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Recent studies have demonstrated that topical application of glycerol on intact skin does not affect its optical scattering properties. Investigators from our research group recently revisited the use of dimethyl sulfoxide (DMSO) as an agent with optical clearing potential. We address the use of optical clearing to enhance quantitation of subsurface fluorescence emission. We employed both in vitro and in vivo model systems to study the effect of topical DMSO application on fluorescence emission. Our in vitro experiments performed on a tissue-simulating phantom suggest that DMSO-mediated optical clearing enables enhanced characterization of subsurface fluorophores. With topical DMSO application, a marked increase in fluorescence emission was observed. After 30 min, the fluorescence signal at the DMSO-treated site was 9× greater than the contralateral saline-treated site. This ratio increased to 13× at 105 min after agent application. In summary, DMSO is an effective optical clearing agent for improved fluorescence emission quantitation and warrants further study in preclinical in vivo studies. Based on outcomes from previous clinical studies on the toxicity profile of DMSO, we postulate that clinical application of DMSO as an optical clearing agent, can be performed safely, although further study is warranted.

Laser Speckle Contrast Imaging (LSCI) plays an important role in studying blood flow, but suffers from limited penetration depth of light in turbid tissue. The strong scattering of tissue obviously reduces the image contrast which decreases the sensitivity to flow velocity. Some image processing or optical clearing methods have been proposed to lessen the deficiency, but quantitative assessment of improvement is seldom given. In this study, LSCI was applied to monitor the blood flow through a capillary embedded within various tissue phantoms at depths of 0.25, 0.45, 0.65, 0.85 and 1.05 mm, and the flow velocity in capillary was controllable from 0 to 4 mm/s. Here, glycerol, a common optical clearing agent, was mixed with Intralipid at different volume ratio to make the reduced scattering coefficient of tissue phantom decrease from 13.00 to 0.50 cm^-1. The quantitative analysis demonstrates that the optical clearing method can obviously enhance the image contrast, imaging depth, and sensitivity to blood flow velocity. Comparing the Laser Speckle Contrast Analysis methods and the optical clearing method, we find that for typical turbid tissue, the sensitivity to velocity estimated by the Laser Speckle Temporal Contrast Analysis (LSTCA) is twice of that by the Laser Speckle Spatial Contrast Analysis (LSSCA); while the sensitivity to velocity estimated by using the two analysis methods has a 10-fold increase, respectively, if addition of glycerol makes the reduced scattering coefficient of tissue phantom decrease by 30%. Combining the LSTCA and the optical clearing method, the sensitivity to flow velocity will be further enhanced.

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 30min and 60min 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 efficiency. Also, an increase in treatment time allows to improve the optical clearing efficiency 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.