1. Saratov State University ______________________________________________ Department of Optics & Biophotonics __________________________________________________ Saratov Fall Meeting 2011 Absorption and scattering properties of human eye sclera Alexey N. Bashkatov, Elina A. Genina, Vyacheslav I. Kochubey, Tatyana G. Kamenskikh, Valery V. Tuchin Department of Optics and Biophotonics Saratov State University Saratov State Medical University e-mail: a.n.bashkatov@mail.rua.n.bashkatov@mail.ru Alexey N. Bashkatov, Elina A. Genina, Vyacheslav I. Kochubey, Tatyana G. Kamenskikh, Valery V. Tuchin Department of Optics and Biophotonics Saratov State University Saratov State Medical University e-mail: a.n.bashkatov@mail.rua.n.bashkatov@mail.ru

2. Saratov State University ______________________________________________ Department of Optics & Biophotonics __________________________________________________ Saratov Fall Meeting 2011 Motivation: Development of optical method in modern medicine in the areas of diagnostics, therapy and surgery has stimulated the investigation of optical properties of various biological tissues, since the efficacy of laser treatment depends on the photon propagation and fluence rate distribution within irradiated tissues. The knowledge of tissue optical properties is necessary for the development of the novel optical technologies of photodynamic and photothermal therapy, optical tomography, optical biopsy, and etc. Numerous investigations related to determination of tissue optical properties are available however the optical properties of many tissues have not been studied in a wide wavelength range. Goal of the study is to investigate of optical properties of human eye sclera in the wavelength range 400-2000 nm Development of optical method in modern medicine in the areas of diagnostics, therapy and surgery has stimulated the investigation of optical properties of various biological tissues, since the efficacy of laser treatment depends on the photon propagation and fluence rate distribution within irradiated tissues. The knowledge of tissue optical properties is necessary for the development of the novel optical technologies of photodynamic and photothermal therapy, optical tomography, optical biopsy, and etc. Numerous investigations related to determination of tissue optical properties are available however the optical properties of many tissues have not been studied in a wide wavelength range. Goal of the study is to investigate of optical properties of human eye sclera in the wavelength range 400-2000 nm

3. Saratov State University ______________________________________________ Department of Optics & Biophotonics __________________________________________________ Saratov Fall Meeting 2011 Materials and Methods:  For this study twenty samples of human eye sclera have been used. The samples keep in saline during 2-4 hour until spectrophotometric measurements at temperature 4-5°C. All the tissue samples has been cut into pieces with the area about 25  25 mm 2. For mechanical support, the tissue samples have been sandwiched between two glass slides.  Measurement of the diffuse reflectance, total and collimated transmittance have been performed using a commercially available spectrophotometer PerkinElmer LAMBDA 950 in the spectral range 400-2000 nm. All measurements were performed at room temperature (about 20°C)  For estimation of absorption and scattering coefficients, and anisotropy factor of the tissue the inverse Monte Carlo method was used.  For this study twenty samples of human eye sclera have been used. The samples keep in saline during 2-4 hour until spectrophotometric measurements at temperature 4-5°C. All the tissue samples has been cut into pieces with the area about 25  25 mm 2. For mechanical support, the tissue samples have been sandwiched between two glass slides.  Measurement of the diffuse reflectance, total and collimated transmittance have been performed using a commercially available spectrophotometer PerkinElmer LAMBDA 950 in the spectral range 400-2000 nm. All measurements were performed at room temperature (about 20°C)  For estimation of absorption and scattering coefficients, and anisotropy factor of the tissue the inverse Monte Carlo method was used.

4. Saratov State University ______________________________________________ Department of Optics & Biophotonics __________________________________________________ Saratov Fall Meeting 2011 Experimental setup The geometry of the measurements in A) transmittance mode, B) reflectance mode. 1 ‑ the incident beam (diameter 1-10 mm); 2 ‑ the tissue sample; 3 ‑ the entrance port (square 25  16 mm); 4 ‑ the transmitted (or diffuse reflected) radiation; 5 ‑ the integrating sphere (inner diameter is 150 mm); 6 ‑ the exit port (diameter 28 mm) The geometry of the collimated transmittance measurements. Diameter of the incident beam is 2 mm.

5. Saratov State University ______________________________________________ Department of Optics & Biophotonics __________________________________________________ Saratov Fall Meeting 2011 Inverse Monte Carlo The computer program package for determination of absorption and scattering tissue properties has been developed. This inverse Monte Carlo method based on the solution of direct problem by Monte Carlo simulation and minimization of the target function To minimize the target function the Simplex method described in detail by Press et al (Press W.H., et al. Numerical recipes in C: the art of scientific computing / Cambridge: Cambridge University Press, 1992.) has been used. Iteration procedure repeats until experimental and calculated data are matched within a defined error limit (<0.1%). Here R d exp, T t exp, T c exp, R d calc, T t calc, T c calc are measured and calculated values of diffuse reflectance and total and collimated transmittance, respectively. with the boundary condition

6. Saratov State University ______________________________________________ Department of Optics & Biophotonics __________________________________________________ Saratov Fall Meeting 2011 Inverse Monte Carlo This method includes inverse adding-doubling (IAD) method developed by Prahl et al (Prahl S.A., et al. // Appl. Opt., 1993, Vol. 32(4), P. 559-568) and inverse Monte Carlo simulations. The IAD method is widely used in tissue optics for processing the experimental data of spectrophotometry with integrating spheres. This method allows one to determine the absorption and the reduced scattering coefficients of a turbid media from the measured values of the total transmittance and the diffuse reflectance. In these calculations the anisotropy factor can be fixed as 0.9, since this value is typical for tissues in the visible and NIR spectral ranges. Based on the obtained values of the tissue absorption and reduced scattering coefficients the inverse Monte Carlo calculations have been performed. The inverse method includes direct problem, i.e. Monte Carlo simulation, which takes into account the geometric and optical conditions (sample geometry, sphere parameters, refractive index mismatch, etc.), and solution of inverse problem, i.e. minimization of target function by an iteration method. In this study, we used Monte Carlo algorithm developed by L. Wang et al (Wang L., et al. // Computer Methods and Programs in Biomedicine, Vol. 47, P. 131-146, 1995). The stochastic numerical MC method is widely used to model optical radiation propagation in complex randomly inhomogeneous highly scattering and absorbing media such as biological tissues. Usually the inverse Monte Carlo technique requires very extensive calculations since all sample optical parameters (absorption and scattering coefficients and anisotropy factor) unknown. To avoid the long time calculations as a guest values we used values of absorption and reduced scattering coefficients obtained from calculations performed by IAD method. For final determination of the tissue absorption and scattering coefficients, and the tissue anisotropy factor minimization of the target function has been performed.

7. Saratov State University ______________________________________________ Department of Optics & Biophotonics __________________________________________________ Saratov Fall Meeting 2011 Inverse Monte Carlo The flow-chart of the inverse Monte Carlo method

8. Saratov State University ______________________________________________ Department of Optics & Biophotonics __________________________________________________ Saratov Fall Meeting 2011 The typical spectra of sample of human eye sclera. Rd is diffuse reflectance; Tt is total transmittance and Tc is collimated transmittance Results:

9. Saratov State University ______________________________________________ Department of Optics & Biophotonics __________________________________________________ Saratov Fall Meeting 2011 The absorption spectrum of the scleral tissue IS, IMC, data averaged for 20 samples The absorption spectrum of the scleral tissue IS, IMC, data averaged for 20 samples Results:

10. Saratov State University ______________________________________________ Department of Optics & Biophotonics __________________________________________________ Saratov Fall Meeting 2011 The reduced scattering coefficient spectrum of the scleral tissue IS, IMC, data averaged for 20 samples The reduced scattering coefficient spectrum of the scleral tissue IS, IMC, data averaged for 20 samples Results:

11. Saratov State University ______________________________________________ Department of Optics & Biophotonics __________________________________________________ Saratov Fall Meeting 2011 The scattering coefficient spectrum of the scleral tissue IS, IMC, data averaged for 20 samples The scattering coefficient spectrum of the scleral tissue IS, IMC, data averaged for 20 samples Results:

12. Saratov State University ______________________________________________ Department of Optics & Biophotonics __________________________________________________ Saratov Fall Meeting 2011 The wavelength dependence of scattering anisotropy factor of the scleral tissue IS, IMC, data averaged for 20 samples The wavelength dependence of scattering anisotropy factor of the scleral tissue IS, IMC, data averaged for 20 samples Results:

13. Saratov State University ______________________________________________ Department of Optics & Biophotonics __________________________________________________ Saratov Fall Meeting 2011 Grant # 10-02-90039 Бел_а of Russian Foundation of Basis Research Russian Federation governmental contacts 02.740.11.0484, 02.740.11.0770, and 02.740.11.0879 Grant # 10-02-90039 Бел_а of Russian Foundation of Basis Research Russian Federation governmental contacts 02.740.11.0484, 02.740.11.0770, and 02.740.11.0879 Grant #224014 Network of Excellence for Biophotonics (PHOTONICS4LIFE) of the Seventh Framework Programme of Commission of the European Communities Acknowledgement: