Supplementary Material for: Biophysical Alterations of the Stratum Corneum Induced by Optical Clearing Agents: Implications for Efficiency and Safety Optimization

Introduction. Optical clearing (OC) of biological objects is a promising technique with broad potential in clinical and diagnostic applications, as it enhances imaging depth, spatial resolution, and contrast of acquired images or spectra. The aim of this study was to study the effects of the common optical clearing agents (OCAs) glycerol, fructose, dimethyl sulfoxide (DMSO) and iohexol (Omnipaque®) solutions at various concentrations and treatment durations on the stratum corneum (SC) of intact skin using confocal Raman microspectroscopy (CRM). Methods. The pig ear was selected for study. Various OCAs were applied topically on the skin, including water solutions of DMSO (100%), fructose (50%), Omnipaque® (90% and 100%), glycerol (40%, 60%, 80%, 90% and 100%). Ex vivo measurements were carried out on porcine ear skin using a Model 3510 SCA confocal Raman microscope (RiverD International B.V., Rotterdam, the Netherlands). Results. It was found that a 25%-25%-50% water-ethanol-fructose solution permeated the SC at all tested exposure times (5, 15, 30, and 45 min). Similarly, a mixture of 80% glycerol and 20% DMSO, applied for 45 min, also demonstrated effective penetration through the SC. Enhanced OC was achieved by combining OCAs with DMSO, ethanol, or distilled water. The highest OC efficiency was observed with a 25%-25%-50% water-ethanol-fructose solution and a mixture of 90% Omnipaque® with 10% DMSO, both applied for 5 minutes, resulting in a 4.1- and 4.2-fold increase, respectively, in signal intensity at a skin depth of 80 µm. It was shown that the optical properties of the skin can be controlled at a depth of approx. 80 μm. Almost all used OCAs, which are not occlusive from the conventional viewpoint, have an occlusion effect on the SC. Conclusion. The findings of this study contribute to the identification of optimal OCA combinations – namely, a 25%-25%-50% water-ethanol-fructose solution and a mixture of 90% Omnipaque® with 10% DMSO, each applied for 5 minutes – for potential in vivo applications. Moreover, the results provide a strong foundation for future multimodal investigations aimed at developing advanced methods for visualizing biological tissues and organs and analyzing their biophysical parameters, in conjunction with controlled OC.