1. COMPARISON OF DIFFERENT REGISTRATION METHODS OF OPTICAL CLEARING OF MUSCLE TISSUE
Marina D. Kozintseva1, Vyacheslav I. Kochubey1, Alexey N. Bashkatov1, Elina A. Genina1, Valery V. Tuchin1,2,3   1 Saratov State University, Department of Optics and Biophotonics, 83, Astrakhanskaya Street, Saratov , Russia 2 Institute of Precise Mechanics and Control of RAS, 24, Rabochaya Street, Saratov , Russia 3 University of Oulu, P.O. Box 4500, Oulu 90014, Finland

2. Slide master Motivation
The possibility of selective translucence of the superficial tissue layers is very useful in developing functional imaging and therapeutic techniques. A potential benefit of the optical clearing is the improvement of laser therapeutic techniques that enable sufficient light penetration to a target embedded in tissue. Combination of optical clearing with laser radiation can reduce the laser fluences required for a therapeutic effect. However, in spite of numerous investigations deal with transport of glucose solution within biological tissue the problem of estimating the diffusion coefficient of the agent in muscle has not been studied in details.
There are several methods of diffusion coefficient measurement. But it is necessary to find the most simple and effective method.
Goal of the study is to compare two methods of measurement the diffusion coefficient of 40% glucose solution into muscle tissue in vitro: OCT and method of Collimated Transmittance.

3. The comparison of OCT and method of Collimated Transmittance1
OCT 2
Collimated Transmittance 3
Results and discussion, conclusion

4. 1. Materials and methods 1. OCT
For this study ten beef muscle tissue samples have been used. All the tissue samples were cut into pieces with the area about 20˟15 mm2. For mechanical support, the tissue samples were placed in bath where the aqueous 40% glucose solution was flowed in.
Experiments were provided with the Spectral Radar OCT system (THORLABS, USA). All measurements were performed at room temperature (about 20°C). The light source used in this system is a low-coherence broadband superluminescent diode (SLD) working at the central wavelength 930 nm.
As a clearing agent 40% glucose solution was used. Refractive index of the solution is 1,391. It has been measured using Abbe refractometer at wavelength 589 nm

5. 1. OCT
Method for determination of glucose diffusion coefficient
where ∆z = |z1 – z2|
Figure 1. Estimation of the total attenuation coefficient μt of the tissue on the different depths: the black line describes the signal at the beginning (0 sec) of the experiment, the red line – on the 600 sec, the green line – on the 5400 sec.

6. Results: glucose diffusion
1. OCT
Figure 2. B-scan of intact muscle tissue sample in vitro
Figure 3. B-scan of muscle tissue sample in vitro treated by 40% glucose solution

7. 2. Materials and methods OCT
For this study ten fresh beef muscle tissue samples have been used. All the tissue samples were cut into pieces with the area about 20˟15 mm2. For mechanical support, the tissue samples were placed in bath where the aqueous 40% glucose solution was flowed in.
Measurement of collimated transmittance have been performed using a commercially available spectrometer USB-4000 (Ocean Optics, USA) in the spectral range nm. All measurements were performed at room temperature (about 20°C), all calculations were done for samples at the wavelength 930 nm.
As a clearing agent 40% glucose solution was used. Refractive index of the solution is 1,391. It has been measured using Abbe refractometer at wavelength 589 nm

8. 2. Collimated Transmittance
Method for determination of glucose diffusion coefficient
Figure 4. The time-dependent transmittance of muscle measured at different wavelength concurrently with administration of glucose solution
Figure 5. The transmittance spectra of muscle measured concurrently with administration of glucose solution at different time intervals

9. 2. Materials and methods OCT
Determination of glucose concentration within muscle tissue has been performed using the assumption of one-dimensional diffusion equation :
where С(x,t) is the concentration of glucose within tissue sample, g/ml; D is the diffusion coefficient, cm2/sec; x is a spatial coordinate, см; t is the time of glucose diffusion in muscle tissue, sec.
The dependence of the collimated transmittance of muscle, that was placed in the hyperosmotic immersion solution, takes the form:
where μa is the absorption coefficient of muscle tissue; µs is the scattering coefficient.
The "complex" method was used to minimize the objective function:
where N is the total number of data points obtained during the registration of temporal dependence of collimated transmittance with the fixed wavelength; Tc (D, t) is the calculated transmittance value, when the value of D is given; Tc * (t) is the experimentally measured transmittance value.

10. 3. The comparison of OCT and method of Collimated Transmittance1
OCT
Averaged thickness: 1,86±0,18 mm2
Averaged diffusion coefficient of 40% glucose solution into muscle tissue: <D> = (2,98±0,94)10-6 cm2/sec 2
Collimated Transmittance
Averaged thickness 1,9±0,1 mm2
Averaged diffusion coefficient of 40% glucose solution into muscle tissue <D> = (2,07±0,14)10-7 cm2/sec

11. 3. Results and discussion, conclusion
The difference between the coefficients of two equal samples can be described by the different methods of diffusion coefficient measurement. During the optical clearing of tissue the diffusion of clearing agent into intracellular space occurs. In case of in vitro muscle tissue the bilateral diffusion takes place. So, the diffusion starts in the direction from the edge to the center. According to the experiments we know that the process of clearing agents diffusion into tissue takes about an hour. During the collimated transmittance measuring the whole sample thickness is involved. On the other hand during the OCT measurements occur at the edges of samples, where diffusion goes first. Thus, the diffusion coefficient numerical value measured by OCT method is high.
Conclusion:
Both methods are used for diffusion coefficient measurement, but the OCT method is a little more precise than the method of collimated transmittance, because it takes place where the clearing agent diffusion goes.