Abstract
The study of agent diffusion in biological tissues is very important to understand and characterize the optical clearing effects and mechanisms involved: tissue dehydration and refractive index matching. From measurements made to study the optical clearing, it is obvious that light scattering is reduced and that the optical properties of the tissue are controlled in the process. On the other hand, optical measurements do not allow direct determination of the diffusion properties of the agent in the tissue and some calculations are necessary to estimate those properties. This fact is imposed by the occurrence of two fluxes at optical clearing: water typically directed out of and agent directed into the tissue. When the water content in the immersion solution is approximately the same as the free water content of the tissue, a balance is established for water and the agent flux dominates. To prove this concept experimentally, we have measured the collimated transmittance of skeletal muscle samples under treatment with aqueous solutions containing different concentrations of glucose. After estimating the mean diffusion time values for each of the treatments we have represented those values as a function of glucose concentration in solution. Such a representation presents a maximum diffusion time for a water content in solution equal to the tissue free water content. Such a maximum represents the real diffusion time of glucose in the muscle and with this value we could calculate the corresponding diffusion coefficient.

Abstract
Optical immersion clearing is a technique that has been widely studied for more than two decades and that is used to originate a temporary transparency effect in biological tissues. If applied in cooperation with clinical methods it provides optimization of diagnosis and treatment procedures. This technique turns biological tissues more transparent through two main mechanisms - tissue dehydration and refractive index (RI) matching between tissue components. Such matching is obtained by partial replacement of interstitial water by a biocompatible agent that presents higher RI and it can be completely reversible by natural rehydration in vivo or by assisted rehydration in ex vivo tissues. Experimental data to characterize and discriminate between the two mechanisms and to find new ones are necessary. Using a simple method, based on collimated transmittance and thickness measurements made from muscle samples under treatment, we have estimated the diffusion properties of glucose, ethylene glycol (EG) and water that were used to perform such characterization and discrimination. Comparing these properties with data from literature that characterize their diffusion in water we have observed that muscle cell membrane permeability limits agent and water diffusion in the muscle. The same experimental data has allowed to calculate the optical clearing (OC) efficiency and make an interpretation of the internal changes that occurred in muscle during the treatments. The same methodology can now be used to perform similar studies with other agents and in other tissues in order to solve engineering problems at design of inexpensive and robust technologies for a considerable improvement of optical tomographic techniques with better contrast and in-depth imaging.

Abstract
To determine the differences between the optical clearing effects created by ethylene glycol in fresh and frozen samples, we have performed several measurements from samples in both conditions. Fresh samples were used after animal sacrifice and frozen samples were kept at -20 degrees C for 72 hours. The different measurements performed with samples from both cases were total transmittance, collimated transmittance, total reflectance and specular reflectance. Considering, for instance, collimated transmittance measurements, we have verified that the spectra measured from both samples before adding the solution present different levels of collimated transmittance. The time-dependence evolution of the collimated transmittance spectrum is similar between both cases of samples, but since they present different levels of "natural" transmittance, the optical clearing effect is observed at different levels if we compare between fresh and frozen samples.

Abstract

Abstract
Part of the optical clearing study in biological tissues concerns the determination of the diffusion characteristics of water and optical clearing agents in the subject tissue. Such information is sufficient to characterize the time dependence of the optical clearing mechanisms-tissue dehydration and refractive index (RI) matching. We have used a simple method based on collimated optical transmittance measurements made from muscle samples under treatment with aqueous solutions containing different concentrations of ethylene glycol (EG), to determine the diffusion time values of water and EG in skeletal muscle. By representing the estimated mean diffusion time values from each treatment as a function of agent concentration in solution, we could identify the real diffusion times for water and agent. These values allowed for the calculation of the correspondent diffusion coefficients for those fluids. With these results, we have demonstrated that the dehydration mechanism is the one that dominates optical clearing in the first minute of treatment, while the RI matching takes over the optical clearing operations after that and remains for a longer time of treatment up to about 10 min, as we could see for EG and thin tissue samples of 0.5 mm. (C) 2015 Society of Photo-Optical Instrumentation Engineers (SPIE)

Abstract
Skeletal muscle dispersion and optical clearing (OC) kinetics were studied experimentally to prove the existence of the refractive index (RI) matching mechanism of OC. Sample thickness and collimated transmittance spectra were measured during treatments with glucose (40%) and ethylene glycol (EG; 99%) solutions and used to obtain the time dependence of the RI of tissue fluids based on the proposed theoretical model. Calculated results demonstrated an increase of RI of tissue fluids and consequently proved the occurrence of the RI matching mechanism. The RI increase was observed for the wavelength range between 400 and 1000 nm and for the 2 probing molecules explored. We found that for 30 min treatment with 40% glucose and 99% EG, RI of sarcoplasm plus interstitial fluid was increased at 800 nm from 1.328 to 1.348 and from 1.328 to 1.369, respectively.