Skeletal muscle dispersion (400-1000 nm) and kinetics at optical clearing.

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.

Diffusion characteristics of ethylene glycol in skeletal muscle.

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 mechanisms­tissue 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.