Hydrogen bound water profiles in the skin influenced by optical clearing molecular agents-Quantitative analysis using confocal Raman microscopy.

Confocal Raman microscopy has been used to measure depth-dependent profiles of porcine skin ex vivo in the high wavenumber region after application of molecular optical clearing agents (OCAs). Glycerol (70%) and iohexol (100% Omnipaque [300]) water solutions were used as OCAs and topically applied to porcine ear skin for 30 and 60 minutes. Using Gaussian function-based deconvolution, the changes of hydrogen bound water molecule types have been microscopically analyzed down to the depth of 200 μm. Results show that both OCAs induced skin dehydration (reduction of total water), which is 51.3% for glycerol (60 minutes), 33.1% for glycerol (30 minutes), 8.3% for Omnipaque (60 minutes) and 4.4% for Omnipaque (30 minutes), on average for the 40 to 200 μm depths. Among the water types in the skin, the following reduction was observed in concentration of weakly bound (51.1%, 33.2%, 7.5% and 4.6%), strongly bound (50.4%, 33.0%, 7.9% and 3.4%), tightly bound (63.6%, 42.3%, 26.1% and 12.9%) and unbound (55.4%, 28.7%, 10.1% and 5.9%) water types on average for the 40 to 200 μm depths, post application of glycerol (60 minutes), glycerol (30 minutes), Omnipaque (60 minutes) and Omnipaque (30 minutes), respectively. As most concentrated in the skin, weakly and strongly bound water types are preferentially involved in the OCA-induced water flux in the skin, and thus, are responsible for optical clearing efficiency.