatively (±10% E-SAF and C-SAF equivalence) unaffected by CESA-staining. PTA-staining with cryopreservation caused loss of nucleic contrast, while 1:2 Hf-POM influenced ECM haematoxylin uptake, reducing nuclei visualisation and cell count accuracy in H&E histology. Both CESAs bind non-specifically but preferentially to elastin, enabling reliable quantification. However, vascular collagen remains difficult to visualise as ECM is non-specifically stained. STATEMENT OF SIGNIFICANCE: Understanding the three-dimensional microstructure of vascular tissues is crucial for revealing how it governs mechanics and disease progression. This study investigates two contrast agents, phospho-tungstic acid and 1:2 hafnium-substituted Wells-Dawson polyoxometalate, for their potential to visualise and quantify two of the key constituents within arterial tissue, collagen and elastin, using Contrast-Enhanced Micro-focus Computed Tomography (CECT). By assessing these agents combined with CECT as alternatives to gold-standard histological techniques for the visualisation and quantification of these key extracellular matrix components, this research advances CECT as a non-destructive alternative to traditional histological techniques. Improved three-dimensional imaging of vascular architecture deepens our understanding of arterial structure-function relationships and supports more accurate computational modelling, disease characterisation, and the development of next-generation cardiovascular devices.