1. Daria K. Tuchina, 1,2 Rui Shi, 1 Alexey N. Bashkatov, 2 Elina A. Genina, 2 Dan Zhu, 1 Qingming Luo, 1 Valery V. Tuchin 1-4 1 Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, China 2 Research-Educational Institute of Optics and Biophotonics, Saratov State University, Saratov, 410012, Russia 3 Laboratory of Laser Diagnostics of Technical and Living Systems, Institute of Precise Mechanics and Control RAS, Saratov, 410028, Russia 4 Optoelectronics and Measurement Techniques Laboratory, P.O. Box 4500, University of Oulu, FIN- 90014, Oulu, Finland Saratov Fall Meeting 2014

2. Optical methods of monitoring and treatments of tissue properties become more visible and widely applicable in medicine, biology and other fields of natural science. The limitation of the optical imaging techniques is mainly connected with strong light scattering in tissue. Optical clearing is a perspective technique for solution of this problem. The method is based on the interaction of tissue and immersion agent that causes dehydration of tissue and partial diffusion of an agent into tissue. Matching of refractive indices of tissue scatterers and their environment takes place. As a result scattering coefficient of tissue decreases and tissue becomes more transparent for light. In this study the experimental results of in vitro investigation of skin optical clearing by topical application of glucose solutions of different concentrations are presented. Saratov State University Department of Optics and Biophotonics Saratov Fall Meeting 2014

3. Saratov State University Department of Optics and Biophotonics Samples Two month white mice Balb/c with body weight of 20 – 25 g were used in the study. The skin samples were obtained ex vivo by autopsy, hairs were removed with depilatory cream, hypodermic fatty layer was removed from the samples. The area of the samples was about 8×15 mm 2. The thickness of each sample was measured by micrometer with a precision of 50 µm in 5 points before and after experiment. 30%-, 43%-, and 56%-aqueous glucose solutions were used in this study. Glucose solutions were prepared using glucose monohydrate powder (SCR, China) and distilled water. Refractive indices of solutions were measured by Abbe refractometer WAY-2S (Optics Ivymen Systems, Spain). Optical clearing agents Glucose concentration30%43%56% Refractive index at 589 nm1.3791.3981.418

4. Saratov State University Department of Optics and Biophotonics Saratov Fall Meeting 2014 The investigations had 3 steps: Measurement of: 1.Collimated transmittance 2.Thickness 3.Weight and area of skin samples in the course of optical clearing by glucose solutions 90 samples of mouse skin tissue have been used in this study: 30 samples for each step

5. The collimated transmittance of 30 skin samples were measured using USB4000-Vis-NIR spectrometer (Ocean Optics, USA) (1) concurrently with administration of the glucose solutions in the spectral range 400-1000 nm. Saratov State University Department of Optics and Biophotonics Saratov Fall Meeting 2014 The sample was fixed in the cuvette (2) filled up with glucose solution using the plastic plate with a square aperture of 5×5 mm 2. The cuvette with sample was placed between two optical fibers (3) with collimators 74-UV (Ocean Optics, USA) (4). Optical fibers (QP600-2-VIS-NIR, Ocean Optics, USA) had 600 μm core diameter. The halogen lamp (HL 2000) was used as a light source (5). The collimated transmittance was recorded every 15 sec during 7-9 min. The measurements were performed at room temperature about 20°C. Collimated transmittance measurement

6. Saratov State University Department of Optics and Biophotonics Saratov Fall Meeting 2014 Thickness of 30 skin samples was measured using confocal microscope (FV 1000, Olympus, Japan) at application of glucose solutions. The sample was placed on the glass substrate by the epidermis side and the thickness of intact sample was measured. Glucose solution was dripped on the sample to cover it. Sample thickness was measured every 20 - 30 sec during 8 min. Thickness measurement

7. Saratov State University Department of Optics and Biophotonics Saratov Fall Meeting 2014 Weight and area measurement Weight of 30 skin samples was measured before immersion in glucose solutions and during action of solutions using weight scale (PL203, Mettler Toledo, China) with the precision of 1 mg. Area of each sample was obtained by processing of successive digital images of the sample. The measurements of sample weight and digital images were made every 5 min during 15 min of immersion.

8. Saratov State University Department of Optics and Biophotonics Saratov Fall Meeting 2014 56%- glucose solution 43%- glucose solution30%-glucose solution τ=1.59 A=0.17 τ =1.25 A=0.32 Typical collimated transmittance kinetics of skin samples A is a constant, which characterizes the maximal degree, τ is the characteristic time τ=1.02 A=0.46

9. Glucose solution Number of samples Wavelength 500nm600 nm700 nm800 nm900 nm 30%104±0.93.3±0.62.9±0.52.6±0.42.38±0.1 43%105.6±2.84.1±1.73.5±1.43.2±1.22.9±1 56%56%1011.9±5.58.4±3.36.7±2.35.6±1.85±1.5 The degree (efficiency) of optical clearing of skin The degree (efficiency) of optical clearing of skin samples was evaluated as the ratio of the maximal transmittance (within the saturation region of the kinetic curve) to the initial transmittance Saratov State University Department of Optics and Biophotonics Saratov Fall Meeting 2014

10. The time-dependences of averaged weight of skin samples under action of glucose solutions The time-dependences of averaged thickness of skin samples under action of glucose solutions The time-dependences of averaged area of skin samples under action of glucose solutions Glucose solution Weight/Dehydration Thickness/ ‘transverse shrinkage’ Area/‘along shrinkage’, min 30%0.223.860.253.750.049.53 43%43%0.166.870.142.150.097.78 56%0.262.700.354.510.199.56 Kinetic parameters of dehydration and shrinkage of mouse skin samples A is a constant, which characterizes the maximal degree, τ is the characteristic time

11. Conclusion Increase of collimated transmittance and decrease of weight, thickness and area of skin samples was obtained for application of all three solutions. Increase of diffusion time of glucose corresponds to increase of glucose solution concentration. The diffusion process takes more time when more concentrated solutions are used because they induce greater degree (efficiency) of skin optical clearing. This can be related to mean total water (  54-60%) and a specific balance of free and bound water in the skin (total 54-60% - bound 16 % = free 38-44%; water content in solution of 56 % = 44%, i.e. is comparable). Less weight loss was obtained when 43%-glucose solution was applied. The less transverse shrinkage was also found for 43%-glucose solution. Saratov State University Department of Optics and Biophotonics Saratov Fall Meeting 2014

12. ACKNOWLEDGEMENTS Saratov State University Department of Optics and Biophotonics Saratov Fall Meeting 2014 We thank Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology (Wuhan, China) for support of this research This work was partially supported by the RF Presidential grant 703.2014.2 “Scientific School Support” and Grant #13-02-91176 of Russian Foundation for Basis Research