In a clinical pilot study performed on 104 patients suffering from bladder cancer it could be shown that intravesical instillation of a solution of 5-aminolevulinic acid (5-ALA) induces a tumorselective accumulation of Protoporphyrin IX (PPIX). Malignant lesions could be detected with a sensitivity of 97% and a specificity of 67%. The Kr⁺-laser as excitation light source could successfully be replaced by a filtered short arc Xe-lamp. Its emission wavelength band (375 nm - 440 nm) leads to an efficiency of 58% for PPIX- excitation compared to the laser. Two-hundred-sixty mW of output power at the distal end of a slightly modified cystoscope could be obtained. This is sufficient for recording fluorescence images with a target integrating color CCD-camera. Red fluorescence and blue remitted light are displayed simultaneously. Standard white light observation is possible with the same instrumentation. Pharmacokinetic measurements were performed on 18 patients after different routes of 5-ALA application (oral, inhalation and intravesical instillation). PPIX-fluorescence measurements were made on the skin and on the blood plasma. Pharmacokinetic of 5-ALA could be performed on blood plasma. Endoscopical florescence spectroscopy showed the high fluorescence contrast between tumor and normal tissue with a mean value of 10.7. Forthcoming clinical multicenter studies require an objective measure of the fluorescence intensity. Monte Carlo computer simulations showed that artifacts due to observation geometry and varying absorption can largely be reduced by ratioing fluorescence (red channel of camera) to remission (blue channel). Real time image ratioing provides false color images with a reliable fluorescence information.

Laser-induced fluorescence spectra were recorded in patients undergoing urinary bladder cystoscopy. The measurements were performed in vivo and the spectra were collected from normal and diseased tissue. The patients were divided into two groups. An instillation of a 1% delta-amino-levulinic acid (ALA) solution was performed 2 - 4 hours prior to the investigation of one group of patients. A second group of patients was investigated without any tumor marking substance. The fluorescence was detected following laser excitation at 405 and 337 nm. Fluorescence emission related to ALA-induced protoporphyrin IX (PpIX) was detected in the ALA group for 405 nm excitation. The data were evaluated at the PpIX emission peak at 635 nm and at 490 nm, which approximately corresponds to the peak of the tissue autofluorescence. The data obtained with 337 nm excitation were evaluated at 400 and 460 nm as well as at 390 and 431 nm. The ratios of the respective wavelength pairs were formed in order to investigate the demarcation between tumor and normal tissue. The tumor demarcation results were better and more consistent utilizing the autofluorescence signal following excitation at 337 nm than the PpIX-related signal excited at 405 nm.

The aim of this paper was to demonstrate the feasibility of routine real time clinical detection of occult urothelial precancerous and cancerous lesions by laser induced autofluorescence (LIAF), that is with no previous instillation of exogenous fluorescent marker. Three different pulsed laser wavelengths were alternately used for excitation: 480 nm (dye laser), 337 nm (nitrogen laser), and 308 nm (XeCl excimer laser). A clinical endoscopic study was performed on 23 patients immediately before transurethral resection of bladder tumor. Spectroscopic results were compared with histological analysis. For 480 nm and 337 nm excitation a single fluorescence broad band was obtained in any case, but for tumors the overall intensity was significantly reduced compared to normal mucosa. For 308 nm excitation, two main broad bands were observed. In the case of neoplasic lesions (including carcinoma in situ), the intensity ratio [I(360 nm)/I(440 nm)] was always greater than 2, but for normal or inflammatory areas this ratio was less than 2. A clear diagnosis could then be achieved for 308 nm excitation without the need of absolute intensity measurements. We get no false positive at this wavelength. XeCl LIAF spectroscopy is therefore a promising technique for the detection of urothelial precancerous lesions and could be used to perform an 'optical biopsy' in a routine mode with real time results.

In vivo spectrofluorometric analysis during photodynamic therapy (PDT) is a tool to obtain information about fluorophore bleaching kinetics in tissue. Using a cylindrical esophageal light distributor for PDT with an integrated sensing fiber, together with a fluorescence detection setup, we can obtain tissue fluorescence spectra endoscopically in a clinical environment. This study was performed on patients with early squamous cell carcinomas in the esophagus. Patients were injected intravenously with 0.15 mg/kg of mTHPC and underwent PDT (lambda equals 514 nm, fluence rate equals 100 mW/cm²) 96 hours after injection. Bleaching kinetics of mTHPC and tissue autofluorescence at different wavelengths were recorded in real time and showed decreases in the observed fluorescence intensity in the 652 nm band of about 60% for light doses around 100 J/cm². Additional information on bleaching kinetics induced at another excitation wavelength, lambda equals 652 nm, was obtained by irradiations at much lower doses in the buccal cavity. The data are analyzed using a simplified mechanism in which singlet oxygen is the hypothetical reactive intermediate which can both bleach the mTHPC and the autofluorescent molecules. The differential equations are solved by applying the quasi stationary state approximation for the reactive intermediate. The experimental data at least do not appear to contradict this oversimplified mechanism.

An instrumentation is being developed to draw a fluorescence life-time map of tissue endoscopically. This fluorescence life-time of an endogenous or exogenous fluorochrome gives information about the physico-chemical environment which is thought to vary between normal and diseased tissue. The excitation light from a cw laser is modulated in amplitude at high frequencies by an electro-optic modulator and delivered to the endoscopic site through an optical fiber. The image of the tissue is spectrally split in two parts, the one being the backscattered excitation light, the other the fluorescence of the fluorochromes. Each image is focused on the photocathode of an image intensifier whose gain is modulated at the same frequency. By acquiring frames at different phases between the excitation and the emission, it is possible to calculate pixel by pixel the apparent fluorescence life-time of the corresponding tissue region.

Five patients with Barrett's metaplastic epithelium were investigated by means of laser- induced fluorescence after low-dose i.v. injection (0.35 mg/kg b.w.) of Photofrin^R in connection with endoscopy procedures. The excitation wavelength was 405 nm. Recorded fluorescence spectra were evaluated by forming ratios with the photosensitizer fluorescence as numerator and the autofluorescence as denominator. Two patients had no evidence of malignancy and their fluorescence ratios were consequently rather small, whereas the other three patients had adenocarcinoma and showed considerably higher ratios. The results indicate that laser-induced fluorescence can be used as an aid in detecting malignant transformations in Barrett's metaplasia.

Fluorescence spectra of primary colon tumors, normal colonic tissue and lymph nodes were recorded ex vivo, following pulsed laser excitation at lambda equals 505 nm. Fluorescence was detected at zero delay and at a delay of 20 ns between the optical pulse and the opening of the intensified diode array detector. Generally, the spectra consist of a broad unspecific background (unspecific autofluorescence) and two characteristic fluorescence bands at lambda approximately equals 630 nm and lambda approximately equals 700 nm (specific autofluorescence). By delayed observation the intensity of the specific fluorescence diminishes less than the intensity of the unspecific background indicating a longer fluorescence decay time of the specific compared to the unspecific fluorescence. The fluorescence excitation and the fluorescence emission spectra of the specific autofluorescence as well as the fluorescence decay time are similar to those of porphyrins. Assuming that the relative amount of specific autofluorescence can be used to discriminate between primary tumors and normal colonic tissue or involved and non-involved lymph nodes, we have analyzed undelayed and delayed fluorescence spectra quantitatively to derive characteristic quantities for discrimination. We have investigated 18 primary colon tumors and associated normal colonic tissue as well as 174 lymph nodes. Out of 18 primary tumors classified by routine pathology we have correctly identified 16 by fluorescence analysis. Because of the rather weak specific autofluorescence in lymph nodes only 22 metastatically involved lymph nodes were detected out of 34 identified by routine pathology. Although the sensitivity of 65% is rather low a discrimination between involved and non-involved lymph nodes is of particular medical importance.

Crohn's disease is an inflammatory bowel disease of unknown etiology. Vasculitis is hypothesized but it was never demonstrated in vivo. This study aimed to evaluate the vascular mucosa perfusion using fluorescence imaging in 13 patients who had previously undergone eileocolonic resection and who agreed to participate in a prospective endoscopic study of anastomotic recurrence. This anastomotic recurrence rate is known to be high (73% after 1 year follow-up) and is characterized by ulcerations. The fluorescence study was started with an I.V. bolus injection of sodium fluorescein. The pre-anastomotic mucosa was endoscopically examined with blue light that stimulates fluorescein fluorescence. Fluorescence emission was recorded with an ultra-high-sensitivity camera connected to the endoscope via an interference filter (520 - 560 nm). A uniform fluorescence was observed a few seconds after the injection and lasted for 15 min in healthy subjects. In case of recurrence, the centers of the ulcerations displayed a very low fluorescence indicating localized ischemia. In contrast, the rims of the ulcers revealed brighter fluorescent images than those of normal mucosa. The anastomotic ulcerations of Crohn's disease recurrence exhibit a high fluorescence intensity at their margins indicating an increased mucosal blood flow and/or enhanced transcapillary diffusion. These findings support the hypothesis of a primary vasculitis in Crohn's disease.

The aim of a dosimetry for the photodynamic therapy (PDT) is to optimize the therapeutic index: destruction of the pathological areas without impairing surrounding healthy tissue. The sensitizer concentration (PS), the global extinction factor (Sigma) _eff and the partial pressure of oxygen P_O2 in the tumor are the parameters to be studied from the backscattered light during irradiation. A spectrofluorimeter, suitable for flexible endoscopes, has been designed for measurement of (PS) in situ; kinetics of fixation of the sensitizer in the tumor and in healthy tissues allow the user to choose the optimal schedule for treatment; follow-up of the variation of (PS) during irradiation gives an idea of the photochemical reactions. To measure (Sigma) _eff in the situation of an interstitial treatment we use the backscattered signal of the excitating light collected through the emitting fiber itself and several contiguous receiving optical fibers. The prototype of this device is 2 mm in diameter. The calculation of the depth of penetration allows computation of light isodoses in the tumor. To have an idea of the available oxygen we measure the absorption of red light by reduced haemoglobin. A general organization chart of the dosimetry shows the various parameters and interactions working towards the therapeutic result.

Indocyanine green has been used to measure cardiac and liver functions. More recently, it has been proposed as a contrast agent in ophthalmic angiography, tumor imaging and as an infrared absorbing dye in the context of laser-induced thermal damage of blood vessels. The aim of the study is to overcome the disadvantage of a very short blood half-time and to participate to a better confinement in blood vessels. Indocyanine green was administered intravenously to Wistar rats at a 7.5 mg/kg dose. Formulations consist in indocyanine green aqueous solution and o/w emulsion. Blood samples were collected and analyzed by spectrophotometry. Fluorescence was recorded in vivo by spectrofluorometry using an optic fiber coupled to an optical multichannel analyzer. The fiber optic was placed at a 4 mm distance from the skin surface. Results show that aqueous solution of indocyanine green leads to a rapid blood clearance. To the administration of ICG emulsion belongs the advantage of increasing the half-time and the residence time of indocyanine green in skin. It may be noted that however the formulation is, the observed blood clearance profiles are quite different from the tissue fluorescence kinetic profiles. The dye could have a longer residence time (20 - 60 min. plateau phase). Moreover, a shift of the maximum emission peak is noted after i.v. administration. The study of ICG fluorescence in the presence of model membranes shows that ICG is able to interact with phospholipid bilayers. These findings may be interesting for therapeutic applications of indocyanine green requiring a high level of dye in tissues for a great period of time and participate to the knowledge of ICG behavior in vivo.

We have already demonstrated the ability of fluorescence spectroscopy and imaging to measure the pH of superficial tissues using pH sensitive fluorescent probes. The purpose of this study was to investigate the in vivo behavior of such fluorescent probes. We report the monitoring of tissue fluorescence after injection of two fluorescein derivatives (carboxyfluorescein and biscarboxyethyl-carboxyfluorescein). The in vivo study was performed on anaesthetized adult Wistar rats. After laparotomy, CF or BCECF solution was injected into the penial vein. Fluorescence spectra were recorded during one hour using an optical multichannel analyzer coupled to a CCD camera. Fiber optic was placed alternatively on the liver area or on the skin. Blood samples were collected and fluorescence was measured in vitro. A clear linear relationship between dose and fluorescence intensity was found in liver for these fluorescent markers. Concerning spectral characteristics, it was found that CF and BCECF spectra show a shift compared to in vivo maximum emission peak and BCECF emission peak was different when recorded in the liver and in the skin. Differences of kinetic profiles are also observed between CF and BCECF. The BCECF derivative displays a fluorescence peak in the liver two minutes after injection, while CF fluorescence peak is observed seven minutes after injection. Clearance of skin fluorescence is slower than the plasmatic one indicating that dye elimination in superficial blood vessels does not follow the same pharmacokinetic behavior. Based on these preliminary findings, fluorescence spectroscopy appears as a tool in pharmacokinetic study in situ and in vivo.

Conventional laser-induced fluorescence spectroscopy (LIFS) of endogenous chromophores like NADH (Nicotineamide Adenine Dinucleotide, reduced form) and PP IX (Protoporphyrin IX) provides information about the relative amounts of these metabolites in the observed cells. But for diagnostic applications the concentrations of these chromophores have to be determined quantitatively to establish tissue-independent differentiation criterions. It is well- known that the individually and locally varying optical tissue parameters are major obstacles for the determination of the true chromophore concentrations by simple fluorescence spectroscopy. To overcome these problems a fiber-based, 2-channel technique including a rescaled NADH-channel (delivering quantitative values) and a relative PP IX-channel was developed. Using the accumulated information of both channels can provide good tissue state separation. Ex-vivo studies with resected and frozen samples (with LN₂) of squamous cells in the histologically confirmed states: normal, tumor border, inflammation and hyperplasia were performed. Each state was represented in this series with at least 7 samples. At the identical tissue spot both, the rescaled NADH-fluorescence and the relative PP IX- fluorescence, were determined. In the first case a nitrogen laser (337 nm, 500 ps, 200 microjoule, 10 Hz) in the latter case a diode laser (633 nm, 15 mW, cw) were used as excitation sources. In this ex-vivo study a good separation between the different tissue states was achieved. With a device constructed for clinical usage one quantitative, in-vivo NADH- measurement was done recently showing similar separation capabilities.

A system for time-gated imaging has been used to measure the spatial distribution of the fluorescence decay time in tumor-bearing mice sensitized with Hematoporphyrin Derivative. Mice were injected with five doses of the sensitizer ranging from 0.1 to 10 mg/kg body weight. Several fluorescence images of each mouse were acquired at different delays after the excitation pulses, and a matrix processing algorithm was applied to calculate the average decay time corresponding to each pixel. It was found that, for any drug dose, the decay time of the exogenous fluorescence in the tumor is always significantly longer than in normal tissues. The images created by associating a gray scale to the decay time matrix of each sample allow a reliable and precise detection of the neoplasia, even with a very low dose of the sensitizer.

Opto-acoustic investigations of copper chloride water solution, milk, suspension of graphite particles in water and milk, and biological tissues were carried out with Q-switched Nd-YAG laser. The pressure course of the leading edge of acoustic pulse is determined by the distribution of the intensity of light in the absorbing medium. The registration of the acoustic transients both at front and rare surfaces of investigated medium was carried out. The distribution of absorption for homogeneously absorbing and micro-inhomogeneous media was reconstructed. The experimental arrangement allows the user to measure the light absorption coefficient in the range of 10 - 300 l/cm with the spatial resolution of the order of 10 microns. Absorbing particles in transparent, homogeneously absorbing and scattering media were detected. The proposed method of time-resolved opto-acoustic diagnostics possess the ability to investigate z-axial distribution of light absorption averaged in lateral directions over the laser spot. For biological tissues it is most promising to use front surface registration of acoustic transients that are well established to detect boundaries of absorbing layers.

With classical fluorescence microscopes, the sample is illuminated by a monochromatic source and the image is recorded through a bandpass filter that selects a portion of the emitted fluorescence. Recently, the spatial resolution and sensitivity of these devices have been considerably increased with the introduction of confocality and large 2D CCD array detectors and it is now possible to perform even 3D mapping of fluorescent markers in single living cells. Unfortunately, small changes occurring in fluorescence spectra cannot be mapped so easily. A set of colored filters is hardly enough even to separate slightly overlapping fluorescence contributions from multiple marking. The number of biological studies requiring a detailed description of fluorescence spectrum is growing continuously. Spectral changes are induced, for example, by environment (hydrophilic/hydrophobic binding), pH, oxygenation, ion concentration (Ca, Mg, Na), conformation of bound macromolecule (A/B DNA, protein folding), metabolism, dimerization of the probe, etc. . . . The classical approach to these problems is to couple a spectrograph to a microscope and obtain, point after point, a set of significative fluorescence spectra from the sample. This is a long procedure which gives incomplete information. We report in this paper on two methods we have developed to quickly record an array of spectra over the sample and to map spectral features such as bandwidth, maxima shifts, or decomposition in multiple overlapping components. We distinguish between the confocal scanning method and the global illumination (non confocal) method.

A new, simple and quick approach, oblique-incidence reflectometry, was used to measure the absorption and reduced scattering coefficients of a semi-infinite turbid medium. An obliquely incident light beam causes the center of the far diffuse reflectance to shift from the point of incidence, where the far diffuse reflectance refers to the diffuse reflectance that is several transport mean free paths away from the incident point. The amount of shift yields the diffusion constant by a simple formula, and the slope of the diffuse reflectance yields the attenuation coefficient. Only the relative profile of the diffuse reflectance is needed to deduce both optical parameters, which makes this method attractive in clinical settings because it does not require a stringent calibration for absolute quantity measurements. This method was tested theoretically by Monte Carlo simulations and experimentally by a reflectometer. Because this method can be used to measure optical properties of biological tissues quickly and requires only inexpensive equipment, it has potential clinical application to the diagnosis of disease or monitoring of treatments.

Photon emission from mammalian cells has been the subject of study for many years. Throughout the history of this field of research the question of a functional biological role of the low intensity emission has been repeatedly raised. The discussion concerns the possible participation of biophotons in intra- and intercellular communication. In this paper we consider the significance of the studies on light-induced photon emission of isolated mammalian cells. Furthermore we report on the source of this light-induced photon emission.

The rapid development of both FTIR and Raman including the use of convenient fiber optics for wide spectral region from visible till far JR are becoming almost routine tools for detailed investigations and the noninvasive monitoring of tissues in vitro and in vivo. Fiberoptic Evanescent Wave FTIR Spectroscopy (FEWS) have been used for the monitoring in vivo ofhuman lips, for cancer diagnostics in human organs in vitro. The main features oftissues were demonstrated. The advantage oftechnique is to receive the possibility ofin vivo cancer diagnostics.

The fluorescence properties of fluorophores relevant in tissue metabolism (NADH, flavines, etc.) are characteristic of the clinical states of tissues. Especially the differentiation of healthy, cancerous, and necrotic tissue states is of large interest in lung-tumor diagnostics, e.g. to ensure that biopsies are taken from non-necrotic areas. In contrast to the common fluorescence detection our approach provides both a combination of spectral and time information from autofluorescence and the simultaneous detection of two fluorophores in order to improve differentiation between various tissues. The basis of analysis of autofluorescence is knowledge of the photophysical parameters of the fluorophores. Aqueous solutions of NADH, flavines and their mixtures have been investigated using the method of time-correlated single photon counting. The fluorescence was recorded with a new 'delay-line' microchannel-plate photomultiplier tube, that enables time- and wavelength-resolved measurements simultaneously. Nicotine-adenine-dinucleotide (NADH) and flavine-adenin-dinucleotide (FAD) display their characteristic temporal behavior (NADH: (tau) ₁ equals 250 ps, (tau) ₂ equals 660 ps; FAD: (tau) ₁ equals 160 ps, (tau) ₂ equals 2.25 ns, (tau) ₃ equals 4.6 ns) in aqueous solution. In a mixture of NADH and FAD both components could be isolated by using global analytical methods. Time-gated fluorescence measurements on lung-tissue samples of 12 patients immediately after surgical resection have been performed with a fiber- based fluorescence detector. It could be demonstrated that NADH measurements are suitable for differentiating tumorous and necrotic tissue while flavine measurements are suitable for differentiating healthy and non-healthy tissue types. Applications of optical fibers facilitate simple combinations of the detection method with common surgical instruments (e.g. biopsy needles).

The laser fluorescent methods are very promising for photosensitizer distribution control in diagnostics and photodynamic therapy of tumors in intracavital organs. We have developed the laser fluorescent imaging system employing high sensitive CCD-camera. The system is adjusted to the standard endoscope (Olympus), cystoscope and can be also used for surface tumor observations. The high sensitivity of the system makes it possible to evaluate the photosensitizer distribution, its dynamic during PDT treatment and control the irradiation process observing tissues in fluorescent light during endoscopic investigations of stomach and lungs. The ways of improving of fluorescent imaging systems are discussed including the choosing of suitable laser source for fluorescent excitation and special filters for enhancing fluorescent contrast.

For research of photochemical processes which take place under the influence of light with definite wavelength it is important to know what quantity of light was absorbed by some or other chromophor. In photochemotherapy this gives an opportunity to choose the power, time and wavelength of light for effective influence. It is perfectly clear that the direct measurements of these contributions to absorption are difficult. So, it is necessary to use the numerical methods with application of effective models of light transport inside the sample. The purpose of the work is to develop the calculation method for partial contribution of separate chromophores based on 5-layer skin model.

On the basis of investigations of tumors with different pathology in vitro, the optical sensor, allowing us to differentiate a malignant tumor from a non-malignant one and from normal tissue, with a high degree of certainty was received. This method is based on the measuring of multiple light scattering by tissue slabs in the visible and near infrared spectral regions and careful analysis of significant chromophores researched tissues, not limited only by the degree of oxygenation. In our mind, the proposed approach has some advantages in comparison with luminescent methods because it allows one to make the quantitative assessment, and also to use them for transcutaneous measurements in vivo.

Results on laser induced fluorescence (LIF) properties of collagen, cholesterol, and chlorophyll a (chl a) are reported, when excited at 337.1 nm. The collagen and cholesterol fluorescence was studied in connection with atherosclerotic plaques diagnostics and/or removal within the cardiovascular system. The fluorescence signal excitation and collection were made using optical fibers, for liquid samples at concentrations between 0.1 mg/ml and 5.0 mg/ml for both collagen and cholesterol, their fluorescence lifetime being 5 nsec and 6 nsec, respectively. CHl a LIF concerned the stimulated emission of radiation considering the pigment as laser active medium. Chl a concentrations varied, in different solvents between 5 multiplied by 10^-3 M/l and 10^-5 M/l. For chl a at 0.62 mM/l and pumping photon densities higher than 5 multiplied by 10¹⁶ photons/cm² and pulse, fluorescence linewidth narrowing from 28 nm to 5 nm in ethanol and from 25 nm to 4 nm in DMSO were obtained. Lasing maxima at 672 nm in acetone, 674 nm in ethanol and 678 nm in DMSO were observed. At lasing conditions for chl a in DMSO (1mM/l) the measured fluorescence lifetime was 2 nsec instead of 5 nsec for normal fluorescence. The results on chl a stimulated emission show a lower lasing threshold at 10¹⁶ photons/cm² and pulse, than previously reported, if DMSO solvent is used.

We offer a two-layer dermis-like model phantom with controlled concentration of absorbers and scatterers; namely, the mixture of whole milk diluted with isotonic solution and suspension of washed human erythrocytes -- as one layer, and teflon base -- as another. The optimum milk dilution was determined, and wavelength dependencies of phantom remittance (R) over the range 480 - 680 nm were obtained. We use the mixtures with physiological concentrations of erythrocytes which corresponded to 2 - 18% per volume blood content in human papillary dermis and upper blood plexus. Phantom remittance spectra for the cases of 2% and 4% blood content virtually coincide with remittance spectra of normal and erythema human skin. Collimated transmittance (T) of blood-milk layer was also measured. At 500 nm we estimated the range of linearity of D and D' (D equals $min1gT, D' equals -1gR) dependence on blood content: offset from linearity was observed near 5 - 6% and 10% of blood, respectively.

A tissue-optical model is presented in which changes in the blood volume fraction, f_v, and tissue saturation, SO₂, are calculated from non-invasively measured intensity changes at two wavelengths during ischemia. Measurements were performed during occlusion and during muscle contraction at the human forearm with a sensor containing two LEDs, (lambda) equals 660 nm and (lambda) equals 940 nm, and photodiodes at 7.0 mm, 9.5 mm, and 20 mm from the LEDs. We used diffusion theory for a homogeneous semi-infinite medium to obtain registrations of (Delta) f_v and (Delta) SO₂ from measured changes in the photon fraction, (Delta) I, during the experiment for each detector separately. As expected, f_v stays nearly constant during occlusion, whereas SO₂ decreases, for each detector. During muscle contraction we observed that the intensity changes at each detector are much smaller than during occlusion. As expected, both f_v and SO₂ decrease at the beginning of the contraction period, but increase before the end of the contraction period. (Delta) SO₂ depends more strongly than (Delta) f_v on the assumed myoglobin concentration, the scattering coefficients, the blood volume fraction and the saturation at the beginning of the session. The success of the homogeneous model in the occlusion experiment is probably caused by simultaneous deoxygenation in the muscle and in the skin. However, during muscle contraction the changes in SO₂ and f_v were different at each detector. The failure of the homogeneous model in that case may be explained by the deoxygenation which is expected to be larger in muscle tissue than in skin tissue.

The in vivo model of the chorioallantoic membrane of fertilized chicken embryos (CAM) was employed for studying the fluorescence characteristics of tumor tissue in comparison with non tumorous tissue. Tumors were grown from the murine fibrosarcoma cell line SSK II and murine 3T3 fibroblasts (clone A31) were used for cultivating non tumorous tissue. Autofluorescence and xenofluorescence intensities induced by 5-aminolaevulinic acid (5-ALA) were compared. Exogenous administration of 5-ALA, an early precursor in haem synthesis, induces accumulation of endogenous photoactive porphyrins, in particular protoporphyrin IX (PpIX). Fluorescence investigations were performed after 3-4d of incubation, when the tissues had reached macroscopically three dimensional stages of growth. Fluorescences were excited with a HBO-X 100 W lamp (Carl Zeiss) at a wavelength (lambda) equals 405 plus or minus 5 nm. Emissions were detected in the spectral range above 630 nm and visualized by real time digital image processing (Argus 10, HAMAMATSU) using an ICCD camera (HAMAMATSU). After administration of 0.4 mmolar 5-ALA solution to the CAM inoculated tissues the SSK II tumors exhibited higher fluorescence intensities than the 3T3 non tumorous tissues. Autofluorescence intensities of both types of tissues were not distinguishable. Furthermore, the effects of several biochemicals on the xenofluorescence intensities of the fibrosarcoma and fibroblast tissues were investigated.

A system for the excitation and detection of autofluorescence induced by N₂ laser light (337.1 nm, 1 divided by 1.5 mJ/pulse) on biological tissues in vivo is described. Spectra obtained with medium spectral resolution spectrograph were detected with a 512 by 512 CCD array. Spectral measurements performed on patients bearing different cutaneous diseases show that, in the wavelength range 420 - 480 nm, pathological skin tissues fluoresce less than healthy ones and these differences might be useful for diagnostic purposes.