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Los Alamos National Laboratory has a long history of exploring unconventional applications for lasers. Three novel applications currently under investigation include using lasers for the analysis of dinosaur gastroliths, for detecting Salmonella contamination in chicken eggs, and for ultra- sensitive, ultra-stable interferometry.
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The emergence of time-resolved fluorescence techniques has been very important to the study of biochemical, biophysical and biomedical research areas. Many of the advances in these fields have been technology driven. The Laboratory for Fluorescence Dynamics has been actively involved in development of frequency domain technology for a variety of applications. Our recent work on microwave super heterodyning detectors, fluorescence lifetime resolved spectroscopy, frequency domain fluorescence imaging microscopy, fluorescence lifetime resolved stopped-flow kinetics and global methods of data analysis is summarized in this report. We provide examples of how these new technologies are applied.
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The description of coherent optical methods including speckle-optics and speckle-interferometry techniques with respect to laser diagnostics methods development is made. The prospective for medical diagnostics schemes of speckle-interferometers and the application of differential speckle- interferometers and coherent optical analyzers for skin vibration and structure investigations is shown.
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The relationships between Mueller matrix elements are considered. The correctness of their application as a criterion for consistency of experimental data and for the estimation of depolarizing ability of real objects are analyzed. Experimental errors have been assessed by means of numerical simulation.
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There is a defmite need to determine accurately optical parameters of tissues. It is required either when planning a laser treatment or trying to make diagnostic conclusions, based on differences in optical properties of healthy and pathological tissues. Scattering phase function is an important characteristic of medium optical properties1.Knowledge of scattering phase function of epidermal layers is necessary for direct calculations of light propagation through skin2. Changes in angular properties of scattered light may reflect pathology of tissue and can be used in investigation of skin diseases, like psoriasis3. Besides the self4mportance measurements of scattering phase function can be used in combination with integrating sphere measurements to determine absorption and scattering coefficients of tissue4. This combination can also be useful for testing of photoprotectors. Direct gomometric measurements of the angular distribution of radiation, scattered by human samples have been reported by several authors5'6. However, known literature data on angular scattering properties of human epidermis are still liniited7'8. In present paper we concentrate on outermost epidermal layers and report measurements, performed on samples, obtained by the novel technique3. We investigated also influence of various chemical's applications to skin surface on scattering phase function of epidermal layers.
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The optical properties of white matter human brain, canine prostate and pig liver were measured in the wavelength range 330 - 1100 nm. The measurements were carried out in native as well as in coagulated tissues. We used the double integrating sphere technique to provide reflection and transmission measurements and a special homogenizing technique to prepare the tissue. The optical properties were evaluated using an inverse Monte-Carlo simulation, considering the geometry of the experimental set-up. All tissues show characteristic absorption bands at 420 nm and 550 nm, related to the strong absorption of haemoglobin. After coagulation the scattering increases drastically while absorption remains nearly unchanged. The anisotropy factor g increases with increasing wavelength and drops down slightly after coagulation. The wavelength behavior of tissue scattering has been compared with theoretical calculations (Mie-theory), showing that ideal spheres with an diameter between 0.6 and 0.8 micrometers fit best to the experimental results.
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An inverse Monte Carlo technique for determination of optical properties of tissues (absorption coefficient, scattering coefficient and anisotropy factor) from spectrophotometric measurements (total transmittance, diffuse reflectance and collimated transmittance) is developed. This technique enables to take into account 2D-geometry of experiment, finite sizes of incident beam and ports of integrating sphere, boundary conditions and sideway losses of light. We performed comparison of inverse Monte Carlo results with predictions of the Kubelka-Munk theory for model media, so estimating validity area of the Kubelka-Munk theory for spectrophotometric data processing. Value of the sideway losses for model media has been evaluated. An example of use of the inverse Monte Carlo technique for tissue properties determination is presented.
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A computer model will be presented which enables the calculation of interstitial temperature distributions during laserinduced thermotherapy. Prerequisite for the simulation is the knowledge of the individual optical and thermal tissue properties. To reach an acceptable duration for the simulation of long laser treatments a simplified spherical geometry was chosen since it fits best to the radiation field of diffusing applicators. Comparison of the calculated temperature behavior with experimental data shows good agreement. A new applicator design with internal cooling will be presented, which enables the coagulation of larger volumes at lower contact temperatures.
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Absence of satisfactory criteria for discrete model parameters choice during computer modeling of thermal processes of laser-biotissue interaction may be the premier sign for accuracy of numerical results obtained. The approach realizing the new concept of direct automatical adaptive grid construction is suggested. The intellectual program provides high calculation accuracy and is simple in practical usage so that a physician receives the ability to prescribe treatment without any assistance of a specialist in mathematical modeling. The real possibility of controlling of the hyperthermia processes exists: the changes of hyperthermia region volume, of its depth and of the temperature levels are possible by means of changing of free convection boundary conditions on the tissue outer surface, of the power, the radius and the shape of laser beam.
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Some biophysical and mathematical aspects of development of computer model of low-intensity laser radiation influence of the dynamics of the X-irradiated bone marrow tissue hemopoiesis are discussed.
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Noninvasive measurement of tissue (human skin) scattering parameters using a mode-locked laser He-Ne (633 nm) and YAG:Nd (1064, 532 nm) is presented. Broadening of output scattered optical pulse was detected by analyzing spectral components on the frequencies of mode beating.
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A yield of hemoglobin and different proteins from erythrocytes in physiological solution was registered by spectrophotometer Specord UV-VIS. This yield was noted after development of dark reactions upon single irradiation of erythrocytes by total light of the mercury lamp DRT-375. It was found that this effect took place in the presence of intact plasma at physiological solution (containing as much as 98% of plasma) too. However the higher percentage of plasma solution presented, the greater doses of irradiation was necessary for demonstration of a yield of different substances from erythrocytes. It was observed definite dependence of this dose versus percentage of plasma. It was indicated that the degree of the damping effect of plasma had time dependence from season, i.e. from a structural composition of plasma.
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Frequency domain spectroscopy provides a quantitative measure of the optical properties, namely (mu) a and (mu) s' coefficient spectra, of multiply scattering, macroscopically homogeneous media or tissue. The diffusion model for photon transport provides the theoretical framework for the analytical expressions of the optical properties. Experimentally, we intensity modulated (60 MHz) a light emitting diode, which emits between 620 - 700 nm. From data sets of relative phase shifts and average intensity at different source- detector separations, we calculated on the basis of the analytical expressions a wavelength resolved absorption (mu) a ((lambda) ) and scattering (mu) s' ((lambda) ) coefficient spectrum. The test material was methylene blue, whose absorption spectrum (maximum 656 nm) closely matches the wavelength profile of the diode source. The multiply scattering, macroscopically homogeneous medium for dissolving the methylene blue was provided by a diluted fat emulsion, Liposyn III. The concentrations of both the absorbing and scattering materials were adjusted to correspond to ranges typical of (mu) a and (mu) s' in tissues. We obtained quantitative agreement between the measured (mu) a ((lambda) ) in the scattering medium and a control solution measured in a spectrophotometer under non-scattering conditions.
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The spectral and image analysis of wound processes performed. It was shown that the intensity of laser induced autofluorescence strongly correlated with the degree of the tissue damage and can be used for the express diagnostics of the tissue status. The values of contrast between different types of wounds and intact tissues were determined and discussed.
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Some living biological objects were investigated with `AiryScan'--computer- aided Phase Microscope with spatial resolution up to 0.05 micrometers and time resolution 1 ms. Influence of ATP on the frequency and intensity of Infuzoria Paramecium Caudatum and Unio pictorium cilia beat were observed.
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Carbonization decreases the optical density in parts of the ultraviolet spectral range while the optical density is increased (up to a factor of 5) in the visible and near infrared spectral range. Optical changes after continuous wave Nd:YAG laser irradiation are inhomogeneously distributed within the irradiated vascular tissue.
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Isotropic and anisotropic Raman spectra of RNA and DNA solutions have been obtained with a scanning monochromator system. The registration of the spectra was optimized for the special purpose of extracting bandwidth information and the non-coincidence effect.
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This paper describes in some detail the analysis of dynamic light-scattering spectra for isolated connectin filaments in solution in terms of a wormlike chain model with the preaveraged hydrodynamic interaction.
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We studied the pH-dependence of the fluorescence and absorption spectra of concentrated solutions of tetrasulphonated aluminum phthalocyanine (AlPcS4)--the potent photosensitizer for the photodynamic therapy of cancer. The possible use of AlPcS4 as the pH-sensitive fluorescent probe for local pH measurements is discussed. By means of numerical Alentsev's procedure the individual spectral components of the fluorescence spectrum main band were resolved.
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The evolution of absorption spectra of blood and bone marrow during keeping has been studied for different sample--anticoagulant--buffer ratios and different anticoagulants. It has been shown that the highest stability of spectra is achieved in the case of delution by isotonic NaCl solution with trylon `B' added. Preliminary experiments on laser irradiation effect on blood and bone marrow in vitro were performed.
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Most methods established so far to estimate red blood cell (RBC) deformability are hard to standardize and include high error of measurement. For our present investigation on the effects of oxygen and ozone on RBC deformability we used the method of laser diffraction in combination of image analysis.
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Quite a number of experimental techniques are used in biomedical research involving the registration of flow velocities of biological fluids. These arc: particle image velocimetry, ultrasonic Doppler velocimetry, speckle microscopy, transmission grating microscopy, etc. Each of these methods has its advantages but none of them provides means to study all the variety of biological objects and dynamic phenomena, via performing noninvasive measurements. An essential alternative method is laser Doppler (LD) spectroscopy and microscopy, based on the registration of Doppler frequency shifts of laser radiation scattered from moving particles. The method yields high spatial and temporal resolution and hence can be used in different fields of biophysics and biomedicine [1,2,3]. The amount of the integral information, averaged over all the particles traversing the probe volume, the real-time mode of measurements, the possibility of registration of the flow velocity profiles -all this makes LD microscopy an efficient method enabling to study biological objects of different levels of complexity with broad range of optical properties. The possibility of fluid flow measurements in live objects with the LD technique was first shown in 1972 [4] and since then the potentialities of this technique have been studied extensively [5, 6, 7J. Nonetheless, though there arc no apparent technical reasons which would prevent from designing an LD microscope (LDM) as a commercial device, LD microscopy has not become yet a conventional technique. There still exist problems of Doppler spectra interpretation and evaluation of data experimentally obtained from biological objects with different opticai properties. These will be discussed below. We describe here an LDM designed in Moscow State University specifically for biophysical and biomedical applications on the basis of our earlier experience in application of LD spectroscopy to the study of intracellular hydrodynamics [8] and of haemodynamics [9). The potentialities of the LDM are illustrated by the results of real time measurements of oscillating flow velocities in relation to two different phenomena: 1 -protoplasmicstreaming in plasmodium of myxomycete Physarum, and 2 -bloodflow in aquarium Danio rerio fish embryo. To carry out measurements in biological objects with different optical properties and a broad range of values of measured parameters the following innovations have been introduced: - inl.roduction of controlled high stability frequency shift in the probing beams; - two-steps formation of optimal probe volume; - generation of the output signal with high signal-to-noise ratio both in analog and in photon ounting regimes; - arrangement of computer controlled fast scanning;- elaboration of the software for signal processing and calculation of parameters under study
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The photometric studies of blood aggregation characteristics were started by the works of Dognon et.al. [1,2] who registered the deviation of the intensity of light scattered from a layer of blood under mixing and after its halt. They have shown the dependence ofthe signal (scattering intensity) on orientational aggregation ofthe erythrocytes. The detailed study of the intensity variation oflight passing through a blood layer in a rheoscope coneplate viscometer was carried out by H.SchmidSchonbein et.al. [3,4,5]. A simplified modification of the registration technique in case of light passing through a special couvette under lOOjn thick after the halt of stochastic mixing of blood, has been elaborated by R.Tukhvatulin (1986) [6]. Backscattering from a shear Couette flow was registered by S.Usami and S.Chien (1973) [7]. This registration mode allowed to considerably raise the probed volume of blood. Detailed analysis has shown that there exists a strong connection between the intensity of the transmitted or backscattered light with the aggregation characteristics ofthe erythrocytes suspension. This has made the technique a major one in clinical and experimental hemorheology (J.F.Stoltz, M.Donner, 1987) [8]. In accord with the primary experiments two approaches have been developed in the aggregometry. The first is based on the registration of light transmission through the StUdied thin (25pi) blood layer in a coneplate type viscometer [3,4,5]. Thesecond is based on the registration ofthe backscattered light from a blood layer in a Couette type viscometer [9, 1 0]. The corresponding commercial apparatuses most commonly used are: the Myrenne aggregometer and the Sefam erythroaggregometer. Anyhow the differences in geometry of the flow and in the detection modes of the outcome light yield different results in the study of the hyperaggregational syndrome even in the in vitro experiments [11]. Attempts of theoretical analysis of multiply scattered light from blood [12,13,14] have not yieldod any important results in the estimation of aggregates dimensions and morphology, and thus could not form the basis to substantiate the optimum construction of the aggregometer. Such optimization includes the optimal choice: of the shear blood layer thickness, ofthe registration mode of scattered light (transmitted or backscattered), of the scattering angle and detection aperture. The ideal design of aggregometer must give reliable information about the sizes of aggregates in each moment of the spontaneous aggregation of erythrocytes and enable to estimate the velocity of aggregation process and durability of aggregates. In this article we shall characterize each of these problems in more detail.
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Modification of optical channel parameters of diode laser-aided analyzer of blood platelets aggregation was offered. Higher, when compared with conventional technique, sensitivity of the method was demonstrated in experimental study of kinetics of cell response to isradipine and `Actovegin' adding to blood platelets suspension.
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The problems of modern polarimetry applications in medical laboratory diagnosis are discussed. An optical train of two channel differential polarimeter is suggested. The possibility of creation of an optoelectronic polarimeter of high sensitivity is experimentally shown. The prototype of an optical differential polarimeter is developed.
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Cyanoacrylate skin surface stripping is a noninvasive method of examination in spatial organization of epidermis. Morphometrical analysis of keratinocytes nuclei in stained epidermal stripping can be used to determination of nuclei size and shape. With the help of karyometrycal analysis the spatial heterogeneity of psoriatic lesions structure was detected. The suggested method of karyometrycal analysis can be used to estimate psoriasis treatment effectiveness and to solve other research problems.
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Investigations of the statistical properties of dynamic speckle fields formed by the diffraction of the scanning focused laser beam on the human epidermis samples have been carried out. The behavior of the second and third order moments of the speckle intensity distributions in the paraxial region of the far diffraction zone has been studied. The comparison of the statistical moments of speckle intensity for the normal and psoriatic skin epidermic strippings has been done.
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The advantages of optical spectroscopy in skin are discussed. The basis for fluorescence spectroscopy (FS) is briefly presented. The potential for in vitro and in vivo FS of biotissues is significant, but not yet well developed. Resolution of autofluorescence spectra into discrete excitation-emission bands is presented, related to various skin fluorophores.
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In the present work the temporal behavior of autofluorescence of upper (30 - 40 micrometers ) layer of human skin under continuous 337 nm laser exposure was investigated. The samples were prepared using epidermis stripping technique. Kinetic curves of autofluorescence decay at 420 nm, 440 nm, and 460 nm have been obtained. In double exponential approximation (I(t) equals a exp(-t/(tau) 1)+ b exp(-t/(tau) 2)+ c) the parameters of autofluorescence decay have been evaluated. Under the assumption that different terms in double exponential approximation correspond to different individual fluorophors the contribution of fast bleaching, slowly bleaching, and non-bleaching components to fluorescence spectrum of the sample at 420 nm, 440 nm, and 460 nm have been calculated.
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The use of fluorescence emission to distinguish between pathologic tissue of tumor and blood vessel was demonstrated. Spectroscopic guidance angioplasty has been reported. These are all based on the difference between normal and pathologic tissue fluorescence. Laser-induced biological fluorescence (LIF) depends on excited wavelength specimen environment. In this work a fourth harmonic from Nd:YAG laser is used as a excitation source. Fluorescence spectra were observed from normal wall of human and animal. Laser-induced fluorescence were also reported from cancerous tissues and cells. We have analyzed the dependence of the fluorescence on specimen environment and exciting intensity. Reasons of causing the difference of LIF were discussed.
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The using of laser spectroscopy methods are proposed for diagnosis of diabetes through the measurements of acetone concentration in respiratory air. Detection of acetone concentration was performed with laser spectrometer operating in the 3 microns spectral range. Minimal detectable concentrations were found. Acetone components in expired lung air of diabetics (for adult as well as juvenile patients) are detectable by absorption spectra measurements in single pass 1m-length cell. Digital smoothing and other methods were used to improve signal to noise ratio.
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Laser induced thermotherapy is a new procedure for local hyperthermia and photocoagulation allowing minimal invasive treatment of tumors and other disorders such as benign prostatic hypertrophia.
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In the present paper the thermal calculation of bone tissue slash modes by intensive IR laser radiation was fulfilled.
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The article is devoted to photocoagulation of the chorioretinal complex by two laser wavelengths (lambda) 1 and (lambda) 2 simultaneously with a controlled ratio of these components intensities. The thermal and biological effects such as mixing under definite conditions is equivalent to the effect of dye laser mono-chromatic radiation with the variable wavelength within the range from (lambda) 1 to (lambda) 2.
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Victor B. Loschenov, Anna I. Volkova, A. I. Volegov, T. A. Kramarenko, Sergey S. Kharnas, Anna M. Posypanova, Alexander A. Stratonnikov, Eugeny A. Lukyanets, P. V. Poleshkin, et al.
Proceedings Volume Cell and Biotissue Optics: Applications in Laser Diagnostics and Therapy, (1994) https://doi.org/10.1117/12.179014
The results of investigations of photosensitizers distribution, accumulation and outputting in the process of photodynamic therapy on the model of Erlikh carcinoma in the experiment are adduced. Phthalocyanines of Al, differing in technology of synthesis and content of sulfa groups, have been used. The character of photosensitizer binding has been determined by the change of its spectral characteristics after binding with albumin in vitro and by analysis of fine structure of fluorescence spectra in vivo.
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Low energy lasers have been successfully applied for the treatment of more than 10 thousand patients in the Sklifosovsky Scientific Research Institute for Emergency Medicine. We have reviewed and generalized the results of clinical investigations of 34 patients with trauma of motor-locomotive system, 15 patients with muscular atrophy occurred as a result of lower extremity fracture immobilization, 124 patients with burns and of 88 post-infarction patients with ischemic heart disease, who underwent laser therapy with He-Ne laser, N2 Ultra-Violet and GaAs Infra-Red lasers.
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Gregory E. Brill, Lyudmila S. Filimonovskaya, Sergei N. Grigoriev, Svetlana G. Petrisheva, Olga V. Proshina, Tatyana P. Romanova, Valentina N. Zhigalina, Tamara M. Zolotarjova
Proceedings Volume Cell and Biotissue Optics: Applications in Laser Diagnostics and Therapy, (1994) https://doi.org/10.1117/12.179017
Positive clinical effect of He-Ne laser usage in different forms of pathology allows to suggest the existence of the influence of low power laser irradiation on certain universal forms of living systems reaction to the action of pathogenic factors.
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The results of the performed experiments revealed that for mechanisms of antioxidant system insufficiency in the wound process, the important part can be assigned to the intensification of glycolytic reactions. Metabolic effects of laser influence on the reparative processes take place at the expense of the fermentative link activation of the antioxidant system, which ensures the maintenance of stability of the biological membranes and thereby of preventive secondary alternative shifts.
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The panel discussion "Nontrivial phenomena in laser light interaction with biotissues and blood" was organized by the Scientific Research Institute of Physical Biomedicine at the GermanRussian Free Uthversity in Saratov. Moderators: Grigory E. Brill, Director of the InstitUte Valery V. Tuchin, Vice-Director of the InstitUte.
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The panel discussion "Interdisciplinary approach to educational problems in biomedical physics" was organized by German-Russian Free University in Saratov. Moderators: Vadim S. Anischenko, Saratov State University Valery V. Tuchin, Saratov State University
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Nowadays in Russia, as well as in Europe, problem of the lack of trained specialists in interdisciplinary fields of science and technology, and among them engineersphysicists and physicists-researchers able to collaborate with biologists and physicians at high today's scientific level, is sharpiy outhned. As a result, problems are evident in efficiency of employment and development of modem medical diagnostic and therapeutic equipment. Skill specialists trained in the field of physics and mathematics with substantial knowledge in biology and medicine are needed. Similar situation has been seen in Russia in the field of biophysics in early sixties'. Today, in medicine, the growing emphasis is placed on less invasive methods of interventions. The demand ofless invasive intervention is best of all met by laser (optical) diagnostic methods and laser treatment. The use of these techniques and laser surgery methods and tools provides shorter hospitalization and rehabilitation time. But the medical personnel and technicians who work now in clinics often can't provide effective, reliable, and safe employment of such intricate equipment as laser units and tools. However, if for the efficient and safe clinical use of lasers training specialists in the field of laser technology and optical units with very general understanding of medico-biological principles oflaser radiation effect on bio-objects seems to be sufficient, in the case of basic medical and biological research the participation of specialist with basic education in biotissue optics, properties of coherent radiation, theory of lasers and fiber optics, laser measurements, and principles of spectroscopy, on the one hand, and physiology, biochemical and biophysical mechanisms of radiation action on living systems, principles of medical diagnosis and objective criteria of treatment, on the other hand, is essential. In recent years, the need for specialists trained in physics and laser technology and at the same time with the knowledge of biology and medical disciplines is evident in industry. Many plants and research-productive amalgamations with modem high technologies lost the bulk of orders for the cutting of budget allocations to defense (in the US the reduction of defense spending takes place too'). Conversion ofindustry, the use ofhigh technology facilities for production oftechnically intricate medical equipment, including laser diagnostic and therapeutical units, is the way out. As a rule, large manufacturers have their own research and design departments, and that is the place where the specialists trained in interdisciplinary field of biomedical optics are needed. It is University with its traditional developed system of basic education in various fields ofnatural sciences (lecturers only for special medical courses should be invited from Saratov Medical University) that is able to train specialists who can on the base of basic knowledge and practical skills perform research and design project on their own.
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The method to determine red blood cell deformability by laser diffraction is presented. It combines ektacytometry (laser diffraction) with image analysis. Laser light is passed through erythrocyte suspensions, which are sheared in a Searle like viscometer. The laser diffraction patterns are photographed by a CCD camera and further analyzed by a computer. Flexibility is characterized by the quotient of minor and major axes of isointensity lines of the elliptically transformed diffraction patterns. The variation coefficient of the measurement is less than 0.5%.
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Laser sclerostomy is currently being evaluated as an approach which can possibly provide a better means of controlling open angle glaucoma than by conventional filtering surgery. This article will introduce various laser systems and their applications in glaucoma filtering surgery.
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Personal monitoring of till-radiation (UVR) is important fo find out both overexposure and underexposure to UVR. At present polysulfone films seem to be most suitable for these purposes. They allow a reliable measurement of till-exposure to wavelengths between 250 and 330 nm if a number of influencing factors is respected. These are described in detail in a technical report of the Commission Internationale de L'Eclairage (CIE). Own investigations with polysulphone films have shown that these polymers can help as well to detect an overexposure to UVR at workplaces as an UVR-deficiency of people living in an old peoples home. It could be demonstrated that this till-deficiency well correlates to the deficiency of serum 25(OH)D3 levels of these people. Furthermore after optimization of a new phototherapeutic equipment in uniformity of UV-exposure round patients body by polysulphone film dosimeters it got useable in therapy.
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