WO2006032981A1 - A method of non-invasive measurement of sugar in blood and construction for its realisation - Google Patents

Abstract

A method of non-invasive measurement of sugar in blood and construction for its realization. The invention refers to technique of measuring devices for noninvasive determination by physical-chemical methods of presence in organisms of human or animal organic and non-organic inclusions and measuring their concentration, for measuring sugar concentration in human blood in particular. The invention is designed for creation of a cheap and portable construction of broad application, using which any person or medical person is able to determine sugar concentration in blood, without sampling said blood. Above mentioned possibilities of the device in proposed device are implemented on the basis of using laser absorption spectroscopy, photo-physical effects of excitation in organic molecules by laser radiation electron and spin-oscillatory transitions, causing big organic molecules of live organism to emit fluorescent and conversion radiation, and resonant absorption of said radiation in solution of substrate under investigation when laser radiation is scattered on human tissues and part of scattered radiation is resonantly absorbed by investigated substrate solution.

Description

A method of non-invasive measurement of sugar in blood and construction for its realisation

The invention refers to technique of measuring constructions for non-invasive measurement by physical- chemical methods of presence in organisms of humans or beasts organic and non-organic inclusions and measuring their concentration, for non-invasive measurement of sugar in human blood in particular.

The invention is designed for creation of a cheap and portable construction of broad application, using which any person or medical person, while inspecting patients, is able to measure the sugar in blood, without sampling said blood. The usage of such construction makes it possible to determine with high enough precision concentration in blood of sugar or any other organic or non-organic components, on the basis of measurements to make a preliminary diagnosis of health conditions of a person and start proper treatment.

Above mentioned possibilities of the construction for non-invasive measurements of presence and concentration of any substrates in human organism in proposed construction are implemented on the basis of using laser absorption spectroscopy, photo-physical effects of excitation in organic molecules by laser radiation electron and spin-oscillatory transitions, causing big organic molecules of live organism to emit fluorescent and conversion radiation, and resonant absorption of said radiation in solution of substrate under investigation when laser radiation is scattered on human tissues and part of scattered radiation is resonantly absorbed by investigated substrate solution. Out of methods, suitable for substrate measurement in general diagnostics, most adequate are modern colorimetric methods by absorption (spectrophotometry) . In majority of them cheap enough pigments are used, and their- procedure of application, precision and selectivity- allow swiftly to obtain the results, sufficient for adequate judgment. Spectrophotometrical methods of substrate measurement may be conditionally divided into two groups: chemical and enzyme(ferment) methods. In first case, reaction consists of a chain of simple chemical transformations, specific for analyte determined (for example, measurement of sugar using its reducing properties) . In second case, chemical conversion of analyte takes place under influence of one or several specific ferments. In colorimetric measurement different types of results registration are used - directly by measuring optical density on the known wavelength

(spectrophotometry) , on the degree of turbidity (for UV range) and on fluorescent and chemiluminiscent marks. Sugar measurement methods are mainly based on using specific for it chemical reactions, but polarographic and electrochemical methods are applied too. On the other side, to achieve precision three main methods are used: reduction methods (based on reducing properties of acyclic glucose) , colorimetrical (ferment and non ferment) and polarographic and electrochemical methods. The first of three is known long ago and is based on property of acyclic glucose to reduce heavy metals acids in alkali medium (the result is obtained by titration) . This method cannot be used as non-invasive, for it needs direct contact with blood samples. Besides, this method is not specific enough, for in blood plasma there are othex- substances, possessing similar properties and adding interference into measurement results. The third group of methods is quite applicable for non-invasive method of measurement of sugar in blood, without its sampling. Especially it refers to electrochemical metb-ods. In said methods different electrochemical or optical sensors are employed, said sensors, being in contact with the skin of a human, allow to non-invasively measure the sugar in blood, by measuring small currents in human skin. Most popular electrochemical sensor - ion-selective electrode. Ion-selectivity is used in potentiometric and ferment electrodes. Their membrane is covered by chemical substance, separated from analyzed solution or gas by second membrane, permeable to substance under investigation. Potenciometrical gaseous electrode registers changes of equilibrium of chemical reaction, taking place in a layer of substance on electrode membrane. In potenciometrical gaseous electrode's membrane is covered by ferment. To monitor sugar content in biological liquids gluoxidase amperemetric sensors are used. In optical sensors specific reagent is applied on the but of fiber thread - light guide. Light beam is directed into light guide butt and light, reflected from the other butt of fiber thread, where the sample is applied, is registered. In metal ion sensors ligands, strongly fluorescenting, when connected with said metal ions, are used. In oxygen sensors oxygen quenching of immobilized fluorofor is used. This equilibrium measurement is less susceptible to temperature and flow rate changes, than amperometric oxygen sensors. Biosensors, using immunological analysis principles, are developed. On the butt of fiber thread of such sensor, antibodies and fluorescent marked antigens are applied. In mass-sensitive transducer (piezoelectric oscillator, for example) selective adsorbent is applied. The substance to be determined precipitates on it and the sensor registers mass change. Similar sensors are applied in measurement of gaseous and volatile matters, aromatic and aliphatic carbon hydrates and pesticides. At the moment such methods of non-invasive measurement of sugar in blood are used in theoretical investigations only and are rarely used on practice, for it is very difficult to develop simple enough construction on their basis. Polarimetric methods need for implementation complicated electro-optical apparatus and even power installations for generating strong magnetic or electric fields. In many modern systems biological objects(ferments, antibodies and receptors) special sensors are applied. Sensors comprise the chemically active matter layer and physical transducer; they are usually applied to selectively determine chemical substances concentration. Besides, they allow to carry out continuous remote measurements. Ferment methods may be applied for analysis as of equilibrium systems and for non-equilibrium systems as well, combine them different methods of detection: spectrophotometry, fluorescence, chemiluminiscence, potenciometry, amperometr;y• Often enough, immobilized ferments are used. In some cases this enhances selectivity of the method, and, besides, allows recycling of ferments and their appLication in flow reactors or in biosensors. Ferments are included in membranes (polymer gel with cross linking) or are adsorbed on a hard substrate. Recently fluorescent, chemiluminiscent, electroactive markers and ferments are widely used. The concentration is measured! of products of reaction of blood interaction with reagent. For measurements changes coloration in visible light range or turbidity for UV light range are used. Colorimetric non-ferment methods are based on reactions of oxidization- reducing and/or complex generation with sxigar participation and colored product formation. In majority of non-ferment methods reducing properties of acyclic glucose are used and most of reagents contain heavy τnetal acids and are toxic. That is why recently in clinic practice ferment methods of measurement of sugar in tilood are most widely used, as most sensitive and selective. Besides, these methods may be applied in automated analyzers and results may be standardized. One group of ferment methods is based on two conjugated reactions: oxidization of glucose to gluconate with hydrogen peroxide released (catalyst- specific ferment gluoxidase) and following measurement of released hydrogen peroxide quantity by coloration of phenol and 4-aminoantipirin mixture, transformed under hydrogen peroxide and glucosidase ferment influence into colored derivatives of chinoimin. The measurements are executed on 546 nm(Hg 492-550nm) wavelength. Another widely used method applies hexocinase measurement of sugar. In this method there is no need to de-proteinize the samples; besides, in measurements (340nm) no dyes are needed: measurement is done by increase of optical density on 340nm. But, it should be noted, that hexocinase measurement of sugar most often is executed in double wave length mode, with obligator-y thermostating at 37⁰C and optical measurements are executed most often in kinetic mode. This restraints the method application in "manual" measurement, but said method is very convenient for semi-automated and automated systems. As follows, there are two border criteria in choosing most adequate method: clinical aims and problems from one side and economics factors from the other.

At the moment, when using colorimetric methods, blood is sampled from organism. But ttαe same method may be implemented without this sampling _ of blood from organism of a human or a beast, e.cj". by non-invasive method. Let us consider these methods as applied in non¬ invasive mode. Of physical-chemical methods of biological tissues content measurement and concentration of substance studied, as modern may be regarded spectroscopic, electrochemical, chromatographic. The spectroscopic method is based on. interaction of electromagnetic radiation with the matter, e.g. on measurement of characteristics of absorbed, irradiated or scattered radiation. Exposed to electromagnetic radiation molecules of the matter transit to higher energy Hevels. Graphically these levels are shown as horizontal, lines and transitions between them as vertical arrows . Energy is absorbed and irradiated in discreet portions (qxiantas) and to exert this absorption falling quanta energy must comply with energy of transition of absorbing molecule into one of excited states. When molecule transits from excited state into lower energy level, the energy is emitted as photon with the energy equal to the energy difference between two this levels. Graphic dependence of absorbance or emission of electromagnetic radiation on wavelength or energy presents the spectrum of the "matter. It comprises of peaks or bands of different amplitude. The difference in positions of peaks, relatively to abscissa (λ or E) shows the difference of energy of these levels. The spectrum character gives information on the nature of the emitting or absorbing matter and the peaks amplitude - on quantity of molecules, which took part in transition (e.g. of matter concentration) . The energy range of electromagnetic radiation is very wicle. The choice of necessary equipment - radiation source, monochromator, detector - depends on the used radiation wavelength and on the character of measurements. A-S light sources in UV and visible range lasers and incarxdescent lamps are usually applied. The slot or prism is "used as monochromator and photoelectron multiplier or photo diodes as detector. Absorption spectrums in UV and visible range contain both quality and quantity information of absorbing matter. Light absorption follows Lambert - Ber law. The samples, used in absorption spectroscopy in UV and visible range, are as a rule strongly diluted solutions. Measurable concentrations depend on molar coefficient of extinction of the matter, under investigation, with maximal value of 10^s (measurements are usually executed on the wavelength of maximal absorption) . To obtain the reliable results optical density to be measured is chosen in 0.01 - 2.0. With absorbing layer of lcm thick it corresponds to concentration of 10^"8 M, which is 1000 times lower, than when titration is used. Usually in process region (linear one) of measurements the concentration may change at least in 100 times. The wavelength is chosen for obtaining of maximal absorption, to exclude the matrix (solvent) influence. Optical density measurements are short in time and that allows to determine the reaction velocity with their help. If the mixture of different substances is explored, then concentration of each of them is measured on different wavelengths, corresponding to maximal absorption of each of them. In luminescent spectroscopy the intensity of radiation, emitted by atoms or molecules, while transiting from excited state to normal one, is measured. There are two types of luminescence: fluorescence and phosphorescence. In fluorescence atom or molecule transit into basic state from excited state, which exists a short time. Fluorescence is monitored nearly immediately after energy absorbance, quickly fades down and disappears because of collisions with other nearby molecules of solution (extinguishing of fluorescence) . Phosphorescence is monitored, when molecule returns into basic state from rather long lived excited state and there may be a long enough interval of time between light absorption and light emission. Phosphorescence is characterized by long wavelength, lower peaks and greater influence of matrix. Fluorescence measurements are more selective than spectrophotometric, due to their dependence on both wavelength: that of absorbed and that of emitted light. Fluorescence intensity is connected with absorbed light intensity by following relation: I_em = klabs• This relation is linear, as concentration is concerned, at small concentrations only:I_em = k^' la_bs C. Here k and k^' - constants, characteristic for molecule absorbing and emitting abilities and C - investigated matter concentration.

Fluorescent analysis allows to measure concentrations in 1000 times less, than spectrophotometric method. It is due to different relations between measured signals: In fluorescent measurements small difference between small signals is determined, while in spectrophotometric measurements big difference between big signals is determined and it is far more complicated. If the light is emitted as a result of chemical reaction, then such process is called chemiluminiscence. Its intensity depends on chemical reaction velocity, which in its turn depends on concentration. Thus, measuring chemiluminiscence intensity, it is possible to determine corresponding reagent concentration. Absorptions spectrums, mentioned above, are the results of electron transitions in atoms and molecules. But absorption in infra-red region is caused by transitions between oscillatory levels, corresponding to different oscillatory energies of functional groups. In IR spectroscopy most often is used the middle of IR-range - 4000-200 cm^"1. The special catalogues and tables are drawn up for interpretation of IR spectrums, where characteristic frequencies of different groups oscillations are shown. The values of molar coefficients of extinction for IR range are smaller than for UV and visible ranges and because of it IR spectroscopy allows to investigate pure matters or very concentrated solutions. Liquids are placed between optically transparent glass plates, where they form a thin film, solid matters are milled and inserted into suspension in optically transparent medium. Investigation of solution is more complicated than of solids, for often the solvent is absorbing in the same wavelength range. To enhance sensitivity" and selectivity of the method in modern equipment IR-spectroscopy with Fourier conversion. Thus, it is clear, that for measurement of sugar concentration in biological tissues by non-invasive method the most suitable is spectrometric method of analysis of scattered light, which passed the biological tissue and created fluorescence by exciting molecules of biological tissue, glucose molecules included. As a result of said excitation of molecules, they emit quantum of light, corresponding with electron transition of every of these molecules. Resulting scattered radiation is analyzed by their wavelengths and intensities. Naturally, each spectrum obtained will characterize corresponding molecule, belonging to biological tissue. But, prior to applying of spectrometric method directly to biological tissue, it is necessary to consider conditions, in which a beam of light, being scattered in such a medium, will be of intensity, sufficient for spectrophotometrical analysis of scattered light and for measurement of sugar in blood on the background of light, scattered by all others molecules. For achieving it is necessary to know approximate biochemical content of biological tissue and concentration of every type of molecules, glucose molecule included. Complex organic compounds play a big role in the nature and are widely used in various technologies. That is why the development of methods and means of their detection was and stays as an actual scientific and technical problem. Complex organic compounds, which are incorporated into natural environment and live organisms, or which are put into these objects accidentally or with some purpose, are of special interest. It is apparent, that most valuable are such methods and means, which allow the nondestructive diagnostics of an object (e.g. in vivo), directly in habitat of complex organic compound(e.g. in situ) , with high sensitivity, quickly and, in some cases, remotely (in case of ecological systems monitoring, for example) . Many organic compounds are able, when optically excited, to fluorescent with quantum output, sufficient for diagnostics. All of them fluorescent in the bandwidths, which position, form and intensity may be used for said compound diagnostics. Fluorescence presents by itself energy, emitted as electromagnetic radiation when molecules transit from excited singlet state into basic one. In fluorescence the life time of excited state for majority of organic molecules lies in the limits from 10^"9 to ICT⁶ s. As fluorescence is the outcome of transition from lower energy level and sublevel, then fluorescence spectrum is practically independent of exciting light wavelength and is always shifted into range of wavelengths longer, than red peak of absorption. All big organic molecules can emit fluorescence, glucose molecules included.

There is known the method (RF patent "The method and the construction to measure the sugar in blood" W 2122208) of measurement of sugar in blood, based on physical principles of excitation and fluorescence (emission at spin-oscillatory and singlet transitions) of molecules, comprising blood, as a result of influence of laser emission of near IR range on blood vessels. Fluorescence is excited, when laser emission is absorbed by molecules of blood. This invention aim is the enhancement of precision of sugar concentration in blood measurements, deleting the blood sampling necessity, creation of cheap, portable, simple in usage construction for sugar concentration in blood measurements. Technical result of the problem is achieved by that electromagnetic radiation of diode laser, absorbed, scattered and reflected by blood vessels of skin, is collected by- spherical mirror, which focuses it on photo-sensor, converting it into electric signal. The signal, thus received, is converted by analog to digital converter into digital code. This code is compared with the code of reference blood sample, saved in computer memory and by their difference the value of sugar concentration in blood is determined. To implement all considered processes and to measure sugar concentration the electric power from the battery is fed to microcomputer, digital screen, power source of diode laser, detector and optical construction. Single crystal microcomputer controls laser diode power source in such a way that it feeds laser diode with constant voltage at the stable temperature, said laser emitting radiation of necessary wavelength and digital-analog converter, which excites laser diode power source and converts digital control signal into analog one. Laser diode light is collimated, divided and directed onto skin region. The light, absorbed, scattered and reflected by blood, is focused by spherical mirror and converted by detector into analog electric signal . Said signal is transmitted to preamplifier and from it into analog to digital converter, which converts analog signal into digital and outputs this digital signal to microcomputer to be compared with electric calibrated signal of healthy human blood sample, which was saved in computer memory.

There is known device (RF patent K? 2122208 "The method of non-invasive measurement of sugar in blood and construction for its realisation") comprising of integration unit in a form of spherical mirror, which focuses the laser light, reflected from vessels surface, diode laser emitter of IR range, detector in a form of photodiode, which converts focused radiation into electric signal, said detector connected to preamplifier, after which the signal is input into analog to digital converter(ADC) and from ADC to single crystal microcomputer to be compared with calibrating signal of blood sample, saved in computer memory and determination by said comparison of sugar concentration in blood, said detector being connected with controlled power source of laser diode, display and the radiation source in a form of laser diode, emitting in IR range. Integration unit and detector are connected by fiber optic line of Im length.

The offered method of non-invasive measurement of sugar in blood based on the comparison of electric signals from the fluorescent emission of molecules of the skin and blood vessels received as a result of scattering and diffuse reflection with the impact of laser emission on the skin surface and electric signals from the fluorescent emission of molecules of calibrating blood, written as a digital code in the computer memory in principle will not match right and absolute meanings of sugar concentration in patient's blood. The scattering and the diffuse reflection of the laser emission from the skin causes excitation and the fluorescent emission of all molecules, forming the skin, blood vessels, blood on vessels and blood sugar, i.e. this is the sum of emissions of different types of molecules having the different atomic composition, molecular weights and their concentrations. At the same time if the average order of molecular weight of blood is thousand, the average weight of skin molecules is thousand times more. That means that the intensiveness and the number of transitions of skin molecules of the spectrum (spectrogram) will so overlap the spectrum of emission of blood molecules, that it will be impossible to identify at this background the spectrum of emission of blood molecules, moreover glucose molecules even by modern spectrometric analyzers. That means that at the modern stage of development of laser spectroscopy there is still no method of the identification of the spectrum of a separate molecule in such a difficult complex of molecules, of which consists the biologic tissue of animals. In modern research of physic photoprocesses for complexes of organic molecules are usually used complexes with very restricted number of types of organic molecules (for example, oils, humus, blood etc.) . So, in order to compare the measured spectrum of emission of blood molecules with the emission spectrum of calibrating blood it is necessary to deduct the spectrum of emission of molecules of biologic tissue, not included in the substrate under review. The comparison of such reflected emission of molecules with the emission of molecules of calibrating blood will give the average meaning of concentration of all skin molecules minus the average meaning of concentrations of molecules of calibrating blood and of the blood of a sick person and the average amount of the difference of concentration of glucose molecules of the calibrating blood and of blood of the sick person, i.e. on the basis of such method one can measure only the relative change of sugar concentration in blood of a sick person, provided that the composition of the calibrating blood exactly corresponds to the blood of a sick person, and that emissions of other skin molecules have been deducted fxom the all spectrum of scattered and diffusely r-eflected emission, not connected with blood molecules. Anyway, when deducting emissions of molecules not connected with the emission of blood molecules, it will be necessary to use, for example, the method of non¬ linear fluorescence by means of the spectrometric analysis, carried out when using the optic spectrophotometric devices, which sizes and complexity will not already allow to consider such a construction as small-scale, simple and not expensive.

The mentioned construction for non-invasive measurement of sugar in blood offers to manufacture the device according to the scheme of emission spectrogram measurement by means of registration of the integral spectrum of the emission of excited molecules of the biologic tissue by the photo pair laser emitter - photo detector and the comparison of the obtained integral spectrum of the emission of molecules of biologic tissue with the spectrum of emission of the calibrating blood as a digital code in the microcomputer memory. Anyway, devices offered in the scheme of the construction for the non-invasive measurement of sugar in blood is not enough, as such device does not have elements, which should deduct the integral spectrum of molecules of biologic tissue, not containing in blood under review, what, naturally, will give the wrong meaning of the measured amount of sugar in blood. Such deduction of the unnecessary spectrum of emission of molecules can be made only by means of differential measurement of the spectrum of emission of molecules. That means that first of all it is necessary to register the emission with separate lines of spectrum of the emission of the biologic tissue provided the high resolution of all spectrum lines, for example, -Ln the IR-diapason and the resolution of lines should match the modern spectrographic and spectro- analyzing devices in order to reveal lines of emission of the substr^*a.te under review from the all emission diapason of the biologic tissue. The deduction from the differential spectrum of the emission of molecules of the biologic tissue of the differential spectrum of the substrate under review by means of spectrum analyzing devices, when integrate the spectrum of the substrate under review, and after only compare the integral spectrum of the substrate under review with the spectrum of emission of the calibrating blood. Anyway, with the amount of organic molecules containing in the biologic tissue transition lines for all kinds of organic molecules will overlap , especially in the IR-diapason, what will not allow to fulfill the resolution of lines not only by means of the offered photo pair, but also with the modern spectrtαm analyzing devices. In such case the regressive mathematic analysis of obtained spectrum of emission of molecules of the biologic tissue will not help to define the concentration when comparing such spectrums, i.e. till now there are no mathematic methods of deduction of spectrums of separate molecules or complexes of molecules from the total spectrum of the emission of the biologic tissue.

The technical achievement of the mentioned invention is the elimination of mentioned shortages of the method and construction, as well as it demonstrated the opportunity to create a. very simple to apply small-scale device for the non-invasive measurement of the concentration of any substrate in human or animal organism, including sugar. The peculiarity of the offered method of measurement of the concentration of a separate substrate in human or animal organism is the use of the water solution of th.e substrate under review as a spectrum analyzer for the analysis of the spectrogram of the fluorescence of sixbstrate molecules in the biologic tissue as the natural spectrometer. The fluorescent emission of the substrate under review in the biologic tissue has got the emission spectrum which corresponds with the resonance spectrum of absorption of the same substrate in the water solution. So, while the fluorescent emission of all kinds of molecules of the biologic tissue through the water solution of the substrate under review will filter this part of the spectrum at the expense of absorption, i.e. at the output of the solution the emission spectrum will consist from transitions corresponding to transitions of all molecules except molecules of the substrate under review. If in such a way we deduct the intensiveness of the scattered light in the biologic tissue acting as a reference beam and passed through the solution of the same scattered light through the water solution of the substrate under review, then the difference of such intensiveness will be equal to the intensiveness of the fluorescent emission of the substrate under review in the biologic tissue. Such problem definition and its solution shows that in such case is applied the so called integral method of definition of the concentration of the reviewed substrate according to the sum of inteixsiveness of lines of spectrum of the emission of substrate molecule, but not the differential method for th.e measurement of the spectrum of the emission of sucli substrate, as it is usually being done, revealing such spectrum by means of measurement of the intensiveness of each transition line by means of a spectrometer. So the construction itself for the determination of the amount of the concentration of the reviewed substrate in human organism is rather simplified. The scheme of such construction is analog to the laser absorption device, but it has not got such device elements as interferometerrs, diffractometers and monochromators, necessary for the resolution of emission spectrum lines. Besides that, first of all the laser beam scatters in the reviewed medium, and after the output scattered light is divided into two beams having the same intensiveness by means of the semi-transparent mirror. One of such beams is a reference beam and passes through the solvent, and the second beam passes through the water solution with the reviewed substrate. Both beams are caught with sensitive photorecei-vers and signals from such photoreceivers enter amplifying devices, and then the logic cell of deduction of such signals. The signal obtained after deduction comes to the measuring device (result indicator) , showing the concentration, amount of the reviewed substrate. The resulting signal is calibrated individually for each person by means of existing devices defining relevant dimensions of the reviewed substrate. The exactness of the definition of the concentration of the reviewed substrate is defined by the sensitivity of photo receivers and with the sensitivity of sensors about 10^"3 mWt will be about ~ 1%. The simple application of the construction, with the individual measurement of necessary dimensions of the substrate for each person, the exactness of measurement of the substrate concentration, as well as its dimensions (5x10x3 cm) quite match the use of such device for each person.

The technical result is achieved by means of registration of the absorbed, scattered and. diffusely reflected by blood emission through the integration block by means of transformation of the emission into the electric signal with the following transformation to the digital code, the comparison of the digital code with the calibration curve of blood and the measurement of sugar from the comparison with the following reproduction of the result at the digital screen, with such a feature, that the emitting surface of the light diode with the laser emission in the visible band to the surface of human or animal biologic tissue pass laser- emissions through the strata of the biologic tissue, scatter the emission at the non-uniformities of the biologic tissue and excite the fluorescent emission of electronic singlet - triplet and rotation-vibration transitions of organic molecules of the biologic tissue, forward the output scattered fluorescent emission to the semi-transparent mirror located on the angle to the scattered emission and divide the output emission into two beams with saτne intensiveness : the reflected (reference) beam and the transmitted (second) beam, the reference beam is transmitted through the basin with a liquid solvent and the second beam is transmitted through the basin with tlie solution of liquid solvent and the reviewed substrate, for example, sugar, absorb with resonance from the second beam the whole spectrum of the fluorescent emission corresponding to the emission of the reviewed substra.te from the biologic tissue, forward bunches of the scattered light of the reference beam and the second beam after passing basins with solvent and with the solution of the reviewed substrate to two separate photo receivers and carry out the integral transformation of energy of intensiveness of the reference beam and of the second beam by photo receivers into electric signals by electronic amplifiers, deducted amplified electrric signals from photo receivers by the electronic logic cell and forward the difference of electric signals to the electronic digital display in order to define the concentration amount of the reviewed substrate.

The technical result is also achieved by the integration block, the detector switched to the analog- digital transformer, which in its turn is connected to the current regulating device in the supply circuit and for data processing and the display, having sαch peculiarity, that the construction is made as separate blocks made of rectangular metallic crossbar with the trapezoid cross-section of the channel cut on the surface, into which is inserted the carriage wdLth trapezoid spikes for possible displacements, where is fixed the light diode of the visible diapason, which emitting surface is closely adjacent on the one side to the surface of the biologic tissue, the carriage, on which is fixed the metallic black diaphragm with a hole, which is closely adjacent from the other side of the biologic tissue and the focusing optic system, located near the diaphragm in order to obtain the parallel and dense optical path after the scattering on the biologic tissue, the carriage, on which is fixed the semi- transparent glass plate, placed under 45° to the direction of the optical path, the plate glass basin for the solvent, located along the beam direction at a certain distance from the semi-transparent plate, which flat surface is perpendicular to the direction of the reflected optical path from the plate, and the sensor of the photo receiver, located along the beam at the other side near the flat surface of the basin, its light- sensitive surface is perpendicular to the optical path, the carriage, on which is fixed the flat glass transparent basin for the solution of the substance under review, which surface is perpendicular to the optical path passing through the semi-transparent plate and the photo receiver, located along the transmitting beam near the basin surface, which light sensitive surface is perpendicular to the transmitting optical path, and from electronic chips for the registration, amplification, logic deduction of electric signals from photo receivers and the electronic digital display for the determination of measurement results.

The character of the invention is explained by technical drawings, pictures and graphs. The fig. 1 shows the structural chemical formula of a glucose molecule, the fig. 2 - the file structure of glucose molecule dimensions, the fig. 3 - the structure of glucose molecule, the fig. 4 - the structure of result file according to coordinates for the glucose molecules, the fig. 5 - energies of α (β) - D - glucose molecule, the fig. 6 - the graph of changing intensiveness of the laser emission in the biologic tissue, the fig. 7 - the scheme of construction for the measurement if the substrate in the biologic tissue, the fig. 8 - the scheme of method of the optic measurement of the substrate in the biologic tissue.

The construction for the implementation of the mentioned method consists of the optical and measuring electronic blocks. The optical block contains the rectangular metallic cross bar 1 as an optical bench with the trapezoid cross section channel cut on the surface along the cross bar, where is inserted the carriage 2 with trapezoid spikes, where is strongly fixed at the front butt of the carriage the light diode 2 with the laser emission 4 in the visible band of 340-640 nm, which ax is directed along the cross bar ax and which emitting surface is pressed to the surface of the biologic tissue 5, through which is transmitted the laser emission 4 and which is transformed at the expense of scattering and the fluorescent excitation of tissue molecules to the scattered light 6, the carriage 7 is inserted at the distance about 0,5 cm from the front butt of the carriage 1 and strongly fix the metallic diaphragm 8 on the one ax parallel to the cross bar ax at the end butt of the carriage, the thickness of such diaphragm will not exceed 1 mm and the hole dimension will not be more than 1 cm, it is strongly pressed to the other surface of the biologic tissue 5, from which comes out the scattered light 6, and at the distance of 2 cm from the diaphragm 8 is placed the optical system 9 consisting of the long- focal and of the short-focal lens with combined focus in order to obtain the dense plan-parallel optical beam 10, insert the carriage 11 about 1 cm away from the carriage 7, where the semi-transparent plate 12 is strongly fixed perpendicular under 45° to the cross bar ax, dividing the optical path 10 into two paths having the same intensiveness : the optical path 13 spreading on the one ax line with the light diode 3 and the diaphragm 8, and the optical path 14 (reference beam) , directed perpendicular to the cross bar ax and parallel to the ax connecting the center of the transparent plate and the transparent glass basin 15 with the solvent, which is located 2 cm from the center of the semi-transparent plate 12, and the photo receiver 16, registering the emission after the path transmission through the basin 15 at the distance of 0,5 cm from the back surface of the basin 15, insert at the 2 cm distance from the center of the semi-transparent plate 12 and the photo receiver 16 registering the emission after the path transmitting through the basin 15 at the 0,5 cm distance from the back surface of the basin 15, insert at 2 cm distance from the carriage 11 with the ax of the parallel ax of the cross bar 1 the carriage 17, on which is strongly fixed consequently along the ax of the carriage the basin 18 with the solution from the reviewed substrate, where takes place the resonance absorption of the fluorescent spectrum of the emission of molecules of the reviewed substrate from the optical path 13 and the photo receiver 19, registering the emission after the optical path 13 of the basin 18, and from the measuring electronic block, where the electronic amplifying block 20 is connected with the photo receiver 16 with the sensitivity about 10^{" 2} mW, the electronic amplifying block 21 is connected with the photo receiver 19 with the sensitivity about 10^{" 2} mW, electronic blocks 20 and 21 are connected with the electronic block of the logic scheme 22, which is connected to the electronic demonstrator 23 in order to obtain measurement results.

The method of non-invasive measurement of the concentration of the reviewed substrate is carried out as follows. The construction is composed of the optical measuring block as an optical bench from the rectangular metallic cross bar 1, with the trapezoid cross section channel cut on the surface along the cross bar, where is inserted the carriage 2 with trapezoid spikes, where is fixed at the front butt of the carriage the light diode 3 with the laser emission 4 in the visible band, which emitting surface of the light diode 3 is pressed to the surface of the biologic human or animal tissue 5, through which is transmitted the laser emission through the strata of the biologic tissue 5, the light is scattered on non-uniformities of the biologic tissue 5 and receive the scattered light 6, insert at the distance of 0,5 cm from the carriage 2 the carriage 7, on which the diaphragm 8 is strongly fixed from the back butt of the carriage, the surface of the diaphragm 8 is strongly pressed to the surface of the biologic tissue 5 and the scattered light 6 is diaphragmed, at the same carriage 8 along the cross bar ax at the distance of 1 cm is fixed the optical system 9, which forms from the scattered light 6 the dense parallel emission path 10, 2 cm from the carriage 7 is inserted the carriage 11, which symmetry axis is perpendicular to the cross bar axis, and at this carriage along the carriage axis is strongly fixed the semi-transparent glass 12, which divides the optical path 10 into two optical paths having same intensiveness: the reflected optical path 13 (reference beam) and the transmitting optical path 14, the basin 15, containing the solvent, through which is transmitted the reflected optical path 14 in order to separate from such path the fluorescent emission of solvent molecules at the expense of the resonance absorption, the sensor of the photo receiver 16 is fixed, which registers and transforms the intensiveness of the optical path 13 to the electric signal, which has passed through the basin 15, 1 cm from the carriage 11 is inserted the carriage 17 with the symmetry axis parallel to the cross bar axis and strongly fix the basin 18 with the solution of the reviewed substance, through which is transmitted the optical path 14 in order to separate the fluorescent emission of solvent molecules and of molecules of reviewed substance at the expense of the resonance absorption, and the sensor of the photo receiver 19, which transforms and registers the intensiveness of the optical path 14, which has passed through the basin to the electric signal, and from the measuring electronic block, which is composed from the amplifying block 20, connected to the photo receiver 16 and amplify the electric signal from the amplifying block 21, which is connected to the photo receiver 19 and amplify the electric signal, connect outputs of amplifying blocks 20 and 21 to the electronic block of the logic cell 22, deducting electric signals from blocks 20 and 21m which output is connected to the electronic demonstrator 23 and obtain measuring results. The efficiency of the method and of the construction for the measurement of sugar in blood is as follows. In order to measure the sugar in blood of an organism without blood taking at the same time are combined several well-known methods, which in the offered combination and with several amendments provide the brand new method, simplifying the obtaining of necessary results unlike existing methods. The information concerning the structure of atoms and molecules and their interaction with medium can be obtained by different methods from absorption, emission or scattering spectrums, arising as a result of interaction of the electromagnetic emission and the substance. In our case the most convenient method is the laser absorption and the fluorescence, which can carry out the registration of photons arising as a result of the ever-emission of excited molecules . Anyway when spreading in the reviewed medium the laser emission scatters, and it is necessary to be taken into consideration, in order the intensiveness of the scattered emission at the output of this medium is enough for the determination of dimensions of the molecule under review. The strong anisotropy of optical characteristics, the structural non-uniformity of biologic tissues stipulate the considerable scattering of the optic emission. The measurement of dimensions of the spatiotemporal distribution of light intensiveness in the strata of biologic tissues allows also increasing the exactness of experimental research in the field of interaction of the laser emission with biologic objects, which, besides that, will allow to know the intensiveness of scattered light coming out from the medium. In order to measure the sugar in the biologic tissue by the non¬ invasive method the most convenient is the spectrometric method of the analysis of the scattered light, which has transmitted the biologic tissue and created at the expense of the fluorescence of the medium excited conditions of molecules of the biologic tissue. Including excited glucose molecules. As a result of the light excitement of tissue molecules such molecules will re- emit to resonance light quantas corresponding with electronic transitions of each such molecule. At the output the obtained scattered emission is spectrometered according to wave lengths and according to the intensiveness. Naturally, each obtained spectrum will characterize the relevant molecule belonging to the reviewed biologic tissue.

The joint moment of the given task is the rational selection of the physic model describing the interaction of the optic emission with the assembly of fluorescent organic molecules or complexes. In the mathematic formulation it defines the number of dimensions to be determined when solving the inverse problem. With rather big meanings of the intensiveness (density of the path of photons) of the exciting F emission the dependence of the registered number of fluorescence photons is equal to N_fi (F) . For the biologic tissue such function will define the fILuorescence of the system of biologic complexes consisting of different types of organic molecules (table 1) . Mathematically it is practically impossible to find such a function for the all biologic tissue, as except the big amount of complexes of such molecules, it is necessary to accept their mutual influence as a result of interaction. So we will consider only the fluorescence of glucose molecules surrounded by all other molecules. For complete organic molecules in case when the interaction between them can be neglected, priority dimensions are: a) cross-section of σ^_s = σ_X3, defining the probability of the molecule transition from the main singlet condition So (from the 1 level) to the first excited singlet condition S_x (level 3) under the influence of the path of photons with F density (l/cm²c) ; b) the life time of T₃ molecule in Si condition, that means at the level 3; c) the quanta output of molecules to the lower triplet condition T_x (at the 2 level) as a result of S_x T_x transition, called intersystem crossing: η_x = k'₃₂/ k₃ where k₃ = k_3X + k'₃₂ + k'_3X k_3X and k'_3X - speeds of emitting and non-emitting transitions from S_x to S₀; k'₃₂ is the speed of S_x→ T_x. The ki_j dimension (its dimension is c^"1) characterizes the probability of the transition from the i level to the j level in such a way, that is n± is the concentration of molecules in the initial i condition, then kij n± is the number of molecules (in the volume unit) , which has passed from I to j condition in 1 second . The reviewed model has got three channels of the desactivation of S_x level, which is characterized by such dimensions as k_3X, k'₃₂ and k'_3X. So the complete number of molecules (in the volume unit) leaving Si level in 1 second is equal to k₃n₃ and, so, T₃ = k^^"1 is the life time of such level . The number of fluorescence photons emitted by the element, wh-ich volume is dV=dx dy dz in the time interval between t and t+dt, dNfi = k₃₁n₃ (t,r,z)dVdt (1) where n₃(t,r,z) is the concentration of fluorescent molecules in excited condition Si (at the level 3) , z - the coordinate along the laser bunch and r = {x, y} in its cross-section. Then the complete number of fluorescence photons emitted by V volume for the time from t=o (beginning of the laser impulse) to t=∞,

N_fi = k₃ r dt [ dr [ dzn₃ (t, r, z) (2) Ja Js J) where S is the beam area, 1 is the thickness of the layer, from which the fluorescent response is registered

(so, V=S_x) • It remains determining n₃(t,r,z) amount. For this purpose is used the system of kinetic equations, which inherently represent the broad class of balance equations. In the same way for our tasks we have to write equations, determining speeds of measuring of n_lt n₂, n₃ molecule concentrations at S₀ , S₁ and Ti levels (they are called level populations) and the equation reflecting the law of conservation of particles:

where n₀ is the concentration of molecules. Let's write for it one of these rate equations for n₃ population (neglecting inter—molecular interactions) :

"³ (*' ^r' = F{t, r, z)σ_i3n_ι (t, r, z) - k₃n₃ (t, r, z) (3 ) ot

The first term in the right part of the equation means the speed of entry of molecules to the Si level at the expense of ttieir transition from the S₀ level under action of the path of photons of the exciting emission; the second term is the speed with which molecules leave the S₁ level as a result of emitting (k₃₁) and non- emitting (k'₃i) transitions Si → S₀ and non-emitting intersystem crossing Si -s> Ti(k'₃₂) . This equation along with analog equations foir n₂ and Xi₁ form the system of linear differential equations which can be solved and, so, the n₃(t,r,z) amount can be found. Meeting such object as human or animal biologic tissue the description of photo-physic processes requires using of more complicated models. The biologic tissue represents the system of organic complexes in the water solution, where the local concentration of molecules, participating in the formation of the fluonrescent response of the complex is so big (>10^~3 M/l) that it is necessary to introduce to the model dimensions describing processes of energy transfer between molecules. Kinetic equations for such objects become rather more complicated, if not by the form, then by the content of the incoming dimensions. Instead the equation (3) we get

where σ* is the cross-section of excitement of fluorescent molecules (flmorophores) in a complex. It is determined not only by the cross-section of absorption of fluorophores σ₁₃ (as it was in (3)) , but also by dimensions defining the transfer of the energy from other molecules of the complex to molecules of fluorophores; k*₃ is the full rate of desactivation of excited fluorophore molecules, including the rate of the inter- molecular deasctivation Ic₃ , as well as the rate of the transfer of energy from fluorophore molecules to other molecules of the complex; y is the constant of speed of singlet-singlet annihilation of excited molecules of fluorophore, stipulated by the effect of the transfer of energy between two excited molecules of fluorophore (each such act of interaction eliminates at least one of two excited conditions of molecules) . So, the equation (4) differs from the equation (3) not only Iby the presence of non-linear term, but also by the rather bigger meaning of other dimensions, as they content the information on the inter-molecular interactions. Finding the n₃ (F) amount by solving the system containing (3) or (4) equations and placing it in the integral (1) we obtain the sought dependence N_fi (F) . Besides that, it is necessary to solve such task for the water solution of glLucose, as well as the task of absorption of the resonance emission (in such case the fluorescence of molecules of glucose of biologic tissue) in the water solution of the glucose. For both cases it is enough to solve the equation (3) , but with different coefficients of rates of photo-physic processes. In order to solve the given problem it is necessary to determine all spectrometric dimensions of the glucose molecule. Anyway, in order to apply spectrometric methods directly to the biologic tissue it is necessary to consider terms, when the light beam after scattering in such medium will have the intensiveness enough for the spectrophotometric analysis of the scattered light and the determination of the concentration of glucose molecules at the background of all other molecules of the biologic tissue, where the light also scatters. For this it is necessary to know the approximate biochemical composition of the biologic tissue and the concentration of each type of molecule, including glucose molecules, which approximate composition is given in the table 1

Table 1

The table 1 shows that glucose molecules located in the blood of a live organism are on the background of the enormous and various quantity of different molecules of the biologic tissue, including other molecules of hydrocarbons, to which glucose also refers. At the same time the water molecule has got the biggest concentration. After water mineral salts and hydrocarbons have got the most concentration. Glucose is the main representative of plasma hydrocarbons. In this juncture it is necessary also to take into consideration that besides photo physic processes of the interaction of laser emission with organic molecules of the biologic tissue will take place processes of scattering of the emission in the biologic tissue at the expense of optic non-uniformity and polymolecularity of the biologic tissue. The biologic tissue represents the heterogenic system containing of cells, which, in its turn consist of the bug number of types of organic molecules (table 1) . In order to define the intensiveness of the outgoing light from the polymolecular medium, which is the biologic tissue, let's use laws of scattering without taking into consideration the light absorption in the medium, i.e. in the Rayleigh approach. In such approach it is necessary to assess in series dimensions of light intensiveness, coming out from the reviewed medium with the given 1 thickness and with given dimensions of the medium (table 1) , enough for using of the obtained light intensiveness in following spectrometric dimensions. The quite uniform medium should not scatter the light - secondary light waves emitted by electrons of organic molecules, excited by the incident wave, are coherent and extinguish each other in all directions, besides ones allowed by laws of geometric optic. Anyway, any other medium always has fluctuations - deviations from the even distribution, positions and orientation of molecules. The light is scattered on density and orientation fluctuations in liquids, on fluctuations of concentrations in the solution. So, for example, the biologic tissue, consisting of cells, which, in its turn consist of different organic molecules (table 1) . So, the tissue represents the non-uniform heterogenic medium, where density fluctuations are defined by the presence of cells, close to each other, between which there is the extracellular fluid. The characteristic dimension of cells of the biologic tissue is changing its amount within 1 μcm. Due to the scattering of light at such biologic non-uniformities the emission, passing the substance layer, weakens. Let's consider the semi- infinite medium with coefficients of absorption and scattering of light μ_a and μ_s accordingly and occupying the half-space z > 0. Let the scattering be isotropic and prevail over the absorption (μ_a << μ_s) , i.e. the medium realizes the regime of multiple scattering of the emission. When the light impulse with the flat wave front (time dependence of the intensiveness I_Qf(t) falls on such medium , the characteristic pulse length τ_L the intensiveness of the emission inside the medium can be represented as the sum of weakened initial beam of photons, still not scattered, I_ri(z,t) and the diffuse field of the scattered light I_dif(z,t) The first of these components gets down quickly with the increase z:

In order to find the diffuse component of intensiveness let's use photon balance equation:

where S and W_dif - is correspondingly the distribution of sources and the volumetric density of the diffuse field in the medium. This equation describes rather well the function I_dif(r,t) far from the border of the scattering medium (with z≥(2÷3)λ_π =1/μ_s ^')) and emission sources. If the period of the photon life in the medium (μ_ac) ^~x much less than the pulse length {μ_acτ_L>>l) , then the process of light scattering in the medium can be considered quasi-stationary. Provided also, that the flat wave falls on the scattering medium, we will finally receive the one-dimensional stationary diffusion equation:

where μ² _eff = Ma /D=3μ_aμ'_s The dimension D= is called

the diffusion coefficient for the photon flow. If there is absorption in the scattering medium and μ_a<<μ_s+μ_a and the scattering is not isotropic, than the expression for D should be corrected provided the indicatrix of scattering and absorption of the minor part of the emission. Usually while considering the diffusion of photons in strongly scattering mediums such expression is used:

where g is the average cosine of the scattering angle, μ=μ_a+μ_s - complete weakness coefficient . In order to calculate the space distribution of the intensiveness of light in the medium it is necessary to supplement this equation with boundary conditions. To make it more simple let's first consider that indexes of the deflection of transparent and scattering mediums are same, i.e. there is no reflection, falling on the emission medium. Then, analog to the boundary condition for the current density in the electrodynamics let's put the equation of normal components of the density of the flow of photons at the division border. So, initially the boundary condition for I_dif(z) at the surface of the medium and the function of distribution of sources S(z) can be represented as:

Idif (z =-z₀) = 0 (6) S (z) = I_oδ (z -Z₁) (7) where Z₀ = 2/3 X^ λ_π = 3D - the transport length of the photon Z₁ « X₇₁- the depth on which the collimated emission falling on the medium is transformed into the diffuse one. The solution of the equation (5) with the boundary condition (6) and provided (7) with z > Z₁ = X^ gives equation (8) :

I_di_f (z) = (cl₀/ (2μ_eff D) )exp{-μ_eff (z-zi) } [l-exp{ -2μ_eff (ZQ+ZJ }]« =I₀(3/2μ_eff XJ {exp(μ_eff XJ -exp [-μ_eff K (2Δ+1] }exp ( -μ_eff z)

So, the light intensiveness inside the scattering medium at distances z > X^

I(z)= I₀ exp[-(jU_a + μ_s)z]+I_dif (z) = I_oh(z)

where the coefficient of light weakening at the expense of scattering is defined by the expression:

M_a =32 TΓ³ (n-1)² /(3 X⁴N₁ ) (9)

First of all let's consider processes of light absorption only as a result of light scattering on non-uniformities of the biologic tissue. The coefficient of light absorption at the expense of excitation of molecules of the biologic tissue and in the solution of the reviewed substrate will be calculated below on the basis of quanta processes of excitation of electronic and vibration- rotation levels of the glucose molecule by laser and resonance emission. According to this condition the solution can be represented as more simplified:

I (z) = I₀ exp (-μ_a -z) In such real medium as the biologic tissue there is interaction between scattering centers, depending on

concentration. In such case (AΛO ^V cannot be used for calculation, in order to calculate μ we should use Einstein theory, explaining the light scattering by liquid. For the polymolecular medium, such as biologic tissue, the average molecular weight for all types of molecules of the medium is defined by the equation

The exposed theory refers to particles, which dimensions, are much less than the wave length λ. If such condition is not fulfilled, it is necessary to take into consideration the difference of phases of the secondary light waves issued by different spots of particles. Waves scattered by the particle are interfered in this juncture the summary intensiveness of the scattered light decreases. The angular distribution of scattering changes - in formulas changes, for intensiveness the surplus, the complicated function P (0) , asymmetric relative to 0. The intensiveness of light scattered in front is more than of one scattered behind - there is a Mie effect. The model of the organism cell can be represented as a static Gaussian coil . Then the considered function for such model will look like this: P(0) = (2/ x² ) (exp(-x)+x -1) (11)

32π² r² where x= -sin 3/2, r being the average square of the 3 λ² particle radius. So the cited intensiveness and the turbidity factor look like: βi = μ P ( 5 ) ( 12 )

Let's make digital calculations in order to define the output intensiveness of emission from the biologic tissue after the scattering in this medium of the laser emission of the visible band (red band) . Let's define first of all the amount of the average molecular weight of the biologic tissue basing on data for the table 1. Putting such data in the formula (7) we get that the average molecular weight of the biologic tissue is

M = 2.10⁷ (g/M)

The mean value of biological parts concentration in biological tissue Ni =6.5 10¹⁵ (cm^"3)

Therefore, mean radius of a biological part equals r = 5-lcr⁵ (cm) - (0.5 μm)

Such a size of a biological part corresponds in value to biological cell of organism . With such mean radius the biological part size is comparable or even less than emission wavelength. In this case forward scattering function P(0) < 1 and it may be neglected. Biological tissue is a water solution of all organic and non-organic molecules, shown in Table 1. In this case it is known, that the solvent (water) in this solution makes 70-80% of all the solution. Thus, mass concentration of biological molecules in such solution as biological tissue amount to c = 0.25 (g/cm³) Mean refraction coefficients for the solution and the solvent are equal, accordingly, n= 1.47 n_o= 1.33 Inserting all, named above, values into expressions (5) and (6) we obtain the turbidity coefficient of biological tissue equal to μ_a = 13.05 (I/cm) For determination of intensity of output from biological tissue scattered light, we shall use laser emission source of 1OmW of power and the output window area of 0.25cm² . Emission of such power is harmless for organism and is widely used in medical practice. The intensity of light at such power in the range 500-lOOOnm is equal

I = 5-10¹⁶ (photon/sec)

Inserting the corresponding values of turbidity coefficient, biological tissue thickness (1=0.5cm) and the initial intensity into formula (12) we obtain forward scattered light intensity in biological tissue equal to

I = 7.4 10¹³ (photon/sec)

The value received by its order allows to suppose, that such intensity is sufficient for using of scattered light in determination of glucose concentration blood of organism. But this value was obtained disregarding light quantum absorption in investigated medium. Below are shown calculations of scattering in modeled biological medium, where the absorption of quantum in biological tissue due to photo-processes and exciting resonant transitions in big organic molecules, glucose molecules included, is taken into account. For determination of processes of light quantum absorption in solution of glucose and in biological tissue and obtaining of the glucose molecule in biological tissue emission spectrum the necessary calculation are done on determination spectrum parameters of glucose molecule (α, (/3) -D- glucose) . Determination of spectrum parameters of glucose molecule and photo-physical processes, taking place, when said molecule is excited by laser emission, is necessary for obtaining of spectrum of resonant molecule emission and laser emission absorption coefficient in biological tissue, wheire glucose molecules are included, and for determination of resonant emission glucose molecules. This data will allow to determine the way and necessary parameters of the device for selection of spectrum of glucose molecule emission from all the spectrum of biological tissue emission and on the basis of said glucose spectrum to determine sugar concentration in investigated biological tissue. The calculation are done on the basis of quantum equations of Heartry-Fok. Theoretical investigation of physical-chemical properties and photonics of multi-atomic molecules by semi-empirical methods of quantum chemistry demands solving of Heartry- Fok -Rutan equation system by method of partial neglecting of differential crossover (PNDC) . Rutan equation in matrix form is written down as:

FC = e SC ( 13 )

Where :

S_μv

integer matrix of atomic orbital (AO) crossover, e— diagonal matrix of real proper values of e_x of matrix F, C - coefficient of resolution of molecular orbital (MO) by AO, which are defined by solving of equation: det (F-eS) = 0

This equation is solved by method of partial neglecting of differential crossover, when approximate uniform equation FC= eC is solved. Electron structure and electron-excited states of proposed molecule is defined by nature off molecular orbitales in it and by electron- excited singlet and triplet states. On the basis of obtained results determination and selection of bands in experimental absorption spectrum, determination of changes of electron charge in molecule atoms, when excited by electrons and comparison of calculated characteristic of basic and electron-excited states with known experimental data is carried out. This semi-empiric method takes into calculations only valence electrons of molecule atoms. Calculations of excitation processes in glucose molecule, of emission spectrum and other photo- physical parameters, that are needed for calculations of processes of absorption of laser emission and resonant emission of biological tissue and in glucose solution, using this method, are shown below. As in every semi- empiric method, in solving of said equation system certain parameters- ±ntegers, defining different types of electrons and nucleus of atoms interactions, are used. Some of these integers are chosen in accordance with experimental data and used in solving of equation as ready ones. Others integers are calculated in the process accordingly to special formulas, and for it the coordinates of atoms , constituting molecule investigated, must be known. As initial data values of chemical bonds length, valence and torsion angles are used. The length of bond - the distance between molecule atoms, connected by chemical bond. Data on length of bonds is obtained in physical researches by roentgen-structural analysis of crystals, by electr-on-graphics and neutron-graphics of vapors and by studying of molecule spectrums. Valence angle - the angle between two neighbor bonds. Its value is determined by valence state of atoms connected. The problems of spatial structure of molecule is investigated by stereo-chemistry, with the basis of directed valence theory. In a flat benzole molecule valence angles between bonds are equal 120° and carbon atom state corresponds to sp2-hybrid. In this valence state carbon makes three symmetrical σ-bonds, lying in one plane and one π-bond. Electron cloud of every iτ - boned is perpendicular to the plane of σ-bonds. That is why in phenol ring 7r-bonds lie in X-Y plane and σ- bonds are perpendicular to it . In other aromatic molecules valence angles are near to 120°. Valence angle between bonds of carbon in sp-hybrids are equal tol80°. Carbon in this state creates triple bond, consisting of one jb-bond and two n-bonds, lying in orthogonal planes. The angles between bonds are equal to angles, corresponding to valence state of atom, in isolated symmetrical molecules only. Valence angles deviate from these values due to spatial distortions. These deviation values are such, that in comparison with deformation of valence angles, atom bonds lengths may be taken as constant . Torsion angle - the angle between two bonds, lying in different planes. The angle between such bonds is an angle between two planes, which can be defined by Newman projections. In every concrete case they are to be defined, judging on spatial structure of molecule. General view of the program for atom coordinates of α-D-glucose molecule is shown on Fig.l . The length of contacts at Van-der-Vaals interaction (A) (Table2)

On fig.2 molecular coordinate system, atoms numbering and direction of molecule tracing around, when initial information is entered. When said direction is stated, then usual bonds become vectors. In the first line of a file three numbers are shown: first- the quantity of atoms in molecule, the second- the number of bonds to be entered, and the third - control bonds quantity. The second line - X,Y, Z - coordinates of the first atom. In this case δ-orbitales of aromatic molecule atoms will be lying in X-Y- plane and π-orbitales - in a plane perpendicular to it (Fig.2) . The lines from 3 to 21 of the file "moco.dat" contain information of structural parameters of chemical bonds in molecuILe (length of bonds, valence and torsion angles) . The fiirst two numbers of every line correspond to numbers of the first and the last atom. R[i] , R[j] - Van-der-Vaals radius of atoms i and j; σ=0.15A for a contact without hydrogen, σ=0.3A, when it is taking part in chemical bond, the third number- this bond length in A; the forth and fifth numbers - valence and torsion angles in (°) . In molecule a. (/3) -D-glucose there are 23 bonds, but bond 6-1 may be neglected, as coordinates of atoml will be calculated from data for bond 5-6 and coordinates of atom 6 - from data for bond 5-6. As the program calculates 22 bonds only, then it is necessary to close bond 6-1. For torsion angle determination, besides considered bond it is necessary to know of two previous, as the direction of tracing around is concerned, bonds. In determination of torsion angles of bonds 1-2; 2-3; 2-10; 7-13; 7-14 and valence angles of bonds 1-2; 1-7, real bonds in molecule are not sufficient. Because of this we shall enter two unit vectors (virtual bonds) , which will be previous for above mentioned, strictly complying with direction so as first unity vector was anti-parallel X-axis and. the second - parallel to Z-axis. This will be vectorrs Il and 12, Torsion angle for 2-10 bond will be defined by rotating of projection of vectorI2 anti-clockwise till alignment with projection of bond 2-10. While determining toxsion angles C-H bonds of methyl group the following triples of vectors are taken: 2-3 3-8 8-15; 2-3 3-8 8-16; 2-3 3 -8 8- 17. The angle between projections of C-H bonds on P-plane equals 120°. Torsion angle for each of C-H bonds wiH.1 be defined as an angle between bond 2-3 (2' -3') projection and projection of each of the bond under consideration, counted anti-clockwise (Fig.3 B-C) . The result is shown below:

Bond Torsion angle Bond Torsion angle

3-8 180 5-9 180

8-15 0 9-18 60

8-16 120 9-19 300

8-17 240 9-20 180

As a result we obtain atom of molecule glucose coordinates, shown on Fig.3

Now let us on the basis of obtained coordinates calculate electron structure and electron spectrurns of molecule a (β) -D-glucose by PNDC method and interpret the results obtained. It is assumed to include in calculations of carbon atom and hetero-atom of 4 atomic orbitals and for hydrogen atom -1 orbital. For cairrying out of calculations of excited electron states of gLucose molecule it is necessary to enter main parameters of molecule a. (/3) -D-glucose. Numbering of atoms of moLecule under consideration and molecular system of coordinates are shown on Fig.3. The molecule is flat, parameter's are singlet. Atoms number -23; Molecule charge -0; Coefficients of expansion of MO in terms of AO: filLed MO 10; vacant MO - 10; Variants of calculations: 0- fundamental state (S₀), 1 - S₀ and excited singlets, 2 - S₀ and excited triplets, 3 - S₀ and excited singlets and triplets, Matrix: of configuration interaction: filled MO - 10; vacant MO - 10; excited states - 10. Array of atoms (1-C; 2- O; 3 - H) - 11111133333333122223332, Array of atom orbitals - 2 2 4. 2 2 6, 1 0 1 The calculations deal with the following characteristics of fundamental state of molecule: MO energy, Coefficients of expansion of MO in terms of AO, electron charge distribution in MO and molecule atoms and the value and direction of dipole momentum of molecule in fundamental state. Now let us process the results: Energy array of MO contains energy of all molecule MO. Overall quantity of molecule MO is defined by molecule atoms type and quantity. PNDC method uses in calculations 4 AO for each carbon and oxygen atom and 1 AO for each hydrogen atom. Therefore for molecule a (β) -D-glucose 59 MO are determined:

4x12 (C₁- C₆, C₁₅, O₁₆-O₁₉, O₂₃) +1x11 (H) =59

Overall quantity of valence electrons of glucose molecule, included into calculations, equals correspondently to to number of electrons in valence shells of atoms: C-2s² 2p², 0 - 2s²3p³ and H - Is¹) : 4x7 (C atoms) +6x5 (0 atoms) +1x11 (H) =69 Taking into account Pauli principle, gives us 34 filled and 25 empty MO. In this case 34 MO is upper filled one and 35 - lower empty one. According to Kupmans theorem ionization potentials of molecule are equal to energies correspondent MO, with their sign inverted. Comparison of measured ionization potentials with calculated energies of filled MO will show reliability of results of calculation of fundamental molecule state.On the basis of above calculations coefficients of expansion of MO in terms of AO define the nature and localization of MO. Upper line of data array - AO number, in accordance to numbering of atoms in this molecule. The first - atom C, with belonging to it AO 1 to A04 - these are 2s, 2p_x, 2p_y, 2p_z AO, To atoms O₁₆ -O₁₉ , O₂₃ - AO 25 -48 ( 2s, 2p_x , 2p _y/ 2p_z AO) . To hydrogen belong AO45-499 (Is AO)

While determining the orbital type coefficients of expansion of MO in terms of AO are compared and the biggest ones are chosen. In glucose molecule upper orbital consists of p_x atomic orbitals of C (phenil ring) and 0, i.e. AO 4, 8, 12, 16, 20, 24 and 28. In this case maximal coefficients of expansion belong to p_x AO of C₄ atom and atoms in bond C₄ O₁₆ - As in creation of MO 34 take part 2p_x AO only, then this AO is 7r-orbital . The same may be said of MO 33, 35, 36. MO 33 - 8, 12, 20, 24, 32, 36 - 7r-orbital MO 34 - 4, 8, 12, 16, 20, 24, 28 - τr-orbital

MO 35 - 8, 12, 20, 24 - τr-orbital MO 36 - 4, 8, 12, 16, 20, 24, 28 - ir-orbital

Coefficients of expansion of MO in terms of AO informs us which fragments of molecule take part in formation of this MO. The degree of occupation of AO shows the distribution of electron density in AO of every atom. For C atom, for example, the distribution is as follows:

In glucose molecule in fundamental state the sum of charges in all AO of this atom is positive, as atoms charge is less than 4e. the values of charges are shown for all atoms of molecule. From this follows, that In glucose molecule in fundamental state negative charge is localized in atoms: C₃, O₁₆ -O₁₉, O₂₃. Dipole momentum and its projections on molecular axis are shown for fundamental state. The comparison of the values and signs of molecule dipole moment projections on molecular axis show the direction of dipole momentum. Dipole momentum of glucose molecule in fundamental state is 9.69D and is directed to X-axe.

Dipole momentum (in D) : D=9.69 DX=6.00 DY=-3.92 DZ= - 6.52. Spectrum of singlet conditions of molecule:

51 condition *energy: .89eV 1396.1 nm 7163.cm-1 *F = .0014818787 * D = 8.16, FX = .000716017 FY = .000492266 FZ = .000273595* DX = 5.54DY = -.68 DZ = -5.95, condition

52 * energy: 1.22eV 1013.8 nm 9864.cm-1 F = .0151369641D = 10.12, FX=.011901426 FY = .001287547 FZ = .001947991*

14331.cm-l*F = .0013560233*D = 14.30, FX = .000000263 FY = .001352943 FZ = 0.000002817* DX = 13.09 DY = -5.23 DZ = -2.39, conditions S4 * energy: 1.90 eV 652.8 nm 15320.cm- 1*F = .0153159267*D = 9.77, FX = .012663934 condition S5 * energy: 2.22eV 558.6nm 17903.cm-l*F = .0060119506*D = 12.53, FX = .002832839FY=.003161600FZ=.000017511*DX = 10.43 DY= - 12.53, FX = .002832839 FY = .00316100 FZ = .000017511*DX = 10.43 DY = -1.07 DZ = 1.50 DY = -3.27 DZ = -4.40, condition S7 energy: 2.32eV 533.4 nm 18747.cm-lF = .0253928142*D = 7.29, FX = .016197885 FY = 0.009032485 FZ = .000162444* DX = 4.43 DY = -2.17 DZ = -5.36, condition S8 * energy: 2.40 eV 517.6nm 19318.cm-1 *F .0186957303 *D = 9.71, FX = .005446416 FY = .006814361 FZ = .006434953 * DX = 6.87 DY = -1.66 DZ = -6.65, condition S9 * energy: 2.65eV 467.6nm 21388.cm-l*F = 0.196913425*D

10.07, FX = 0.01115767 FY = .009357185 FZ .009218390*DX = 7.30 DY = -1.07 DZ = 6.85

The spectrum of singlet conditions contains the energy of singlet conditions, the oscillator power S₀-> S₁ - transitions (f) , the dipole moment of molecule (D) in excited conditions, as well as projections f and D at the axis X, Y and Z.

Experimental data on spectral characteristics and photo-processes of interaction of light quantum with glucose molecule Pure sugar crystals are transparent and colorless. There were found several bands in absorption spectrum in IR region: 0.98; 1.44; 1.51; 1.58; 1.7 μm. Said crystals possess triboluminiscence properties - sugar crystals emit very bright bursts of light, when crushed. This crystal is an insulator, its dipole momentum equals 3.1-10^"18 din⁰"⁵ cm², piezoelectric, diamagnetic, its dielectric permeability is 3.5 - 3.85, magnetic permeability -0.57 • 10^"6 .

Accordingly to results of calculations parameters of the first singlet state of molecule a (β) -D-glucose 59 are as follows:Si state energy equals 7.163 cm^"1, oscillatory force at transition into this state from the fundamental (S₀- >Si) equals 0.0015, dipole momentum in S₁ state equals 8.16 D, electron transition is polarized mainly in direction X, as projection f_x on this axe exceeds the other two in 2-3 times. All others excited states and electron transitions may be characterized in similar way. Coefficients of expansion of MO in terms of AO allow to determine orbital nature of electron transitions (into excited state) . From analysis of expansion coefficients for once excited configurations for Si - state, follows, that this excited state is created by superposition of two configurations: -0.232 |34->35 and 0.075 | 33->36. Orbital nature of S₀- >Si is determined from nature of MO, taking part in generating of S₁ state, i.e. MO 33-36. As all MO of once excited configurations , forming this excited state, belong to iτ- type, then S₁ state will be i ir^* - type state. Orbital nature of any excited state is defined in similar way. Some data on occupation of MO and atom charges values allow to obtain information of electron density distribution and charges of each of excited states.

Information on triplet states are obtained the same way as for singlet ones. The triplet states spectrum of glucose molecule is as follows :

Energy -0.37eV ; wavelength - 3344.4nm; oscillatory force - 2990cm^"1 *F = 0.0028614161; *D = 8,37 Comparison of experimental absorption spectrum with the calculated one, allows to interpret electron absorption spectrum of molecule a(/3) -D-glucose the following way: long wave absorption band of investigated molecule is made up of one electron transition of 7T7r* - type of average intensity and is polarized along the short molecule axis. The absorption band in the middle of wavelength range includes several electron transitions of different orbital nature, polarization and intensity. The intensity of this region is formed by S₀->S₃ IT 7r* - type and exceeds that one in long wave range for an order of value approximately. Possible difference between measured and calculated values of electron transitions energies may amount to 1700 cm , that can be connected with omission in calculations the influence of the solvent (spirits or water, for example) . Taking into account in calculations specific interaction of glucose molecule and the solvent may decrease this difference.

From the obtained parameters for emission and absorption spectrums, characterized by oscillatory force, it is possible to determine the value of coefficient of absorption of emission, passing the medium, where is glucose molecule. The ratio of intensity of the light I, passed through absorbing layer to incident light intensity is connected with absorbing matter concentration (C) and absorbing layer thickness by Bugger-Lambert-Bar law, which in logarithmic form is as follows:

lg(—) = D =ecl (14)

A> where D - optical density, e- molar extinction coefficient (1 -cm; c - mole/liter) . Molar extinction coefficient and oscillatory force of electron transition in their turn are connected by relation f=432Λ0-⁹$ε(v)dv (15)

Integration is executed in full absorption band. Taking into account, that mean half-width of separate band in absorption spectrum is approximately 5000 cm^"1, in accordance with the theorem of mean, the expression (11) is transformed into

Z=IO-⁵^(F_1113x) (16)

Therefore, calculated oscillatory electron transition force may be compared with molar extinction coefficient for maximum in absorption band. Full dipole momentum of molecule equals to the sum of electric momentums of core and electrons.

Using the relation r_r = R_A + r_A we obtain

M = ∑Z_AR_A - ∑(R_A + r_A)

A r Using the relation (2.12) and wave functions of electron states it is possible to determine dipole

—> momentum D for different molecule electron states.

Among the characteristics of calculated electron transitions is the value of transition dipole momentum and its projections on axe of coordinates. In case of transitions between π ir-states in flat molecular structures transition momentum lies in molecule plane and its projection on Z-axis equals 0. Thus, of π TΓ* -type transitions may be said, that they are polarized in molecule plane along X-axis or along Y-axis. For transitions of mr*-type or σ7r*-type in flat molecules will be equal to zero the projections of transition dipole momentum on X-axis or Y-axis, i.e. electron transitions of this type are polarized along Z-axis

(per-pendicular to molecule plane) . In experiments, when measuring electron bands polarization in absorption or luminescence spectrums, they determine along which molecular axis this electron transition is oriented. The calculated polarization of molecule electron transitions is to be compared with this characteristic. In other words, comparing between each other calculated values of osc-Lllatory forces f_x,f_y,f_z and selecting the biggest, we determine along which axis is electron transition polarized. Intensity of light, which passed through absorbing layer, J at the given initial intensity of ligh-t J is connected with glucose molecule concentration in absorbing layer and with this layer thickness 2 accordingly to Buger-Lambert -Bar law , that is shown in relation (14) . From (15) - (16) follows, that calculated electron transition oscillatory force may be compared with. molar extinction coefficient for maximum in absorption band. In calculated spectrum of singlet states for glucose molecule the oscillatory force value at mind-tnal wavelength of transition 544nm is f=0.003. Hence, a mean extinction coefficient equals e_cp=300 1-cm/mole. At passing of laser emission in biological tissue, due to scattering processes, excitation of electron transitions and fluorescent radiation of organic molecules, glucose molecules included, in said tissue is generated output rad±ation on the frequencies, corresponding to frequencies of excited electron transitions in visible range and to spin-vibratory transitions in IR range in all organic molecules, contained in said tissue. Exc±tation cross-section of fluorescent molecules of glucose for laser emission in visible range (640nm) is about σ*=10^"15 cm² , velocity of singlet-singlet annihilation of excited state of molecules of biological tissue is about γ=10^i:L /n₀ cm³/s. Let us consider (accordingly to equation (4) ) a stationary case of concentration of glucose in excited state, being excited by laser radiation scattered in biological tissue. Solving quadratic equation at laser radiation flow F-IO¹⁷ l/c-cm², that corresponds to laser power of 1OmW, we obtain expression

This means, that practically all glucose molecules will be in excited state. Therefore, scattered radiation from biological tissiie will contain luminescent radiation of glucose molecule, which makes about 1% of all scattered radiation, i.e. about 10 1/s-cm², what correlates with above calculations of scattering of laser emission in biological tissue and the calculations to follow. If such radiation is passed through water solution of glucose, then, due to resonance fluorescence, excited in molecules by emission frequencies, coinciding with frequencies of electron and spin-vibratory transitions, resonant absorption on these frequencies takes place. Thus, in correspondent absorption bands of glucose molecule takes place nearly complete absorption of that part of emission, whichi matches to fluorescent emission of glucose molecule in biological tissue. This means, that in this way spectrometry of scattered radiation may be done using instead of complex spectrometric optoelectronic apparatus only thin transparent solution of investigated matter. After passing resonant fluorescent rad±ation through water solution of glucose of concentration c=0.1 mole/1 and thickness l=lcm, the ratio of output intensity to initial intensity will be l/l_o=exp(-ecl)= 10^"13 (18) μ_s=e . c=30 cm^"1

Therefore, previously scattered in biological tissue emission, after passing through water solution of glucose, will not contain photons, matching fluorescent emission of electron and spin-vibratory transitions of glucose molecule in biological tissue. After exposure of water solution of glucose to fluorescent emission of glucose molecules in biological tissue resonant excitation of electron and spin-vibratory levels in water solution of glucose takes place. In the limits of expanding lines of transitions the energy of excitation is spend on thermal processes in the medium. Thus takes place the resonant nearly complete absorption of fluorescent emission of glucose molecules in biological tissue in considered water solution of glucose.

Supposing, that at laser radiation scattering on optical non-homogeneity in biological tissues, the intensity of output scattered in turbid, colloidal or disperse medium light exceeds or at least is comparable with intensity of fluorescent emission, then for determination of coefficient of absorption of laser radiation, due to photoplaysical processes in molecules, it is possible to compare these intensities, using Buger- Lambert equation. Using above obtained value of fluorescent emission of glucose molecule in biological tissue I^* = 10¹¹ 1/s cm from its ratio to scattered light flow value I=IO¹³ 1/s cm we determine the value of coefficient of absorption of emission due to photoph/ysical processes, using Buger- Lambert equation for light propagation in biological medium. With biological tissue thickness 1=0.5cm we obtain:

=4 (19) Thus it is clear, that coefficient of absorption of laser radiation due to scattering on optical non-homogeneity is greater than coefficient of absorption of laser radiation due to excitation of molecules of glucose. It may be supposed, that presence in biological tissue others organic molecules will lead to approximately same value of coefficient of absorption, as that of glucose molecule. If to sum all others organic molecules influence on processes of laser radiation absorption on the basis of their overall concentration in water solution of biological tissue, then we obtain, that overall concentration of all organic molecules makes about 10^"4 mole/1, but glucose concentration in blood makes 1.3 10^"4 mole/1. Hence, we obtain that intensity of fluorescent emission of all others orrganic molecules in biological tissue will be of same orcLer that of glucose molecule. Comparing coefficient of laser emission absorption due to excitation of molecules in biological tissue( 15), when fluorescent emission is generated, with coefficient of resonant absorption ( 19) of fluorescent emission of glucose molecules in biological tissue in water solution of glucose, it is seen, that resonant fluorescent absorption exceeds on order of value laser absorption in biological tissue. For determination of quantity of excited glucose molecules in biological tissue and consideration of resonant emission propagation in glucose solution it is necessary to calculate velocities of radiative and non- radiative processes in multi-atom molecule and to determine donor-acceptor properties of: separate parts of molecule in fundamental and excited state., to compile initial data for of calculations of constants photophysical processes. Besides, it is necessary to calculate the constants of velocities of photophysical processes and changes of electron density in fragments of molecule and to analyse the results of of calculations of constants of velocities of photophysical processes and to make energy schematic of electron excited states of glucose molecule. Donor-acceptor properties of separate parts of molecule in fundamental and excited state and their changes in singlet excited states must be characterized too.

Processes, decreasing electron excitation energy, i.e. energy deactivation processes, by their nature may be radiative and non-radiative. Spontaneous emission, accompanying the transition between two states is called luminescence and radiative transition S₀-> Si is called fluorescence and spontaneous transition Ti—» S₀ phosphorescence. For the non-radiative deactivation processes are taken non-optical (without dirrect photon emission) transitions between electron states, when electron excitation energy is transformed into vibratory system energy completely or partially. Two types of non- radiative conversion are differentiated — internal conversion and inter-combinational one. The f±rst occurs between electron states of one multiplet (S_n-> S_m, S₀->Si, Ti->Tk) and the second - between different multiplet states (S_n-» T_m , T_x-> S₀) . Non radiative transition is often called degradation (Si—> S₀ , Ti-> S₀) . Let us mark one more type of non-radiative transitions - oscillatory relaxation, which contrary to the internal conversion occurs inside of one electron state and leads to establishing of balanced distribution of energy in vibratory levels of electron state of excited molecule as a result of internal molecule interactions and /or interaction with ambient medium. The probability of occurring of each of these processes energy deactivation of excited electron state (radiative and non-radiative) are characterized by constants of velocities of these processes. k_r , k_Bκ, k_sτ - this are the constants of radioactive disintegration, internal and intercombinational conversions correspondingly. Velocity- constant is equal to the quantity of electron transitions, occurring in a time unit (second, for example) . But it is difficult to directly measure these values . In experimental researches of photoprocesses velocity constants are evaluated by measuring of life time and luminescence (fluorescence or phosphorescence) quantum outputs and dynamics of population of excited electron states in absorption spectrums. Here we shall research only fluorescence process, where quantum output equals : y= k_r / (k_r + k_BK + k_sτ ) (20)

Let us note, that this formula is valid for the case of absence of photochemical conversions in molecule under investigation. From formula (20) follows, that if non- radiative conversion is absent: y=l. Thus, by value of quantum output we may judge on relation between velocities of radiative and non-radiative processes. The constant of radioactive disintegration may be calculated by formula: k_r = (f (So→Sj/l.ΞjEtS_!)² (21)

Where f -oscillatory force of electron transition Sχ-»

E(S₁) - this transition energy in cm^"1 .The constant of radioactive disintegration is connected with another important characteristic of fluorescence - radiation life time of excited state t_r, which characterizes duration of fluorescence in absence of non-radiative conversion:

Where t - real (natural) life time or fluorescence duration (duration of fluorescence in presence of non- radiative conversion) , which may be measured experimentally. Accordingly to (22) by changes of y and t values of k_r and (k_Bκ + k_ST)) may be determined. Parallel with deactivation processes of energy of electron excited molecule it may absorb energy, what leads to the increase of energy and to transition on higher level excited states, i.e. to occurring of re-absorption processes or absorption spectrums from excited state (Si-> S_n and Ti-» T_m ) . Fluorescence spectrum of multi-atom molecules is a wide structured or non-structured band , shifted to longer waves relatively absorption spectrum. This property of fluorescence was discovered and formulated by J. Stoxe. Stoxe rule says that molecule does not radiate all absorbed energy, part of which is spent on inside molecule transformations. In other words, this rule says: emitted quant ( V_f) frequency must be less, than that of absorbed quantum (v_a) , i.e. h v_f< h v_a . Earlier it was discovered that contour of fluorescence band is a mirror reflection of long wave absorption band (both spectrums are built on frequency scale) . For complying to mirror reflection rule the balanced distribution of absorbed energy on oscillatory freedom degrees in a state S is needed, i.e. high velocity of oscillatory relaxation is needed, what is characteristic for molecules of complex structure only. Is simple to show, that mirror symmetry of luminescence and absorption spectrums is proper to complex molecules with similar distribution of excited molecules by oscillatory levels in fundamental and excited states. It should be marked, that mirror symmetry will be naturally spoiled, if electron nature of Frank- Condon (non-balanced) and fluorescent (balanced) states do not coincide. It is known, that if, prior to emission, in fluorescent state of molecule balanced distribution of absorbed energy on oscillatory freedom degrees (in condensed phase) is established, then the form of fluorescence spectrum will coincide with the form of the heat emission spectrum at the temperature of the experiment. Universal Stepanov relation in logarithmic form is shown as follows:

In k_v n³/l_v = hn/kT ÷const (23)

Where k_v - absorption coefficient, I_v - emission intensity, T - temperature of experiment, k - Boltsman constant. In such form Stepanov relation is easily checked and its validity was shown. This relationship shows onto thermally balanced nature of spectral distribution in fluorescence band. This means, that despite of short life time of fluorescence (t>> 10^"9 s) , balanced distribution of energy on oscillatory freedom degrees establishes in time. On the other hand, disobeying to Stepanov universal distribution means that relaxation process in electron excited molecule state is slowed down. It is known, that quantum output of fluorescence stays constant at the excitation by different wavelength along the all absorption band. At first this relationship was established for fluorescent molecule and then for plenty of other compounds . Vavilov law for condensed mediums is based on the fact, that due to inter-molecular interactions the excess over balanced value of oscillatory energy in electron excited state is transmitted into surrounding medium and balanced distribution of energy on oscillatory freedom degrees is established. Violations of Vavilov law takes place in rarefied vapors, at some peculiarities of molecular structure, in presence of photochemical reactions, with conformers present. Analysis of experimental data has shown, that for organic molecules the state, in which luminescence occurs, is the lower excited state of this multiplet, despite of its state prior to excitation, i.e. fluorescence corresponds to Si—> S₀ transition and phosphorescence Ti-> S₀ transition. This relationship, discovered by M. Casha, may be explained by that in medium (solvent, solid matrix) the difference of energies of excited states S₃, S₂, S₁ is redistributed in oscillatory freedom degrees inside of the molecule and after that may be emitted into medium. Thus, at the moment of emission electron excited molecule comes into state Si. At that it is inexplicitly supposed, that probability of internal conversion process greatly exceeds the probability of radiative transitions S_n-» S₀ . At the moment Cash rule is formulated as follows: in majority of molecules internal conversion processes between electron excited states occur in time , less than 10^"11 s and perceptible quantum output of fluorescence (Ύ^ICT³) may be observed for transitions from lower excited state of this multiplet. As marked above, information on non-radiative processes in experiments obtain from fluorescence quantum output measurements. Experimentally was established, that in many photostable molecules in condensed phase singlet-triplet conversion is the main channel for non-radiative energy deactivation. Earlier it was shown, that processes of internal Si—> S₀ and intercombinatory T₁-*- S₀ conversions take place with participation of C-H bonds. "The law of energy slot" was established, according to which velocity of non-radiative transition swiftly falls down with the increase of energy of state T_1.. Similar conclusion was made by J.Birks for Si-> S₀ conversion. To all said we add, that in molecules, apt to photochemical reactions, non-radiative transitions have some peculiarities and that is why we shall consider photostable molecules only. The expression for the constant of internal conversion velocity was determined by many ways. Most completely the nature of given process is taken into account by the model shown above. In this model the expression for the constant of internal conversion velocity in aromatic molecules is as follows : k_BK

o|d/aψ_v >|²π_v|< o|«_v >|² where E_if - energy difference of electron states, combining in the process of internal conversion JV - the quantity of p-electrons in molecule; n - the set of oscillatory quantum numbers of final state f ;

<0 d/dxn > =a²/2xr(n+l)r(2s+l-n) (s-n) /r(2s) where a- potential Morse, s=2D/ω (D- dissociation energy C-H bond, ω - frequency of zero oscillations of this bond) , r - gamma-function; I <0 |n_v> |²=exp(-y)yⁿ (n!)^"1, where y=l/2 Δ² μω ( Δ - the change of equilibrium state of normal coordinate of given oscillator at the electron transition between electron states J and f ) . Using the above expression for the case of internal conversion between states p-p type the dependence of k_Bκ from energy interval between combining states J and f. From this dependence follows: in case, if Ei_f £ 10⁴ cm^"1, the probability of internal conversion is about 10¹¹ s^'1 , what coincides with the evaluation of internal conversion velocity by Cush rule and confirms it theoretically. For E±_f values, lying in the interval 10⁴ - 2 10⁴ cm^"1, internal conversion velocity constant approaches the values of radiative disintegration of initial state i, what allows the process of radiation from this state to compete with internal conversion process, leading to the breach of Cush rule. As an example of this type of molecule we may take azulen molecule, for which

E₁₂ =1.4 10⁴ cm^"1 and the fluorescence from S₂ state is observed. In the region Ei_f >_ 2 10⁴ the value k_Bκ is less than radiative disintegration velocity. It was shown also, that at Ei_f <_ 10⁴5 10³ cm^"1the constant of velocity of internal conversion between states of different orbital nature are approximately of same order (« 10¹¹ cm^{" x}) • k_BK= Gpg N_XH k_pg (Ei_f ) where k(E_if ) - the value of internal conversion velocity, evaluated by Plotnicov formula; N_XH - quantity of bonds in molecule; X - heavy atom (C, 0 ...)

N G_n ⁼∑^' P?_pq where the sum on a means summing on a oscillators, corresponding X-H bonds in molecule and expression under the sum sign depends on nature of wave functions of interactive electron states (coefficients of expansion of molecular orbitals on atomic ones and coefficients of configurational expansion) . It allowed, taking into consideration dependence of k_Bκ from energy interval and of the type of accepting oscillations, to take explicitly into account orbital nature of wave function of interactive electron states. Optical transition between different multiplet states are banned. This ban can be partially removed by interaction between magnetic fields, arising from spin and orbital electron movement. Interaction of such type is called spin-orbital interaction. Most simple model of such interaction is as follows: In description of one electron atom it is supposed, that electron is spinning around itself and rotates around the core. With the help of simple conversion it is possible to go to the system with fixed electron. In this coordinates system the core is rotating around electron. As the core possess electric charge and acceleration, then relativistic magnetic field should arise, encompassing electron, which is directed perpendicular to orbit plane. Another magnetic field arises from electron spinning. This field is directed along axis of electron spin. Interaction of two these fields is called spin-orbital interaction or spin-orbital bond. That is the spin-orbital interaction, which starts singlet-triplet conversion process. The expression for evaluation of velocity of singlet -triplet conversion constant is as follows :

Here Y=l/2 μ ω² Δi_f ² where Δi_f - the change of equilibrium position of oscillator at electron transition, μ and ω - reduced mass and frequency of oscillator; n - is determined from condition of minimal defect of resonance of initial and f inal state of electron transition A_if S ^ Π_VOJV

Generalized evaluation of matrix elements of spin-orbital interaction for electron states, taking into account their orbital symmetry, produces

<|H_S0|> = 0.3cm^"1 for interacting states pp* and np* types. In this case expression (18) is transformed into:

for states of similar orbital nature

for states of different orbital nature

Here n_v ≡Δ E_sτ cm^"1 = 1000 cm^"1, where ΔE_ST - the difference of energies of interacting states, 1000 cm^"1 - accepting oscillation frequency

It was shown, that as accepting serve valence oscillation of C-C bonds of phenil ring. The empirical rule was accepted, according to which k_Sτ between states of similar orbital nature (pp* -> pp*, np* -> np*) is about 2-4 orders smaller than between states of different orbital nature (pp* ->np*, np* -> np*) . Out of expressions (20-23) the dependence k_sτ from the value of energy slot ΔE_ST between interacting singlet and triplet states. Comparison of formulas (18) and (19) leads to the conclusion, that probability of intercombinational conversion between states of different orbital nature is about of three orders higher than of conversion between states of similar orbital nature. Thus, for example, at k_Sτ ~ 10¹⁰- 10¹¹ s^"1 efficiency of singlet -triplet conversion exceeds on 1-2 orders the constant of velocity of radiative disintegration of fluorescent state even for the case of most efficient luminophors (k_sτ ~ 10⁹ s^"1 ) and may lead to suppression of fluorescence. At ΔE_Sτ = 3000 cm^"1 only in case of efficient luminophores conversion between states of similar orbital nature may be neglected. Therefore, interconmbinatory conversion between states of different orbital nature must be taken into account at any value DE_ST . Expression (17) for k_sτ is obtained for the case of flat aromatic molecule, where there is no atoms heavier, than C (carbon) . Thus the algorithm of: calculation of

< IH₃₀1 > is realized as for the case of flat molecules with atoms C, O and H, and for non-flat ones as well. At that matrix elements are calculated for every concrete case, automatically taking into account orbital nature of interacting electron-excited states. On the basis of results, obtained above, let us determine expressions for coefficients of absorption and emission of photon resulting at different optical transitions in organic molecule. From these expressions cross-sections of emission an<d absorption between two states of molecule are determined. Using these cross-sections the value of coefficient of absorption of photon in given medium is determined. The starting point for determination of cross-section of emission and absorption of photon are the values of probability of emission and absorption, determined by oscillatory forces of optical electron transition ±n dipole approximation. Oscillatory force of electron optical transition from state Ψ_p into state Ψ_q is calculated by formula: 2mCO

/(P→q)= M(p→q) = 0.0875-E 'pq M(p→qj e²h

Where E_pg = E_p - E_g = hω - energy of transition in molecule M - dipole momentum, calculated for excited state of electron transition of organic molecule. When exciting from fundamental singlet state (Ψ_o) oscillatory force of electron transition is calculated by formula

In general case ttαe probability of dipole emission of photon by quantum system is determined from expression for oscillatory forrce of molecule and matrix expression for operators generation and annihilation of photons:

where n_ω - photon number, u_p - photon unity vector of polarization p.

Taking the cross-section of stimulated emission as the ratio of probability of stimulated emission in unity of time to photon flow density, we obtain for stimulated emission of photon cross-section:

Similar way the cross-section of absorption is determined:

Let us iinnttrroodduuccee coefficient of absorption, which characterizes the probability of absorption of photon on the unity of the way length as a result of excitation of given transition in molecules, being in thermal equilibrium with gi^ren medium 2π²e²

K = N_pσ_pq -N_qσ_qp = N_pf_pq - g_pa_ω(\ - ^_V{-hωl kT)) (28) όmc

Estimated value of radiative absorption coefficient, for example, for glucose molecule with concentration molecule in solution N_p=6 10¹⁹ cm^"3 , according to calculations data from above, relatively to oscillatory force f_pq -3 ICT³, is equal k_R = 10 cm^"1 . Minimal width of spectral line corresponds to maximal cross-section of absorption. Minimal width occur at radiative expansion, when function a_ω is as follows:

Complete coefficient of absorption is determined by summing not only the processes of emission and absorption but processes of internal conversion, when non-radiative transition of photon energy to spin-oscillatory transitions of organic molecule occurs, too. Coefficient of absorption of photon, which energy goes into spin- oscillatory transitions is about k_sτ = ICT¹ - 10^"2 cm^"1 k_ω = k_R +k_sτ

Laser radiation, while propagating in a system of organic molecules, will be partially absorbed due to processes of electron excitation and photoprocesses in organic molecules. Balance equation for photon density is shown below:

^- = - K Np - 1/1 IT Φ (29) dx the first term on the right side of equation characterizes the change of photon quantity due to emission and absorption of molecules, and the second term describes decrease of photon quantity as a result of their leaving the medium due to scattering. From this we obtain the condition of excited state of medium: k_ω >= kc _g fc

Now we determine : N_q — - N _p = — —

S q ^ p

Which is defining relation of concentration of molecules in fundamental and in excited state. For- determination of density of flow of photons, leaving tb_e medium and the quantity of excited molecules in the medium, let us use balance equations for atom densities in fundamental and excited state

N_qv_qp +

Where τ_g , τ_p - - life times of excited and fundamental states correspondingly; Vgp - frequency of transition from excited to fundamental level at collision and spontaneous emission; M_q - quantity of molecules in excited state, generated in a unity of volume, σ_ω -cross- section of stimulated emission of photons. Solving a system of equations for stationary mode, we obtain that the quantity of molecules in excited state, generated in a unity of volume in a unity of time equals:

g where τ — τ / λ — — τ P (1-r 9v IP >) For the flow of emission in a

S p medium photon flow increases due to reemission processes and decreases due to scattering processes at ω> ω₀ and that is why we obtain the equation: dJ_ω/dx = F_ω + (k_ω - k_c)J_ω (32) Here the first term presents the quantity of photons of spontaneous emission, generated on a given frequency in a unity of volume in a unity of time, the second term corresponds to increase of emission of molecules in excited state due to transitions from excited state into fundamental. Supposing k_c>>k_ω , i.e. processes of scattering prevail on processes of absorption in given medium, we obtain the value of photons flow, propagating in the medium

J= F₁₁₁/ (k_ω - k_c) (33)

Where F_ω=I_ω N₀ , N₀ - concentration of molecuLes in a medium, I₀,- intensity of spontaneous emission. Now let us determine intensity of spontaneous emission for transition of molecule system from state q into state p. Accordingly to formula of probability of emission we obtain

In a classical case emission occur with system transition into close by value of energy states. Summing on all the states we obtain expression

I_ω = 2/ (3c³) (Mω²)² Hence, photon density in the medium , taking into account processes of scattering and reemission, will be defined by expression

Inserting all the spectrometric parameters from above and photon density value into expression for density of molecules in excited state, we obtain the value of volume intensity of molecules, which excited, being exposed to light radiation, M_g =10²⁷ cm^"3 s^"1. In all this quantity there are 3-10²³ cm^"3 s^"1 of glucose molecule. The photon flow, exposing the medium, is J_o1=IO¹³Cm^-2S^"1 Such a value of the flow coincides with calculated above the flow of photon, leaving the given medium, but neglecting absorption due to reemission. At that glucose molecules get the photon flow of about 10¹¹ Cm^-2S^"1 This calculations confirm obtained above value of coefficient of absorption of laser radiation in a biological tissue

(19) , equal μ*_a =4cm^~1 Let us now consider in connection with this scattering of laser radiation in a biological tissue, taking into account absorption processes due not only to scattering on optical nonhomogeneity, but to photophysical processes, occurring in organic molecules of biological tissue, when laser radiation propagates in said tissue, too. The following relation between coefficients of absorption and scattering is characteristic for biological medium μ_a<< μ_g ^' β_s ^' = μ_s(l-g) g=(cos(θ)}

The intensity of light in a turbid medium is presented by equation system (8) I(z,t)=I_cohr(z,t)+I_diff(z,t);

Diffusive approximation z > 1

Modified boundary condition:

Idif(z= -zo ) =0 z = Δl Δ = 2/3 (1-R)/(1+R) Inserting proper coefficients of absorption of laser radiation (9) , (18) , (19) we obtain the root in diffusive approximation (Fig.6)

Calculated parameters of device for non-invasive measurement of sugar concentration in blood

Laser light source:

Capacity -1OmW Wavelength - 640nm (red light) Basin with water solution of glucose:

Basin thickness - lcm, solution concentration -0.1 mole/1 Photosensors: sensitivity - no less than 10^"3 mW

Claims

Claims

1. A method of registration of absorbed, scattered and diffusive reflected by blood radiation by integration unit by way of conversion of said radiation into electric signal, with following conversion of said signal into digital code, by comparison of said code with calibration curve of calibration blood sample and determination of value of concentration glucose by said comparison with following displaying of the result on digital screen, differing in that emitting surface of laser diode, emitting in visible wave range, is placed in contact with surface of biological tissue of a human or a beast, transmit laser radiation through the thickness of biological tissue, scatter light on optical non homogeneity of biological tissue, absorb laser radiation in molecules of said tissue and excite fluorescent emission of electron singlet-triplet and spin-oscillatory transitions of organic molecules of said tissue, focus and form output radiation from said tissue in parallel light beam, input said focused parallel light beam of output scattered fluorescent emission on a semi- transparent mirror, which is placed at an angle to said beam, divide said beam on two beams of equal intensity: reflected beam (reference beam) and passing beam (second beam) , pass the reference beam through basin with solvent and resonantly absorb fluorescent emission of biological tissue, corresponding with solvent molecules spectrum, pass the second beam through basin with the solution of investigated substrate, glucose, for example, and resonantly absorb fluorescent emission in the second beam, corresponding to the spectrum of emission of solvent molecules and investigated substrate of biological tissue, input scattered light of reference beam and of second beam after passing basins with solvent and with solution of investigated substrate onto two separate photosensors, integrally register all the emission spectrum left and convert in photosensors the energy of reference and second beams into electric signals, amplifier said signals, subtract amplified signals in the electron logic cell and input the difference of electric signals onto display for determination of concentration of investigated substrate.

2. The method according to claim 1, differing in that laser wavelength lies in visible wave range.

3. A device posses an integration unit, detector, connected to analog-to digital converter, said converter being connected to device for current regulation of power source and for data processing and display, differing in that the device is made of two separate units: optical one and electronic one, said optical unit comprising of rectangular metal optical bar with scaffold cut-out along the surface of the bar, into which are inserted the pins of the movable carriage are inserted, with laser diode of visible wave range fixed on the said carriage, said laser diode emitting surface tightly fixed to one side of biological tissue surface, the carriage with metal blackened diaphragm with a hole in said diaphragm tightly fixed from the other side of biological tissue and focusing optical system, placed near the diaphragm, for obtaining parallel focused light beam after scattering on biological tissue, carriage with fixed on it semi- transparent glass plate, oriented at 45° to light beam direction, flat glass transparent basin for solvent, said basin placed across the light beam, perpendicular to it, at some distance from semi-transparent plate, and photosensor, placed on the other side of basin, with said sensor sensitive surface perpendicular to light beam, carriage with flat glass transparent basin for solution of investigated substance, said basin flat side perpendicular to light beam, and photosensor, placed after the basin with its surface perpendicular to light beam and electron units for photosensors electric signals processing and unit for displaying results of measurements.