Ex-vivo confocal Raman microspectroscopy of porcine skin with 633/785-NM laser excitation and optical clearing with glycerol/water/DMSO solution

Confocal Raman microspectroscopy (CRM) with 633- and 785-nm excitation wavelengths combined with optical clearing (OC) technique was used for ex-vivo study of porcine skin in the Raman fingerprint region. The optical clearing has been performed on the skin samples by applying a mixture of glycerol and distilled water and a mixture of glycerol, distilled water and chemical penetration enhancer dimethyl sulfoxide (DMSO) during 30 min and 60 min of treatment. It was shown that the combined use of the optical clearing technique and CRM at 633nm allowed one to preserve the high probing depth, signal-to-noise ratio and spectral resolution simultaneously. Comparing the effect of different optical clearing agents on porcine skin showed that an optical clearing agent containing chemical penetration enhancer provides higher optical clearing effciency. Also, an increase in treatment time allows to improve the optical clearing effciency of both optical clearing agents. As a result of optical clearing, the detection of the amide-III spectral region indicating well-distinguishable structural differences between the type-I and type-IV collagens has been improved.