National Academy of Sciences of Belarus B.I.Stepanov Institute of Physics of the National Academy of

Sciences of Belarus

International Conference Optical Techniques and Nano-Tools for Material and

Life Sciences

(OTN4MLS-2010)

Minsk, Belarus, June 15-19, 2010

CONTRIBUTED PAPERS

In two volumes Volume II (Section 7, Poster Section)

Minsk Kovcheg

CONFERENCE ORGANIZERS

B.I.Stepanov Institute of Physics of National Academy of Sciences of Belarus, Minsk, Belarus

International Science and Technology Center, Moscow, Russia

Fraunhofer Institute for Non-destructive Testing, Dresden/Saarbrucken, Germany

Editorial Board

N.S.Kazak (managing editor), V.N.Belyi, S.N.Kurilkina, A.G.Smirnov, R.A.Vlasov

SPONSORED BY:

International Science and Technology Center Belarusian Republican Fund of Fundamental Researches

The reports in the proceedings of the International Conference are presented by the individual authors. The views expressed are their own and do not necessarily represent the views of the Publishers or Sponsors. Whilst every effort has been made to ensure the accuracy of the information contained in the book, the Publisher or Sponsors cannon be held liable for any errors or omissions however caused. All rights are reserved. No part of this publication may be reproduced, stored in a retrieval system, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without prior permission of the copyright owner.

ISBN 978-985-6950-31-2 (vol 2) ISBN 978-985-6950-31-8

B.I.Stepanov Institute of Physics, National Academy of Sciences of Belarus, 2010

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CONTENTS CONFERENCE COMMITTEE 7 Section 7. Medical diagnostics and Point of Care Testing Application of OCT in Dermatology Y.-H. Shin, J.-S. Lee, Department of R&D, NUGA Medical Co., LTD. (Korea); T.Stautmeister, MICRO-EPSILON Optronic GmbH (Germany); E.Koch, Clinical Sensor and Monitoring, Dresden (Germany), B. Fischer, J.Schreiber, T.-Y. Han, Fraunhofer Institute for Non-Destructive Testing, Dresden (Germany)

Augmentation in osteoporotic bone quality through minimally invasive laser therapy C.Y. Ko, D.Kang, D.H. Seo, Y. Ryu, B. Jung, H. S. Kim, Yonsei University (South Korea)

Colloidal gold based immunoassay for high-sensitivity NTx detection in urine M.H.Lee, Korean Electronics Technology Institute, Medical IT Research Center (Korea); S.Lee, W.Seong, IM-Electronics (Korea)

Laser kinetic method for determination of the molecular oxygen affinity of human hemoglobin and polyhemoglobin, artificial blood substitute product S.V. Lepeshkevich, B.M. Dzhagarov, B.I. Stepanov Institute of Physics, NAS of Belarus (Belarus); R.A. Roziev, V.K.Podgorodnichenko, Research and Production Company Medbiopharm (Russia)

Laser - optical method of detection and elimination the local tissue hypoxia and its application in oncology M.M. Asimov, A.N. Rubinov, B.I. Stepanov Institute of Physics, NAS of Belarus (Belarus), R.M. Asimov, "Sensotronic Ltd." (Belarus)

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POSTER SESSION Section 1. Characterization of physical and functional performance materials

The analysis of laser thermosplitting of materials by using of crescent- shaped beams S.Shalupaev, A.Serdyukov, Y.Nikitjuk, A.Sereda, Francisk Skorina Gomel State University (Belarus)

Stable birefringence recording in benzaldehyde polymers by UV radiation U.Mahilny, A.Stankevich, A. Trofimova, Belarusian State University (Belarus)

Non-linear optical structures in glasses by phase inhomogeneities formation and local crystallization under laser irradiation V.N. Sigaev, S.V. Lotarev, N.V. Golubev, V.S. Ryzhenkov, Optical Glass Center of Mendeleev University of Chemical Technology (Russia); G.E. Malashkevich, B.I. Stepanov Institute of Physics, NAS of Belarus (Belarus); B. Champagnon, D. Vouagner, University Claude Bernard Lyon-1 (France); Yu.S. Priseko, Research and Production Enterprise VELIT (Russia)

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Diamonds differentiation with simultaneous registration of Raman spectra and photoluminescence Ya.I. Didkovskij, M.N. Kovalenko, .. Minko, .N. Poklonskaya, .R.Posledovich, Belarusian State University (Belarus)

About the capability of water-sand mixture microstructure monitoring in petroleum production systems A.B. Gavrilovich, B. I. Stepanov Institute of Physics, NAS of Belarus (Belarus); I.P.Shingaryov, Belarusian State University (Belarus)

Laser diagnostics and control over formation of the fractal structures A.B. Gavrilovich, B.I. Stepanov Institute of Physics, NAS of Belarus, Minsk State High Radiotechnical College (Belarus)

New polymeric material for phase optical recording in spectral range 350-370 nm U.Mahilny, D.Marmysh, A.Stankevich, Belarusian State University (Belarus)

Solution composition influence on the optical characteristics of liquid-containing composite materials T.Pulko, P.Safrankov, N.Nasonova, Belarusian State University of Informatics and Radioelectronics (Belarus)

Precise control of three dimensional objects S.A. Korenjako, A.M. Leonov, United Institute of Informatics Problems, NAS of Belarus (Belarus)

Peculiarities of ultrasound excitation in magnetic fluids by laser pulses A.R.Baev, M.V.Asadchaya, Institute of Applied Physics, NAS of Belarus (Belarus); A.A.Karabutov, M.Lomonosov Moscow State University (Russia); V.G.Gudelev, A.I.Mitskovets, B.I. Stepanov Institute of Physics, NAS of Belarus (Belarus)

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Section 2. On-line control of quality of products and fabrication processes

An improved method of nondispersive correlation spectroscopy for the total atmospheric sulphur dioxide column measurements N. S. Miatselskaya, V. P. Kabashnikov, B.I. Stepanov Institute of Physics, NAS of Belarus (Belarus)

Portable elemental spectroanalyzer based on erbium glass laser M.V.Bogdanovich, K.Yu.Catsalap, E.A.Ershov-Pavlov, G.I.Rybtsev, M.A.Shchemelev, B.I. Stepanov Institute of Physics, NAS of Belarus (Belarus)

Transformation of Gaussian light beam into Bessel one for control systems G.V. Kulak, A.E. Anisimova, Mozyr State Pedagogical University (Belarus); P.I. Ropot, B.I. Stepanov Institute of Physics, NAS of Belarus (Belarus)

Methods for narrow hollow light beams formation A. A. Ryzhevich, S.V.Solonevich, A.G.Smirnov, N.S.Kazak, B.I.Stepanov Institute of Physics, NAS of Belarus (Belarus); Turki S.M. Al-Saud, Soliman H.Al-Khowaiter, Muhanna K.Al-Muhanna, King Abdulaziz City for Science and Technology (Saudi Arabia)

Remote sensing of the Earth S.A. Korenjako, A.M. Leonov, United Institute of Informatics Problems, NAS of Belarus (Belarus)

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Section 4. Tools for nanoimaging and nanocharacterization

A modeling of band structure and a formation of spontaneous polarized LB-clusters of carbon nanotubes H.V. Krylova, B. G. Shulitsky, L.V. Baran, Belarusian State University (Belarus), Belarusian State University of Informatics and Radioelectronics (Belarus)

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Section 5. Application of functionalized nanoparticles

Genetic effects of carbon nanoparticles on mouse and human cells I.B. Mosse, P. M. Marozik, L. N. Kostrova, D.A. Ushakova, Institute of Genetics & Cytology, NAS of Belarus (Belarus)

Multicolor compositions of cadmium selenium nanoparticles and dye molecules with - cyclodextrin inclusion complexes M.M. Asimov, B.I. Stepanov Institute of Physics, NAS of Belarus (Belarus)

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Session 6. Physical simulation of sensors and control systems

Transformation of topological structure of singular light beams by multiwave mixing in resonant media O.G. Romanov, A. L. Tolstik, D.V. Gorbach, Belarusian State University (Belarus)

Amplitude modulation of radiation using two connected resonators V.A.Pilipovich, .I.Kanojka, A.I.Mitkovets, B.I.Stepanov Institute of Physics, NAS of Belarus (Belarus)

Laser beam apodization with biaxial crystal A. A. Ryzhevich, S.V.Solonevich, N.S.Kazak, B.I.Stepanov Institute of Physics, NAS of Belarus (Belarus); Turki S.M. Al-Saud, Soliman H.Al-Khowaiter, Muhanna K.Al-Muhanna, King Abdulaziz City for Science and Technology (Saudi Arabia)

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Session 7. Medical diagnostics and Point of Care Testing

Features of tissue probing by conic light beams E.S. Petrova, Gomel State Technical University (Belarus); L.I. Kramoreva, Gomel State Medical University (Belarus)

Intracellular Accumulation of chlorin e6 and chlorin e6 dimethylester by activated T- Lymphocytes V.P.Savitski, M..Logatskaya, Belarusian Center for Pediatric Oncology and Hematology (Belarus); V.P. Zorin, Belarusian State University (Belarus)

Laser radiation influence on transport phenomena in a bio-tissue A. A. Ryzhevich, S.V.Solonevich, B.I.Stepanov Institute of Physics, NAS of Belarus (Belarus); T. A. Zheleznyakova, Belarusian State University (Belarus)

Control of light absorption distribution in blood for the extracorporated diagnostics systems . B. Gavrilovich, Minsk State High Radiotechnical College (Belarus)

Nanosensing based on LB-CNT-clusters to register a functioning of membrane-dependent biological reactions A.I. Drapeza, H.V. Grushevskaya, V. V. Hrushevsky, I. V. Lipnevich, Yu. M. Sudnik, G. N. Semenkova, N. G. Krylova, T. A. Kulahava, Belarusian State University (Belarus); T. I. Orekhovskaya, B. G. Shulitsky, , Belarusian State

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University of Informatics and Radioelectronics (Belarus)

Novel indolocarbazoles and oligophenylenevinylenes fluorescence probes N. A. Nemkovich, B.I.Stepanov Institute of Physics, NAS of Belarus (Belarus); H. Detert, Institute of Organic Chemistry, J. Gutenberg University of Mainz (Germany)

Visualization of eye structure under the condition of tissue scattering in optical coherence tomography L.I. Kramoreva, Gomel State Medical University (Belarus); Yu.I. Rozhko, Republican Research Center for Radiation Medicine and Human Ecology (Belarus)

Investigation of structurally-functional properties of erythrocytes by optical methods E. K. Naumenko, B. I. Stepanov Institute of Physics, NAS of Belarus (Belarus)

Time-lapse microscopy of living cells in vitro O.V. Kvitko, I.I. Koneva, Ya.I. Sheiko, S.E. Dromashko, Institute of Genetics and Cytology, NAS of Belarus (Belarus); S. V. Gloushen, N.A. Balashenko, A.S.Sapun, Belarusian State University (Belarus)

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KEY TO AUTHORS 224

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CONFERENCE COMMITTEE

INTERNATIONAL ADVISORY COMMITTEE

CHAIR .N.Rubinov (Belarus)

MEMBERES V..Orlovich (Belarus), V. Giurgiutiu (USA), D. Inman (USA), N. Takeda (Japan), M.Scott

(Australia), .S.Soskin (Ukraine), B.Wilhelmi (Germany), V.I.Aksenov (Russia), W.Gudowski (Russia), .Kroening (Germany), J.-.K. Krueger (Germany), A.V. Rogachev (Belarus), . I. Belous (Belarus), A.Forbes (South Africa), Mohanna Al-Mohanna (Saudi Arabia), M. Kujawinska (Poland), Young Ho Kim (South Korea), B. Culshaw (UK), M. Kaschke (Germany), V.S. Kamyshnikov (Belarus)

INTERNATIONAL ORGANIZING COMMITTEE

CHAIRS N.S.Kazak (Belarus) Ch.Boller (Germany)

VICE-CHAIRS V.N.Belyi (Belarus) J.Schreiber (Germany)

SECRETARY .G.Smirnov (Belarus)

MEMBERS V.V.Kabanov (Belarus), S.N.Bagaev (Russia), V.S.Pavelyev (Russia), V.A.Lapina

(Belarus), I.V.Semchenko (Belarus), G.S.Mityurich (Belarus), V.Kotlyar (Russia), U.Cho (Korea), .S.Kondratiev (Russia), G.Salomon (Germany), .P.Shkadarevich (Belarus), S.N.Cherenkevich (Belarus), V.L.Vengrinovich (Belarus)

BELARUSIAN ORGANIZING COMMITTEE

CHAIR V.N.Belyi

VICE-CHAIR S.N.Kurilkina

SECRETARY .G.Smirnov

MEMBERS V. Yu. Plavskii, I.Mukhurov, ..Ryzhevich, V.G.Gudelev, B..Bushuk, S.B.Bushuk,

P.I.Ropot, .E.Kunina, V..Adamenko, N.. Khilo, .G.Maschenko, R.Y.Vasilyev, S.E. Kozik, S.V.Solonevich, R.A.Vlasov, A.V.Gorelik, V.N.Yushkevich

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SECTION CHAIRS

1. Characterization of physical and functional performance materials

M.Kroening, QNET, Saarbrcken, Germany J.Schreiber, Fraunhofer Institute for Non-destructive Testing, Germany

2. On-line control of quality of products and fabrication processes

V.Belyi, B.I.Stepanov Insitute of Physics, NAS of Belarus, Belarus A.Smirnov, B.I.Stepanov Insitute of Physics, NAS of Belarus, Belarus

3. Sensors for Structural Health Monitoring

Ch.Boller, Fraunhofer Institute of Non-Destructive Testing, Germany V.Vengrinovich, Institute of Applied Physics, NAS of Belarus, Belarus

4. Tools for nanoimaging and nanocharacterization

B.Wilhelmi, CTB WILHELMI, Germany S.Kurilkina, B.I.Stepanov Insitute of Physics, NAS of Belarus, Belarus

5. Application of functionalized nanoparticles

N.Tarasenko, B.I.Stepanov Insitute of Physics, NAS of Belarus, Belarus V.Lapina, B.I.Stepanov Insitute of Physics, NAS of Belarus, Belarus

6. Physical simulation of sensors and control systems

A. Tolstik, Belarusian State University, Belarus N.Khilo, B.I.Stepanov Insitute of Physics, NAS of Belarus, Belarus

7. Medical diagnostics and Point of Care Testing

A.Konstantinova, Shubnikov Institute of Crystallography, RAS, Russia B.Dzhagarov, B.I.Stepanov Insitute of Physics, NAS of Belarus, Belarus

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APPLICATION OF OCT IN DERMATOLOGY

Young-Hoon Shin1, Jong-Soo Lee1, Torsten Stautmeister2, Edmund Koch3, Bjrn Fischer4, Jrgen Schreiber4, Tae-Young Han4

1Department of R&D, NUGA Medical Co., LTD.

2MICRO-EPSILON Optronic GmbH, Dresden-Langebrck

3Clinical Sensoring and Monitoring, TU Dresden

4Fraunhofer Institute for Non-Destructive Testing, Dresden branch of IZFP

ABSTRACT

Optical Coherence Tomography (OCT) is a relatively new optical imaging modality. OCT performs high resolution, cross-sectional tomographic imaging of the internal microstructure in materials and biological samples by measuring the echo time delay and magnitude of the backscattered light. The first clinical application of OCT was ophthalmology. OCT enables noncontact and noninvasive imaging of the anterior eye as well as imaging of morphologic features of human retina including the fovea and optic disc. Advances in OCT technology have made it possible to image nontransparent tissue, thus enabling OCT to be applied in a wide range of medical applications. Imaging depth is limited by optical attenuation from tissue scattering and absorption. However, imaging up to 1 to 2 mm in depth can be achieved. In this scale many interesting phenomena take place in the skin. Imaging studies have also been performed in vivo to investigate applications in dermatology. Recently, many requirements on quantitative analysis and high-resolution cross-sectional imaging of the skin are raised from the clinical application in dermatology and the cosmetic industry. NUGA Medical collaborates with MICRO-EPSILON Optronic, TU Dresden and Fraunhofer institute to develop a first commercial OCT system for these requirements. Fundamental research has been carried out by the group. NUGA Medical will develop the commercial product based on the research and numerous clinical studies will be performed to optimize the performance of OCT. In this paper, we review the key specification of OCT for dermatology and the scope of the joint development project.

Keywords: Optical Coherence Tomography, Dermatology

1. INTRODUCTION

Optical Coherence Tomography (OCT) is a relatively new optical imaging modality. OCT performs high-resolution, cross-sectional tomographic imaging of the internal microstructure in materials and biological samples by measuring the echo time delay and magnitude of the backscattered light. Different to ultrasound, OCT uses near-infrared light instead of sound waves and employs interferometric methods to detect light reflected from within tissue. OCT images are generated by dividing light from an optical source into two paths, one is directed to the tissue sample, and the other to a reference mirror as shown in Fig. 1. Light reflected from the sample is recombined with light from the reference mirror and detected, forming an interference signal only when the length of the sample and reference path are matched to within a short distance. This distance is termed the coherence length of the light source. By varying the path length of the reference arm, a profile of reflected signal amplitude is obtained as a function of depth in the sample. By acquiring such depth profiles or A-scans as the beam is scanned across the tissue sample, a two-dimensional image or B-scan can be generated.

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Fig 1. Configuration of the OCT

The first clinical application of OCT was ophthalmology. OCT enables noncontact, noninvasive imaging of the anterior eye as well as imaging of morphologic features of human retina including the fovea and optic disc. Advances in OCT technology have made it possible to image nontransparent tissue, thus enabling OCT to be applied in a wide range of medical applications. In terms of resolution and penetration depth, OCT fills the gap between ultrasound and confocal microscopy as shown in Fig. 2. Imaging depth is limited by optical attenuation from tissue scattering and absorption. However, imaging up to 1 to 2 mm depth can be achieved. In this scale many interesting phenomena take place in the skin. Imaging studies have also been performed in vitro to investigate applications in dermatology. Imaging and visualization of tissue structure and function is of fundamental importance in the field of dermatology. Conventional optical microscopy, fluorescence microscopy, and confocal microscopy are established imaging modalities, each with individual strengths and weaknesses. OCT has emerged in recent years to provide subsurface tissue imaging with resolution at the 10 m scale, and a field-of-view covering several millimeters. Early systems indicated potential applications of OCT in dermatology. Additional information, complementary to tissue structure, is provided by the simultaneous display of polarization-sensitive and flow-sensitive (Doppler) images, with all three images displayed in real time. Previous reports describing OCT imaging of skin demonstrated resolution superior to high-frequency ultrasound. Epidermal and dermal layers could be distinguished and skin appendages including hair follicles and eccrine ducts were identified in normal skin [3].

Fig 2. OCT vs. Standard imaging [1]

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Effects of topical and sub-dermal treatments on skin structure and dimension have also been monitored over time using OCT. Systems based on 830 nm light sources demonstrated imaging to depths of around 700 m in skin with reduced scattering in the 1310 nm wavelength region enabling imaging to depths of around 1.2 mm [3]. To maximize the penetration depth of light in highly scattering tissues such as skin, optical sources are commonly chosen with emission centered at around 1310 nm. Imaging in this spectral region takes advantage of the 1/4-dependent reduction in scattering with increasing wavelength, before absorption by the water content of tissue becomes the dominant attenuation mechanism at wavelengths in the mid-infrared. The reduced imaging depth achievable with 830 nm sources permits the use of focusing optics with higher numerical apertures, with a correspondingly smaller beam size resulting in higher transverse resolution, though over a reduced depth range. Earlier OCT systems typically required many seconds or minutes to generate a single OCT image of tissue structure, raising the likelihood of suffering from motion artifacts and patient discomfort during in vivo imaging. To counter such problems, techniques have been developed for scanning the reference arm mirror at sufficiently high speeds to enable real-time OCT imaging [4]. In addition to obtaining images of internal tissue structure based purely on the amplitude of the reflected signals, information concerning tissue functionality may be extracted by considering the wave properties and the polarization state of light. Regions of blood flow have been identified by both Doppler [5] and phase-resolved techniques [6], enabling vessels and capillaries to be located and flow velocity determined. Certain components of the skin possess an ability to change the polarization state of light by virtue of being birefringent. In skin, birefringence is attributed to the regular arrangement of collagen fibers in the dermis. Polarization-sensitive OCT allows polarization effects to be quantified and related to the structural integrity of the collagen scaffold, as demonstrated by PS-OCT imaging of thermally damaged skin [7]. These Doppler and polarization-sensitive functions provide distinct, complementary information to structural OCT images. These capabilities can be integrated in a high-speed multifunctional OCT system specifically tailored toward dermatology. In order to produce such a system suitable for clinical research, several technical obstacles needed to be resolved. The simultaneous display of multiple structural and functional OCT images was precluded by prohibitively long computation times in combination with non-optimal interferometer arrangements.

2. PROJECT OVERVIEW

Recently, many requirements on quantitative analysis and high-resolution cross-sectional image of the skin are raised from the clinical application in dermatology and cosmetic industry. OCT can provide the imaging and analysis tool for dermatology. In cosmetic industry, high resolution imaging and quantization of the chemical effect on the skin is highly required for the research. For regulatory office, even though the cosmetic is one of widely used chemicals for the human, an accurate tool for controlling its quality and effect does not exist. NUGA Medical will collaborate with MICRO-EPSILON Optronic, TU Dresden and Fraunhofer institute to develop a commercial OCT system for these requirements. Fundamental research has been carried out by the group. NUGA Medical will develop the commercial product based on the current research. The target system is supposed to take such images of in vivo human skin, demonstrating advances in both qualitative and quantitative OCT imaging in dermatology. Biopsy and histological processing remain the gold standard for tissue diagnosis, and we are not proposing that OCT will replace such techniques.

The project will be supported partially by Korean and German government research funds. It is planned as two stage project. In the first stage, an OCT device for monitoring the skin with high resolution will be developed. It provides very high resolution images for dermatology and cosmetic applications with the target application of supervisory offices & national authority organizations/ institutes for standardization, and cosmetic companies. In this stage, all the basic technologies are developed and clinically evaluated. In the second stage, the cost-effective OCT device for commercial use will be developed. It provides high-resolution images with low costs for skin-care shops and dermatology-oriented business units. Four parties will participate in this project. TUD will design the OCT system and build up the prototype. Principle research will be conducted together with the dermatology department of the Dresden University Hospital and TUD will organize and manage clinical tests together with tests in Korea. MICRO-EPSILON Optronic will develop the hardware prototype and the software for the hardware control. IZFP-D will manage the whole project and develop the software for the evaluation and integrate the OCT prototype. NUGA Medical will conduct the clinical tests in Korea and commercialize the OCT system.

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3. PROJECT SETUP

The optical coherence tomography (OCT) system consists of 5 sub-blocks. They are light source, beam delivery and probes, OCT imaging engine, computer control and image & signal processing. Fig. 3 shows the system perspective of OCT. The target OCT system for dermatology will be developed based on a prototype OCT system which has been developed for general purposes by MICRO-EPSILON Optronic, TU Dresden and Fraunhofer institute.

The prototype OCT system shown in Fig. 4 consists of a main unit incorporating light source, spectrometer and specific electronic parts. Different scanner heads or hand pieces, optimized for various applications, can be connected by a combined electrical and fiber optical cable. For easy orientation on the sample, the scanner heads incorporate a high-resolution color camera. The system is controlled by a standard PC.

Fig. 3. System Perspective of OCT [1]

Fig. 4. Photo and schematic of prototype OCT system developed by TU Dresden

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From the many options to build an OCT system, the wavelength of the light source (800 or 1300 nm) is the most important. Depending on the wavelength of the light source, penetration depth and target skin structure can be different as shown in Fig. 5. In case of 1300 nm, it penetrates a little bit deeper than 800 nm but the costs are higher than for an 800 nm systems. On the other hand, the 800 nm range allows a higher resolution.

Fig. 5. Skin image from prototype OCT system by TU Dresden. Left side shows image at 800 nm with the higher resolution. Right side shows image at 1300 nm with the higher penetration depth.

Fig. 6.Wavelength of the light source of OCT vs. Related Skin Structure [1]

A system combining the two wavelengths ranges (dual band OCT) is also available. The combination of both images gives the high resolution, the high penetration depth and reduces the speckle noise. In order to apply the device to investigate the skin surface like roughness, size and density of sweat glands and its skin near layers, a 3D OCT system should be considered. Two prototypes will be built in the first stage of the project and clinical studies will be conducted at the TU Dresden as well as in Korean. From these clinical studies, optimized system requirements can be obtained. The clinical study should look at the penetration of cosmetic products but should also look for long term effects on skin thickness, roughness and further parameters. After successful results of the prototype will be achieved, the commercialization of the prototype can be planned.

4. CONCLUSION

Recently many requirements on quantitative analysis and high-resolution cross-sectional images of the skin are raised from the clinical application in dermatology and the cosmetic industry. NUGA Medical collaborates with MICRO-EPSILON Optronic, TU Dresden and Fraunhofer institute to develop a first commercial OCT system for these

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requirements. Fundamental research has been carried out by the group. NUGA Medical will develop the commercial product based on the research and numerous clinical studies will be performed to optimize the performance of the OCT system. We review the key specification of the OCT system for dermatology and the scope of the joint development project.

REFERENCES

[1] Courtesy of Prof. P.E. Anderson, Ris, Technical University of Denmark [2] J. M. Schmitt, Optical coherence tomography (OCT): A review, IEEE J. Select. Topics Quantum Electron. 5, 1205-

1215 (1999) [3] J. M. Schmitt, M. J.Yadlowsky, R. F. Bonner, Subsurface imaging of living skin with optical coherence

microscopy, Dermatology, 191: 93-98 (1995) [4] G.J. Tearney, B.E. Bouma, J.G. Fujimoto, High-speed phase- and group-delay scanning with grating-based phase

control delay line, Opt.Lett 22: 1181 1183 (1997) [5] Z. Chen, T.E. Miller, S. Srinivas, X. Wang, A.Malekafzali, M.J.C. Van Germer, J.S. Nelson, Noninvasive imaging

of in vivo blood flow velocity using optical Doppler tomography, Opt. Lett 22:1119-1121 (1997) [6] Y. Zhao, Z. Chen, C. Saxer, Q. Shen, S. Xiang, J.F. de Boer, J.S. Nelson, Doppler standard deviation imaging of in

vivo human skin blood flow, Opt. Lett 25: 1358-1360 (2000) [7] P.C. Pierce, J. Strasswimmer, B.H. Park, B. Cense, J.F. de Boer, Advances in optical coherence tomography

imaging for dermatology, J Invest Dermatol 123:458-463 (2004)

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AUGMENTATION IN OSTEOPOROTIC BONE QUALITY THROUGH MINIMALLY INVASIVE LASER THERAPY

C.Y. Ko, D. Kang, D.H. Seo, Y. Ryu, B. Jung, H. S. Kim*

Department of Biomedical Engineering, Yonsei University, South Korea

ABSTRACT

The aim of this study is to verify the augmentation of osteoporotic bone quality through minimally invasive low level laser therapy (LLLT) system. Twelve female ICR mice were allocated into 2 groups; SHAM and LASER group. Osteoporosis in mice was induced by denervation on right hind limb. The mice in LASER group were treated by using the minimally invasive LLLT system (3J/cm2, 5 days/week over 2 weeks). The tibiae were scanned before LLLT and at 2 weeks after LLLT by using in-vivo -CT at a 18um of resolution. Structural parameters and distributions of bone mineral density (DBMD) were calculated from acquired -CT images. Relative variations of bone volume fraction, trabecular thickness and trabecular number in LASER group were significantly bigger than those in SHAM group (p

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bone surface to volume ration (BS/BV, 1/mm), trabecular separation (Tb.Sp, mm) and trabecular bone pattern factor (Tb.Pf, 1/mm) were used. In addition, the distributions of bone mineralization density (DBMD) were calculated.

3. RESULTS

The % relative variations of BV/TV, Tb.N and Tb.Th in the LASER group were 370%, 334% and 112%, respectively, higher than those in the SHAM group (Fig. 1, p

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those in the SHAM group. These data indicated that the LLLT might diminish a continuous progress of bone perforation, thinning and disconnection.

Fig. 3 Comparison for 3D microarchitectures of trabecular bone (X-ray attenuation coefficient in LASER and SHAM group)

At 2 weeks after LLLT, the distributions of bone mineralization density in the LASER group were higher compared with those of the SHAM group. This result indicated that LLLT might increase bone mineralization and thereby improve a mechanical characteristic of osteoporotic bone.

As a conclusion, our results suggest that LLLT may have an effect on an improvement in osteoporotic bone quality.

REFERENCE

1. David, V., Lafage-Proust, M. H., Laroche, N., Christian, A., Ruegsegger, P. and Vico, L., "Two-Week Longitudinal Survey of Bone Architecture Alteration in the Hindlimb-Unloaded Rat Model of Bone Loss: Sex Differences," Am J Physiol Endocrinol Metab, 290(3), E440-7 (2006).

2. Aguirre, J. I., Plotkin, L. I., Stewart, S. A., Weinstein, R. S., Parfitt, A. M., Manolagas, S. C. and Bellido, T., "Osteocyte Apoptosis Is Induced by Weightlessness in Mice and Precedes Osteoclast Recruitment and Bone Loss," J Bone Miner Res, 21(4), 605-15 (2006).

3. Warden, S. J., Bennell, K. L., Forwood, M. R., McMeeken, J. M. and Wark, J. D., "Skeletal Effects of Low-Intensity Pulsed Ultrasound on the Ovariectomized Rodent," Ultrasound in Medicine and Biology, 27(7), 989-998 (2001).

4. Renno, A. C., de Moura, F. M., dos Santos, N. S., Tirico, R. P., Bossini, P. S. and Parizotto, N. A., "Effects of 830-Nm Laser, Used in Two Doses, on Biomechanical Properties of Osteopenic Rat Femora," Photomed Laser Surg, 24(2), 202-206 (2006).

5. Renno, A. C., de Moura, F. M., dos Santos, N. S., Tirico, R. P., Bossini, P. S. and Parizotto, N. A., "Effects of 830-Nm Laser Light on Preventing Bone Loss after Ovariectomy," Photomed Laser Surg, 24(5), 642-645 (2006).

6. Ninomiya, T., Hosoya, A., Nakamura, H., Sano, K., Nishisaka, T. and Ozawa, H., "Increase of Bone Volume by a Nanosecond Pulsed Laser Irradiation Is Caused by a Decreased Osteoclast Number and an Activated Osteoblasts," Bone, 40(1), 140-148 (2007).

7. Diniz, J. S., Nicolau, R. A., de Melo Ocarino, N., do Carmo Magalhaes, F., de Oliveira Pereira, R. D. and Serakides, R., "Effect of Low-Power Gallium-Aluminum-Arsenium Laser Therapy (830 Nm) in Combination with Bisphosphonate Treatment on Osteopenic Bone Structure: An Experimental Animal Study," Lasers Med Sci, 24(3), 347-352 (2009).

8. Muniz Renno, A. C., "The Effects of Infrared-830 Nm Laser on Exercised Osteopenic Rats," Lasers in medical science, 21(4), 202-207 (2006).

9. Ruffoni, D., Fratzl, P., Roschger, P., Phipps, R., Klaushofer, K. and Weinkamer, R., "Effect of Temporal Changes in Bone Turnover on the Bone Mineralization Density Distribution: A Computer Simulation Study," J Bone Miner Res, 23(12), 1905-14 (2008).

10. Boivin, G., Farlay, D., Bala, Y., Doublier, A., Meunier, P. J. and Delmas, P. D., "Influence of Remodeling on the Mineralization of Bone Tissue," Osteoporosis International, 20(6), 1023-6 (2009).

18

COLLOIDAL GOLD BASED IMMUNOASSAY FOR HIGH-SENSITIVITY NTX DETECTION IN URINE

Min-Ho Lee*a, Sangdae Leeb,Wookyeong Seonga

aKorean Electronics Technology Institute,

Medical IT Research Center, 68 Yatap dong, Bundang Go, Seongnam Si, Gyeonggi Do, Korea;

bIM-Electronics, Planning Department, Ingye Dong, Pal Dal Gu, Suwon Si, Gyeonggi Do, Korea

ABSTRACT

A simple and highly specific immunoassay for the screening of osteoporosis has been developed using gold nanoparticles. The assay is based upon the absorption changes due to aggregation of gold nanoparticles conjugated with antibodies in the presence of target antigen proteins. For the study, antibodies against bone turnover marker was manufactured and immobilized onto nanoparticle surface. The biomarker cross-linked N terminal telopeptide of type I collagen (NTx) is a well-accepted indicator of osteoporosis and numerous studies have demonstrated their reliability by displaying interactions between the progress of osteoporosis and NTx. Anti-NTx conjugated gold nanoparticles were used to determine the level of NTx in urine samples by measuring the changes of absorption using UV/vis absorption spectroscopy. Their corresponding NTx was leveled and the amount of NTx presence was determined with commercially available ELISA kits for the quantification. Our findings show that the changes in absorption well corresponds with the levels of NTx when tested with NTx kits and it is expected that gold based osteoporosis analysis could be used for fast and simple screening of NTx in clinical applications.

Keywords: Osteoporosis, urine, gold nanoparticle, bone turnover marker

1. INTRODUCTION

Osteoporosis is often diagnosed by evaluating structural bone mass densities using a radiographic technique. However, this method cannot discriminate between the predominance of bone resorption and formation process at the microstructural level, and requires a long time to detect structural changes. Increased understanding of bone physiology has led to the discoveries of several bone turnover markers in urine or blood related to bone formation and resorption. These bone turnover markers can be used to detect biochemical changes before structural changes occur. In addition, they could be potentially used to screen for osteoporosis and to monitor response to treatment. Cross-linked N-telopeptides (NTx) are specific breakdown products of type I collagen which can be used as specific markers of bone resorption, and can be measured in urine or in serum. Previous studies have used NTx to monitor estrogen and bisphosphonate treatment for osteoporosis, and have found that NTx detects response to treatment earlier than other bone turnover markers in samples of patients with osteoporosis. Currently, NTx levels in urine are measured using a commercially available ELISA kit for diagnosis and research purposes (Osteomark; Ostex International, USA). In this study, we used gold nanoparticle based absorption to measure the urinary NTx. Urine samples were collected from the first void of the day and their measured figures were systematically compared with ELISA kit.

2. MATERIALS AND METHODS

2.1 GOLD NANOPARTICLES

The Gold nanoparticles (17 nm) were prepared using the sodium citrate reduction procedure as previously described in the literature. Briefly, 0.01% of tetrachloroauric acid trihydrate (HAuCl43H2O, Sigma Aldrich) was dissolved in 250 ml of boiling ultrapure water by vigorous stirring. Sodium citrate solution (15ml; 1% solution) was then rapidly added,

19

which caused the faintly blue solution to become dark red, indicating the formation of monodispersed spherical particles. The gold nanoparticles formed were spherical and had a diameter of approximately 17 nm (Figure 1). Colloid suspensions were stored in the refrigerator for further use.

Fig. 1. High-resolution scanning electron microscopy (SEM) images of gold nanoparticles.

The gold nanoparticles were conjugated with antibodies against NTx using well known method1 with some modification. Absorption spectra of anti NTx conjugated gold nanoparticles were then measured using an Optizen 322 spectrophotometer. Antibodies against NTx have been used in numerous researches and proven to be reliable marker of bone resorption 2-4.

2.2 URINE SAMPLE PREPARATION

This clinical study was approved by Institutional Review Board at Seoul National University Bundang Hospital (SNUBH) and urine samples were collected during the period between September 2009 and January 2010. All patient provided informed consent. The inclusion criterion was scheduled orthopedic surgery.

3. RESULTS

The gold nanoparticle based immunoassay is designed that if there is NTx present in the urine sample where antigen-antibody interaction leads to the aggregation of gold nanoparticles and eventually lead to the changes in absorption. Antigen-antibody induced aggregation results in the overall size increases of gold nanoparticle which eventually leads to the absorption changes from original value

Figure 2 shows the absorptions of gold nanoparticles conjugated with anti-NTx upon different concentrations of NTx. As the level of NTx is increased, one can see that absorption peak at 535nm decreases interactively.

20

Fig. 2. The CRP concentrations from six serum samples were measured with three different SiNW FET chips. A total of 18 chips were used to measure the samples. Each samples has the average (n=3) and standard deviations.

As shown in figure 3, data points corresponds to optical density changes for anti-NTx gold nanoparticles alone (GNP) and anti-NTx conjugated gold nanoparticles in the presence of NTx antigens different concentrations of 1, 20, 50, 100, 300 g/ml in distilled water. Absorption peak was measured to be at 535 nm.

Fig. 3. Calibration curve based on absorption changes at 535 nm as the function of NTx concentration in phosphate buffer solution. (GNP: 0 g/ml of NTx, NTx: 10, 20, 50, 100, 300 g/ml)

Each data point represents the average of 5 runs. Relative standard deviations ranged from 0.14~ to 1.6%.

For the clinical feasibility tests, a total of 60 urine samples were collected and measured and it turned out that they had serial NTx values due to random collection from the subjects of a comprehensive medical testing center. Total elapsed time of repeat NTx assessment was 3 ~ 4 months after initiation of sample collection and measurements.

When the levels of NTx in male ages under twenty were compared with those of female, both bone formation and absorption rate represented by bone turn-over rate tend to increase up to maximum and start to decrease eventually to the

21

values of grown-ups 5,6. In addition, the results show that differences in bone metabolic rate exist between male and female adolescence (Figure 4).

Fig. 4. Urine levels of NTx in male and female, according to their age. Round represents the levels of male and diamond represents the levels of female.

Of these subjects, seven samples were taken and their spectrum absorption variations were measured with gold nanoparticles (Figure 5).

Fig. 6. Absorption changes in urine NTx samples (right) as well as their corresponding urinary NTx (left, nmole/mmoleCr)

4. DISCUSSION

Gold nanoparticle based immunoassay for osteoporosis has been developed. The variations of absorption upon gold aggregation were measured as a function of NTx concentration. The results were verified using conventionally used ELISA. Although we could not obtain statistically significant estimation from both measurements, their proportional correlation of absorption change to the NTx concentration was clearly observable from the results. The new gold

nanoparticle based assay is capable of measuring the concentration of as low as 1 which is believed to be comparable with the conventional ELISA. In addition, this aggregation based assay needs relatively simple steps in detecting as well as short time in assay process. In order to evaluate the feasibility of our newly developed method, we prepared broad concentration range of NTx by randomly selecting study subject samples aged less than 3 to above 60. Therefore, we could obtain large span of NTx concentration dependent upon their age related bone turnover rate, for example, subjects under 20 years old are measured to have high NTx concentration due to their high turnover rate. Methodically, some issues have to be considered. Although the results have good correlation with ELISA, our experimental results obtainable have relatively short dynamic ranges due to the power limit of detecting equipments. Therefore, in order to enhance simple and inexpensive measurement as well, we have to consider laser diode based

22

system to guarantee power and detection stability. In addition, for the clinical stability, number of subjects should be increased as well as subjects with osteoporosis or paget disease should be considered and monitored for the future studies.

REFERENCES

[1] Jennes L, Conn M, Stump WE 1986 Synthesis and use of colloidal gold coupled receptor ligands. Methods in Enzymology 124:36 - 47.

[2] Hanson DA, Weis MAE, Bollen A-M, Maslan SL, Singer FR, Eyre DR 1992 A Specific Immunoassay for Monitoring Human Bone Resorption: Quantification of Type I Collagen Cross-linked N-Telopeptides in Urine Journal of bone and mineral research 7(11):1251 - 1258.

[3] Garnero P 2008 Biomarkers for Osteoporosis Management. Mol Diag Ther 12(3):157 - 170. [4] Clemens JD, Herrick MV, Singer FR, Eyre DR 1997 Evidence that serum NTx (collagen-type 1N-telopeptides) can

act as an immunochemical marker of bone resorption. Clinical Chemistry 43(11):2058-2063Hirsch LR, Jackson JB, Lee A, Sershen SR, Rivera B, Price RE et al. Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance. Proceedings of the National Academy of Sciences 2003;100(23):13549-54.

[5] Blumsohn A, Hannon RA, Wrate R, Barton J, al-Dehaimi AW, Colwell A, Eastell R 1994 Biochemical markers of bone turnover in girls during puberty. Clin. Endocrinol 40(5):663 - 670.

[6] Mora S, Prinster C, Proverbio MC, Bellini A, Poli SCLd, Weber G, Abbiati G, Chiumello G 1998 Urinary Markers of Bone Turnover in Healthy Children and Adolescents: Age-Related Changes and Effect of Puberty. Calcified Tissue International 63:369 - 374.

23

LASER KINETIC METHOD FOR DETERMINATION OF THE MOLECULAR OXYGEN AFFINITY OF HUMAN HEMOGLOBIN AND POLYHEMOGLOBIN, ARTIFICIAL BLOOD SUBSTITUTE

PRODUCT

Sergei V. Lepeshkevicha, Boris M. Dzhagarova, Rakhimdzhan A. Rozievb, Vladimir K. Podgorodnichenkob

aB.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, 68 Nezavisimosti Ave, Minsk 220072, Belarus;

bResearch and Production Company Medbiopharm, 24a Kurchatova str., 249031Obninsk, Russia

ABSTRACT

Development of blood substitutes that fulfil the vitally important respiratory function is a topical problem of medicine. The major strategy for developing molecular oxygen (O2) carriers is based on chemical modifications of hemoglobin, the natural O2-carrying protein. To find effective carriers, one needs a rigorous method to study O2 binding properties. An approach is proposed to provide answers to such problem as the O2 affinity difference between and subunits in human hemoglobin and polyhemoglobin, artificial O2 carrier. The approach is based on determination of the bimolecular association rate constant for O2 rebinding and the apparent quantum yield of photodissociation from single flash photolysis experiment. The potential of the approach is demonstrated with and subunits within human hemoglobin and polyhemoglobin in the R-state. The affinity of O2 binding to polyhemoglobin is found to be in 2.20.8 times lower than that for native hemoglobin. Plasticity associated with the tertiary structure within R-state hemoglobin is explored through measurements that focus on the functional properties of hemoglobin under conditions designed to tune the tertiary structure without inducing the R to T transition. The possible structural changes, which modify the O2 rebinding properties of human hemoglobin and polyhemoglobin, are discussed.

Keywords: blood substitute, human polyhemoglobin, laser spectroscopy, photodissociation, quantum yield, bimolecular recombination

1. INTRODUCTION

Human hemoglobin (HbA) is the molecular oxygen (O2) carrier of the blood. HbA is an ensemble of two dimers formed by pairs of and subunits, each containing heme b. This protein is known to be able to bind four O2 molecules, one molecule per one heme in each subunit [1]. A saturation curve of O2 binding to HbA exhibits a sigmoid behavior, i.e., the protein binds four ligands cooperatively. During the course of oxygenation, the tetramer can form 10 different molecular species that constitute the particular structural combinations of liganded and unliganded subunits. Each reaction stage is characterized by a corresponding ligand affinity, K, that is the ratio of the bimolecular association, k, to the monomolecular dissociation rate constant, k

kkK '= . (1)

Molecular oxygen affinity, K, is enhanced as HbA is oxygenated and, consequently, the protein itself regulates the ligand affinity [1]. A low-affinity HbA structure is called T-state, and a high-affinity one is called R-state. Since the and subunits differ in structure, knowledge of individual properties (ligand affinities) of each subunit type in the different conformational forms of tetrameric HbA is necessary to completely describe the sigmoid curve of O2 binding to HbA.

24

Human hemoglobin has been evaluated [2] as an ideal blood substitute in surgical procedures. Blood substitutes, also referred to as artificial O2 carriers or artificial blood, have long been studied for their possible use as safe and effective alternatives to blood transfusions [2,3]. With the concern over possible HIV and hepatitis infection, blood substitutes could have a major impact in ensuring the safety of blood transfusion. Typically, the stroma-free HbA as therapeutics suffered from high O2 affinity [4] and showed renal toxicity caused by tetramer-dimer dissociation. To overcome these disadvantages, polymerization and cross-linking of HbA have been proposed [5,6]. Polymerization or surface decoration with long chain polymers is used to increase the size of the molecule and reduce extravasation. On the other hand, chemical cross-linking is used to increase tetramer stability. HbA molecules purified from a natural source are derivatized using bifunctional reactants, such as glutaraldehyde-based chemicals, that form covalent bonds with specific exposed side chains, particularly lysines and histidines. The cross-links promoted by these reactants are usually both intra-tetramer and intertetramer, with multiple side chains involved. Conformational constraints introduced by cross-linking and polymerization perturb the sigmoid curve of O2 binding to HbA by hampering structural relaxations that follow ligand and effector binding.

A detailed comprehension of the molecular mechanism of HbA cooperative oxygenation as well as the mechanism of polymerization effects on the O2-binding properties are fundamental to understand and, to a certain extent, to foresee and control the multifaceted functional properties that will determine the success of a perspective blood substitute. To provide answers to such problem as the difference in O2 affinity for each subunit within human hemoglobin and polyhemoglobin in different conformational forms, an approach based on the time resolved spectroscopy is proposed.

2. MATERIALS AND METHODS

2.1 Sample preparation

Oxygenated human hemoglobin was purified according to a previously described procedure [7]. Human polymerized hemoglobin (PolyHbA) was synthesized according to protocols reported recently [5,8]. Bifunctional agent, modified glutaraldehyde (Fig. 1) was used to cross-link the reaction amino groups of HbA to assemble HbA molecules into soluble PolyHbA of nanodimension.

Fig. 1. Modified glutaraldehyde

Hemoglobin and modified glutaraldehyde solutions were mixed and allowed to react at pH 7.1 0.1 for 1 h with constant stirring. The reaction between amino groups of HbA and aldehyde groups on modified glutaraldehyde yields chemically unstable imine bonds. To stabilize the imine bonds present in PolyHbA dispersions, the reducing agent NaBH4 was used to reduce the imine bonds into stable amine bonds. NaBH4 also terminates the polymerization reaction by reducing free aldehyde functionalities into alcohols. A quenching agent NaBH4 was added to the reaction vessel with constant stirring. The reaction was quenched at pH 8.25 0.25 for 30 min. All PolyHbA production steps were carried out under nitrogen at 7 1C. The molecular weight distribution of PolyHbA ranges predominantly from 250 to 360 kDa, indicating that the majority of this PolyHbA dispersion is mostly composed of species with sizes ranging from 4 to 6 HbA tetramers.

The O2 rebinding experiments were carried out in TrisHCl buffer, pH 7.4.

2.2 Time-resolved spectroscopy

Time courses for the O2 rebinding with hemoglobin were measured on a homemade nanosecond laser spectrometer [9]. Samples were excited by the second harmonic (532 nm) of an Nd:YAG laser (LS-2132U, LOTIS TII, Minsk, Belarus), pulse duration being 8 ns. A halogen lamp KGM24-150 (Brest Lamp Plant, Belarus) fed by a stabilized power supply was used as a probing light source. The output of the lamp was focused onto the sample, then collimated, and finally

25

entered into the entrance slit of monochromator equipped with a FEU-84 photomultiplier as a light detector. The signals measured by the photodetector, I(t), were digitized and visualized by a digital oscilloscope BORDO110 (Unitechprom BSU, Minsk). The photoinduced change in sample absorbance A(t), which reflects the change in the transmitted light from the probe beam after laser excitation, is calculated using the formula:

+=

0

)(1lg)(I

tItA . (2)

The value of I0 in Eq. (2) determines the light level before the laser fires. The transient absorption decays were analyzed with a standard leastsquares technique using a homemade software for PC. The quality of fitting was determined by examining the Student's coefficients for fitted function parameters and also by visual examination of residues. The sensitivity of the detection system allowed us to measure, in the microsecond time range, photoinduced absorption changes down to 1105 absorbance units per 2000 shots.

2.3. Measurements of oxygen rebinding with human hemoglobin and polyhemoglobin

The O2 rebinding with native hemoglobin and polyhemoglobin was examined using a previously described methodology [10]. Transient absorption changes were monitored in the Soret band (430 nm). Monochromator slits were adjusted to a fixed value corresponding to a spectral bandwidth = 10 nm compared with the Soret onehalf width of 16 nm. Such measurements do not distinguish among different conformational substates, but yield information about the average O2 rebinding behavior. The maximal change in the absorbance of an oxygenated sample after photodissociation at the initial time moment, A0, is in direct proportion to the primary quantum yield of photodissociation, 0, i.e. to the ratio of the number of photodissociated 2 molecules to that of absorbed light quanta:

NlVA

=

00 (3)

where is the difference in the extinction coefficients for deoxygenated and oxygenated proteins at the wavelength of observation, l is the optical path, is the fraction of absorbed light, and N is the number of photons striking upon the working volume V. Following the oxygenation recovery, the number of deoxygenated binding centers produced after photodissociation decreases. The ratio of the number of deoxygenated binding centers to that of absorbed light quanta will define the apparent quantum yield of photodissociation. It is obvious that this ratio will be a function of time elapsed after the dissociation event and its value will always be smaller than 0. After the O2 rebinding from within the protein matrix (geminate recombination) is completed, the apparent quantum yield is properly responsible for the number of O2 molecules which escape from the protein. In this case, the apparent quantum yield can be defined as the quantum yield of bimolecular recombination, . In their turn, the ratio /0 represents the efficiency of O2 escape from the protein matrix after photodissociation, . The quantum yield of bimolecular recombination, , was determined using a relative method described previously [11]. Human hemoglobin HbA in 10 mM TrisHCl buffer, pH 7.4, was used as a reference standard, for which = 0.023 0.003 was obtained [10].

3. RESULTS

The photoinduced O2 rebinding to both HbA and PolyHbA was studied when a small amount (0.30.5%) of O2 was released from fully saturated tetramers. Bearing in mind the contribution from geminate recombination, we assumed that the primary photodissociation level does not exceed 5% [10,12]. Such a photoexcitation level was used to ensure the experimental conditions when, statistically, each photo-deoxygenated tetramer loses only one molecule of oxygen. In fact, after photodissociation in the protein solution, two reactions are initiated simultaneously. One occurs with the participation of the subunit within tetramer and the other with participation of the subunit:

( )( ) ( )( ) ( )( )( )( ) ( )( ) ( )( )2222

'

22222222

2222'

22222222

,,,,,,

,,,,,,

OOOOOOOOOOOO

OOOOOOOOOOOOkhv

khv

+

+ (4)

where (O2,O2)(O2,O2) denotes the oxyhemoglobin molecule. In Eq. (4), the oxygenated subunits are shown together with O2. The central terms in Eq. (4) represent the case of free 2 motion in the solution. Here k and k are,

26

respectively, the bimolecular association rate constants for the O2 rebinding to the and subunits within triliganded tetramer.

Time courses for the bimolecular O2 rebinding are presented in Fig. 2. After kinetic normalization, analysis shows that the time courses for the protein reoxygenation over the microsecond (04000 s) time range are fitted with a biexponential function:

Anorm = aexp(k[O2]t) + aexp(k[O2]t) (5)

where Anorm is a normalized change in the absorbance of the sample; a, a, k, and k are the amplitudes and rate constants of bimolecular processes. The quantity [O2] is the concentration of O2 dissolved in the surrounding medium. Based on considerations described previously [10,11], these two exponential processes were assigned to the bimolecular O2 rebinding to the and subunits within tetramer (Eq. (4)). The quantum yields of these bimolecular processes are defined as ()=2a() [13].

Fig. 2. Time courses for the bimolecular O2 rebinding to human hemoglobin (a) and polyhemoglobin (b) in buffer solutions at pH 7.4. Buffer: 1025 mM TrisHCl (a), 10 mM TrisHCl (b). Temperature, 21 C. Excitation wavelength, exc=532 nm; detection

wavelength, det=430 nm (=10 nm). Protein concentration, 100 M in heme.

The parameters for the bimolecular O2 rebinding to hemoglobin and polyhemoglobin in buffer solutions are given in Table 1. The rate constant k and quantum yield for the subunits within HbA fall in the range 30 3 (Ms)1 and 0.0115 0.0035, respectively. The rate constant k and quantum yield for the subunits are found to lie in the range of 66 3 (Ms)1 and 0.036 0.006, respectively. The parameters for the bimolecular O2 rebinding to PolyHbA can be considered as follows (Table 1). The rate constant k and quantum yield for the subunits within polymerized oxyhemoglobin fall in the range 6.8 0.2 (Ms)-1 and 0.0062 0.0006, respectively. The rate constant k and quantum yield for the subunits are found to lie in the range of 49.6 1.6 (Ms)1 and 0.058 0.005, respectively. The data show an essential ligand rebinding difference between hemoglobin and polyhemoglobin.

27

Table 1. The kinetic parameters for the bimolecular O2 rebinding to the and subunits within oxygenated hemoglobin and polyhemoglobin in buffer solutions.

Protein Buffer k

M-1s-1

k

M-1s-1

a

%

a

%

, a

10-2,10-2

, a

10-2,10-2

, a

10-2,10-2

HbA b

525 m

Tris HCl,

pH 6.88.5

30 3 66 3 23.5 5.5 76.5 5.5 1.15 0.35

[4.9 1.7]

3.6 0.6

[15.5 3.5]

2.35 0.45

[10.3 2.2]

PolyHbA

10 m

Tris HCl,

pH 7.4

6.8 0.2 49.6 1.6 9.7 0.2 90.3 0.2 0.62 0.06

[2.7 0.4]

5.8 0.5

[25 4]

3.2 0.3

[14 2]

Protein concentrations are 100 M on a per heme basis. Temperature: 21C. For the kinetic parameters the uncertainties are presented as 95% confidence intervals.

a The efficiency of O2 escape from the protein matrix, , is presented in square brackets.

b Data were taken from Lepeshkevich et al. [12].

Based on considerations described recently [12], the observed O2 rebinding properties of hemoglobin and polyhemoglobin can be ascribed to a family of conformational substates of human hemoglobin in the R-state. In other words, the observed variations in the O2 rebinding are due to the tertiary structural changes of R-state hemoglobin. It is likely that we have regime where the R-state conformational substates are present and are easily interconverting through cross-linking and polymerization of tetrameric HbA. We suggest that, at the hemoglobin modification, the observed decrease in k at the increase in (Table 1) is resulted from a decrease in the rate constant of the O2 rebinding from within the primary docking site. It is suggested that Val(E11) moves closer to the heme iron. It can lead to lowering the access of the dissociated ligand to the heme. Consequently, it can result in an increase of the inner barrier controlling a bond formation between the ligand and heme iron.

4. DISCUSSION

Laser time resolved spectroscopy is a powerful method for studying the protein oxygenation, as it enables a very fast dissociation of O2 and allows to follow for the ligand rebinding process. In the present work, we propose to use the flash photolysis technique for determination of the protein subunit affinity for O2, K, as well as the rate constant for O2 dissociation from fully liganded protein, k, from single experiment. The bimolecular association rate constant, k (Eq. (1)), is measured directly using the flash photolysis technique. The ligand binding to heme proteins corresponds to a complex phenomenon, which may be described in several stages: the ligand diffusion in the solution, the ligand penetration from the solvent phase into the protein matrix, transport across the polypeptidic matrix to the coordinate position, and, finally, the binding process itself, which involves the reactivity of the metal center as well as electronic, structural distal and proximal effects. Under natural conditions the monomolecular dissociation rate constant, k, is determined by the thermal heme-ligand bond-breaking rate, kth, and the efficiency of O2 escape from the protein matrix, th, after the thermal heme-ligand bond rupture. The thermal bond-breaking rate, kth (s1), defines the number of ruptures of the heme-ligand bonds in a unit of time. In the general case the dissociation rate constant is determined as

ththkk = . (6)

A major difference between photodissociation and thermal dissociation is that there is a large amount of excess energy deposited in the heme as a result of absorption of one or more photons [14]. It was demonstrated [15] that the excess

28

energy transfers to the surrounding protein and, eventually, to the water layer in ~20 ps. The time constant of a fast component of the geminate O2 rebinding to hemoglobin (~140 ps) [10] and the time constant of protein thermalization (~20 ps) [15] differ by an order of magnitude, i.e., the ligand movement in protein occurs with a considerable time delay after the protein thermalization. Consequently, it is unlikely that the temporal protein heating influences barriers, which O2 must overcome to leave the protein. Therefore, the ratio /0, representing the efficiency of O2 escape from the protein matrix after the photodissociation, can be considered to be equal to the value th. As a result, the dissociation rate constant, k, can be presented by the following equation:

0

= thkk (7)

Hence, the subunit relative dissociation rate constant, kkref , and the subunit relative affinity, KKref , can be calculated using the formulas:

refth

ref

refthref kkk

k

=

1

00, (8)

ref

refth

ref

refthref

kkkk

KK

=

''1

00 (9)

respectively. Here, the subscript ref corresponds to the reference subunit. The only value in Eqs. (8) and (9) that cannot be determined by the flash photolysis technique is the thermal bond-breaking rate, kth, which is the reciprocal of the strength of Fe-ligand bond. In the case of O2-bound heme proteins, the frequency of the FeO2 stretching mode (FeO2) directly reflects the strength of the FeO2 bond. This stretching mode has been identified for several O2-bound heme proteins by Resonance Raman spectroscopy [16]. Previous experiments [17,18] demonstrated that the FeO2 frequency does not differ between the T- and R-states of HbA within the experimental uncertainty. Therefore, using the Morse potential [17] and assuming the dissociation energy of the Fe-O2 bond equaled to 15 kcal/mol [19], we may conclude that, for each subunit in oxygenated proteins, the thermal bond-breaking rate, kth, can be considered constant with an accuracy of 9%. Based on considerations described previously [20], the primary quantum yield, 0, can also be considered constant and equaled to 0.23 0.03. Therefore, the second important conclusion is that the ratio of 0thk in Eqs. (8) and (9) can be considered constant for each subunit in oxygenated hemoglobin and polyhemoglobin. This is a very fruitful conclusion, because the knowledge of the association rate constant, k, and the quantum yield of bimolecular recombination, , are only required to find the relative dissociation rate constant as well as the relative affinity.

The tetramer total affinity, Kt, for the last ligand binding step is determined using the formula:

KKKK

Kt +

= 2 (10)

where K and K correspond to the affinity of O2 binding to the and subunits within the triliganded tetramer, respectively. Hence, the relative total affinity of the protein is determined as:

refreft

ref

KK

KK

KK

KK

+

+

=

1

. (11)

29

Using Eqs. (9) and (11), the difference in the affinity of the and subunits within hemoglobin as well as polyhemoglobin can be investigated in detail. The presented O2 rebinding measurements indicate that, for two considered proteins, the / affinity ration, K/K, does not exceed 1.4 0.4 (Table 2). However, the affinity of O2 binding to oxygenated polyhemoglobin is found to be in 2.2 0.8 times lower than that for native hemoglobin (Table 2).

Table 2. The relative affinity for the last step in O2 binding to protein, KKref , and the relative rate constant for O2 dissociation from fully liganded protein, kkref

Protein Buffer KK ( kkref ) ( refkk ) ( KKref ) ( KKref ) ( KKref )t

HbAa 525 m Tris HCl, pH 6.88.5, at 21C 1.4 0.4

PolyHbA 10 m Tris HCl,

pH 7.4, at 21C 1.3 0.3 1.9 0.7 1.6 0.5 2.4 1.0 2.1 0.6 2.2 0.8

Protein concentrations are 100 M on a per heme basis.

a The bimolecular oxygenation parameters for hemoglobin in Tris HCl buffer (Table 1) were used as reference.

5. CONCLUSIONS

In the present approach, the laser flash photolysis method is used to measure directly the bimolecular association rate constant, k, and to obtain the efficiency of O2 escape from the protein matrix after photodissociation. The latter value is in direct proportion to the dissociation rate constant, k. Consequently, using a reference standard, for which the dissociation rate constant was found previously, Eqs. (8), (9), and (11) can be modified for calculating absolute values. The affinity of O2 binding to human polyhemoglobin in the R-state is found to be in 2.2 0.8 times lower than that for native hemoglobin. It should be stressed that the represented approach is a way of direct comparison of values determined by the same technique on the same materials. The simultaneous measurement of the individual parameters of the bimolecular oxygenation for each subunit type within proteins avoids mistakes that may arise from an independent determination of the association and dissociation rate constant if the same protein conformation is not measured. Within the framework of the approach, the laser flash photolysis technique allows us to monitor the O2 rebinding to the and subunits within hemoglobin-based blood substitutes as well as to study the influence of various hemoglobin modifications on the O2-binding. We suppose that the represented approach will help to control the O2 binding properties that will determine the success of a perspective blood substitute.

REFERENCES

[1] M.F. Perutz, A.J. Wilkinson, M. Paoli and G.G. Dodson, The stereochemical mechanism of the cooperative effects in hemoglobin revisited, Annu. Rev. Biophys. Biomol. Struc. 27, 1-34 (1998).

[2] T.M.S. Chang, Future prospects for artificial blood, Trends Biotechnol. 17(2), 61-67 (1999). [3] L. Ronda, S. Bruno, S. Abbruzzetti, C. Viappiani and S. Bettati, Ligand reactivity and allosteric regulation of

hemoglobin-based oxygen carriers, Biochim. Biophys. Acta. 1784, 1365-1377 (2008). [4] R.M. Winslow, Blood substitute, Adv. Drug Del. Rev. 40, 131-142 (2000). [5] N.P. Kuznetsova, L.R. Gudkin, R.N. Mishaeva, E.F. Panarin, I.M. Bystrova and E.A. Selivanov, The use of

polycondensed hemoglobin as the basis of a blood substitute capable of transporting oxygen, Doklady Biochemistry and Biophysics. 386, 257-259 (2002).

[6] J.H. Eike and A.F. Palmer, Effect of glutaraldehyde concentration on the physical properties of polymerized hemoglobin-based oxygen carriers, Biotechnol. Prog. 20(4), 1225-1232 (2004).

30

[7] E. Bucci and C. Fronticelli, A new method for the preparation of and subunits of human hemoglobin, J. Biol. Chem. 240(1), 551-552 (1965).

[8] A.Ya. Goncharova, V.K. Podgorodnichenko, R.A. Roziev, V.V. Homichenok, A.F. Tsyb , O.B. Bruskova, Oxygen-transferring blood substitute and a pharmaceutical composition (Variants), Russian patent 2361608, 2008.

[9] S.V. Lepeshkevich and B.M. Dzhagarov, Effect of zinc and cadmium ions on structure and function of myoglobin, Biochim. Biophys. Acta 1794(1), 103-109 (2009).

[10] S.V. Lepeshkevich, J. Karpiuk, I.V. Sazanovich and B.M. Dzhagarov, A kinetic description of dioxygen motion within alpha- and beta-subunits of human hemoglobin in the R-state: geminate and bimolecular stages of the oxygenation reaction, Biochemistry 43(6), 1675-1684 (2004).

[11] S.V. Lepeshkevich, N.V. Konovalova and B.M. Dzhagarov, Laser kinetic studies of bimolecular oxygenation reaction of and subunits within the R state of human hemoglobin, Biochem. (Russian) 68(5), 676-685 (2003).

[12] S.V. Lepeshkevich, M.V. Parkhats, I.I. Stepuro and B.M. Dzhagarov, Molecular oxygen binding with and subunits within the R quaternary state of human hemoglobin in solutions and porous solgel matrices, Biochim. Biophys. Acta 1794(12), 1823-1830 (2009).

[13] S.V. Lepeshkevich and B.M. Dzhagarov, Mutual effects of proton and sodium chloride on oxygenation of liganded human hemoglobin: oxygen affinities of the and subunits, FEBS J. 272(23), 6109-6119 (2005).

[14] L.P. Murray, J. Hofrichter, E.R. Henry and W.A. Eaton, Time-resolved optical spectroscopy and structural dynamics following photodissociation of carbonmonoxyhemoglobin, Biophys. Chem. 29(1,2), 63-76 (1988).

[15] Y. Mizutani and T. Kitagawa, Direct observation of cooling of heme upon photodissociation of carbonmonoxy myoglobin, Science. 278(5337), 443-446 (1997).

[16] S. Hirota, T. Li, G.N. Phillips, Jr., J.S. Olson, M. Mukai and T. Kitagawa, Perturbation of the Fe-O2 bond by nearby residues in heme pocket: Observation of 2OFe Raman bands for oxymyoglobin mutants, J. Am. Chem. Soc. 118(33), 7845-7846 (1996).

[17] K. Nagai, T. Kitagawa and H. Morimoto, Quaternary structure and low frequency molecular vibrations of haems of deoxy and oxyhaemoglobin studied by Resonance Raman scattering, J. Mol. Biol. 136(3), 271-289 (1980).

[18] K. Adachi, P. Sabnekar, M. Adachi, L.R. Reddy, J. Pang, K.S. Reddy and S. Surrey, Polymerization of recombinant Hb S-Kempsey (deoxy-R state) and Hb S-Kansas (oxy-T state), J. Biol. Chem. 270(45) 26857-26862 (1995).

[19] C. Rovira and M. Parrinello, First-principles molecular dynamics simulations of models for the myoglobin active center, Int. J. Quantum Chem. 80(6) 1172-1180 (2000).

[20] B.M. Dzhagarov and S.V. Lepeshkevich, Kinetic studies of differences between - and -chains of human hemoglobin: an approach for determination of the chain affinity to oxygen, Chem. Phys. Lett. 390(1-3), 59-64 (2004).

31

LASER - OPTICAL METHOD OF DETECTION AND ELIMINATION THE LOCAL TISSUE HYPOXIA AND ITS

APPLICATION IN ONCOLOGY

M.M. Asimov *a, R.M. Asimov b, A.N. Rubinov a

a B.I. Stepanov Institute of Physics, National Academy of Science of Belarus,

68 Nezavisimosti Ave., 220072 Minsk, Belarus.

b "Sensotronic Ltd.", 11Kulman str., 220100 Minsk, Belarus

ABSTRACT

Hypoxia - the deficit of oxygen - in solid tumors is the major factor that limits the efficiency of radiotherapy, chemotherapy and photodynamic therapy. In this report new approach to solving the problem of local tissue hypoxia based on phenomenon of laser-induced tissue oxygenation is proposed. Noninvasive laser-optical method of local tissue hypoxia detection is developed. It is shown that by analyzing surface distribution of the intensity of incident and transmitted light it is possible to restore the image of cutaneous blood vessels. Different application of laser-induced tissue oxygenation and the method of visualization the local net of blood vessels both in diagnosis of earlier risk tumor formation, it prevention and increasing the efficiency of therapeutic methods in modern Cancerology is discussed.

Keyword: Tissue oxygenation, hypoxia, oxyhemoglobin, photodissociation, solid tumor, oncology

1. INTRODUCTION

Hypoxia in solid tumor is the major problem [1-3] that limits the efficiency of therapeutic methods, in particular, the method of photodynamic therapy (PDT). Hypoxia in cancer tissues occurs due to the fast growth of cancer cells and disordered angiogenesis. That why solid tumor is located in the regions with reduced oxygen concentration [3]. Large hypoxic area within human solid tumor has been detected in measurements of oxygen tension directly inserting the oxygen electrode in tumor tissue [4].

In the method of PDT mechanism of tumor destruction is based on photochemically generation of singlet oxygen (1O2) and its toxicity for tissue cells. It makes oxygen a key component in the method of PDT. Generation of 1O2 involves the following process:

[1S0]+h [1S1]+[3S1]

[3S1]+[3O2] [

1S0]+[12]

were, [1S0] - is the concentration of photosensitizer in solid tumor; [3O2] and [12] - are concentrations of molecular oxygen and its singlet form in tumor cell. The first stage of photochemical reactions involves excitation of the molecule of sensitazer by laser irradiation in its triplet state 3S1. Second stage includes generation of singlet oxygen - 12 due to interaction of triplet molecules of sensitizer 3S1 with the oxygen molecules in ground state 3O2. It makes oxygen a key component in the method of PDT. Rapid decrease of oxygen concentration in cancer tissue during the generation of 1O2 in PDT is the main factor that induces a local tissue hypoxia [5,6]. Improving oxygenation of the solid tumor masses to eliminate tissue hypoxia remains as actual problem in modern Cancerology.

In present two main methods of oxygenation are used. The oldest method is based on ventilation of lung with pure oxygen at normal pressure. This method is not selective in terms of local tissue oxygenation. An alternative the method of hyperbaric oxygenation (HBO) has been developed. The method of HBO based on inhalation of 100% oxygen greater than one atmosphere [5]. This method is not selective and may cause oxygen toxemia that limit its application.

32

In this paper the results of evaluation of our basic conception [7,8] and experimental study of laser-induced photodissociation of oxyhemoglobin is presented. Special attention is paid to investigation of dependence between the saturation values, laser parameters and optics of human skin.

2. LASER-OPTICAL METHOD OF VISUALISATION THE LOCAL NET OF BLOOD VESSELS

The fast algorithm for light-tissue interaction is based on probabilistic approach. In highly disperse optical media like skin tissue, the photon emitted by light source can reach the receptor by enormous variety of non-trivial paths.

To find out light intensity registered by the receptor all possible paths and their probabilities has to be taken into account. The complexity of such calculations leads to immanent compromise between simplification of the model and the accuracy it can provide.

Classical approach to solve this complexity vs. precision problem is to use Monte Carlo simulation method. It is well known that this method can deliver sufficiently accurate results but the major disadvantage of the Monte Carlo method is high consumption of the processor power. In other words, Monte Carlo is far to slow for usage in real-time applications. We evaluated an alternative approach that deals with complexity problem in another way. A basic of this approach is to separate model generation and model usage parts into separate modules (Fig.1). The most processor resources consuming part is the model generation one. But the generated model itself structured in a way that allows fast obtaining of the calculation results (Fig.2).

Fig.1. The model the tissue structure represented by two layers with homogeneous optical properties

Fig.2. The result of calculation of back scattered light intensity in dependence of detector positio

The model represents the tissue structure by several layers with homogeneous optical properties inside. In this model space within the layer was divided into smaller portions that forming uniform mesh of the nodes. A following simplification was done: Each photon that travels inside the tissue can propagate from node to node only. The probability that photon can move from one node to another is Pn = Pt*Ps. Here Pt is probability that transmittance parameter of the media allows photon to travel the distance between the nodes and Ps is probability that scattering parameters of the media allows photon to propagate in a direction of chosen node. When composing the probability of more complex path each individual node has to be taken into account: Pp=Pn1*Pn2*Pn3 The final probability that photon emitted by the light source can reach the detector can be obtained by summing all possible paths in the media Pd=Pp1+Pp2+Pp3

The input for whole process is a text file with tissue model description. It contents following parameters: number of layers, density of the mesh, light source and detector positions.

Layer1

Layer2

IncidentlightLightdetection

12

Pd=Pt1*Ps1*Pt2*Ps2*Pt3*Ps3

Calculation of back scattered light Intensity

0

0.005

0.01

0.015

0.02

0.025

0 2 4 6 8

Detector position [rel]

Rela

tive

Inte

nsity

33

3. LASER-INDUCED TISSUE OXYGENATION

The molecular oxygen is generated due to laser-induced photodissociation of HbO2 in blood vessels allows control the local increase of oxygen concentration at irradiating region (fig. 3). The possibility of additional oxygen supply allows develop a new method of tissue hypoxia elimination that restores normal cell metabolism.

Basic principle of measuring the oxygen tension 2 in arterial blood is direct method of registration of gas that dissolved in blood plasma. For this usually is used Clark-type oxygen sensor (TcPO2 electrode at Fig.3). The amount of current flowing in electrochemical cell is proportional to the value of oxygen tension 2 in blood plasma or TcPO2 that indicates oxygen tension in tissue. Transcutaneous oxygen monitor (TCOM) - "Radiometer -2 - is used for measuring the value of tissue oxygenation tension.

Amount of oxygen available for cell metabolism delivered by microcirculation is the function of:

2 (TcPO2) = f{F(HbO2)*[O2]} (1)

were HbO2 is the value of oxyhemoglobin arterial blood and [O2] - is the concentration of oxygen released into plasma. In the case of deterioration of the blood microcirculation extra oxygen supply is critical to provide the demands of cell for normal metabolism. This could be reached by in vivo laser-induced photodissociation of HbO2 directly at the zone were necessary to increase the local concentration of free molecular oxygen.

As a result we obtain average concentration of oxygen that releasing in conventional way and due to laser-induced photodissociation of HbO2 in arterial blood:

[O2] = [O2] + [O2h] (2)

For direct in vivo measurements of the tissue oxygen tension TcPO2 electrode was placed on human skin in shoulder area. First, initial oxygen tension was measured. Then He-Ne laser radiation at the power of 1mW was applied. Kinetic of oxygen tension in tissue was investigated. Obtained results were normalized to initial oxygen tension value. Three set of measurement was carried out with the three voluntaries. This method allows measuring and controlling the process of releasing extra oxygen from HbO2 under laser irradiation directly to arterial blood plasma and future it diffusion into tissue.

4. RESULTS AND DISCUSSION

In fig. 3 kinetic of local tissue oxygenation directly at laser irradiation for normal blood microcirculation and disturb one are presented. As it seen the level of tissue oxygenation increases under the laser irradiation and around ten minutes of exposure riches its maximal value exceeding about 1.6 times compare the initial one.

34

0 2 4 6 8 100,9

1,0

1,1

1,2

1,3

1,4

1,5

1,6

1,7

,

.

.

,

1

2

Fig.3. Kinetic of local tissue oxygenation tension under the laser irradiation for normal blood circulation - (1) and artificial ischemia of cutaneous blood vessels - (2).

The comparison of calculated results with experimental data [8] demonstrates that kinetic of tissue oxygenation in dependence of the time of laser radiation gives possibility to determine diffusion coefficient of 2 into tissue. This means that one could calculate and determine how to reach desirable level of 2 in zones with the disturbed blood microcirculation such as solid tumor, burn or wounds [9]. So it's possible to determine optimal parameters of irradiation taking into account the volume that has to be oxygenated and the time of interaction.

Calculations demonstrate equal increase more than for times of extra oxygen released into blood plasma under the irradiation by laser light. For all three cases, calculated diffusion coefficient of oxygen in tissue, differs significantly (up to two times). This demonstrates individual differences in density of skin. The efficiency of laser-induced oxygenation by means of increasing the 2 in blood plasma is comparable with the method of hyperbaric chamber oxygenation at the same time gaining advantages in local action. Proposed novel optical method can be used to selective supply oxygen in hypoxic region to support cell metabolism until therapeutic effect is reached

Thus unique possibility in selective and local increase of the concentration of free molecular oxygen into tissue that enhances metabolism of cells is developed. Laser-induced enrichment of tissue oxygenation stimulates cell metabolism and allows develop new effective methods for laser therapy as well as phototherapy of pathologies where elimination of local tissue hypoxia is critical.

5. CONCLUSION

Optical model of laser - tissue interaction and algorithm of mathematical calculation of optical signals is developed. The model combines spatial and spectral analysis of optical tissue properties. In perspective, time based analysis can be added.

Proposed algorithm of light-tissue interaction modeling deals effectively with complexity of calculations; real-time reconstruction of tissue optical structure became possible. This approach could be applied in developing different laser-optical systems in Biometry.

Besides it the same approach can be used for non-invasive detection of hypoxic regions in order to apply novel technology of laser-induced tissue oxygenation.

It is shown that the efficiency of laser-induced oxygenation is comparable with the method of hyperbaric oxygenation (HBO) at the same time gaining advantages in local action.

35

Method of determination of oxygen diffusion coefficient into tissue based on kinetics of tissue oxygenation 2 under the laser irradiation is developed.

Laser-induced tissue oxygenation in combination with the visualization the local net of tissue blood vessels may become helpful instrument both in therapy and surgery of many skin diseases such as wounds, burns, ulcers, burns and bedsores.

ACKNOWLEDGEMENTS

This work partially was supported by the Belorussian Fond of Fundamental Research, project F08BH-008.

REFERENCES

[1] M.M. Asimov, R.M. Asimov, A.N. Rubinov, "Laser Application in Medicine: On mechanism of biostimulation and therapeutic effect of low energy laser radiation," Proceeding of III Conference "Laser Physics and Spectroscopy". Minsk, l(1), 169-.172 (1997). (Russian).

[2] M.M. Asimov, R.M. Asimov, A.N. Rubinov, "Investigation of the efficiency of laser action on hemoglobin and oxyhemoglobin in the skin blood vessels," SPIE Proceedings Laser - Tissue Interaction 1X. 01.27 - 01.29. 98. San Jose. CA. USA, 3254, 407 - 412 (1998).

[3] M.M. Asimov, R.M. Asimov, A.N. Rubinov, "Action spectra of laser radiation on hemoglobin of skin blood vessels," Journal of Applied Spectroscopy. 65(6), 877 - 880 (1998).

[4] M.M. Asimov. "Laser-induced Photodissosiation of Hemoglobin Complexes with Gas Ligands and its Biomedical Applications," Proceedings of "LTL Plovdive 2005", IV International Symposium Laser Technologies and Lasers, October 8.10 - 11.10. Plovdive, Bulgaria. 3 - 11 (2005).

[5] P.S. Grim, "Hyperbaric Oxygen Therapy", JAMA, 263, 2216 - 2220 (1990). [6] B.A. Teicher, and. C.M Rose, "Perfluorochemical Emulsion Can Increase Tumor Radiosensitivity," Science 223,

934 - 936 (1984). [7] M.M. Asimov, A.N. Korolevich, E.E. Konstantinova. "Investigation of the kinetics of tissue oxygenation under the

effect of low intensity laser radiation," J of Appl. Spectr. 74, 120 -125 (2007). [8] G. Anneroth, G. Hall, H. Ryden, L. Zetterqist, "The effect of low-energy infrared laser radiation on wound healing

in rats," Br J Oral Maxillofacial Surg., 26, 12 - 17 (1988). [9] J.D. Whitney, "Physiologic Effects of Tissue Oxygenation on Wound Healing," Heart and Lung. 18, 66 - 474

(1989).

36

THE ANALYSIS OF LASER THERMOSPLITTING OF MATERIALS BY USING OF CRESCENT-SHAPED BEAMS

S. V. Shalupaev, A. N. Serdyukov, Y. V. Nikitjuk, A. A. Sereda

Francisk Skorina Gomel State University,

104Sovetskaya str., 246019 Gomel, Belarus

ABSTRACT

In this paper the numerical modeling of allocation of thermoelastic fields which are formed during controllable laser thermosplitting in fragile nonmetallic materials is executed within the limits of theory of elasticity. Modelling is executed for laser beams with a cross-section in the form of an ellipse, a ring, and semi-ring and crescent beams. The classical circuit of realization of the given method consists in the superficial heating of a material by laser beam and the aftercooling of this zone by means of a refrigerating medium. Thus, the microcrack, which is organized in the zone of refrigerating medium supply, follows for a laser beam along a treatment line. On the basis of the analysis of allocation of thermoelastic fields it is displayed, that application of the classical circuit of the given method realization with the use of elliptic and ring-shaped beams possesses a number of the disadvantages, one of which is the quick deflection of a microcrack from a line of influence of a laser beam and refrigerating medium at treatment close to collateral border of the sample. Thus, the microcrack is progressed in a direction to collateral border of the sample. It is displayed, that application of crescent beams allows diminishing degree of effects of treatment line closeness to boundary line of the sample on microcrack development. The positive effect is attained due to forming a compression stress zone not only ahead and under the field of a refrigerating medium effects where the microcrack is initialized and explicated, but also on each side of zone of a refrigerating medium effects, that in its turn does not allow a microcrack to be deflected aside.

1. INTRODUCTION

Method of controllable laser thermosplitting is one of the most effective and exact precision methods of separation of products of brittle nonmetallic materials. A number of papers [1-4] is devoted the given method. The classical scheme of realization of the given method is presented in figure 1 and consists in the surface heating of a material by a laser beam 1 and the subsequent cooling of this zone by means of a coolant 2. As a result in the area of coolant feeding the microcrack which follows a laser beam along a treatment line is organized [1, 2]. Final division is fulfilled by mechanical, thermal or ultrasonic finish chopping.

Previously authors had been carried out finite-element solution for a problem on allocation of the thermoelastic fields arising in a sheet silicate glass in the process of controllable laser thermosplitting with use of 2- laser [5-9] which scheme is presented in figure 1. The problem is solved in quasistatic statement according to [10].

X

Y

ZA

B D