The performance of a fluorescence imaging device was compared with conventional white-light bronchoscopy in 100 patients with lung cancer, 46 patients with resected State I nonsmall cell lung cancer, 10 patients with head and neck cancer, and 67 volunteers who had smoked at least one pack of cigarettes per day for twenty-five years or more. Using differences in tissue autofluorescence between premalignant, malignant and normal tissues, fluorescence bronchoscopy was found to detect more than twice as many moderate-severe dysplasia and carcinoma in situ sites than conventional white-light bronchoscopy. The use of fluorescence imaging to detect small peripheral lung nodules was investigated in a micro metastatic lung model of mice implanted with Lewis lung tumor cells. Fluorescence imaging was found to be able to detect small malignant lung lesions. The use of (delta) -aminolevulinic acid (ALA) to enhance fluorescence detection of CIS was investigated in a patient after oral administration of 60 mg/kg of ALA four hours prior to bronchoscopy, although ALA enhanced the tumor's visibility, multiple sites of false positive fluorescence were observed in areas of inflammation or metaplasia.

Laser-induced fluorescence spectroscopy is a promising technique for detecting colonic dysplasia in vivo but, at present, the biological basis for the success of the method is poorly understood, and little information is provided as to the morphological/molecular origin of tissue fluorescence. We present a methodology for establishing this, taking as a starting point a recently completed prospective laser-induced fluorescence (LIF) clinical study of colonic polyps. The method is based on a morphological model of tissue fluorescence with three components: the intrinsic lineshapes of the fluorophores, the spatial distributions of their intensities, and the optical parameters of tissue. We measure these using fluorescence microspectroscopy, microscopic imaging and tissue optics, respectively. The model predicts the features of the clinical spectra, and quantifies the respective intrinsic and architectural contributions. The results can be inverted to extract biological parameters from the spectra, and used to select optimal excitation wavelength(s) and guide probe design. Implications to detection of nonpolypoid dysplasia are discussed.

Laser induced fluorescence (LIF) is a technique which can perform an 'optical biopsy' of gastrointestinal mucosa. LIF was performed in resected specimens using a pulsed N₂-laser coupled fiberoptically to a probe. Fluorescence was measured using a 0.2 meter spectroscope with an intensified photodiode array. Measurements were made on fresh (<30 minutes after resection) esophageal specimens containing normal mucosa, Barrett's esophagus, and adenocarcinoma. Each tissue section was examined using an optical probe consisting of a central fiber for delivering the excitation energy and a 6 fiber bundle surrounding the central fiber for detection of the fluorescence. An excitation wavelength of 337 nm was used which generated 3-ns pulses while fluorescence intensities were acquired from 300-800 nm. Spectra were obtained from each section in a standardized fashion and background spectra subtracted. Fluorescence readings were taken from 54 normal esophageal sections and 32 sections of adenocarcinoma. A fluorescence index obtained from the tumor sections was 0.68+/- 0.01 compared with 0.51+/- 0.01 for the normal sections (p<0.001). Using a discriminant value of 0.65, this technique had a sensitivity of 81% and a specificity of 100% for detection of malignant tissue. The positive predictive value was 100% and the negative predictive value was 90% for an overall accuracy of 93%. LIF is a promising technique which has the capability of distinguishing normal versus malignant tissue in the esophagus with good accuracy.

Instillation of a solution of 5-aminolevulinic acid (5-ALA) into the urinary bladder leads to a tumorselective accumulation of fluorescing Protoporphyrin IX (PpIX) within hours. Upon fluorescence excitation using a Kr⁺- laser, cystoscopy provides high contrast images even of early stage tumors, that are invisible or hardly detectable by routine white light cystoscopy. Fluorescence can simply be judged by naked eyes or recorded with a target integrating camera in real color. Histological and fluorescence data of 91 patients were evaluated statistically, showing a sensitivity of 97% and a specificity of 68% for the detection of dysplastic lesions or malignant tumors. The detectability of a sufficient fluorescence contrast of suspicious versus normal tissue is not affected significantly by either short incubation times of less than 1 hour or prolonged retention times without 5-ALA in the instillation liquid of up to about 6 hours. The fluorescence intensity detected from the tissue surface is not only dependent on PpIX concentration. The additional influence of optical parameters of tissue and fluorochrome distribution on the fluorescence signal was determined using Monte Carlo computer simulations. Results show that 5-ALA induced fluorochrome detection is superior to the detection of fluorochromes that do not exclusively stain the epithelium. Using the ratio of fluorescence intensity to backscattered excitation light corrects for geometrical and absorption effects but would introduce a dependence on the scattering coefficient.

The feasibility of detection of skin cancer using in vivo NADH fluorescence is investigated. The results of a series of measurements on normal skin and cancerous skin suggest that the major chromophore is keratin.

A noninvasive probe has been devised and clinically used to perform in vivo measurements of the optical properties of the esophageal wall. The absorption coefficient and the effective scattering coefficient are determined from the observation of the spatial distribution of the diffuse reflectance at the tissue surface. Results, obtained at 514 and 630 nm, i.e. wavelengths of interest in photodynamic therapy with actual clinically used photosensitizers are summarized. An efficient and easily-built isotropic microprobe is also presented which is suitable to measure in situ the radiant energy fluence rate distribution in tissue.

The Los Alamos National Laboratory has continued the development of the Optical Biopsy System (OBS) for noninvasive, real-time in situ diagnosis of tissue pathologies. Our clinical studies have expanded since the last Biomedical Optics Europe conference (Budapest, September 1993), and we report here on the latest results of clinical tests in gastrointestinal track. The OBS invokes a unique approach to optical diagnosis of tissue pathologies based on the elastic scattering properties, over a wide range of wavelengths, of the tissue. The OBS employs a small fiber-optic probe that is amenable to use with any endoscope or catheter, or to direct surface examination. The probe is designed to be used in optical contact with tissue under examination and has separate illuminating and collecting fibers. Thus, the light that is collected and transmitted to the analyzing spectrometer must first scatter through a small volume of the tissue before entering the collection fiber(s). Consequently, the system is also sensitive to the optical absorption spectrum of the tissue, over an effective operating range of <300 to 950 nm, and such absorption adds valuable complexity to the scattering spectral signature. More detailed discussions of the technology have appeared in earlier publications.

Ocular fluorometry has been recognized as a relevant noninvasive technique in ophthalmology diagnosis and research. Fluorometric methods are widely used namely for measuring some of the major physiological parameters of clinical significance: measurement of corneal epithelial and endothelial permeability, use of sodium fluorescein (a well known exogenous fluorophore) to estimate the aqueous flow, and, most important, to measure the permeability of blood-ocular barriers that can give an early indication of diabetic retinopathy, etc. Ocular fluorometry instrumentation relies on the performance of light detectors based on different technologies. The trend, over the past few years, seems to be the increasing use of solid state ones, namely the so-called photodiode arrays (PDA). Good quantum efficiency, wide spectral range, inherent spatial resolution, good reproducibility and low dark current make them appropriate to measure ocular fluorescence light intensity distribution along the eye optical axis. When the light levels to be measured are very low, special care must be focused on the parameters and the working conditions of these devices so that their quantitative results can be meaningful and reliable. Cooling to reduce dark current, slow data rates to allow for longer exposure times increasing sensitivity, high gain and low noise amplification can be listed as some of the operation requirements. The study of the specific nonuniformity profile of the sensor used is another important procedure.

In vitro or in situ single spot or area excitation of human tissue with either 337 nm or 365 nm results in a fluorescence spectrum at about 470 nm. Since in most tissue samples the spectra obtained look alike and differ in intensity only, the 2D tomographical distribution of the fluorescence intensity was also investigated. It could be shown that the fluorescence images of unfixed and unstained cryosections match the histological images (HE stained cryosections). They exhibit a fluorescence pattern unique for the different types of diseases investigated (basalioma, melanoma, nevi, psoriasis, seborrheic keratosis). Additional information can be obtained by elastica van Gieson stained images and by illuminating the HE stained cryosections with 365 nm. Since the cryosections can be prepared and evaluated in less than 5 minutes, this technique might be used as a fast cut technique for determining e.g. the diagnosis of a biopsy sample, also during surgery.

In an attempt to evaluate the role of the histological and histochemical features of adenocarcinoma and normal tissue on their fluorescence spectral patterns, we developed a Monte Carlo model to simulate the fluorescence emission from tissue. According to histology, normal colon and adenocarcinoma were considered composed of three and two tissue layers, respectively (i.e., superficial epithelium, mucosa and submucosa; tumor mass and submucosa). Characterization of the fluorescence pattern of the tissue layers as well as of several histological components was performed by spectrofluorometric analysis on ex vivo samples. Results obtained from differently modelling the morphology and histological arrangement of colon suggest the following remarks. The decrease in fluorescence intensity observed in passing from normal to abnormal tissue could be ascribed mainly to a modification in morphology. When carcinomatous tissue is involved a modification in the spectral shape is also expected.

In vivo spectrofluorometric analysis represents a tool to obtain information about fluorophore distribution in tissue. Based on a Peltier-cooled CCD we designed a fluorescence excitation and emission spectrograph which allows to obtain tissue spectra endoscopically and in a clinical environment. Clinical studies were performed on patients with positive cytology or tumor recurrence in the urinary bladder. Patients received a 50 ml instillation of 3% ALA solution at pH 5.5 during 3 to 4 hours and underwent a normal white light cystoscopic examination together with light induced fluorescence photodetection at 5 to 8 hours after the beginning of the instillation. Local fluorescence measurements with a single fiber were performed before photodetection. These showed fluorescence ratios between tumor and normal tissue of 1.5 to 20 with the strongest ratios for exophytic papillary tumors. Fluorescence excitation between 380 nm and 450 nm revealed that the higher Protoporphyrin IX (PPIX) signal on tumor tissue is accompanied by a decrease of the autofluorescence at the emission wavelength of 500 nm.

Golden Syrian hamsters are evaluated as an animal model for phototherapy of early squamous cell carcinomas of the mucosa of the upper aerodigestive tract, the esophagus and the tracheobronchial tree. Carcinomas of this type are induced on the hamster cheek pouch mucosa by the application of the carcinogen 7,12 DMBA. For phototherapeutic experiments on the animals we utilized meso- (tetrahydoxyphenyl)chlorin (mTHPC). The same drug is currently in phase I, II clinical trials for ENT patients with superficial squamous cell carcinomas. By means of light induced fluorescence (LIF) we measured in vivo the kinetics of the uptake and removal of mTHPC in the normal and tumoral cheek mucosa and in the skin. The photodynamic therapy (PDT) reaction of the tissue after excitation of the photosensitizer by laser light at 652 nm was studied. Both pharmacokinetics and PDT efficacy are compared between animal model and clinical results with special emphasis on selectivity between normal and tumoral mucosa. These first experiments show that this tumor model in the hamster cheek pouch seems to be suitable for tests of a number of PDT variables of new photosensitizers preceding their clinical application as well as for optimization of the multiple parameters of clinical phototherapy.

The absorption spectrum of disulphonated Aluminum Phthalocyanine (AlS₂Pc) was measured in vivo in a murine tumor model by time-resolved reflectance (TRR). Mice bearing the L1210 leukemia were administered intraperitoneally 2.5 mg/kg body weight of AlS₂Pc. Reflectance measurements were performed in the 650-695 nm range before and 4 h after the drug administration. The absorption coefficient at each wavelength was obtained by fitting the experimental curve using the diffusion equation. The spectrum of the photosensitizer was obtained as a difference between the absorption of treated and untreated mice. The absorption spectrum of AlS₂Pc was peaked at 685 nm, showing a red-shift of approximately 15 nm with respect to the spectrum in aqueous solution.

The absorption and scattering of light through a taut flexible tube containing flowing samples of whole blood and haemolysed blood are studied by high resolution linear imaging of the transmitted intensity. A 16-bit photodiode array is used to obtain transmission profiles which are compared to a theoretical model developed from Twersky's multiple scattering theory. Profiles from near the edge of the vessel cavity are shown to be especially important in classifying the concentration and nature of the suspensions. The distribution of red blood cells across the vessel diameter is shown to be a function of the haematocrit and cell morphology. The measurement technique is relevant to blood vessel physiological studies and noninvasive blood analysis.

A noninvasive NIR instrument for diagnostic purposes is presented. It is specifically designed for clinical tests during exercise. The instrument makes use of the most modern optical components and a high performance front-end electronics to enhance the signal to noise ratio and to reject stray light effect. A new signal analysis is proposed. The main features are low noise, high stability and readability.

We are developing a method to quantitatively analyze the biochemical composition of human coronary artery in situ using near-infrared Raman spectroscopy. Samples of normal artery (intima/media and adventitia) and noncalcified and calcified plaque from coronary arteries, obtained from explanted recipient hearts during heart transplantation, were illuminated with 830 nm excitation light from a CW Ti:sapphire laser. Raman spectra were collected in seconds using a spectrograph and a cooled, deep-depletion CCD detector, and calibration and background corrections were made. Artery samples in different stages of atherosclerosis exhibited distinct spectral features, providing clear histochemical indicators for characterizing the type and extent of the lesion. Spectra were analyzed by means of a Raman biochemical assay model to determine the relative weight fractions of cholesterols, triacylglycerol, proteins and calcium minerals. Such information, when obtained clinically, promises to be useful in diagnosing and studying human atherosclerosis, its progression and response to drug therapy.

An in-situ holographic technique, involving the use of a flexible miniaturized endoscope (diameter less than 1 mm) coupled to a CCD camera, to record the hologram, has been developed for medical applications and more particularly in-vivo biopsy. The hologram is formed, by reflection, on the tip of a multimode, multicore fiber (MCF), sampled, and then treated electronically. The image is reconstructed numerically, providing more flexibility to the holographic process. Reconstructed images show the capability of the microendoscopic system to restore 3D informations of the observed scene. Our predictions and experimental results have shown that the hope to achieve tissue observations at the cellular level is realistic. Furthermore, the different sources of noise of the experimental device were analyzed and their influence on the quality of the reconstructed image quantified. Images of simple cell models such as epithelial cells easily taken in the oral cavity, have been taken and analyzed. The possibility of using the microholographic technique for in-vivo biopsy is discussed both from theoretical considerations and experimental observations.

Reflectance and fluorescence spectroscopy are successfully used for skin disease diagnostics. Human skin optical parameters are defined by its turbid, scattering properties with nonuniform absorption and fluorescence chromophores distribution, its multilayered structure, and variability under different physiological and pathological conditions. Theoretical modeling of light propagation in skin could improve the understanding of these conditions and may be useful in the interpretation of in vivo reflectance and autofluorescence spectra. In the present work the temporal behavior of in vivo reflectance and autofluorescence (excitation wavelength 337 nm) spectra of human skin under ultraviolet irradiation and external mechanical pressure were investigated. Experimental results and Monte Carlo modeling of light distribution in skin were compared and demonstrated good agreements. Combination of diffuse reflectance and autofluorescence measurements is a very promising technique for precise erythema and pigmentation of the human skin evaluation.

Fluorescent intracellular pH indicators have been widely applied in recent years to monitor changes in intracellular pH. More recently, we have proposed the use of dual-excitation pH-fluorescent indicators (5,6-CF: 5,6- carboxyfluorescein or BCECF: 2',7'-bis-carboxyethyl-5,6-carboxy-fluorescein) to monitor pH on animals. This study aimed to evaluate a dual-emission fluorophore (C-SNAFL-1: 5' (and 6') carboxy-seminaphthofluorescein) for pH monitoring in-vivo. In that case, one excitation wavelength and two emissions are chosen to give a pH-dependent emission intensity ratio.

The diagnostic exploitation of fluorescence spectroscopy and imaging has been largely used in experimental and clinical oncology. Only a few studies concern the ability of these techniques to study the in vivo behavior of fluorescent drugs or dyes. We have proposed recently a spectroscopic and imaging method to monitor pH in living tissues using pH-sensitive dyes. The tumor pH was depressed by previous glucose injection. We describe in this study the effect of glucose injection on the pharmacokinetic behavior of a fluorescent dye (calcein) by noninvasive fluorescence spectroscopy.

A new spectrometer (EMPHO IIb) for measurements of NADH fluorescence spectra in tissue was developed which enables recording of 100 spectra per sec. by use of a rotating filter disc. The excitation of fluorescence is induced by a mercury lamp (HbO100 W/2) at 366nm. For the optical coupling of the instrument to the surface of tissues quartz microlight guides are applied.

A new method to detect and quantify ischemic states of tissue is presented. One important indicator of the metabolic and ischemic status of a cell is the concentration ratio of NADH : NAD⁺. The use of time-resolved laser- induced fluorescence techniques (LIF) in combination with previous biochemical measurements of NADH (Nicotineamide Adenine Dinucleotide) concentrations in tissue offer the possibility of quantitative NADH detection. The essential of the new method is a double-rescaling procedure of the fluorescence signal taking into account both the optical limitations of the device and the individually and locally varying optical tissue properties which are the scattering coefficient (mu) _s, the absorption coefficient (mu) _a and the anisotropy factor g. All the data presented in this paper were acquired from experiments with in vivo guinea pig hearts.

Fluorescence spectroscopy experiments were focused on circulating monocytes and endothelial cells, two cell types that play a central role in the development and progression of atheromatous lesions. Our aim was to gain new insights into the cellular physiology of atherosclerosis and allow the development of diagnostic tests for the evaluation of atherosclerosis risk.

Based on novel time-resolving methods, the autofluorescence of saccharomyces, cultivated endothelial cells and biopsy specimens of human bladder was measured. The emission was found to be composed by the coenzymes NADH (free and protein-bound) and flavins with a concentration ratio of 100:1 between free NADH and flavin molecules. The fluorescence intensity of free NADH appeared to be a measure of the respiratory function. In addition, epithelial and connective tissues could be distinguished by the intensity ratio at 435 and 460 nm, which may be affected by the relation of bound and free NADH, but also by the extracellular fluorescence of elastin.

The spectroscopic and microscopic characteristics of skin tissue autofluorescence emission were studied using a combined fluorescence and reflectance spectroanalyzer and a fiber optic microspectrophotometer. The autofluorescence spectra of in vivo human skin were measured over a wide excitation wavelength range (350 nm - 470 nm). The excitation emission matrices (EEMs) of in vivo skin were obtained. An excitation-emission maximum pair (380 nm, 470 nm) was identified. It was revealed that the most probable energy of skin autofluorescence emission photons increases linearly with increasing excitation photon energy. It was demonstrated that the diffuse reflectance R can be used as a first order approximation of the fluorescence distortion factor f to correct the measured in vivo autofluorescence spectra for the effect of tissue reabsorption and scattering. The microscopic in vitro autofluorescence properties of excised skin tissue sections were examined using 442 nm He-Cd laser light excitation as an example. It was found that the fluorophore distribution inside the skin tissue is not uniform and the shapes of the autofluorescence spectra of different anatomical skin layers vary. Using the obtained in vitro microscopic data, published skin optical parameters, and Monte Carlo simulation, the in vivo fluorescence emission spectrum was reconstructed and compared with the measurement results.

N-(1-Pyrene)maleimide is highly fluorescent upon covalent binding with sulfhydryl and amino groups of the proteins. Multiexponential fluorescence decays were observed for the dye bound to different proteins even when a single binding site is involved. The lack of information about the fluorescence decay of free dye does not allow to define the variations of fluorescence parameter following the conjugation and their correlation with the binding properties of the fluorophore. In this work, a study of the fluorescence of the probe, free in solution, bound to different antibodies and to the antigen-antibody complex both in solution and in cell, has been performed. The experimental results showed that chemico-physical properties of the medium influence the fluorescence decay of the probe in both the free and bound forms, although to a different extent. The variations of fluorescence decay and anisotropy of the bound probe are related to the electronic characteristics of microenvironment and show an increased stabilization of the probe binding site with the increasing complexity of the substrate. The sensitivity of the fluorescence properties of the probe to the binding site environment opens interesting perspectives concerning the application of Py- maleimide fluorochromization to assess the degree of specificity of immunocytochemical labelling.

The complete process, image acquisition, deblurring, 3D reconstruction and analysis has been tested on 160 normal T lymphoid cells marked with monoclonal antibodies CD4-CD8. The images are acquired from a conventional optical microscope illuminated by a mercury vapor lamp. A set of 16 serial image cuts is first grabbed by a high sensitivity Silicon Intensified Target (SIT) camera fitted on the microscope. The 2D image cuts are then deblurred using an original deconvolution method developed in the laboratory. The 3D reconstruction is processed slice by slice using a variant of the 2D Delaunay triangulation. To get a correct triangulation in the case of our specific application the following stringent requirements must be met. First, we exclude the possibility of joining two contours if we meet an empty cut between them, and secondly we must avoid branching points and finally, we state that reconstructed shapes may not have holes. The 3D reconstruction highlights some qualitative characteristics of the cells. So we can show that the fluorescent spots are either scattered on the membrane surface and present a regular radial arrangement or on the contrary are confined in a restricted area. Accordingly we propose to introduce a spatial coefficient in order to characterize such a volume distribution. In a further development we plan to obtain discriminant descriptors of the immunological status of hematopoietic cells.

The aim of our study is to improve the performances of an optical microscope by image deconvolution technique to attain that of a confocal one in the field of laser cytofluorescence. The fluorescence of antigen lymphocyte sites marked by rhodamine is induced by a laser ((lambda) equals 543 nm) or a mercury vapor lamp. A set of scanned fluorescence images is acquired at different focal planes by a SIT camera, fitted on an optical ZEISS microscope (N.A. 1.25, X100). The entire system is controlled by a PC equipped with a MATROX-MVP-AT image processing card. Since an optical microscope has a more important focal depth than a confocal one, an improved iterative deconvolution algorithm of Van Cittert's has been used to artificially reduce the focal depth. In order to ensure the convergence of such an iterative algorithm, a new criterion for relaxation coefficient is defined through a theoretical study. The experiments show an improvement of 50% in the spatial x-y resolution of 30% in optical z axis gain. As regards our application, this processing brings our system to a comparable performance level as a confocal microscope.

The importance of the intracellular concentration and transmembrane flux of cations has led scientists to develop various techniques for the evaluation of intracellular concentrations of H⁺, Ca²⁺, Mg²⁺. Quantitative microfluorometry appears to be a powerful and noninvasive technique for it allows kinetic studies or mapping of ionic concentration in different cellular compartments as long as fluorescent markers specific of given cations are disposed of. Nevertheless, the previously published results sometimes look controversial. To minimize problems encountered by some scientists the following protocol is proposed: (1) careful and exhaustive determination of the physicochemical properties of the probe(s), (2) use of microspectrofluorometric techniques to resolve the complex intracellular fluorescence spectra into its components and calculate the molar fraction of each compound participating in the cellular fluorescence, (3) use of the same kind of algorithm to perform numerical image analysis.

A major problem in clinical laser therapy and laser based diagnosis is the prediction of the light distribution in tissue. Phantom models with homogenous and reproducible optical properties have been used in the past in order to investigate methods of measuring optical properties.

Resonant four-photon scattering and excited molecules depopulation/rotational diffusion are considered. Scattering pulse is generated due to three ultrashort pulses interacting with molecules. First pulse prepares excited molecules and two coincident probe pulses, which have a delay in time relative to excitation pulse and in resonance with absorption band of excited molecules, control a transient nonlinear susceptibility. High sensitivity of proposed nonlinear optical measurements to orientational motion of excited molecules is shown.

Dimers of the fluorescent dye Oxazole Yellow (YOYO and POPO) are used for high sensitive DNA detection in gel electrophoresis. Upon binding to DNA they show a 3000 to 5000 fold enhancement of fluorescence. The binding constant of those dimers to dsDNA is between 10⁸ M^-1 and 10⁹ M^-1. This is due to the dye's ability to bisintercalate between adjacent DNA basepairs. We investigated the occurring forms of intercalation of YOYO to dsDNA in solutions of different ionic strength by fluorescence lifetime methods.

Erythema and pigmentation are the most important visible phenomenons in the ultraviolet irradiated skin. The diffuse reflection has long been used to in vivo monitor skin chromophores (hemoglobin and melanin). In this paper, the temporal behavior of the autofluorescence spectra (excitation wavelength 337 nm) of the human skin under ultraviolet irradiation (4 MED) are presented. A simple model of the skin was used for analysis of blood content in different skin layers. In vivo autofluorescence measurements are suggested for improving the precise detection of blood and melanin content in the human skin.

This paper descsribes the development of an apparatus, method, and practical recommendation on using fluorescence diagnostics in alimentary-intestinal tract surgery and analyses of blood serum and plasma for investigating influence of various drug preparations on a human organism. The report of the firm Israel Aircraft Industries on the high efficiency of using fluorescent analysis in early diagnostics of rectum, lung, and breast cancer has stimulated our publication.

The most frequently used drug for the fluorescent visualization and photosensitization of tumors is haematoporphyrin derivative (HpD). However, the mode of action of this sensitizer in vivo is not yet entirely understood. From the Photodynamic Therapy it is well known that their efficiency and selectivity increases with increasing amounts of oligomers/aggregates. The 'highly aggregated' HpD (Photofrin II) is also used in the fluorescence diagnostics. However, from the investigation of porphyrins it is known that the oligomers/aggregates fluorescent weakly compared with monomers. The monomers as well as linear chains of monomers (linear dimers and special forms of trimers) have longer fluorescence lifetimes and hence higher quantum yields required for effective fluorescence diagnostics. This discrepancy stimulates the studies of the interaction of HpD containing high concentrations of tumor localizing fraction with membranes and membrane like-structures. The interaction has been investigated by means of fluorescence (OMA-system) and absorption spectroscopy. The results obtained lead to the conclusion that the monomers form the aqueous phase and/or self-associated aggregates which are in equilibrium with the monomers are taken up by the membranes only to a small degree compared with chemically bound oligomers. On the other hand, oligomers are incubated and defolded, and this leads to an increasing amount of linear polymers/monomers in the membranes versus incubation time. This seems to be the dominating process in the fluorescent visualization of cells and tissue, including tumor tissue, when HpD, Photosan or other amphiphilic porphyrins are applied.

The metabolism of cancer cells frequently shows higher glycolytic activity than normal tissue. This glycolytic activity can result in changes of the NAD⁺/NADH equilibrium and variations of NAD^{+$ and NADH levels. The objective of this study has been to monitor changes in NADH fluorescence spectra of cancerous and normal bronchial tissue. Spectra were taken from in- vitro samples of normal and carcinogenic bronchial tissue in a microspektrofluorometric setup and in a spectrofluorometer, the latter giving the Emission-Excitation-Matrix (EEM} of the tissue fluorescence. Two numerical approaches were taken to determine changes in the NADH spectra. (1) Graphical comparison of normalized emission spectra shows a blue shift of the maximum for carcinogenic tissue. There is a good correspondence between the form of the spectrum and the spectrum of LDH-bound NADH. (2) Numerical analysis of the EEM from freeze slices showed an increased signal in the case of normal tissue and a decreased one in the case of cancerous samples in the spectral range that is typical for NADH.

We examined the relation between cytochrome aa3 (cyt) oxidation level, oxygen delivery (QO2) and oxygen uptake (VO2) during progressive hindlimb ischemia in pigs. The right hindlimb of 6 anesthetized- ventilated pigs was subjected to progressive ischemia over 100 min by 10 stepwise decreases in pump-controlled blood flow (Q). Hindlimb QO2 was Q x arterial content and VO2 was Q x arteriovenous difference. Cyt oxidation level was measured by multiple wavelength NIRS (Hamamatsu). Measurements were done very 9 min after each change of Q. O2 extraction (ER)2) was determined as VO2/QO2. Critical values for both VO2 and cyt oxidation level as a function of QO2 were determined in each animal and compared by t-test. As QO2 was progressively lowered, VO2 remained constant (3.5 +/- 0.1 ml/min per kg) until QO2 reached a critical value of 5.4 +/- 0.4 ml/min per kg, not statistically different than the QO2 (5.9 +/- 0.4 ml/min per kg) critical for maintaining cyt oxidation level. Critical ERO2 determined using the QO2-VO2 relation (0.60 +/- 0.09) and by the QO2-cyt oxidation relation (0.57 +/- 0.03) were also not significantly different. At the lowest QO2, VO2 was 1.0 ml/min per kg and cyt oxidation level was -7 (mu) M. NIRS appears capable of accurately and noninvasively describing the O2 supply dependency relationship during local ischemic hypoxia.

By time resolved fluorescence spectroscopy in the psec range the fluorescence behavior of flavines as important endogenous fluorophores was investigated. The substances were examined under various conditions (e.g. pure solutions and cellular suspensions; different buffer systems and pH values). Particular attention was dedicated to the properties of these coenzymes under in situ conditions. The results were applied to test numerical methods for characterization of complex mixtures of fluorophores and for discrimination of different states of flavines.

The neutrophil is the cell in which phagocyting and transforming of some exogeneous agents results in marked stimulation of nonmitochondrial respiratory chain activity (respiratory burst). In our experiment we focused on determining the level of chemiluminescence (CL) of stimulated neurotrophils during and after irradiation, measuring the photon emission intensity in 6 second's intervals. We used Ga-Al-As pulsed laser (wavelength 904 nm, mean power 8,9 mW, Alpha-Electronics GmbH, Germany) which was placed over the tube containing the suspension of guinea pig peritoneal neurotrophils (2X10⁶ cells/ml). The sensitivity range of used photomultiplier (9514s, THORN EMI, Middlesex, England) was 300-600 nm, which allowed us to measure the CL of neutrophils while being irradiated. The neutrophils were stimulated by phorbol myristate acetate (PMA) and CL intensified by luminol. The decay of luminol-dependent CL of neutrophils may be described by hyperbolic function curve. We switched the laser radiation on for 20 s, 60 s and 300 s and each time we observed the same reaction: the about 20% decrease of intensity of CL immediately after beginning the irradiation. The CL remained on decreased level during the whole period of irradiation reaching immediately the level of CL intensity characteristic for decay curve (20% increase), just after switching off the laser. Only after the longest irradiation time (300 s) we observed CL being higher and inconsistent with decay curve for several minutes. The type of reaction was always the same, regardless to the point of CL decay curve at which laser radiation was applied. The same changes of Cl we obtained irradiating the enzymatic system: horseradish peroxidase (HRP)-luminol - H₂O₂.

A comparative study on phagocytosis of P. Aeruginosa by human neutrophils in diabetic patients and healthy volunteers was carried out by means of the monolayer of neutrophils in optical microscopy and ultrastructural observation in electronic microscopy. The results demonstrated that the level of phagocytosis in diabetics is lower than health people. The impairment in phagocytosis of neutrophils may be the important cause of severe and repeated infection in diabetic patients.

A proper interpretation of the signals from fluorescent indicators in imaging technology implies a knowledge of the processes in the excited state. This work focuses on the pH probe C.SNAFL-1. The kinetics of the excited-state processes are investigated by global compartmental analyses of fluorescence decay surfaces obtained by time-correlated single photon counting. Within the pH range 5-12 only two species have to be considered in the ground and the excited state. The two fluorescent decay times do not depend on pH. Therefore, the process of protonation in the excited state is very slow as compared to the deactivation rate of the excited states. A proper identifiability study has been performed to determine the rate constant of the deprotonation process. The rate of deprotonation is also small and its upper value is estimated to be 0.05 ns^-1. It can be concluded that there is negligible interference of the excited-state reaction on the determination of intracellular pH by C.SNAFL-1.