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Optical sensing techniques have obtained remarkable attention in developing biosensors. Especially, methods based on evanescent field probing are suitable for direct immunosensing. However, miniaturized immunosensors are more difficult to realize when compared with catalytic sensors. Miniaturization and the small amounts of reagents will speed up detection but they are also a potential source of errors and instability. Different aspects of miniaturization of immunosensors are discussed. Some modeling methods of the optical biosensing interface are presented. Modeling is important, but there is a lack of detailed optical data of biomolecules. Two integrated optics detectors are described and their use in biosensing applications are evaluated.
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We have developed a (bio)chemical analysis system based on luminescence generation and detection in the evanescent field associated with light guiding in an optical fiber. Our intention was directed towards optimization of not only the sensor, including the sensor handling and the immobilization of biochemical recognition elements, but also of the assay chemistry, with special emphasis on methods used for sensor regeneration, of the fluidic system, and of the experimental control software. Goals of this optimization process were not only to achieve high sensitivity, reproducibility and the related precision of the results, but also maximum regenerability of the sensors and system flexibility for a variety of different applications. Four examples of different bioaffinity assays, established on our sensor system, are presented: a competitive immunoassay for atrazine, a sandwich immunoassay for hirudin, a DNA hybridization assay, and first studies for the development of sensors based on membrane- bound receptors. In the atrazine assay, the sensor could be regenerated more than 300 times. In the hybridization assay, a detection limit of 7.5 multiplied by 10-14 M complementary fluorescein-labeled DNA was achieved. The performance of our system is compared with an established enzyme-linked immunosorbent assay (ELISA) on the example of the hirudin assay. In the concluding section of this paper, advantages and disadvantages of our fiberoptic, luminescence-based system, compared with commercialized systems, based on detection of changes of the effective refractive index, are discussed.
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The interaction between double-stranded DNA and a luminescent Pd-porphyrin complex has been studied by room temperature phosphorescence (RTP). Intercalation of the Pd complex into DNA in deoxygenated solution is accompanied by an observed enhanced RTP emission centered at 680 nm. Analysis of the RTP spectral data gave a value of 1.0 multiplied by 106 M-1 for the Pd complex-DNA association constant and the complex seemed to bind DNA at the GC rich environments. The spectroscopic features of this interaction and the analytical performance characteristics of the RTP method for DNA determination are evaluated. The implication of the use of the Pd-complex RTP probe in combination with time resolved luminescence measurements are discussed.
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The use of an immunosupport microreactor for a competitive flow-through fluorescent immunosensing device is shown. In the competitive assay format chosen, fluorescein-labeled and non-labeled IgG in solution compete for the binding sites of anti-IgG [F(ab') fragments] immobilized on Agarose activated beads and packed into a flow cell.
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In the work reported here, surface concentrations of 0.027 and 0.073 molecules nm-2 of the fluorescent membrane probe molecule nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE) were shown to yield optimum sensitivity for fluorimetric transduction of membrane structural perturbations for lipid membrane-based biosensor development. These optima were obtained through correlation of experimental data with theoretical predictions of optimum surface concentrations based on a model for NBD-PE self quenching previously published by our group. It was also determined that membrane structural heterogeneity improves the sensitivity of NBD-PE labeled membrane transducers. Together with fluorescence microscopy, observations of surface potential change upon compression or expansion of phosphatidylcholine (PC)/phosphatidic acid (PA) monolayers were used to qualitatively indicate the degree of structural heterogeneity in these membranes. It was determined that sub-microscopic domains must exist in microscopically homogeneous egg PC/egg PA membranes in order to facilitate the observed NBD-PE self-quenching responses upon alteration of bulk pH and therefore, membrane surface electrostatics and structure.
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Cell movement is vital to normal tissue growth and regulation. Although not often realized, cells are continually moving relative to each other, and many physically migrating from their original site. Blood and immune cells rely on migrating through other tissues to perform their function. Cell division also needs physical separation of daughter cells, and other cells, e.g. muscle cells have developed their mechanical machinery to perfection. Substrate interactions are complicated. The ability to migrate and stop migrating when needed is a vital part of tissue regeneration. Understanding cell migration and movement is very important to being able to discover how cancer cells are able to continue dividing and why they abnormally migrate. Much other work has established that cells contact substrates through specific attachment points, but it is almost impossible to visualize these in three dimensions since cell cytoplasm is translucent and resolution limited by wavelength of light. We have developed a small senor device of a metalized glass substrate on which we have electron beam lithographically produced arrays of sub-micron circular apertures. A surface plasmon resonance (SPR) wave is set up in the metal by excitation with an incident laser beam. The circular apertures act as discrete centers of optical scattering of the SPR wave and the associated light emanating from these points can be detected and studied using an associated image analysis system. The intensity of the light scattered from each of these apertures is a strong (exponential) function of the changes of local refractive index close to (within 250 nm) the aperture. The contact of the underside of the cell with the aperture bearding surface modulates the intensity pattern of the aperture matrix allowing high resolution of the spatial distribution of the contact points between the cell and the metalized surface.
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A prototype fiber optic fluorosensor for measuring low (radiobiologically relevant) levels of oxygen is described. The fluorescence of an excited fluorophor immobilized in a polymer at the end of an optical fiber is quenched by oxygen. The prototype fiber optic sensor operates most effectively in the 0 - 2% O2 range with fast response and settling times. This approach shows promise in overcoming some of the limitations of existing oxygen sensor systems.
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Several methods have been developed for the qualitative and quantitative measurement of breathing effort. The most useful kind of breathing pattern monitor includes devices for recording chest and abdomen dimension changes, such as impedance plethysmography and respiratory induction plethysmography. These devices can measure the tidal volume in relative terms, and even measure it in absolute terms after suitable calibration. In this study a novel method for measuring chest circumference based on an optical fiber is presented. The sensor is based on the measurement of light transmitted through a bent optical fiber, which is connected to an elastic band, wrapped around the chest, and whose radius of curvature changes due to the respiratory act. The amount of transmitted light is related to the radius of curvature of the fiber which depends on the chest circumference. The output of the respiratory sensor was checked qualitatively by changing the respiration rate and depth. The changes in breathing effort were clearly demonstrated in the sensor output recording. The respiratory effort was also correlated with the heart rate, measured by photoplethysmography. Statistically significant correlation was found between the lungs' volume and the heart rate, but the correlation coefficient was not high.
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Observation of the intradental blood supply is important in cases of dental trauma, but difficult. As the methods used by dentists to measure pulp vitality are not very reliable, a dental pulp vitalometer based on fiberoptic reflectance measurement and measurement of the absorption of blood has been designed and built. In addition to the fiber optic probe and reflectance sensor electronics, the vitalometer includes a data acquisition card, a PC and data processing programs. The thick dentin and enamel layers and the small amount of blood in a tooth are major problems for optical measurement of its vitality, and scattered light from the enamel and the dentin surrounding the pulpa also causes a problem in measurements based on reflectance. These problems are assessed here by means of theoretical models and calculations. The advantage of reflectance measurement is that only one probe is used, which is easy to put against the tooth. Thus measurements are simple to make. Three wavelengths (560 nm, 650 nm, 850 nm) are used to measure photoplethysmographic signals, and these should allow the oxygen saturation of the blood in a tooth to be measured as well in the future. Series of measurements have been performed on vital and non-vital teeth by recording photoplethysmographic signals, using the vitalometer and using a commercial laser-Doppler instrument. Verifications of the laser-Doppler and vitalometer results are presented and deduced here.
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Several parameters of the cardiovascular system such as heart rate, arterial blood pressure and blood flow fluctuate spontaneously. These fluctuations are related to the autonomic nervous system activity. In particular the low frequency fluctuations are mediated by the sympathetic nervous system. In the cuttent study, the low frequency fluctuations of the tissue blood volume and the blood volume pulse in the fingertips of healthy subjects were investigated using photoplethysmography (PPG). The baseline of the PPG signal (BL) is inversely related to tissue blood volume so that the parameter BV, defined by: BV equals Const. minus BL is directly related to the blood volume. The amplitude (AM) is directly related to the systolic blood volume increase. For most of the examinations BV and AM show positive correlation but in some examinations the two parameters were inversely correlated. PPG measurements performed in near infrared radiation, showed better correlation between BV and AM than for red light PPG. The results show that several mechanisms are involved in the low frequency fluctuations in the tissue blood volume. The analysis of the PPG signal provides, therefore, a potential tool for studying the mechanism of the regulation of the microcirculation by the sympathetic nerves.
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Vjacheslav G. Artioushenko, Alexander A. Lerman, Alexander P. Kryukov, E. F. Kuzin, V. N. Ionov, Natalia I. Afanasyeva, V. S. Letohov, Victor V. Sokolov, George A. Frank, et al.
Prototypes of fiberoptic sensors for Fourier transform infrared (FTIR) spectrometers, based on commercially available middle infrared (MIR)-fibers, are developed. Their use in the currently central field of medical diagnostics is demonstrated. The non-toxic, flexible MIR-fibers, transparent in the middle infrared region (from 4 mm to 16 mm), are used in these sensors as remote attenuated total reflection (ATR) elements. These sensors, in which FTIR spectrometer combined with remote fiberoptic sensors could be used in cancer diagnostics, glucose content measurements as well as in other areas of medical diagnostics, in which spectroscopic analysis of human tissues and human extra-cell liquids could be used for diagnostic purpose. The additional minimal invasive medical diagnostics in vivo will become possible in the future with such fiberoptic sensors.
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The hydrophobic bases tetradodecylammonium hydroxide (TDAOH) and tetrakisdecylammonium hydroxide (TKAOH) are to be used to solubilize the anionic form of m-cresol purple in ethyl cellulose to create a dry colorimetric thin polymer film sensor for CO2 in the gas phase or dissolved in solution. When used in aqueous solution, both TDAOH and TKAOH appear significantly more resistant to interference by protons or other ions at high concentration when compared with tetraoctylammonium hydroxide (TOAOH), the hydrophobic base which has been used for such work in previous studies. The TDAOH films are used as carbon dioxide sensors in aqueous solution at high ionic strength (e.g. 1 mol dm-3) and still appear blue at pH 1 after 1 h.
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The near-field interaction between an optical aperture of sub-micrometer diameter and a sample surface can be exploited to spectroscopically probe a biological medium with a resolution unattainable with traditional far-field techniques. A simple, low-cost technique is described for producing a chemical sensor which is based on the principles of near-field optics, and preliminary results obtained using this device to measure pH are reported. Singlemode optical fibers are drawn into sub-micrometer optical fiber tips and then coated with aluminum, to form a subwavelength aperture. By using laser illumination through this aperture, samples can be studied with a resolution better than the wavelength of light. Sub- micrometer pH sensors have been prepared by incorporating the pH sensitive dye fluorescein into a silica based sol-gel glass which is then coated in the form of a thin film onto the fiber tip surface. These sensors were used to monitor the pH of buffer solutions inside micron-size holes in a polycarbonate membrane, and to probe the intracellular environment of mouse embryonic fibroblast cells. It was found that the pH response is reversible in the range 3 - 10. In addition the design potential of the sol-gel process for optimization of the sensor by careful control of the coating thickness is demonstrated.
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Fiber-optic sensors are attractive for biomedical applications primarily because of their non- electrical and non-conductive nature. This secures the safety of use and immunity to electromagnetic interference. In addition to these advantages, the optical-fiber level-sensor presented here has a small size, high accuracy, and fast time response. The sensor probe is chemically passive and can be easily sterilized. These features make the sensor attractive for numerous applications in physiology, biochemistry and medicine. The level-sensor presented here is a multipoint discrete optical-fiber device employing an array of small-size liquid- sensitive optical refractometric transducers of new type. The array of such transducers is connected via multi-fiber optical cable to the optoelectronic transmitter-receiver unit of the sensor. It performs an intelligent quasi-continuous level monitoring of a fluid. The sensor accuracy, resolution and level measurement range depend on the array length and the total number of the transducers employed. In case of the most close spacing of the transducers, the sensor level-resolution is better than plus or minus 0.2 mm.
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Ion-selective bulk optode membranes have been combined with enzymatic reactions for the determination of neutral analytes in clinical applications. A new development in membrane technology has been introduced, using reverse micelles to entrap the biocatalyst in bulk polymeric membranes. The use of such reverse micellar systems allows the design of a single layer biosensor where the recognition process as well as the chemical transduction into an optical signal take place in the same sensing layer. Urea-sensitive micellar bio-optode membranes have been realized; dynamic range, response behavior, long-term stability as well as the operational lifetime are discussed.
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For the first time crystalline fibers with optical losses 0.05 dB/m at 10.6 micrometer, 0.2 dB/m at 5 - 6 micrometer and 0.7 dB/m at 2.94 micrometer have been fabricated from solid solution AgClBrI by hot extrusion. Optical losses and numerical aperture for fibers have been measured throughout infrared spectra. Power delivery of carbon dioxide-, Er3+- lasers has been investigated. Fibers with diameter 0.5, 0.7 mm are able to deliver linearly at least up to 80 W power of cw carbon dioxide laser. Fibers demonstrate stable delivery of 40 W power for 200 hours. Fibers were not damaged by 0.5 J/pulse in msec pulse operation of Er3+ laser at 2.94 micrometer. Fibers have low mode mixing and low bending losses. Mechanical properties are the same like for ordinary AgClBrI strong solution fibers. Fibers have long shell-life. Clad crystalline AgClBrI fibers with optical losses 0.2 dB/m at 10.6 micrometer and diameter 0.7/0.9 mm have been fabricated. Clad fibers have numerical aperture 0.3 - 0.5. Clad fibers stable deliver 20 W power of cw carbon dioxide laser on the distance of 2 m with total transmission 73% for 200 hours.
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The direct or waveguide transmitted beam shape of carbon dioxide laser was recorded by irradiating perspex blocks. The influence of waveguides materials, geometry, defects and roughness on the crater produced in the blocks was observed. Fused silica transmitted beams are narrower and sharper than the plastic transmitted beams, and the energy distribution can be clearly observed. The main effect on the beam shape comes from the wall roughness. The moving tip method was applied for non-destructive attenuation measurements.
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Fused silica flexible hollow wave guides developed at TAU were used to deliver Er-YAG laser radiation to a biological membrane model. The model was composed of fibrotic (inner egg shell) membranes that were either brushed with or immersed in physiological saline solution. Waveguides used included fibers with an inner diameter of 0.7 mm and 0.5 mm. Membrane aperture size and fiber output power density were obtained. Results for the 0.5 mm waveguides yielded average apertures of 100 micrometer length at a power density of 5.35 W/cm2 and threshold fluence of 10 mJ/cm2 for exposed membranes and 35 mJ/cm2 for wet membranes. The results indicate that this waveguide can be used to cut dense membranes, finally, we have succeeded in delaminating immersed membranes using a sealed waveguide. These results show the applicability of using fused silica flexible hollow waveguides for Er-YAG surgical applications of dense membranes in aqueous surroundings such as vitrectomy and posterio-copsulectomy in ophthalmology, and possibly for procedures involving pleural membranes.
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For most applications in laser medicine suitable delivery systems are required. We developed fiber optic based diffusing tips especially for photodynamic therapy (PDT) and laser induced thermotherapy (LITT). To realize an adequate emitting cylindrical diffuser the fiber core was abraded by a precision cutter. Hence, the use of scattering media such as TiO2-doped polymers is avoided. Because the diffuser size is mainly determined by the manipulated fiber and a surrounding glass capillary, one can realize small diameters ((phi) approximately equals 3 mm). The laser light is distributed mainly by surface scattering and total reflection at the fiber air boundary. Because the use of absorbing media is avoided, it is possible to apply high laser power as necessary in LITT and pulsed PDT. We produced diffusing tips with lengths of several centimeters and typical diameters of 3 mm. By controlling the fiber-shaping process, a homogeneous intensity profile or even special designs can be achieved. The control is done by either on-line camera surveillance or calculated predictions. A delivery system especially for the photodynamical treatment of female cervix dysplasia has been designed.
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Hubert van den Bergh, Jerome C. Mizeret, Jean-Francois Theumann, Alain Woodtli, Roland Bays, D. Robert, P. Thielen, J. M. Philippoz, Daniel Braichotte, et al.
A brief overview is given of light distributors developed by our group in Lausanne for photodynamic therapy (PDT) of cancer. We focus on fiberoptic devices which have to a large extent been tested over the years in the clinic for PDT of the upper aerodigestive tract, the tracheobronchial tree, the esophagus, the uterus, and the skin. Both surface and interstitial light distributors are discussed. Several different physical principles for obtaining the desired light intensity distribution in tissue are demonstrated, including the use of specially shaped reflecting surfaces, light scattering and refraction by particles, the use of flexible highly reflecting balloons, controlled fiber core surface roughening, and microlenses. PDT can be improved using 'intelligent' light distributors, which permit the measurement of the light intensity reflected from the irradiated surface, as well as the dye fluorescence signals. Both are measured in situ and in real time during the treatment. The use of such devices, which can measure photobleaching kinetics, and enable one to adjust the light dose to the observed dye fluorescence signals, thus giving better PDT control, is discussed.
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Germanium dioxide GeO2 forms glasses having a good transmission in the near and middle infrared region up to 4.5 (mu) . Various modifiers as PbO, TeO2, Sb2O3, etc. added to GeO2 produce binary or ternary glasses having the similar transmission spectra and are more stable than pure GeO2 glass. This paper deals with the study of GeO2-PbO glassy system, with the preparation of these glasses with the low concentration of OH groups, with the study of optical properties such as absorption spectra, extrinsic and intrinsic absorption, refraction index, absorption edges, etc. The attention is also focused on preform preparation and the fiber's drawing. These optical fibers will be able to be used for power delivery of Ho:YAG and Er:Yag laser energies in medicine.
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Laser power transmission, luminescence, mechanical behavior and effects of various thermal treatments have been investigated on mixed sliver halide crystals. This knowledge may suggest ways of optimizing the fabrication of optical fibers from such crystals. Anti-reflection coating for high-power 10.6 micrometer light transmission was studied both on crystals and fibers. Laser power transmission and mechanical properties of the fibers also have been investigated. On the basis of these experiments the prototype of the medical catheter was produced and surgery experiments with this catheter have been performed.
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Optical fibers with low transmission losses are very useful in medical endoscopic laser surgery. In the past we developed unclad silver halide IR fibers for the transmission of carbon dioxide laser energy. We have recently developed core/clad polycrystalline silver halide optical fibers with a loss of roughly 0.3 dB/m at 10.6 micrometers. Such fibers, with a core diameter 0.35 - 0.6 mm and length of 1 to 2 meters are capable of continuously delivering output power densities as high as 14 KW/cm2. We have studied the transmission properties of these fibers for different launching conditions such as the acceptance angle at the input end and the near and far field distributions at the output end. We have also investigated the effects of bending on the optical transmission. We show that by properly designing the core/clad structure we obtain significant improvements with respect to unclad IR fibers.
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Bent multimode optical fibers were studied using a 3D ray tracing program. Effect of fiber bending increased with smaller input aperture beams. Transmission of fibers decreased for the longer proximal straight part of the fiber. Significant focusing effect and output light redistribution were detected if a proximal straight part of the fiber was less than 1 fiber diameter. Transmission of hollow waveguides considerably depended on the inner surface quality. Calculated data were in accordance with experimental measurements of fiber transmission and output light distribution. Ray tracing is a useful approach to simulate different delivery systems using optical fibers and hollow waveguides.
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An analysis of injuries and risks using high frequency (HF) and lasers in medicine based on a literature search with MEDLINE was performed. The cases reported in the literature were classified according to the following criteria: (1) Avoidable in an optimal operational procedure. These kind of injuries are caused by a chain of unfortunate incidents. They are in principle avoidable by the 'right action at the right time' which presupposes an appropriate training of the operating team, selection of the optimal parameters for procedure and consideration of all safety instructions. (2) Avoidable, caused by malfunction of the equipment and/or accessories. The injuries classified into this group are avoidable if all safety regulations were fulfilled. This includes a pre-operational check-up and the use of medical lasers and high frequency devices only which meet the international safety standards. (3) Avoidable, caused by misuse/mistake. Injuries of this group were caused by an inappropriate selection of the procedure, wrong medical indication or mistakes during application. (4) Unavoidable, fateful. These injuries can be caused by risks inherent to the type of energy used, malfunction of the equipment and/or accessories though a pre-operational check-up was done. Some risks and complications are common to high frequency and laser application. But whereas these risks can be excluded easily in laser surgery there is often a great expenditure necessary or they are not avoidable if high frequency if used. No unavoidable risks due to laser energy occur.
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