In recent years, the tissue optical clearing (OC) technique in the biomedicine field has drawn lots of attention. Various physical and chemical methods have been introduced to improve the efficacy of OC. In this study, the effect of the combination of glycerol and ultrasound treatment on OC of in vitro porcine muscle tissues has been investigated. The refractive index (RI) matching mechanism of OC was directly observed based on the derivative total reflection method. A theoretical model was used to simulate the proportion of tissue fluid in the illuminated area. Moreover, the total transmittance spectra have been obtained by a spectrometer over the range from 450 nm to 700 nm. The administration of glycerol and ultrasound has led to an increase of the RI of background medium and a more RI matching environment was achieved. The experimental results support the validity of the ultrasound treatment for OC. The RI matching mechanism has been firstly quantitatively analyzed based on the derivative total reflection method.

We analyze the existence of non-uniformity at the boundary of turbid media, and develop a gradient complex refractive index multilayered model in terms of this fact. Our model reveals the physics mechanism of the discrepancies between experimental data above the critical angle and the fitting curve with Fresnel's Formula. Also, from the perspective of the energy flow, reflectance R is obtained by the simplified models. We get complex refractive indexes and reflectance curves by fitting experimental data of 20% and 30% Intralipid solutions and rutile TiO₂ powder suspension with two different methods. Compared with Fresnel's Formula, our model can fit experimental data better.