## 3 Results and Discussion  
### Laser Irradiation and Thiazone Treatment-Induced Changes in Reflectance Spectra of Skin In Vivo  
Figure 2a-c show the typical reflectance spectra of a rat skin at native state after 0.5 J/cm\({}^{2}\) laser irradiation and 10 and 60 min after the followed topical application of PEG-400 with or without thiazone; Figure 2d shows the relative change of the reflectance in 60 min at 0.5 J/cm\({}^{2}\) with different treatment. No change in reflectance without laser irradiation is observed. Figure 2a indicates that the mono-treatment of thiazone slightly changes reflectance statistically. The mono-treatment of thiazone did not enable PEG-400 to exert its optical clearing effect without laser irradiation. In Figure 2b,c compared with the reflectance spectrum of native skin, the decrease in reflectance after laser irradiation were 2.5%, 0.8%, and 1.4% at 475, 575, and 615 nm, respectively. After the laser irradiation, OCA is applied to the skin. The significant decrease is observed after OCA treatment. Figure 2c indicates that Nd:YAG laser irradiation reduces the reflectance of short wavelengths (400-600 nm), and the following treatment of PEG-400 with thiazone can further decrease the values by approximately 10% in 10 min and 25.2%, 23.7%, and 17.6% at 475, 575, and 615 nm wavelength in 60 min, respectively. Figure 2b indicates that the reflectance only decreases by 17.9%, 20.8%, and 13.8% without thiazone. Compared with mono-treatment of laser irradiation, combined penetration enhancement makes the penetration rate of the OCA fast, and the final optical clearing efficacy is improved. Combined Treatment of Different Doses of Laser Irradiation and Thiazone-Induced Changes in Reflectance Spectra of Skin In Vivo  
Figure 3a,b show the effect of laser irradiation dose on the combined penetration enhancement; Figure 3c,d show the relative change of the reflectance in 60 min at 0.7 and 0.9 J/cm\({}^{2}\) with different treatment. As shown in Figure 3a, when the laser irradiation dose increases to 0.7 J/cm\({}^{2}\), the decrease in reflectance after laser irradiation were 5.2%, 5.1%, and 7.0% at 475, 575, and 615 nm, respectively. The combined penetration enhancement method achieves the best optical clearing effect at 10 min (the green solid line), and the reflectance of the skin decreases significantly, between 450-600 nm, by 30.9%, 28.7% and 28.2% at 475, 575, and 615 nm, respectively. By comparing the reflectance spectrum with the combined penetration enhancement method with that of the mono-treatment of laser irradiation at 10 min (the red solid line with square symbols), the penetration rate of the combined penetration enhancement method becomes increasingly fast. By comparing the combined penetration enhancement method with thiazone mono-treatment (the green solid line with triangle symbols), the thiazone mono-treatment cannot achieve any optical clearing effect. This effect is similar to the situation at 0.5 J/cm\({}^{2}\) laser irradiation dose. The situation is different when the laser irradiation dose is further increased to 0.9 J/cm\({}^{2}\). Figure 3b indicates that elevated laser dose causes more significant changes in reflectance, and further treatment of PEG-400 with thiazone cannot reduce the reflectance. Figure 2: Reflectance spectra of in vivo rat skin with (**a**) mono-treatment of thiazone without laser irradiation, (**b**) mono-treatment of laser irradiation at 0.5 J/cm\({}^{2}\) without thiazone, (**c**) combined treatment at 0.5 J/cm\({}^{2}\). (**d**) The relative change of the reflectance in 60 min at 0.5 J/cm\({}^{2}\) with different treatment, * \(p<0.05\); ** \(p<0.01\). Optical clearing effect is almost ineffective statistically, but the reflectance of the skin decreases more significantly after laser irradiation than other doses, especially at 575 nm with an 8.9% decrease. Though increased irradiation dose decreases the reflectance in short wavelengths, the reflectance is not further reduced after the treatment of the OCA.