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Second-harmonic generation (SHG) imaging is combined with coherent anti-Stokes Raman scattering (CARS) microscopy to follow the process of optical clearing in human skin ex vivo using dimethyl sulfoxide (DMSO) as the optical clearing agent. SHG imaging revealed that DMSO introduces morphological changes to the collagen I matrix. By carefully measuring the dynamic tissue attenuation of the coherent nonlinear signal, using CARS reference signals during the clearing process, it is found that DMSO reduces the overall SHG response from dermal collagen. Evidence is provided for a role of DMSO in compromising the structure of collagen fibers, associated with a reduction of the tissue's scattering properties.

Following dimethyl sulfoxide skin optical clearing dynamics with quantitative nonlinear multimodal microscopy