1. Daria K. Tuchina, 1 Alexey N. Bashkatov, 1,2 Polina A. Timoshina, 1 Elina A. Genina, 1,2 Valery V. Tuchin 1-3 1 Research-Educational Institute of Optics and Biophotonics, Saratov State University, Saratov, Russia 2 Interdisciplinary Laboratory of Biophotonics, Tomsk State University, Tomsk, Russia 3 Laboratory of Laser Diagnostics of Technical and Living Systems, Institute of Precise Mechanics and Control RAS, Saratov, Russia Saratov Fall Meeting 2015

2. Strong light scattering in tissues limits application of optical methods in medicine Immersion optical clearing (IOC) can solve problem by using optical clearing agents (OCAs) OCAs have hyperosmotic properties and higher refractive index compared to tissue interstitial fluid (ISF) OCA induces tissue dehydration and partial replacement of ISF by the OCA Better tissue homogeneity and packing cause decrease of light scattering and increase of its penetration depth IOC can be used for determination of tissue permeability for agents Saratov Fall Meeting 2015 OCA

3. Glucose-water solutions are the widely used OCAs for skin and blood optical clearing. Penetration of glucose into the skin may affect blood cutaneous and subcutaneous microcirculation. The goal of this study is to investigate 40%-glucose solution impact on skin optical properties and morphology. Weight, thickness, area and collimated transmittance of ex vivo rat skin samples immersed in aqueous 40%-glucose solution were measured The kinetics of collimated transmittance, weight, thickness and area of skin samples was used to estimate glucose diffusion coefficient in rat skin ex vivo Experimental study of 40%-glucose solution impact on blood microcirculation in rats in vivo was conducted with Laser Speckle Contrast Imaging (LSCI) Saratov Fall Meeting 2015

4. Samples White laboratory rats weighing 200-300 g were used in the study. The investigation of optical clearing of skin was performed on rat skin samples obtained ex vivo by autopsy. The area of the samples was about 10 × 15 mm 2. Hairs were removed from samples before measurements with depilatory cream before measurements; the hypodermic fatty layer was removed by tweezers. The monitoring of blood microcirculation in rat underlying tissues at 40%-glucose solution impact was carried out on 10 experimental animals. Aqueous 40%-glucose (n=1.391) solution was used as an OCA. Saratov Fall Meeting 2015

5. Ten samples of rat skin were used to measure collimated transmittance kinetics of skin during the optical clearing and to measure glucose diffusion coefficient in skin ex vivo. Intact samples were placed in cuvette with 40%-glucose solution and collimated transmittance spectra were measured using USB4000-Vis-NIR spectrometer (Ocean Optics, USA) in the spectral range 500-900 nm every 2-10 min during 120 min. The spectrometer was equipped with optical fibers (QP400-1-VIS-NIR, Ocean Optics, USA) with 400 μm core diameter and collimators 74-ACR (Ocean Optics, USA). Halogen lamp (HL-2000) were used as a light source. The measurements were performed at room temperature about 20°C. Collimated transmittance measurement

6. Saratov Fall Meeting 2015 Measurement of weight, area and thickness kinetics Twenty samples of rat skin were used to measure the weight and thickness (ten samples) using the electronic balance (SCIENTECH, SA210, USA) and a micrometer, respectively, and area (ten samples) by analysis of digital images of samples under action of 40%- glucose solution. The measurements were conducted before immersion and every 5 min during the immersion. The color hue component of sample image was obtained by the READ_HLS_HUE function of MathCad software (Parametric Technology Corporation, USA). To reduce the differential brightness, glare and noise, the median filter was used. The pixels, which were out of sample area were defined as 0. To calculate the number of pixels occupied by the sample and to convert them into the square millimeters the following equation was used: where F is the function counting the pixels occupied by the skin sample, rows is the number of rows, cols is the number of columns, H s is the processed image of the sample, H is the original digital image, z is the width of the image.

7. Obtained kinetics of weight, thickness and area during action of 40%-glucose solution was approximated by the following empirical equation: where W(t) and W(t=0) are the values of measured parameter (weight, thickness or area) at t and t=0, respectively, t is the time, A and B are the maximum of dehydration and swelling degree, respectively, and are the characteristic diffusion time constants of water and glucose, respectively, and y 0 is the residual value of the parameter. Measured kinetics of collimated transmittance was used for estimation of glucose diffusion coefficient. The problem is solved in the framework of the free diffusion model. The obtained kinetics of sample weight, thickness and area was included in the estimation algorithm of the glucose diffusion coefficient via change of scatterer packing. Estimation of glucose diffusion coefficient Saratov Fall Meeting 2015

8. The one-dimensional diffusion equation of the immersion liquid transport has the form: Diffusion equation Initial condition Boundary conditions C(x,t) is glucose concentration in skin sample, g/ml; D is the diffusion coefficient, cm 2 /sec; t is time of glucose diffusion, sec; x is the spatial coordinate of sample thickness, cm; C 0 is concentration of glucose in the cuvette, g/ml; l is the thickness of the sample, cm. The average concentration of glucose in the tissue sample has a form: The temporal dependence of the refractive index of the sample interstitial fluid is: The scattering coefficient of the tissue sample is estimated as: N is a number of scattering particles in unit of tissue volume; n I is a refractive index of interstitial fluid; λ is a wavelength, nm; a is a radius of scattering particles; n c is a refractive index of scattering particles.

9. Collimated transmittance is estimated as: r s is a specular reflection coefficient. Estimation of diffusion coefficient of glucose in skin tissue is based on measuring of collimated transmittance kinetics of tissue samples placed into agent. The solution of the problem is minimization of the target function: N t is the number of time points obtained at registration of kinetics of collimated transmittance; T c (D,t) and T c *(t i ) are the calculated and experimental values of the time-dependent collimated transmittance. Saratov Fall Meeting 2015

10. Monitoring of blood microcirculation He–Ne laser GN­5P (Russia) was used as a light source (wavelength 632.8 nm). The monochrome CMOS camera (Baslera802f, number of pixels in the matrix 656×491, pixel size 9.9×9.9 µm, 8 bit per pixel) with the fixed exposure time of 20 ms, combined with the LOMO objective (10×, St. Petersburg, Russia) was used as a detector. To calculate the contrast of speckle images, the following relation was used: where k is the number of frames in a sequence of speckle- modulated images, and are the scattered light intensity averaged over the analyzed frame and the root- mean-square value of the fluctuation component of the pixel’s brightness, respectively: where M and N are the number of pixels in rows and columns of the analyzed area of the frame, respectively; is the brightness of the -pixel of the k-frame.

11. The typical temporal dependence of collimated transmittance of rat skin sample ex vivo immersed in 40%-glucose solution measured at different wavelengths is presented. Increasing of collimated transmittance of the skin sample is observed. The increase of collimated transmittance is saturated after 30-90 min of immersion. Saratov Fall Meeting 2015

12. The kinetics of weight (a), thickness (b) area (c) and volume obtained by multiplication of thickness and area (d) of ex vivo skin samples under the action of 40%-glucose solution

13. All measured parameters (weight, thickness and area) decreased at the beginning of immersion: The decrease of weight (dehydration) continued about 1 hr It took about 20 min for thickness decrease (transverse shrinkage) area decrease (along shrinkage) For the longer time the subsequent slight weight increase (but not to initial value), as well as transverse and along swelling of skin samples were observed. During 2 hrs sample thickness was go back more or less to the initial state, but sample area was increased slowly and did not reach the initial state after 2 hrs. Decrease of weight (water lost) is resulted by outflow of free water contained in ISF from the sample induced by hyperosmotic action of glucose. Transverse and along shrinkage and swelling are connected with the rearrangement of fiber packing in the sample during immersion in glucose solution. Saratov Fall Meeting 2015

14. The greater change in both shrinkage and swelling stages was observed for thickness The changes of the samples area is less expressed Weight decrease takes more time than for both types of shrinkage Since the decrease of the distance between skin fibers is much more likely than the change of fiber length, more strong transverse shrinkage compared to along shrinkage is observed. Kinetic coefficients of dehydration and shrinkage/swelling of rat skin samples under the action of 40%-glucose solution The measured kinetics of skin collimated transmittance and the skin shrinkage/swelling parameters were used for estimation of glucose diffusion coefficient. The glucose diffusion coefficient averaged by all samples was estimated as (1.06±0.88)×10 ‒ 6 cm 2 /s. Saratov Fall Meeting 2015

15. Result of measurements of the contrast of speckle images are shown. Figure (b) shows histograms of the measured diameter of blood vessel without agent and after action of 40%-glucose solution. The results show that applying 40%-glucose solution produces the increase of the diameter of blood vessels and reduces blood flow velocity (increase of contrast of speckle images).

16. Conclusion The increase of collimated transmittance of rat skin ex vivo during action of 40%- glucose solution was obtained Weight loss and transverse and along shrinkage of skin immersed in 40%-glucose solution at the beginning of optical clearing process; and subsequent weight increase and transverse and along swelling were observed The glucose diffusion coefficient in skin was estimated basing on the results of these measurements as (1.06±0.88)×10 ‒ 6 cm 2 /s It was obtained that 40%-glucose solution produced a vasodilator effect, expressed in the increase of microvessel size and the decrease of average rate of microcirculation (increase of the value of speckle contrast image) The obtained results can be used for optimization of tissue optical clearing and drug delivery techniques, improvement of biophysical and mathematical models describing interactions between tissues and optical clearing agents. The work was supported by grant No.14-15-00186 from the Russian Science Foundation and in part of speckle contrast imaging methodology by grant of Russian Ministry of Education and Science No. 3.1340.2014 / K.