## 2 Materials and Methods  
### Experiments In Vivo  
All experimental protocols and animal handling procedures were approved based on the comments of the laboratory animal care committee of Xi'an Jiaotong University Health Science Center. The corresponding ethical approval code is NO.XJTULAC2018-540. As shown in Figure 1, VIS-NIR spectrometer was used to monitor the changes in reflectance spectra of the rat skin to study the enhancement of optical clearing effect by laser irradiation in conjunction with a chemical penetration enhancer. The system was composed of the light source (SL-1), reflection probe (R400-7), spectrometer (USB-2000, Ocean Optics, USA), and the computer software Spectra Suit. The probe consisted of a bundle of seven optical fibers with six illumination fibers around one read fiber, each of which was 400 mm in diameter. The core-to-core distance between the illumination fiber and the read fiber was 487 mm. Before the experiment, the system was preheated for approximately 30 min and then standardized by a reflectance standard made up of barium sulfate. During the experiment, the probe was vertical and soft touched to in vivo dorsal skin, and the reflectance was then saved. The average value of each measurement was determined three times. In this in vivo experiment, a total of 36 female Sprague-Dawley rats with 100 g to 120 g body weight were obtained from Xi'an Jiaotong University Health Science Center and fed under standard rodent feeding condition. Before the experiment, animals were intraperitoneally anesthetized using 10% chloral hydrate. The dorsal hair was shaved, and the residue hair was removed using 8% NaS applied for 1-2 min until the hair could be easily wiped off. The back of the rats was then cleaned with water. Measurements were delayed after 15 min of hair removal to dry the skin. To study the effect of the chemical penetration enhancer, we prepared the OCA as follows. First, thiazone (99% purity, Wuhan Hengwo Technology Co., Ltd., China) was heated and melted in a 40 \({}^{\circ}\)C water bath. The chemical reagent PEG-400 (analytical reagent, Tianjin Kermel Chemical Reagent Co., Ltd., China) was then mixed with melting thiazone at a volume ratio of 9:1. PEG400 is a high refractive reagent with a refractive index of 1.47, which is similar to the refractive index of collagen fibers in the skin, and has amphipathic molecules. Thiazone is a new type of chemical penetration enhancer that can damage the SC to increase skin permeability. Thiazone is a white crystal at room temperature with a melting point of 40 \({}^{\circ}\)C and a refractive index of 1.576. Q-switched 1064 nm Nd:YAG laser (WON-COSJET TR, WON Technology Co., Ltd., Korea) was adopted for the investigation of conjunction effect with thiazone, and the parameters were set, as shown in Table 1.  
The specific steps of the experiment were as follows. First, the left side of the back skin of the rats was the experimental group, and the right side as the control and both were divided into three areas. The original reflectance spectra of six areas of the rat skin were then measured for reference. Second, the reflectance spectra of the skin were measured in the left experimental area after laser treatment, and then the OCA was applied to the three parts for 10 min, after that the agent was wiped off to eliminate \begin{table}
\begin{tabular}{c c} \hline \hline
**Parameter** & **Value** \\ \hline Wavelength/nm & 1064 \\ Pulse width/ns & 6 \\ Speckle/mm & 2.5\({}^{2}\)\(\times\)\(\pi\) \\ Irradiation dose/J/cm\({}^{2}\) & 0.5, 0.7, 0.9 \\ Pulse number \(n_{p}\) & 60 \\ Frequency/Hz & 2 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Laser parameters. Figure 1: Reflection spectrum measurement system diagram. the specular reflection, measured for a total of 6 times for 60 min. The control area on the right side was treated with non-laser treatment or without the addition of thiazone in PEG-400. Each different experiment was repeated at least 6 times in 6 different rats.