Sergey S. Model

1. SYSTEM FOR DETERMINING THE CONCENTRATION AND VISUALIZATION OF THE SPATIAL DISTRIBUTION OF PHOTOSENSITIZERS BASED ON TETRAPYRROLE COMPOUNDS IN THE TISSUES OF THE FUNDUS Sergey S. Model Laser Biospectroscopy Lab.,A.M. Prokhorov General Physics Institute,Russian Academy of Sciences,Moscow, Russia E-mail: biospec@nsc.gpi.ru Web: www.nsc.gpi.ru/lbs.html, www.biospec.ru

2. A.M. Prokhorov General Physics Institute RAS • Laser Biospectroscopy Lab. • research of spectral fluorescent properties of biological tissues; • development and manufacture of equipment for fluorescence diagnostics and photodynamic therapy; • development of methods for targeted drug delivery using nanoparticles and cellular technologies

3. Introduction In modern ophthalmology there are a number of diseases whose treatment is difficult. One such disease is senile macular degeneration (SMD) with neovascularization, have a devastating effect on the central vision. The probability of loss of vision in the SNM varies greatly and depends on the stage of disease, age, race, and sex. Currently, there are multiple optical, conservative (drug therapy), surgery (removal of subretinal membrane translocation of the retina) and laser treatment of the severe group of patients, indicating the absence of a common approach to the treatment of disease. A few years ago, the only available method of treatment, which is quite debatable could be called a success was retinal laser photocoagulation. Currently the most successful method of influence on SMD with subfoveal neovascularization is photodynamic therapy (PDT) because of the lack of laser coagulation. PDT is a much more secure than laser coagulation because the energy levels used for PDT is significantly lower. Thus, the development of equipment for the FD and PDT is the current trends in modern medical. During the creation of such systems must overcome a number of difficulties.

4. Interaction of laser radiation with the eye tissues Tissues of the human eye is a multicomponent structure. It is necessary to consider the nature of their interaction with radiation of different spectral composition. Even a transparent fabric, the cornea of ​​the human eye, diffuse light, and therefore the total and axial (collimated) transparency are not identical. Water absorption peaks are clearly visible at 300, 980, 1180, 1450, 1900, and 2940 nm due to the weak scattering. They provide low transmission through the cornea in the UV and IR spectral regions. In the visible region the normal crystalline lens is less transparent than the cornea, because in addition to the scattering absorption by different chromophores, including 3-hydroxy-L-kynurenine-O-β-glucoside and age protein (responsible for the yellowing of the lens with age) is important.   The sclera is weakly transparent fabric due to the strong scattering of light on the structural elements (polydisperse package system of irregular collagen cylinders embedded in a base material with a lower index of refraction). For effective treatment and diagnosis of diseases of the fundus absorption and fluorescence spectra of photochemical agents (photosensitizers (PS)) should be consistent with the corresponding spectra of chromophores and fluorophores in the eye tissues.

5. The absorption spectra of major chromophores and fluorophores, and the absorption spectrum of PS "Photosense"

6. The fluorescence spectra of the major fluorophores and PS "Photosense"

7. Quantitative information obtaining methods We use the following method to improve efficiency. To obtain accurate quantitative information about the concentration of photosensitizer in certain points of the biological tissue is a series of point measurements by micro spectral fluorimetric confocal method is used. Concentration map is constructed by illuminating the surgical field by laser beam with constant power density in the cross section and shooting a fluorescent cell tissue response in the appropriate spectral range on a highly sensitive camera. Confocal microspectrofluorometers produces quantitative information about the concentration of the photosensitizer in the small localized amount of tissue. As it was shown above, the precise position information of diagnostic spots in fundus is very important because of it`s complex composition. We developed a system that enables the operational control of the spatial distribution of the concentration of PS "Photosence" and other PS based on tetrapyrrole compounds in the fundus. The drug "Photosens" was developed in State Research Center "NIOPIK“ for FD and PDT in different fields of medicine including ophthalmology. This drug is better in comparison with other FS because diagnosis and treatment can be performed in one procedure, which reduces treatment time. Traditionally, FD and PDT were conducted with different PS. Because of this it was necessary to wait for one PS will be excreted from the organism before entering another PS.

8. Phantom medium As a phantom medium for construction of calibration curves we used a solution of "Photosence" in Intralipid 1% (Fresenius Kabi Austria (Austria)). Concentrations used: E(-5) g / L, 5 *E(-5) g / l, E(-4) g / l, 5 *E(-4) g / l, E(-3) g / L, 5 *E(-3) g / l.

9. System setup System for determining the concentration and visualization of the spatial distribution of photosensitizersbased on tetrapyrrole compounds in the tissues of the fundusis based on slit lamp XCEL 250 (Reichert, USA) with a laser adapter for FD and PDT and fundus imaging system. System setup of the complex is shown below. To obtain quantitative information on the concentration of PS we made an additional measurement channel in the visualization system (dashed line).

10. Equipment SPECTROMETER Fluorescent radiation is incident on the spectrometer LESA-2 (ZAO "BIOSPEC"). The integrated power in the range of fluorescence in relative units is calculated by using the software of the spectrometer. To make these measurements correct, it is necessary to construct the calibration curves in the same relative units. Thus, the calibration curves were constructed using a set of operational and diagnostic equipment, only the eye tissues were replaced by phantom media. Video Analytics To analyze the video images we used monochrome camera Videoskan 415-USB (NPO "Videoskan") witch allows to set the exposure time and gain. Interference filter that suppresses the exciting laser radiation and broadband emission slit lamp and transmits fluorescence in the red and near-infrared region of the optical spectrum was installed before the camera . To determine the optimal reception performance, we analyzed the images obtained with different exposure times and gain values.

11. Algorithm of calculation of the penetration depth The depth of light penetration into the tissue can be calculated using the following expression: , where μa - the total absorption coefficient, μs `- transport coefficient of the medium. For the retina it is 230 cm-1. The absorption coefficient of «Photosence» varies depending on its concentration from 0.24 cm-1 to 120 cm-1. Aspect Ratio: . Introduce a dependence on the concentration of PS, now the formula for calculating the depth of penetration of radiation into the fabric of the fundus is: , Where χaft = 24 * 103 cm-1 * dm3/g, μs `= 230 cm-1.

12. The calibration curves

13. Results For the construction of calibration curves, we performed measurements of the fluorescence power for different concentrations of "Photosence" in the phantom medium. The calibration curve obtained micro spectral fluorimetric method allows to determine the concentration of photosensitizer "Photosence" in the range of 10-5 g/l to 5 * 10-3 g/l. To improve the accuracy of the calibration curve one needs to perform more detailed measurements. The accuracy of this method of determining the concentration of 10-5 g/l. In the analysis of spectrally-resolved images we shooted all the surgical field. Since each pixel of the matrix is gained information about the power of fluorescence from a small volume of tissue. However, the sensitivity and accuracy of this method is lower than that of the previous method because each pixel gets some radiation from other areas of tissue, as well as its depth. The accuracy of this method is 10-4 g/l. Optimal use of the spectral-analysis method allowed us to get generalized distribution of photosensitizer in the fundus, and using the micro spectral fluorimetric method provided us with accurate measurement of borders of tumors.

14. Conclusion A system for the analysis of the spatial distribution of the concentration of photosensitizer "Photosense" in the fundus, which allows you to control the accumulation of PS in the affected and healthy tissues was developed. The combination of micro spectral fluorimetric method implemented in one of the receiving channels of the system, with analysis of the spectral-resolution images, implemented in the other channel, enables the precise delimitation of the affected area and the study of the interaction of PS with biological tissue at the molecular level. For both methods, the calibration curves are constructed on the basis of experiments with phantom media. Knowing the concentration of PS in each point of the operative field, we can determine the depth of penetration of radiation in biological tissue by the proposed algorithm. The system is easy to operate and can be used in daily medical practice, to speed up diagnosis and improve the safety of treatment.

15. Work was performed under the grant RFBR "Study of interaction of laser and broadband light with tissues fundus in hypoxia“ • Equipment was produced on manufacturing facilities of ZAO "BIOSPEC"