We often use contrast-enhanced micro-CT imaging to peer inside biological tissues like the tendon-to-bone interface, leveraging agents such as iodine, PTA, or mercury chloride to improve visibility. But we realized that we knew little about how these contrast agents might be altering the tissue itself, aspects like thickness, mechanical properties, and critical microstructures were overlooked.

Could these agents be reshaping the very structures we hoped to observe? We aimed to systematically assess how four commonly-used staining solutions (Iodine in DMEM, PTA in water, PTA in ethanol, and HgCl₂ in water) affect not only image quality but also the micro- and nano-mechanical integrity of the murine Achilles tendon-to-calcaneus interface. We combined high-resolution in situ micro-CT, digital volume correlation, nanoindentation, and structural analysis to gain a comprehensive view

Key Findings: All contrast agents significantly altered mechanical behaviour and structural features of the enthesis. No contrast agent perfectly preserved both tissue structure and mechanical integrity. Among the four Iodine in DMEM offered the best compromise between clear visualization and preservation of mechanical properties.

"Why it matters"

With structural-function relationships at the heart of biomechanics, understanding how imaging preparations skew observations is vital. Researchers relying on contrast-enhanced imaging for strain analysis, tissue modelling, or mechanobiology must know the trade-offs. Our work makes clear which agents preserve fidelity, and which introduce bias.

The research underscores the need for methodological rigor and critical evaluation of sample preparation protocols in biomechanical imaging. We hope this study inspires the development of improved staining protocols and agents that enhance visibility while minimally altering tissue microarchitecture.