We introduce and validate a framework for imaging and quantifying active molecule penetration into human skin in vivo. Our approach combines nonlinear imaging microscopy modalities, such as two-photon excited auto-fluorescence (TPEF) and coherent anti-Stokes Raman scattering (CARS), together with the use of deuterated active molecules. The imaging framework is exemplified on topically applied glycerol diluted in various vehicles such as water and xanthan gel. In vivo glycerol quantitative percutaneous penetration over time is demonstrated, showing that, contrary to water, xanthan gel vehicle acts as a film reservoir that releases glycerol continuously over time.
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