Simultaneous spatial and spectroscopic characterization of native and biomimetic skin systems by hyperspectral Stimulated Raman Scattering (SRS) microscopy

Understanding molecular organization across complex biological interfaces is essential for evaluating biomimetic tissues and identifying pathological remodeling. Here, hyperspectral stimulated Raman scattering (SRS) microscopy combined with spectral deconvolution was employed for label-free characterization of native human skin, reconstructed three-dimensional (3D) skin models, and melanoma skin model. Layer-specific analysis across the stratum corneum, epidermis, and dermis enabled investigation of biochemical organization and compositional heterogeneity. In the stratum corneum, variations in the C–H stretching region revealed differences in lipid chain ordering and lipid-to-protein balance, with native human skin exhibiting higher lipid ordering and greater compositional homogeneity than reconstructed models. Amide I analysis demonstrated altered keratin-associated secondary structure organization in reconstructed skin systems, including increased β-sheet and disordered spectral contributions relative to native tissue. Melanoma-associated skin models exhibited enhanced nucleic-acid-related spectral contributions together with reduced collagen-associated signals in the dermal compartment, consistent with extracellular matrix remodeling. These findings demonstrate that hyperspectral SRS microscopy combined with spectral deconvolution provides a sensitive and label-free platform for molecular-level characterization of complex skin biointerfaces and comparative evaluation of native, engineered, and pathological skin systems.