COLD LASER QUANTUN THERAPY INTERACTION WITH BIOMATTER AND BIOMEDICAL APPLICATIONS

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COLD LASER QUANTUN THERAPY INTERACTION WITH BIOMATTER AND BIOMEDICAL APPLICATIONS

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LASER LIGHT INTERACTION WITH BIOMATTER AND BIOMEDICAL APPLICATIONS


V.S. LETOKHOV
Institufe of Spectroscopy Russian Academy of Sciences,
142092 Troitsk, Moscow Region, Russia

Any new achievement in science and technology was always finding some useful application in biology and medicine. And naturally the invention of the laser was not an exception to this rule.

All the special properties of the laser light, namely its (1) spatial coherence (directivity and focusability), (2) high intensity in both continuous-wave and pulsed modes of operation, (3) monochromaticity (temporal coherence) and wavelength tenability, and (4) short (controllable) pulse duration proved valuable in biomedical applications. Progress in laser technology leads to subsequent progress in biomedical laser applications, both experimental and clinical. Speaking of the progress made in recent years, one should single out the following trends in laser and photonic technologies: (1) femtosecond lasers, (2) solid-state tunable lasers, (3) laser diode arrays and their application to the pumping of solid-state lasers, (4) soft X-ray lasers, and (5) IR fiber optics.

At the root of all biomedical laser applications is the interaction of laser light with matter at various biological organization levels: (1) molecules, (2) cells, (3) biotissues, (4) organs and (5) the whole organism.

It is convenient to subdivide the biomedical laser applications as follows:

    1. Laser diagnostics based on resonant (spectrally selective) and nonresonant laser-matter interaction.

    2. Laser therapy based on laser-induced molecular processes consequent upon resonant excitation of certain molecules.

    3. Laser surgery based on laser induced destructive macroeffects.

When a biomolecule absorbs laser light, the expenditure of the excitation energy can follow various pathways, depending on the species of the molecule, its surroundings, and radiation intensity. Some absorbed energy degrades to heat in a radiationless manner, some may be transferred to the neighboring molecules.

The excited molecules may take part in chemical reactions, and finally, they may be raised to higher quantum states, provided the light intensity is high enough (ultraviolet pulses), and then be involved in photochemical processes. Almost all these excitation pathways are now being successfully utilized in laser biomedicine: laser thermal surgery, laser fluorescence diagnostics, laser photodynamic therapy, and laser phototherapy. The sum of the quantum yields attained in the particular channels is equal to unity. Depending on the irradiation conditions and the state of the medium, the quantum yield in any desired channel can be varied with a view to improving the efficiency of its utilization.

One can vary the laser radiation parameters (intensity, duration) over very wide limits, thus making it possible to implement various types of light-biomatter interaction (linear and nonlinear, single- and multiple-photon, coherent and noncoherent) and thereby induce various effects in biotissues (photochemical modification, thermal destruction, explosive ablation, optical breakdown, shock pressure waves, etc.) as qualitatively.

When considering the interaction between laser radiation and biotissues, one should take into account (and potentially utilize to advantage) the inhomogeneous character of their various properties: biological, chemical, and physical (optical, acoustical, electrical etc.). The characteristic sizes of these inhomogeneities varies over very wide limits: from the size of individual molecules to that of blood vessels and organs. As an illustration, presents a qualitative view of the spatial spectrum of these inhomogeneities. The spatial characteristics the inhomogeneities probably have a fractal structure [1] which has yet to be studied quantitatively (the fractal dimension in particular).


The spatial inhomogeneity of the optical characteristics of biotissue (specifically its absorption characteristics) leads to a spatially nonuniform distribution therein of heat liberated as a result of absorption of pulsed laser radiation. The following three characteristic times are of importance in this case: tp the laser pulse duration, ts=a/C0 - the time it takes for sound to propagate with a velocity of С0 beyond an inhomogeneity of size a, i.e., the relaxation time of the pressure resulting from the pulsed local heating, and thd=a2/kc - the time it takes heat to propagate by diffusion beyond the inhomogeneity (k- is a numerical coefficient depending on the shape of the inhomogeneity and с- is the thermal diffusitivity), i.e., the relaxation time of pulsed heating. Shows regions of enhanced pressure DP (pressure confinement) and enhanced temperature DT (temperature confinement) in the case of microsphere of radius R (R=a, k=27) for pulses differing in duration. Both these effects are important in the pulsed irradiation of real biotissues and should be not only taken into consideration, but also used to advantage.

All laser biomedical diagnostic can conveniently be subdivided into two classes: (1) macrodiagnostics of various objects (topography, detection of pulsation, blood flow measurements, etc.) and (2) micro (spectro) diagnostics at various biological organization levels: organs, cells, organelles and biomolecules.



The central problem is the development of the methods for the laser diagnostics of inhomogeneities (tomography of biotissue which is an absorbing and highly scattering medium, i.e., a very "awkward" object for optical techniques. The tomography of biotissue is important for medical diagnostics, laser therapy and surgery. To choose the optimal regime and correct dose of laser irradiation it is necessary to know the values of light absorption and scattering of tissue Moreover, it's important to know the spatial distribution of light absorption and scattering nonhomogeneities over irradiated area. For example, the determination of sizes and the depth of location of tissue injures, blood vessels, inclusions. pigments etc. is of real importance. The various methods of diagnostics of light absorption inhomogeneities in turbid media are widely discussed: photon migration [2], time-resolved photon migration [3], optical coherence tomography [4-6] and others. Therefore they are the most effective for transparent media and are sensitive to the refraction coefficient inhomogeneities mainly. The most exciting results are reached by using these methods for diagnostics of eyes injuries [7]. The achieved spatial resolution is tens of microns both in lateral and z-axial directions (in the depth of tissue).

The light propagation in inhomogeneous medium is determined by scattering and absorption processes. It is the case for biological tissues the scattering coefficient is 10-100 times higher than the absorption coefficient [8]. However, for medical use of lasers the value of light absorption coefficient ma and it's distribution over the irradiated region is of great importance. Therefore, the problem of working out accessible and simple methods of diagnostics of light absorption inhomogeneities in biological tissues is still actual.


One of the most perspective methods of measurement of absorbing inhomogeneities in turbid media is opto-acoustic method. It is based on excitation of thermal or acoustic waves (thermooptical excitation) by absorbing of time-modulated or moving laser radiation (see, for example, [9-11]). The excitation of acoustic waves (opto-acoustic signals) takes place due to nonsteady and uniform thermal expansion of the heated area. The advantage of opto-acoustic method is the direct dependence of amplitude of excited wave on the light absorption coefficient, that is, there is no opto-acoustic signal is the turbid medium without absorption.

Time-resolved laser opto-acoustic tomography is powerful and perspective method of investigation of light absorption in inhomogeneous media [12]. At the same time this equipment is based on widely-used laser sources and electronic devices. The realized experimental methods allow to get spatial resolution of 10-15 mm by the probing depth up to several millimeters. The value of light absorption coefficient varies from unites to several hundreds of reversed centimeters. The probing depth is determined by the light penetration depth in the medium. To achieve high lateral resolution is still actual problem. At the same time the ways of it's solution are quite clear and don't require qualitative complication of signal processing. The measurements of light absorption by means of time-resolved laser opto-acoustic tomography may be carried out in real time. Therefore, this method is useful in medical diagnostics.


Spectral diagnostics on molecular level makes use of a wide variety of laser spectroscopy methods which enable us to obtain extremely high parameters inaccessible without using laser light. Here they are with some of their biomedical applications.

1. Spectral resolution may reach any desired values (from nm to MHz). A high spectral resolution in combination with a high sensitivity is used, for example, to detect gas-phase isotopically-labeled products of metabolism and to diagnose, on this basis, certain diseases (see [13]).

2. Temporal resolution, with development in short and ultrashort pulse lasers, was improved from ns to ps and then from ps to fs. The ultrafast laser spectroscopy methods contribute greatly to understanding fundamental processes in biological systems (see [14]). The list of intrinsically fast processes in biological systems is very long: energy and electron transport in photosynthesis and reaction centers, proton transfer and isomerization in vision pigment, bacterial membranes, etc.

3. Sensitivity of detection of atoms or molecules [15] can reach its limiting values, i.e. single-particle level. Highly-sensitive methods of detecting traces of microelements are important for understanding their pathways in environment, food products and human body. Highly-sensitive fluorescence methods of detecting single molecules are used to diagnose diseases, starting from cellular level.

4. Selectivity of laser spectroscopy methods in detecting atoms can be very high that, in fact, enables detecting traces of radioactive isotopes and their pathways in environment and human body. The selectivity of detection of biomolecules usually characterized by wide absorption bands is rather limited though. Nevertheless, quite good results have been obtained in some cases, for instance, in measuring the blood and tissue oxygenation level [16]. There is a vast field here for new methods and their biomedical applications.

5. Spatial resolution on light wavelength level is standard in biomedicine, in particular with the use of confocal microscopy. Now methods are being searched to increase the spatial resolution up to the nanometer range: near-field laser microscopy [17], laser photoelectron spectromicroscopy [18], etc. This will make it possible to use laser radiation for mapping of biomolecules, DNA sequencing [19] which is of great importance for genetic biomedicine.

It seems quite realistic to develop optical spectroscopy methods for noninvasive measurement of concentration of such molecules as glucose, cholesterol, etc. in living biotissue "in situ" including the use of portable low-cost instruments accessible to every patient.

All the methods of laser therapy can be conventionally subdivided into two classes: photodynamic therapy and photothermotherapy.

Photodynamic therapy is class of phototherapeutical methods is based on photochemical reactions of exogenous and endogeneous chromophores. It comprises well-elaborated laser photodynamic therapy [20] and rapidly developing laser low-power biostimulation [21].

Photothermotherapy is class of the methods of this class are based on the effect of heating of biotissue due to nonradiative relaxation of laser excitation. This means nonderstructive local transident heating under short-pulse laser radiation [22].

There are at least three mechanisms that are responsible for the transient local heating of biotissue exposed to laser radiation.

First, even a CW laser radiation is absorbed as separate quant by individual chromophores in the tissue. In the laser intensity range typical of biomedical applications, the excited chromophores are distributed in the tissue thinly enough, and so there must inevitably occur a slight overheating of the molecular chromophores for a very short period of time.

Secondly, the absorptivity of the tissue at a certain radiation wavelength varies over sufficiently wide limits throughout the tissue volume. A most simple example is the enhanced absorption of hemoglobin in blood which is distributed all over the bulk of the tissue by vessels in a great variety of diameters and lengths (fractal structure with a dimension of D=2.7 [1]). The spatial variations of the tissue absorptivity are especially great in the visible and near IR regions of the spectrum, but are smoothed out in the UV and IR regions because of the strong absorption of all organic molecules in the UV region and of water in the IR region.

Thirdly, account should be taken of the nonuniform spatial distribution of the laser radiation intensity. For example, when coherent light propagates through an optically inhomogeneous, scattering biotissue, random interferences there inevitably give rise to speckle structures [23] with irregularities characteristically measuring up to 1/2. If the given effect, for example, local heating, lasts for a time shorter than the correlation time of the speckle pattern fluctuations (which depends on the laser bandwidth and internal motions in the exposed medium), this speckle-like laser intensity distribution will be fairly substantial.

The spatial spectrum of absorption inhomogeneities of biotissues being very wide, one can cause, by properly selecting the duration and wavelength of the laser pulses used, the local transient heating of the desired micro- and macrostructures in the exposed biotissue.

It can be expected that the pulsed heating of the desired kind of microvolumes in biotissue with laser pulses of a certain wavelength, pulse duration, and intensity opens up entirely new possibilities for photothermotherapy. This is due to the fact that the local transient overheating of microvolumes may be substantial (DT= (1, 100) deg), the time- and space-averaged macroscopic heating of the entire irradiated region being much lower. Secondly, by varying the irradiation wavelength, one can effect the pulsed heating of the desired biologically important microvolumes, the relationship between the locally absorbing regions and the wavelength being studied beforehand by a similar method. It can be expected that the locally measured (with reference to DT) pulsed heating of microvolumes of a certain kind will have a material effect on the course of biochemical processes, and so will form the basis for new therapeutic techniques based in principle on the use of tunable ultrashort laser pulses. But their actual types can only be revealed through experimental investigations into local absorption regions, their nature and size, and the characteristics of the pulsed overheating and temperature gradients produced. All this should be the aim of special systematic studies in the near future.

Laser surgery use well established fact that the heating of biotissue causes its irreversible damage. As the tissue temperature grows higher, there takes place the denaturation of enzymes and loosing of membranes (40-45°C), coagulation and necrosis (60°C), drying out (100°C), carbonization (150°C), and finally, pyrolysis and vaporization (above 300°C).

Pulsed laser radiation allows developing finer surgical techniques involving minimal thermal injuries [24]. First, by using pulsed laser radiation of certain wavelength, one can selectively heat local, restricted areas featuring an elevated absorption (this is achieved when operating in region 2. It is this selective photothermolysis technique that is at the root of precise microsurgery. The effect of local laser heating of spatially nonuniform (granulated) biotissue has rather long been discussed in connection with the problem of pulsed-laser-induced retinal injuries [25]. This brings up following important question. What is essential is that the maximum permissible temperature to which biotissue can be heated without running the risk of destruction depends on the irradiation time? With the traditional heating methods. short-term heating conditions are difficult to realize, but laser radiation makes it possible to deposit energy in a small. tissue volume which cools rapidly as a result of thermal diffusion. It therefore becomes possible to study the limits of permissible tissue overheating during time periods from 10-3 to 10-12 s. This is obviously the object of future studies.

Secondly, high-power pulsed laser radiation can help effect the pulsed ablation (expositive evaporation) of the surface of soft and hard biotissues [26]. This process occurs in region 3 of Fig. 4, where heating is strong enough and is accompanied by a substantial pulsed pressure rise. The mechanism and conditions of the laser ablation of various materials, including biotissues, were discussed in [51].

Concluding this fragmentary analysis of laser light interaction with biomatter with applications which is a supplement to my old review often years standing [27], I clearly see the rapidly of development and fruitfulness of this field of application of lasers. As far as these this field can probably only be compared with the laser fiber science and technology for telecommunications. Both these areas will have a great effect of the further development of the human society. Incidentally, when the laser just made its appearance, neither one not the other field was considered a potentially serious area of its application. This is one more evidence of how subjective and unreliable all sorts of predictions can be even in science and technology.

References:

1. Mandelbrot, B. (1983) The Fractal Geometry of Nature, Freeman, New York.

2. Nossal, R., Kiefer, J., Weiss, G.H., Bonner, R., Taitelbaum, H., and Havlin, S. (1988) "Photon migration in layered media", Appl.Opt. 27, 3382-3391.

3. Patterson, M.S., Chance, B., and Wilson, B.C. (1989) "Time-resolved reflectance and transmitance for the noninvasive measurement of tissue optical properties", Appl.Opt. 28, 2331-2336.

4. Patterson, M.S., Moulton, J.D., Wilson, B.C., Berndt, K.W. and Lakowicz, J.R. (1991) "Frequencydomain reflectance for the determination of the scattering and absorption properties of tissue", Appl.Opt. 30, 4474-4476.

5. Hebden, J.C. and Wong, K.S. (1993) "Time-resolved optical tomography", Appl. Opt. 32, 372-380.

6. Нее, M.R., lzatt, J.A., Jacobson, J.M., Fujimoto, J.G. and Swanson, E.A. (1993) "Femtosecond transillumination optical coherence tomography". Opt. Lett. 18, 950-952.

7. lzatt, J.A., Нее, M.R., Huang, D„ Fujimoto, J.G., Swanson, E.A., Lin, C.P., Schuman, J.S. and Puliafito, C.A. (1993) "Ophthalmic diagnostics using optical coherence tomography", Ophtalmic Technologies III, Spie Proc. 1877, 136-144.

8. Cheong,W.F.,Prahl, S.A. andWelch,A.J. (1990) "A review of the optical prоperties of biological tissue",IEEEJ. Quant. Elec. 26, 2166-2185.

9. Zharov, V.P., Letokhov, V.S. (1986) Laser Opto-acoustic Spectroscopy, Springer, Berlin.

10. Tam, A.C., (1986) "Applications of photoacoustic sensing techniques". Rev. Mod. Phys. 58, 381-431.

11. Gusev, V.E. and Karabutov, A.A. (1993) Laser Optoacoustic, AIP, New York.

12. Karabutov, A.A., Podymova, N.B., Letokhov, V.S. (1996) "Time-resolved laser optoacoustical tomography of inhomogeneous media", Appl. Phys. B63, 545-563.

13. Murnick, D.E., and Peer, B.J. (1994) "Laser-Based Analysis of Carbon Isotope Ration", Science 263, 945-947.

14. Hochstrasser, R.M. (1989) "Biological applications of ultrafast laser methods", Ber. Bunsenqes. Phys. Chem. 93, 239-245.

15. Kliger, D.S. (1983) (ed.). Ultrasensitive Laser Spectroscopy, Academic Press, New York. Letokhov, V.S. (1986) (ed.), Laser Analytical Spectrochemistry, Adam Hilger, Bristol and Boston.

16. Wilson, B.C., Sevick, E.M., Patterson, MS. and Chance, B. (1992) "Time-dependent optical spectroscopy and imaging for biomedical applications", Prос. IEEE 80, 918-930.

17. Betzig, E., Chichester, R.J. (1993) Single Molecules Observed by Near-Field Scanning Optical Microscopy, Science 262,1422-1425.

18. Letokhov, V.S, (1989) "Prospects of Laser Spectroscopy of Biomolecules with Nanometer Spatial Resolution", in Feld, M.S., Thomas, J.E., Mooradian, A. (eds.). Laser Spectroscopy IX, Academic Press, Boston, 494-499.

19. Keller, R.A., Katzir, A., (1993) (eds,). Proceedings of Advances in DNA Sequencing Technology, SPIE Рubl. 1891.

20. Jori, G., and Peria, C. (1985) (eds). Photodynamic Therapy of Tumors and other Deseases, Libreria Progetto, Padova.

21. Каru, Т. (1989) Photobiology of Low-Power Laser Therapy, Harwood Acad. Publ., Chur.

22. Letokhov, V.S. (1991) "Effects of transient local heating of spatially and spectrally heterogeneous biotissue by short laser pulses:, Nuovo Cimento 13D, 939-948.

23. Dainty, J.C. (1984) (ed.) Laser Speckle and Related Phenomena, Springer, Berlin.

24. Anderson, R.R. and Parrish, J.A. (1983) "Selective photothermolysis: precise microsurgery by selective absorption of pulsed radiation", Science 220, 524-527.

25. Hansen, W.P., and Fine, S. (1968) "Melanin granule models for pulsed laser induced retinal injury", Appl. Optics 7, 155-159.

26. Srinivasan, R. (1986) "Ablation of polymers and biological tissue". Science 234, 559-563.

27. Letokhov, V.S. (1985) "Laser Biology and Medicine", Nature 316, 325-330.

 

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