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The emerging need for a fast, safe economical approach to global and localized measures of desaturation of hemoglobin with oxygen (HbO2) in the human brain motivates further research on time-resolved spectroscopy in four areas of study. (1) To afford quantization of hemoglobin saturation through time-resolved spectroscopy in the time domain (TD) and in the frequency domain (FD). Evaluation of dual-wavelength TD and FD spectrometers for determining quantitatively hemoglobin desaturation and blood-volume changes by calculations that are insensitive to mutual interference is proposed. The diffusion equation, as it applies especially to TD studies, and the absorption ((mu) a) and scattering ((mu) s) coefficients provide their independent determination from the late and early respective portions of the kinetics of the emergent photons in response to a short input pulse (50-100 psec). (2) The identification of the photon-pathlength change due to the arterial pulse in the brain tissue by FD methods with Fourier transformation affords an opportunity to employ principles of pulse oximetry to vessels localized deep within the brain tissue. (3) Localization of desaturation of hemoglobin in portions of the brain can be achieved through dual-wavelength scanning of the input/output optical fibers across the head for an X-Y coordinate and varying the distance between input and output ((rho) ) or the time delay in data acquisition to afford an in-depth Z scan. Localizations of shed blood, which have an effective concentration of over 10 times that of capillary-bed blood, are identified by X, Y, Z scans using only a single wavelength. (4) Independent measurements of absorption ((mu) a) and scattering ((mu) s) coefficients, particularly by TD techniques, affords structural mapping of the brain, which can be used to diagnose brain tumor and neuronal degeneration. Two experimental systems are used to critically evaluate these studies; the first, a hemoglobin/lipid/yeast model in which intermittent oxygenation gives saturation/desaturation effects and addition of hemoglobin simulates increased blood volume. These models can be global or may contain localized ''black'' absorbers simulating brain bleeds or model-stroke volumes in which oxygenation/deoxygenation simulates normoxia/hypoxia. Secondly, animal brains are used to model the following changes in vivo: global or localized hypoxia, brain bleeding, and hematomas by epidural blood injection, and physiological changes by epilepsy. Neuronal degeneration causing scattering effects is modeled by injection, epidurally or into the animal model brain, highly scattering material such as polystyrene spheres. The proposal envisages a basic science study of photon migration in the brain with important applications to stroke, epilepsy, brain trauma, and neuronal degenerative disease.
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Time-gated fluorescence spectroscopy provides a very useful tool to evaluate the photophysical properties and the incorporation mechanisms of drugs interacting with biological substrates. In particular, taking into account that different fluorophores, even if overlapped in fluorescence spectrum, present different fluorescence lifetimes, it is possible to evidence the emission of a single molecular species by choosing a suitable observation window in the time domain. Using this technique, the effect of systemic administration on the uptake of Hematoporphyrin Derivative (HpD), its tumor localizing fraction (TLF), and disulphonated Aluminum Phthalocyanine (AlS2Pc) at the cellular level was evaluated on a murine ascitic tumor. The results obtained indicate that the TLF is the part of HpD actually retained by the cells and that AlS2Pc is incorporated more rapidly with respect to porphyrins. The observation of the gated spectra of HpD also evidenced the possibility of improving the contrast between the fluorescence of the cells and that of the drug. Thus, an imaging system has been developed which utilizes a gated, intensified, CCD camera synchronized with a subnanosecond laser- pulse excitation. The gate can be set to a minimum width of 5 ns and arbitrarily delayed with respect to the laser pulse. By optimization of the gate parameters, porphyrin fluorescence images in single cells and microscopy sections of tumor were obtained with a valuable signal- to-noise ratio.
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Time-resolved reflectance measures the escape of photons that have been injected into a scattering medium by a very short laser pulse. In this paper, such measurements are discussed in terms of the basic equations governing the escape of photons from turbid tissue, and an experimental example of a prototype endoscopic device is given.
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The preceeding paper by von Bally describes a wide range of holographic techniques which may be applied within biomedicine. One of the most promising techniques in terms of research and (potentia) clinical applications was hologram interferometry whereby minute global displacements of surfaces were displayed as contour maps. The noncontact, nondestructive, and high-sensitivity nature of the technique allows studies of even the most delicate specimen. Conventional hologram interferometry is, however, hampered by the necessity of using film or similar media for the registration process. The development introduces a time delay and restricts the sampling rate in the measuring process. TV-holography (ESPI) circumvents these drawbacks by replacing the conventional recording media (film, thermoplastic, etc.) with the photosensitive target of a video-camera. Using analogue or digital electronic processing, the reconstruction process is simulated to give a real-time presentation of interferometric images on the video monitor. The techniques shows promise in the biomedical field, especially as computers are incorporated into the system to aid the operator in analyzing the fringe patterns. The technique and its advantages for biomedical applications are discussed and illustrated with examples of computerized image processing.
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In frequency-domain photon migration (FDPM), amplitude-modulated light is launched into a turbid medium, e.g., tissue, which results in the propagation of density waves of diffuse photons. Variations in the optical properties of the medium perturb the phase velocity and amplitude of the diffusing waves. These parameters can be determined by measuring the phase delay and demodulation amplitude of the waves with respect to the source. More specifically, the damped spherical-wave solutions to the homogeneous form of the diffusion equation yield expressions for phase ((phi) ) and demodulation (m) as a function of source distance, modulation frequency, absorption coefficient ((Beta) ), and effective scattering coefficient ((sigma) eff). In this work, analytical expressions for the variable dependence of (phi) and m on modulation frequency are presented. A simple method for extracting absorption coefficients from (phi) and m vs. frequency plots is applied to the measurement of tissue phantoms. Using modulation frequencies between 5 MHz and 250 MHz, absorption coefficients as low as 0.024 cm-1 are measured in the presence of effective scattering coefficients as high as 144 cm-1. The results underscore the importance of employing multiple modulation frequencies for the quantitative determination of optical properties.
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Optical measurements represent a valuable tool for in vivo analysis of tissue properties, for example, the average level of oxygenation of perfusing blood. A general problem in turbid medium, such as in the case of most tissues, is distinguishing between phenomena caused by absorption and those due to scattering. These problems can be overcome by using either time- or frequency-domain techniques. Frequency-domain measurements are based on evaluation of the phase and amplitude information of transmitted amplitude-modulated optical beams. These types of measurements might prove to be a valuable tool for in situ evaluation of tissue properties, for example, fluorescence and absorption. Irradiation of turbid media by harmonically-modulated optical beams initiates density waves of diffusely propagating photons. The phase velocity of these waves is quite different from the velocity of light. The velocity, which is strongly dependent on the modulation frequency, can typically vary from the velocity of light as an upper limit down to about 10 of this value in highly scattering, moderately absorbing tissue. This presentation gives a brief discussion of the general properties of these kind of waves in tissues.
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From the various capabilities of holography for image processing and measuring purposes, holographic interferometric techniques have found more extended application in biological and medical research. Due to their special properties the different methods of holographic interferometry are applied to characteristic fields of biomedical investigations where--similar to nondestructive testing--vibration and deformation analysis is of interest. Features of holographic interferometry, such as the possibility of noncontactive, three-dimensional investigations with a large field-of-depth, are used with advantage. The main applications can be found in basic research e.g., in audiology, dentistry, opthalmology, and experimental orthopedics. Because of the great number of investigations and the variety of medical domains in which these investigations were performed this survey is confined to some characteristic examples. As in all fields of optics and laser metrology, a review on biomedical applications of holography would be incomplete if military developments and utilization were not mentioned. As demonstrated by selected examples, the increasing interlacing of science with the military does not stop at domains that traditionally are regarded as exclusively oriented to human welfare--like biomedical research. The term ''Star Wars Medicine'', which becomes an increasingly popular expression for laser applications (including holography) in medicine, characterizes the consequences of this development.
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To describe the fluorescent intensity behaviour at a certain wavelength or waveband that is emitted out of tissue and which is caused by excitation of a shorter wavelength or waveband, requires solving the transport equation of radiative transfer twice. The first time for finding the spatial distribution of the excitation light inside the tissue that is caused by incident irradiance, the second time for finding the spatial distribution of fluorescent light where, in this case, the excitation light energy fluence rate spatial distribution is the source of producing fluorescent photons. It is assumed that a fluorescent photon, when created, has an equal probability of propagating in any direction. Conse quently, when the incident irradiance consists of a waveband of light, the resulting spatial distribution of the fluence rate inside the tissue may not be identical for all wavelengths within the band. In addition to this, when fluorescence occurs at several wavelengths, there may be differences in propagation behaviour for the different wavelengths. Diagnostic methods that use tissue fluorescence should therefore be used with great care because the absorption and scattering behaviour of the tissue can substantially complicate the interpre tation of the fluorescence signal. Recently, this problem was solved with a Monte Carlo numerical method by Keijzer et al. [1] for a finite laser beam that was perpendicularly incident on the tissue. Such a problem cannot be solved analytically and, as a result, does not produce great insight into the influence of e. g. penetration behaviour of excitation light on the final fluorescence outcome. In this report a simpler approach is chosen in which the problem of fluorescence caused by laser excitation is fully solvable in analytical form. This produces insight into the influence of changes in optical parameters on the fluorescence behaviour albeit at the cost of a loss in reality. To solve the problem in an analytical form requires chosing a strictly one-dimensional tissue.
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The transmission of light from a sinusoidally modulated source to a detector through a turbid medium such as tissue can be described in terms of the phase angle and modulation of the detected signal. Such measurements provide information about the optical properties of the tissue. This paper outlines the relationship between tissue optical properties and the phase angle and modulation of the detected signal.
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The development of the laser opened new and exciting avenues in man''s effort to study the properties of matter, understand the effects of intense electromagnetic radiation on inorganic and organic substances, and utilize light to improve quality of life. The development, especially of powerful lasers in the ultraviolet, visible, and near-infrared spectral regions, led to the precise removal of biological tissue (optical ablation) from a laser-irradiated surface to depths and widths in the submicron domain even in the case of transparent tissues. Although this technology has been applied in many important areas and currently presents a new dimension in laser biomedicine, the mechanisms leading to precise tissue removal and the effects on neighboring tissues are still under investigation. In this paper, the basic photophysical and photochemical phenomena and processes related to precise cutting of biological tissue are discussed.
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Pulsed UV and mid-IR lasers have a great potential in various medical disciplines for precise and sparing tissue removal. Beside the study of special applications, the investigation of the underlying ablation mechanisms has become of increasing importance. This paper summarizes the basic models to describe the ablation efficiency and discusses applications as well as limitations.
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Tetrapyrroles obtain very good photophysical properties making them efficient photosensitizers in photodynamic therapy. Most of them have high triplet quantum yields, long triplet lifetimes and triplet state energies suitable for singlet oxygen generation. These photophysical parameters differ depending on the chemical structure and interactions with the environment. Today a large variety of tetrapyrroles are synthetized or chemically separated from natural substances for investigation of their photophysical properties with the aim of using them in photodynamic therapy. In this paper, some general aspects of energy conversion and energy transfer of tetrapyrroles in relation to their photophysical and biophysical properties relevant for photodynamic activity are discussed. Essential optical methods for the investigation of these parameters, as well as the determination of segmental mobilities of sensitizer-carrier complexes, are discussed using the example of phthalocyanines.
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Experimental work on stone-fragmentation rates using a pulsed dye laser has revealed strong differences in the pulse energy dependence of biliary and salivary stones. A simple mathematical model is described relating these differences to a stochastic process governing the generation of shock waves.
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Lasers are well established as the treatment of choice in certain types of cancer, particularly the use of carbon dioxide laser for eradication of early tumors of the cervix and for lesions of the upper airways. More recently, the high-power Nd:YAG laser has proven itself of value for endoscopic palliation of advanced obstructing tumors of the gastrointestinal tract and major airways in patients who are unsuitable for surgery. However, current techniques are only scratching the surface of the potential applications of lasers in medical and surgical practice, and this article outlines two ways in which laser therapy for cancer may develop--low power interstitial hyperthermia and photodynamic therapy.
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The chick chorioallantoic membrane (CAM) model was used to study synergistic effects of photodynamic therapy (PDT) and hyperthermia (HPT). Since HPT is known, and PDT is believed, to involve a vascular mechanism, the CAM is an ideal medium to study the synergism of these modalities. Moreover, the CAM is a particularly convenient model to manipulate the PDT and HPT parameters and to monitor the modifications of the vasculature: (1) It is possible to view individual blood vessels in the CAM and to examine structural changes in real time. (2) The CAM is a closed system in which HPT can be performed quantitatively and to a selected depth, using different lasers. And (3) variations of surface temperature during PDT + HPT can be readily monitored by noninvasive radiometric techniques. A porphyrin-type photosensitizer solution was applied to areas of the CAM, defined by teflon O-rings placed on the surface. Uptake of the sensitizer into the CAM was determined by monitoring its fluorescence. Excitation light at 405 nm from a spectrofluorometer was directed onto the CAM surface using a bifurcated fiberoptic light guide which also transmitted the fluorescence from the CAM area. The fluorescence-emission spectrum (630-730 nm) and intensity at different times following sensitizer application was measured in vivo. This technique permitted the determination of the uptake dynamics of the sensitizer in the CAM and the establishment of the optimal time for irradiation. After an equilibrium time of 30 minutes, to allow for uptake of sensitizer in the CAM, the area was irradiated with a dual-wavelength system composed of a dye laser at 644 nm (to induce PDT) and a CO2 laser at 10.6 micrometers (to bring about HPT). Damage to the CAM vasculature, due to combined PDT + HPT, was compared to the outcome of the separate modalities. The observed synergistic effect of about 30 was interpreted by invoking various physiological processes. The egg, being a closed in vivo system, lends itself to mathematical modeling of the temporal and spatial temperature profile. The importance of heat dissipation due to diffusion, radiation, and blood perfusion was shown to be small compared to that of heat dissipation due to evaporation of water from the CAM.
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Irradiation with 86 J/cm2 of cultures of Fisher-rate thyroid cells (FRTL5) in the presence of daunomycin derivatives at wavelengths between 488 and 595 nm i.e., in the visible- absorption bands of these drugs, is shown to enhance their cytotoxicity. Daunomycin, its 4- demethoxy derivative, 5-iminodaunomycin, and two amino-substituted 4-demethoxy derivatives of daunomycin are tested. While a 2-h exposure to the drugs in the dark produces 50 short-term cell mortality at dosages (LD50) in the range 23 to 138 (mu) g/ml, irradiation administered during the cell exposure to the drugs is found to lower the LD50 values down to the range 45 to 289 ng/ml. Furthermore, while the LD50 values for all drugs in the absence of photoactivation are similar, if light is administered those for the 4- demethoxy compounds are lowered by 3 orders of magnitude and those for the other derivatives by 2 orders of magnitude. Microfluorimetric investigations reveal that photoactivation causes fading of the drug fluorescence in the perinuclear cytoplasm. The effect is more pronounced for drugs with higher photosensitizing properties. The nonfluorescent photoproducts which are formed in the cells during photoactivation exhibit a cytotoxic activity that is, at long term, lower than that of the original drug. The authors cannot yet assess which excited-state property of anthracyclines plays the key role in the photosensitized reaction(s) responsible for both short-term cell kill and long-term toxic effects. The show, however, that such property is strongly affected by the removal of the methoxy group from the C4 position.
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Zn(II)-phthalocyanine (ZnPc) is a tetraazaisoindole pigment which can be prepared by chemical synthesis with a high degree of purity, and it efficiently absorbs 680 nm of light. These properties, associated with its ability to generate activated oxygen species (e.g., singlet oxygen) upon photoexcitation, make ZnPc a potential phototherapeutic agent. Actually, upon delivery of ZnPc to tumor-bearing animals after incorporation into liposomal vesicles, the dye was uptaken and retained by the tumor tissue in significant amounts and with a good degree of selectivity. Irradiation of the ZnPc-loaded tumor area with ca. 680 nm of light caused tumor necrosis to an extent which was related to the dye concentration in the tumor.
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There are three principal classes of laser tissue interactions. All three can be used to remove malignant tissue either by biological digestion or immediate removal i.e., photovaporization or photodecomposition. This paper discusses a semiempirical theory of the so-called photoablation process and the thermal side effects of the surrounding tissue. Describing the photoablation process requires differentiation to be made between very short laser pulse durations (
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The uptake and distribution of the photosensitizer aluminum sulphonated phthalocyanine (AlSPc) has been studied. In a variety of experimentally induced gastrointestinal tumors the photosensitizer is retained between 24 - 48 hours after intravenous administration compared with the adjacent normal tissue in which the tumor arose. However, the maximum tumor-to- normal-tissue ratio was only 2:1. Quantitative fluorescence photometry using digital image processing, with a CCD camera and helium neon laser, was used to probe the microscopic localization of the photosensitizer in tissue sections of tumor and normal tissue. Selective localization of the photosensitizer was nonspecific in tumor stroma and there was never any significant difference between normal and neoplastic cells. Exploitation of the small differences in photosensitizer concentration, photodynamic threshold effects, and photosensitizer photodegration allows up to 2 mm of selective tumor damage to be produced in a tumor, when a similar light dose will produce no damage in adjacent normal tissue. However, selective eradication of a tumor without adjacent tissue damage will not be possible by using these methods. This paper reviews this previously reported data.
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Two laser types are going to find a place in refractive surgery of the cornea: the excimer laser (193 nm) and mid-infrared YAG lasers, such as Ho:YAG (2.1 micrometers ) and Er:YAG (2.94 micrometers ). Whereas the excimer laser used for photorefractive keratectomy (PRK) and phototherapeutic keratectomy (PTK) is currently studied in clinical trials, Ho:YAG and Er:YAG lasers are still in the state of preclinical evaluation. For myopic corrections excimer laser PRK has shown to be safe and effective in the range up to -7.0 D. The results compare favorably with conventional procedures such as radial keratotomy. Complications are rare. Hyperopic and astigmatic corrections using the Ho:YAG laser (HOT) are effective, but safety and stability has yet to be proven. Er:YAG laser photoablation yields a healing response in animal eyes similar to the excimer laser.
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The infrared-laser systems like the Er:YAG, the cw CO2, the Nd:YAG, and the UV- excimer lasers are being investigated for preparing tooth-hard substances. The infrared lasers cause thermal damage to the enamel, the dentin, and the pulp with the exception of the Er:YAG laser. No thermal damage occurs using the Er:YAG laser under practical conditions because of the special thermomechanical ablation process. The ablation rates of the UV- excimer lasers are to low for practical use. Enhancing the ablation efficiency by high- repetition rates causes thermal side effects to occur. Therefore, only the Er:YAG laser has the potential to partially replace the mechanical drill.
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The optimum laser-system parameters are being selected for several types of surgical operations using ablation techniques. The choice is based on the specific demands of the operation performed, knowledge of the ablation laws, limitations on laser-beam intensity which come from the necessity to transport high-intensity light through flexible fiber, and the peculiarities of different laser systems. At present it is more expedient to develop laser medical setups oriented to the solution of one task or a limited number of problems. The choice of a concrete installation should be based on the investigation results of interaction of radiation with biological tissues and its transmission through the fiber, the analysis of the level of development of laser and fiber technique, specificity of the operation, and compatibility of laser facilitates and traditional medical equipment. The paper illustrates such an approach by way of several concrete examples and notes the corresponding laser systems, which were developed or are in the developmental stage in the General Physics Institute of the USSR Academy of Sciences and in organizations connected with the Institute.
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Therapeutic heating goes back as far as 3000 B.C., but the therapeutic use of a local temperature rise of 5-20 degree(s)C above normal body temperature, called hyperthermia, was carried out only in the past few decades, if moxibustion is disregarded. Results obtained by various treatment modalities such as optical waves (laser light), microwaves (MW), radio frequency current (rf), or ultrasound (US) are compared, and a theory based on the assumption that tumor formation can be regarded as some sort of disturbance in the signal processing system of the given biological structure is presented. The author suggests that not the locally generated heat alone but the elevated temperature in relation to several environmental factors is responsible for cell killing. Therefore, the modality of creating hyperthermia may be of significance since various methods may change the thermal behavior of the cell environment in a specific way, which means that using two or more modalities the synergetic behavior could be exploited for a better result.
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The autofluorescence of human arterial tissue with varying degrees of atherosclerosis was studied in vitro to develop a diagnostic tool for tissue differentiation simultaneously to tissue ablation induced by a XeCl-excimer laser (wavelength 308 nm). Healthy vessel walls and artery segments containing lipid-rich or calcified areas were investigated in air, saline solution (0.9 NaCl), and in blood. The fluorescence spectra in the wavelength range from 320 nm to 650 nm were recorded with an optical multichannel analyzer, and they allowed for a clear discrimination between plaque and healthy vessel wall even in blood. For each single laser shot with an energy density of about 4-5 J/cm2, well above the ablation threshold, a complete spectrum was recorded. The fluorescence spectra were analyzed in terms of their contributions from normal arterial tissue, lipid-rich and calcified plaques. The results clearly show the feasibility of controlling the ablation process by fluorescence spectroscopy in order to avoid vessel-wall perforation which is one of the main drawbacks in laser angioplasty.
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A technique to improve signal-to-background ratio in fluorescence images of superficially growing tumors marked with photosensitizers is described. Time-delayed detection of fluorescence following pulsed-laser excitation allows suppression of the autofluorescence background falling into the fluorescence band of the photosensitizer. This technique exploits the longer fluorescence-decay times of porphyrin-based photosensitizers compared to average decay times of tissue autofluorescence. The feasibility of time-delayed fluorescence imaging of tumors has been demonstrated in vitro. From time-delayed fluorescence spectra the authors infer that the ratio between photosensitizer signal and autofluorescence background can be improved by about one order of magnitude.
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A time-gated technique to reduce the effect of light scattering when transilluminating turbid media such as tissue is demonstrated. The concept is based on transillumination with picosecond laser pulses and time-resolved detection. By detecting only the photons with the shortest travelling time, and thus the least scattered photons, the contrast can be enhanced. Measurements on a tissue phantom as well as breast tissue in vitro are presented. It is demonstrated that the spatial resolution can be enhanced by using the time-gated technique. It is also shown that differences in scattering properties may be more pronounced than differences in absorption properties when demarcating tumor from normal tissue.
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Fluorescence imaging is widely used in biological and medical science, and more recently has application to in-vivo tumor detection. The ability to generate image contrast on the basis of luminescence decay time, as well as intensity, opens up new contrast mechanisms and improves image quantization by compensating for most quenching processes. Decay-time imaging using modulated excitation and homodyne detection is a sensitive and versatile technique, which presently has application to fluorescence microscopy, analytical fluorescence spectroscopy, and sensor technology. Apart from fluorescence applications, the technology potentially is valuable for noninvasive time- and phase-resolved spectroscopy of tissues. Two approaches to implement frequency domain imaging are discussed. One of these uses a custom-built modulated intensified CCD system, while the other uses a modified imaging single-photon detector (IPD) in conjunction with a multichannel photon correlator. The CCD system is best suited to microscopy and nanosecond measurements, while the IPD has the potential for much higher time resolution with extremely high sensitivity. Time- and frequency-domain methods for biological and medical imaging are now the subject of considerable research effort worldwide. Up to the present time most studies have been aimed at establishing the principles involved, rather than producing robust devices of the sophistication expected for routine medical applications. In particular, measurements have been made by scanning a single detector across the region of interest--building up an image point-by-point. Scanning methods of imaging have their virtues, and have been widely implemented in biological microscopy. However, the imaging-light emerging from a scattering sample, single-point detection is highly inefficient since information from the sample is necessarily dispersed over an area even when the excitation or illumination is locally concentrated. In this paper, the alternative of using spatially resolving area detectors for time- and frequency-domain imaging is discussed and an attempt is made to identify the advantages and present limitations of the technology.
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Ablation and tissue removal of normal and atherosclerotic arterial tissue by UV excimer-laser radiation were probed by taking photographs with a dye laser as a flash light-source. The ablating pulses were transmitted through a fused silica fiber into a cuvette with the samples exposed to saline solution. The delay time of the probing dye-laser pulse with respect to the ablating excimer-laser pulse was varied in the nanosecond range up to several hundred microseconds. The ablation process and the resulting plume above the tissue surface were recorded with a CCD camera attached to a PC-based image processing system. All samples under investigation were fresh human cadaver aortic and femoral artery specimens which had been shock-frozen for less than 48 hours. The arterial segments showed different types of lipid-rich and calcified plaques. Big cavitation bubbles and small tissue particles emerging from the irradiated area have been recorded.
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The method proposed in this paper is based on the detection of resonantly enhanced fluorescence emission induced by a tunable dye laser. First test on anorganic samples exposed to air and to saline solution demonstrate the potential of this technique. A XeCl excimer-laser ((lambda) equals308 nm) pulse, guided by quartz fibers, causes an efficient ablation of the irradiated samples. The specific species to be detected in the ablation plume determines the wavelength of the narrow-band dye-laser radiation. Preferably, it is set to a strong transition of the selected ablation product. Taking into account the formation of the plume, the dye-laser pulse is applied with a certain delay in order to excite resonantly the chosen species in the plume. The resulting resonance fluorescence is then guided by optical fibers to an OMA system. Compared to the broad-band excimer-laser-indiced fluorescence during the ablation process, the resonance fluorescence signal shows a distinct and easily detectable sharp peak. The signal-to-background ratio is improved by one order of magnitude. The achieved increase in sensitivity as well as selectivity is for the benefit of a reliable identification of ablated tissue.
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The Raman laser fiberoptics (RLFO) method using Raman spectroscopy for determination of chemical composition and optical fibers allowing multiplex, in situ, and remote possibilities, enabled chemical analysis of various human urinary and renal calculi. Raman spectra of about 40 constituents (synthetic or natural) in the authors''s possession and its 437 various binary and ternary mixtures are recorded using 1.06 micrometers radiation of a Nd:YAG laser and a FT Raman interferometer. These spectra--most of them are fluorescence free--constituted the calculi library. In the presence of urine, unknown stones can then be identified by RLFO method using an automatic computer procedure (at the present time, the Bruker IR search program is used). The results obtained for the identification of the stones are satisfactory. Major constituents of a complex calculus (
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Photodynamic therapy, developed since 1961 with Lipson''s studies, is now limited in its clinical applications by the lack of knowledge about light comportment and the action of hematoporphyrin in tissues. Using human tumor models in mice, the intratumoral light flux was measured during an interstitial illumination (cylindrical diffusor 5 mm of length) by an argon dye laser emitting continuously at 630 nm (Spectra-Physics 375 B). The flux measured was captured by a plane-cut fiber (400 micrometers ) linked with an optical power meter (Newport 815). The light decrease in tissue had an exponential shape, and k, the global attenuation coefficient, was easily calculated as well as the depth penetration (1/k). Control measurements were performed in beef muscle, and the k value was very consistent with published data. In small tumors (3), the results presented a good reproducibility for the same histology (ksarcoma equals 0.48 +/- 0.08 mm-1, kcholangiocarcinoma equals 0.67 +/- 0.01 mm-1). The intraperitoneal injection of hematoporphyrin derivative (HpD at 10 mg/kg) did not seem to significantly influence the light evolution in tissues compared with control measurements without HpD. The simplicity and the reproducibility of this technique raises hopes of a coming clinical application and a possible comparison between different studies with measurable references.
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This method is destined for use in connection with photochemotherapy. It consists of two steps: (1) Each tumor is characterized individually by two parameters which are obtained by goniometric measurement of the light crossing a small sample (1 mm3). These parameters can be easily determined in a clinical environment but provide no direct information on the light distribution in the bulk. (2) The above parameters are related with the effective penetration depth for geometries and wavelengths of practical relevance. The relationship is established experimentally by multiple measurements in phantoms which are composed so as to closely approach the optical properties of tumors.
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Attached to an optical fiber, this device delivers light approximately perpendicular to the fiber axis. Matching of the irradiated area to the target area is accomplished by choosing distributors covering sectors with a central angle of 90 degree(s), 180 degree(s), or 360 degree(s), respectively, and by axial scanning. Since the deviation of light is obtained by total reflection the transmission efficiency is high, about 91 at 630 nm. This allows one to apply high intensities (test power 7 W cw from a YAG laser). Light of shorter wavelengths can be used if a slight decrease of the transmission efficiency is acceptable. Further advantages are the small diameter of 2.6 mm (there is also a miniature version having 1.6 mm diam) and the mechanical strength. The clinical effectiveness of the device has been demonstrated for PDT. It might be useful for laser hyperthermia as well.
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The efficiency of optical to acoustical energy conversion during laser-induced optical breakdown has been examined. A point-explosion model has been studied to determine the value of laser-induced shock wave energy. The influence of incoming laser-pulse energy on conversion efficiency has been studied for several absorber materials.
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Photographic studies of pulse infrared laser irradiation of a pure-water target illustrate two aspects of mass removal: (1) surface evaporation, and (2) explosive vaporization. A pulsed Erbium:YAG (Er:YAG) laser provided radiation at a 2.9 micrometers wavelength for delivery to the target site and triggered a second visible laser (nitrogen/dye laser) for illumination of the target site for photography. A variable time delay between the Er:YAG and dye lasers allowed selection of the time of the photograph (
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A point-monitoring fluorescence diagnostic system based on a low-energy pulsed laser, fiber transmission optics, and an optical multichannel analyzer was used for diagnosis of patients with bladder malignancies. Twenty-four patients with bladder carcinoma, carcinoma in situ, and/or dysplasia were injected with Photofrin (0.35 or 0.5 mg/kg body weight) 48 hours prior to the investigation. The ratio between the red sensitizer emission and the bluish tissue autofluorescence provided excellent demarcation between papillary tumors and normal bladder wall. Certain cases of dysplasia could be also be differentiated from normal mucosa. Benign exofytic lesion such as malakoplakia appeared different from malignant tumors in fluorescence. Flat suspicious bladder mucosa such as that seen in infectious diseases or after radiation therapy appeared normal in terms of fluorescence.
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Light-sensitive liposomes incorporating a photochromic phospholipid (Bis-Azo PC) have been developed which exhibit light-activated release of entrapped contents and intervesicular fusion. The trapping and light-induced release of inorganic ions, fluorescent market dyes, and the antitumor drug methotrexate have been demonstrated. These results are discussed together with some of the potential therapeutic applications of light-sensitive liposomes.
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Sequence-specific nonlinear photomodification of a ''target'' oligodeoxynucleotide pAGAGTATTGACTTA (14-mer) has been carried out with the aid of a complementary fluorescent probe, consisting of oligodeoxynucleotide pAATACTCT and a chromophore group (EtBr) attached to its 5-ft end. An approach is used which is a combination of the methods of two-quantum sensitized excitation and oligonucleotide-directed modification. It is based on the following: A chromophore is covalently linked to the probe oligonucleotide which is complementary to a given region of nucleic acid (NA). Irradiation is carried out with laser light which is not absorbed directly by NA but is quasiresonantly absorbed by the chromophore. At sufficiently high intensity of light stepwise excitation of the chromophore to a higher singlet state takes place, and radiativeless transfer of the excitation energy to the NA occurs within the radius of several angstroms around the chromophore. The transferred energy corresponds to the far-UV region and it sufficient for induction of different modifications of NA, including phosphodiester-bond breakage.
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A single multimode optical fiber is used to excite and collect tissue autofluorescence as well as the fluorescence of an IV-injected fluorescent tumor marker. Measurements of the relative fluorescence intensity of a tumor marker as a function of the time after IV injection permit measurement of the kinetics of this substance in tumor, normal tissue, and skin. The authors believe that these are the first measurements of this kind in patients. Furthermore, the autofluorescence spectrum generated at several excitation wavelengths in different tissues is compared, for instance in the oesophagus, the bronchi, and the tongue. The measuring system is based on an optical multichannel analyzer which measures the fluorescence excited by monochromatic radiation from a spectrally filtered Xe lamp. A correlation between the observed pharmacokinetics and tumor properties like the degree of vascularization is of fundamental importance for each selected tumor marker. Also, the results of these measurements are used for the optical detection of tumors.
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Roland Bays, L. Winterhalter, H. Funakubo, Philippe Monnier, Jean-Francois Savary, Georges A. Wagnieres, Daniel Braichotte, Andre Chatelain, Hubert van den Bergh, et al.
Two methods for clinical optical light dosimetry are developed. In the first method, which is invasive, a fluorescent probe attached to an optical fiber is inserted by means of a thin hypodermic needle and measures light transmitted through the cheek as a function of the penetration depth. In the second noninvasive method, the diffusely reflected light intensity, from a small illuminated spot on the surface of the tissue to be investigated, is measured as a function of the radial distance along the surface. Preliminary results with both methods are presented. Simulations of the second measurements, which allow for a simplified extraction procedure of the relevant optical data from such measurements, are also shown.
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Georges A. Wagnieres, Daniel Braichotte, Andre Chatelain, Christian D. Depeursinge, Philippe Monnier, Jean-Francois Savary, Charlotte Fontolliet, J.-M. Calmes, Jean-Claude Givel, et al.
The performance of a fluorescence endoscope for the detection of early cancer is clinically evaluated. the apparatus is based on the imaging of the laser-induced fluorescence (LIF) of a dye which localizes in the tumor after IV injection with a higher concentration than in the surrounding normal tissue. The tests are carried out in several of the hollow organs, such as the upper aerodigestive tract, the bronchi, and the colon. In the two former cases the dye used is photofrin II, whereas in the latter case conjugates between monoclonal antibodies (Mab) directed against carcinoembrionic antigen (CEA) and fluorescein molecules are injected. The fluorescence contrast between tumor and surrounding tissue is enhanced by real-time image processing which eliminates most of the tissue autofluorescence as well as the fluorescence due to the relatively small amount of dye localized in the normal tissue. This is done by recording the fluorescence image in two spectral domains, after which these two images are digitized and manipulated with a mathematical operator (lookup-table). The sources of false positives and false negatives are evaluated in terms of the fluorescent dye and tissue optical properties.
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Present techniques and new perspectives of microscopic fluorescence spectroscopy in cellular diagnosis are outlined. Recent applications include the detection of mitochondrial respiratory deficiencies and of the intracellular locatio and light-induced reactions of photosensitizing porphyrins.
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Using efficient pulse lasers as excitation sources, low-loss flexible light pipes for signal transmission, very sensitive photodetectors, fast electronic circuits, and computing techniques the authors built a versatile measuring apparatus. It consists of a main frame for housing the optical and electronic components, a personal computer, and a fluorescence-measuring head which is connected to the main frame by a flexible fiber cable. The paper shows examples of how such a device can be applied for different diagnostic purposes.
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For the first time the method of two-quantum affinity modification has been employed to probe the structure of an enzyme, bacterial luciferase. Position of the flavin-binding site of this enzyme, which was previously unknown, has been established. The obtained data indicate that the flavin site is positioned on the (alpha) -subunit. The closest contact of the protein chain of the enzyme with the chromophoric group of the flavin takes place near 80 +/- 10 and 120 +/- 10 amino acid residues; the regions 50 +/- 10 and 215 +/- 10 are also close to the flavin. The established localization does not contradict suggestions on positions of the flavin and phosphate sites of the bacterial luciferase, which had earlier been made from the data on evolutionary stability of various luciferases. The present method can, in principle, be applied to a great number of enzymes, including all flavin-dependent enzymes. Enzymatic catalysis has high speed and specificity. Creation of a method of determination of the elements of the primary structure of a protein, making up the active site (in which substratum conversion occurs), could be a significant advance in clearing up mechanisms of enzymatic catalysis. It was proposed to localize active sites of the enzymes, whose substrata are chromophores, using this method of two-quantum affinity modification. An enzyme- substratum complex is irradiated with laser light of sufficiently long wavelength ((lambda) 300 nm) which is not directly absorbed by the enzyme. Two-quantum quasiresonant excitation of the substratum activates it to the state with energy 5-7 eV, which is then radiativelessly transferred to neighboring protein groups. This energy exceeds the energy of activation of peptide bond breakage. Therefore, the enzyme will be disrupted in the vicinity of its active site. In the present paper the above approach has been implemented for the first time. Information has been obtained about the position of the flavin-binding site of bacterial luciferase. In these experiments an enzyme from luminous bacteria photobacterium leiognathi was used. It consists of two subunits having molecular weight of 41,000 and 38,000 correspondingly. The enzyme catalyzes the reaction of oxidizing aldehyde and reduces the flavin (FMN). The enzyme specimen was prepared by the multistep clearing technique. Protein concentration was measured.
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Fourier-transform (FT) Raman spectroscopy using near-infrared (IR) excitation has been studied as a mean to diagnose pathology in human tissues by differentiating between the six types of atherosclerotic and healthy tissues. The importance of this technique lies in the fact that with minimal surgical invasion of the Raman scattering can be collected remotely through optical fibers with a near-IR FT Raman spectrometer. To date, 17 fresh autopsic human-aorta samples were studied in vitro using Nd:YAG laser and a power varying from 50 to 80 mW. Raman spectroscopy indicated a broad-continuum emission between 100 and 3500 cm-1 with Raman peaks of almost equal intensity at wavelengths of 1450 and 1659 cm-1 (R equals ratio; 0.96 +/- 0.06) for healthy intima. Fatty streaks (type I lesion), uncalcified atheromas (type II: lipid, fibrous, and mixed), ulcered lesions (type IIIu) and ulcero-calcified lesions (type IIIu+c, all with low Raman intensity, were found to have lower Raman intensity peak ratio values of 0.74 +/- 0.040, 0.79 +/- 0.022, 0.88 +/- 0.017, and 0.83 +/- 0.016 (p c) were characterized by very strong overall Raman intensity, very low peak ratio (0.57 +/- 0.062, p -1 allowing for precise diagnosis. This diagnostic information, which was based on intensity ratios (I1659/I1450), correlated well with histologic and biochemical compositions. These findings were not only crucial for the achievement of successful laser angioplasty but also promising for the in vivo studies of atherogenesis.
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Stereotactic laser-induced interstitial hyperthermia using a low power Nd:Y1G laser was performed in an experimental study on normal rat brain and on F-98 glial transplantation tumors. 1 new fiberoptic delivery system, the Interstitial Thermotherapy (ITT) laser fibre, was connected to a 1.06 um Nd:YPG laser and introduced stereotactically into the basal ganglia of the rat. Histological results of rat brains removed immediately after laser irradiation revealed a sharply demarcated lesion with a small peripheral edema. One week later, a typical necrosis became evident, while the edematous zone subsided by peripheral spread in the white matter. Four weeks following laser application, there was only a circumscribed cystic lesion without any effects in the periphery. Histological findings in the tumor model were similar. In first clinical trials, laser-induced hyperthermia was performed in patients with cerebral gliomas. Results were controlled by MR imaging and PET scan showing irreversible necrotic effects in the center of the tumor and reversible edematous changes in the periphery of the tumor. With the equipment used it is possible to map the spatial distribution of laser-tissue effects by "real time" MR imaging.
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Human skin shows a strong autofluorescence in the red spectral region caused on the porphyrin production of the Gram positive lipophile skin bacterium Propionibacterium acnes. Irradiation of these bacteria reduces the integral fluorescence intensity and induces the formation of fluorescent photoproducts. The fluorescence band at around 670 nm and the decay times of around 1 ns and 5 ns are typical for protoporphyrin products. The photoproduct formation is connected with an increased absorption in the red spectral region. However the photodynamic activity of these photoproducts determined by scattering measurements on human erythrocytes is lower than that of protoporphyrin IX. 1:
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A fluorescence-detection technique for cancer in human hollow organs has been developed based on endoscope-assisted two-wavelength excitation of the polyporphyrin Photofrin II in tissue. Due to the photosensitizing capability of the fluorescent tumormarket the applied dose is reduced to 0.4 mg/kg bw which avoids photosensitization of the patient''s skin. Fluorescence is detected either spectrally resolved by means of an optical multichannel analyzer or by imaging of diseased tissue areas with an intensified video system. The fluorescence pattern in the final image is presented with high contrast due to realtime subtraction of nonspecific background signals.
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