Magnetic resonance and X-ray contrast agents in the optical clearing of dermis - insights from ex vivo confocal Raman micro-spectroscopy.

Confocal Raman micro-spectroscopy (CRM) has developed into a versatile tool that is frequently used to determine skin penetration of various compounds. Topically applied optical clearing agents decrease skin scattering, improving CRM imaging abilities. The use of magnetic resonance (MR) and X-ray contrast agents to increase transparency of biological objects has attracted more attention recently. By analysing Raman bands at 1003 and 1663 cm-1 and applying Fick's second law model, we calculated the diffusion coefficients of 3.51 × 10-8 cm2/s for Gadovist and 3.20 × 10-7 cm2/s for Omnipaque, with a slower diffusion regime for Dotarem. These diffusion kinetics directly governed penetration depth profiles across the investigated 0-240 µm dermal region and dictated optical clearing efficiency. Gadovist yielded the strongest short-term (30 min, up to 52% signal increase), whereas Omnipaque reached the highest and most depth-uniform optical clearing (60 min), consistent with its one-order-of-magnitude higher diffusivity. In contrast, Dotarem exhibited limited penetration and negligible optical clearing. Autofluorescence enhancement scaled with diffusion-driven refractive index matching, confirming that optical clearing is mechanistically coupled to molecular transport; it may also arise from protein structural modifications, oxidative stress, or increased protein density following contrast agent interaction. Together, these findings establish a quantitative link between contrast-agent diffusivity, dermal penetration depth, optical transparency, and tissue autofluorescence, providing a framework for integrating Raman, MR, and X-ray modalities in multimodal depth-resolved skin imaging.