Sergei F. Kolyakov

Absorption Measurements of Cell Monolayers Relevant to Mechanisms of Laser Phototherapy: Reduction or Oxidation of Cytochrome c Oxidase Under Laser Radiation at 632.8 nm

OBJECTIVE The objective of this work was a further investigation of redox mechanisms of laser phototherapy on the cellular level. BACKGROUND DATA Cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, is believed to work as the photoacceptor to modulate cellular metabolism in laser phototherapy. MATERIALS AND METHODS The changes in the absorption spectra of HeLa-cell monolayers before and after irradiation at 632.8 nm using fast multi-channel recording were evaluated by the intensity ratio between the peaks at 770 and 670 nm (intensity ratio criterion). RESULTS By the intensity ratio criterion, the irradiation effects (reduction or oxidation of the photoacceptor) depended on the initial redox status of cytochrome c oxidase. The irradiation (three times at 632.8 nm, dose = 6.3 x 103 J/m(2), tau(irrad.) = 10 sec, tau(record.) = 600 msec) of cells initially characterized by relatively oxidized cytochrome c oxidase caused first a reduction of the photoacceptor, and then its oxidation (a bell-shaped curve). The irradiation by the same scheme of the cells with initially relatively reduced cytochrome c oxidase caused first oxidation and then a slight reduction of the enzyme (a curve opposite to the bell-shaped curve). CONCLUSION The experimental results of our work demonstrate that irradiation at 632.8 nm causes either a (transient) relative reduction of the photoacceptor, putatively cytochrome c oxidase, or its (transient) relative oxidation, depending on the initial redox status of the photoacceptor. The maximum in the bell-shaped dose-dependence curve or the minimum of the reverse curve is the turning point between the prevailing of oxidation or reduction processes. Our results are evidence that the bell-shaped dose dependences recorded for various cellular responses are characteristic also for redox changes in the photoacceptor, cytochrome c oxidase.

Fourier transform infrared evanescent wave (FTIR-FEW) spectroscopy of tissue

A new Fourier transform infrared fiberoptic evanescent wave (FTIR-FEW) spectroscopy method has been developed for tissue diagnostics in the middle infrared (MIR) wavelength range (3 to 20 micrometers). Specific novel fiberoptical chemical and biological sensors have been studied and used for spectroscopic diagnostic purposes. These nontoxic and nonhygroscopic fiber sensors are characterized by (1) low optical losses (0.05 to 0.2 dB/m at about 10 micrometer) and (2) high flexibility. Our new fiber optical devices can be utilized with standard commercially available Fourier transform spectrometers including attenuated total reflection (ATR) techniques. They are in particular ideally suited for noninvasive, fast, direct, sensitive investigations of in vivo and ex vivo medical diagnostics applications. Here we present data on IR spectra of skin tissue in vivo for various cases of melanoma and nevus in the range of 1480 - 1800 cm-1. The interpretation of the spectra of healthy and different stages of tumor and cancer skin tissue clearly indicates that this technique can be used for precancer and cancer diagnostics. This technique can be designed for real-time and on-line computer modeling and analysis of tissue changes.

Remote skin tissue diagnostics in vivo by fiber optic evanescent wave Fourier transform infrared (FEW-FTIR) spectroscopy

The new method of fiber-optical evanescent wave Fourier transform IR (FEW-FTIR) spectroscopy has been applied to the diagnostics of normal tissue, as well as precancerous and cancerous conditions. The FEW-FTIR technique is nondestructive and sensitive to changes of vibrational spectra in the IR region, without heating and damaging human and animal skin tissue. Therefore this method and technique is an ideal diagnostic tool for tumor and cancer characterization at an early stage of development on a molecular level. The application of fiber optic technology in the middle IR region is relatively inexpensive and can be adapted easily to any commercially available tabletop FTIR spectrometers. This method of diagnostics is fast, remote, and can be applied to many fields Noninvasive medical diagnostics of skin cancer and other skin diseases in vivo, ex vivo, and in vitro allow for the development convenient, remote clinical applications in dermatology and related fields. The spectral variations from normal to pathological skin tissue and environmental influence on skin have been measured and assigned in the regions of 850-4000 cm-1. The lipid structure changes are discussed. We are able to develop the spectral histopathology as a fast and informative tool of analysis.

Irradiation with a diode at 820 nm induces changes in circular dichroism spectra (250-780 nm) of living cells

A sensitive method for measuring the circular dichroism (CD) of living cells in visible-near IR region is developed. The changes in CD spectra from 250 to 780 nm of HeLa cell suspension after the first and second irradiation at 820 nm in dose 9 J/cm2 are investigated. The CD spectrum of the intact cells is well structured and characterized by a positive signal in the UV (250-290 nm) and visible-near IR (500-780 nm) regions as well as by a negative signal in 300-450 nm region. Distinct maxima in the visible-near IR region are recorded at 566, 634, 680, 712, and 741 nm. As a rule, the peak circular dichroism signals decrease in the irradiated cells except of the area 750-770 nm. Peak positions (exception: the peak at 680 nm) shift as a rule to the long-wavelength direction. The most remarkable changes in peak positions as well as in CD signals are recorded in the region 750-770 nm: an appearance of the new peak at 767 nm after the first irradiation and its shift to 752 nm after the second irradiation. The peaks at 712 and 741 nm disappear after the irradiation. A new peak appears at 601 nm. It is assumed that the changes in the degree of oxidation of the chromophores of cytochrome c oxidase caused by the irradiation are accompanied by conformational changes in their vicinity. It can be suggested that these changes are occurring in CuB environment.

Investigation of normal human skin tissue and acupuncture points of human skin tissue using fiberoptical FTIR spectroscopy

An innovative spectroscopic diagnostic method has been developed for investigation of different regions of normal human skin tissue. This new method is a combination of Fourier transform IR fiberoptic evanescent wave (FTIR-FEW) spectroscopy and fiber optic techniques for the middle IR (MIR) wavelength range. The fiber optical sensors we have used are characterized by low optical losses and high flexibility for remote analysis. Our fiber optical accessories and method allows for direct interaction of the skin tissue with the fiber probe and can be utilized with a diversity of standard commercial Fourier transform spectrometers. The FTIR-FEW technique, using nontoxic unclad fibers in the attenuated total reflection regime, is suitable for noninvasive, fast, sensitive investigations of normal skin in vivo for various medical diagnostics applications including studies of acupuncture points. Here we present the first data on IR spectra of skin tissue in vivo for normal skin and several acupuncture points in the range of 1300 to 1800 cm-1 and 2600 to 4000 cm-1.

Diagnostics of cancer by fiber optic evanescent wave FTIR (FEW-FTIR) spectroscopy

The fiberoptic evanescent wave Fourier transform IR spectroscopy (FEWS) using fiberoptic sensors operated in the attenuated total reflection (ATR) regime in the mid-IR region of the spectrum (4 to 16 micrometer) has recently found application in the diagnostics of biotissues. The silver halide fibers used are non-toxic, non-hygroscopic, flexible and soft and are characterized by a low optical loss. The method allows for non-invasive and rapid (seconds) direct measurements of the spectra of normal and pathological tissues in vitro, ex vivo and in vivo with the aim of express testing of various tumor tissues at the early stages of their development. The method is expected to be further developed for endoscopic and biopsy applications.

New method for investigations of normal human skin surfaces in vivo using fiber-optic evanescent wave fourier transform infrared spectroscopy (FEW-FTIR)

Fiber-optic evanescent wave Fourier transform infrared spectroscopy (FEW-FTIR) is a new method developed for different applications for surface analysis of materials, including the diagnostics of skin and living tissues. Our technique allows for the detection of inconsistencies in the molecular structure of normal skin surface non-invasively and in vivo. This FEW-FTIR method is direct, non-destructive and fast (seconds). Our optical fibers for the middle infrared (MIR) range are non-toxic, non-hygroscopic, flexible and characterized by extremely low losses. The combination of Fourier transform infrared spectroscopy and advanced fiber technology in the MIR range has opened the door for new powerful diagnostic tools for investigations of many skin surface phenomena, including normal skin, process of aging, allergies and precancerous conditions. Our method could be extended to applications involving the detection of the influence of environmental factors (sun, water, pollution and weather) on the skin surfaces of children and adults. In this study, we have investigated non-invasively more than 118 cases of normal skin in vivo in the range of 1450-1800 cm -1 . We compare the FEW-FTIR spectra of palm, hand and elbow tissue. The results of our surface analysis of skin tissue are discussed in terms of structural similarities and differences on a molecular level.

Various new applications of fiber optic infrared Fourier transform spectroscopy for dermatology

Fiberoptical evanescent wave Fourier transform infrared (FEW- FTIR) spectroscopy has been applied in the middle infrared (MIR) wavelength range (3 to 20 micrometer) to the in vivo diagnostics of normal skin tissue, acupuncture points as well as precancerous and cancerous conditions. The FTIR-FEW technique, using nontoxic unclad fibers, is suitable for noninvasive, sensitive investigations of skin tissue for various dermatological studies of skin caner, aging, laser treatment, cosmetics, skin allergies, etc. This method is direct, nondestructive, and fast (seconds). Our optical fibers are nonhygroscopic, flexible, and characterized by extremely low losses. In this study, we have noninvasively investigated more than 300 cases of normal skin, acupuncture points, precancerous and cancerous tissue in the range of 1400 to 1800 cm-1. The results of our analysis of skin and other tissue are discussed in terms of structural and mathematical similarities and differences on a molecular level. In addition, we have also performed cluster analysis, using principal component scores, to confirm pathological classifications and to discriminate between genders. We have found good agreement with prior pathological classifications for normal skin tissue and melanoma tumors and normal females were distinctly separate from males.

Changes in the circular dichroism spectra of a suspension of live cells exposed to low-intensity laser radiation (lambda = 820 nm).

Circular dichroism (CD) spectroscopy in the UV spectral region, where absorption bands of proteins and nucleic acids are located, is a well-known method of study of structure of biological macromolecules [1]. The goal of this work was to study the CD spectra of cells in the visible and near infrared (near-IR) spectral regions, where absorption bands of the respiratory chain enzymes are dominant [2–4]. We studied the changes in the CD spectra of live cells induced by exposure to radiation of a semiconductor laser (820 nm).

Investigations of normal skin tissue using fiber optical FTIR spectroscopy

An innovative spectroscopic diagnostic method has been developed of investigation of different regions of normal human skin tissue. This new method is a combination of Fourier transform infrared fiberoptic evanescent wave (FTIR-FEW) spectroscopy and fiber optic technique for the middle infrared (MIR) wavelength range (3 to 20 micrometer). The fiberoptical sensors we have used are characterized by low optical losses and high flexibility for remote analysis. Our new fiber optical accessory can be utilized with a diversity of standard commercial Fourier transform spectrometers. FTIR-FEW, using nontoxic unclad fibers in the attenuated total reflection (ATR) regime, is suitable for noninvasive, fast, sensitive investigations of normal skin in vivo for various medical diagnostics applications including studies of acupuncture points. Our method allows for direct interaction of the skin tissue with the fiber. Here we present the first data on infrared spectra of skin tissue in vivo for several acupuncture points in the range of 1300 to 1800 cm-1 and 2600 to 4000 cm-1.