We designed a laser spectrofluorimeter to determine the optimal delay between drug injection and laser irradiation in which both excitation and fluorescence emission are transmitted through a single optic fiber. Preclinical studies were performed in nude mice after ALA administration. The maximum fluorescence (632 nm) in HT 29 tumors was obtained after 90 min (excitation 488 nm, 20 mW, integration time 3 sec, ALA 250 mg/kg), after 180 min in liver. Iron chelators as EDTA (10 or 20 mg/kg) or desferrioxamine (20 or 40 mg/kg) increased the delay for reaching maximum fluorescence to 2 hours as well as fluorescence intensity in tumors but decreased intensity in liver. Desferrioxamine induced the appearance of protoporphyrin IX in the bladder or kidneys, normally absent in urines. These results indicate that drug distribution varies with tissues and also adjuvant therapies that are often administered together with cancer drugs.

The fluorescence response of biological tissue (excited at 366 nm or 337 nm) depends on its condition: the intensity is rather low in cancer tissue and slightly higher at the edge of the tumor than in healthy tissue. This technique is non-invasive, non-destructive and is based on the native fluorophore NADH. Fluorescence images obtained with unstained, unfixed cryosections match the histological images. Since the cryosections can be prepared and evaluated in less than 5 min, this technique can also be used as a fast cut technique to determine e.g. the diagnosis of a biopsy sample. Additional investigations have shown that the fluorophore seems to be attached to elastic fibers.

An ex vivo microspectrofluorometric analysis has been performed on samples from normal tissue and adenocarcinoma of human colon, to define the biological background of the autofluorescence-based approach to the diagnosis of neoplastic lesions. The fluorescence properties of the biological tissues have been investigated in relation with their morpho-functional and biochemical characteristics, in thin sections and extracts from surgical resections. Differences in the fluorescence of the normal and the neoplastic tissue have been found as to both intensity distribution and spectral shape are concerned, that can be related to the histologic organization of the tissues and/or to the differences in the relative amount of the fluorophores. The results obtained could provide indications useful to improve the in vivo diagnostic procedure, as to the excitation and emission conditions are concerned that can maximize the differences between normal and neoplastic tissues.

A quantitative technique based on fluorescence microscopic detection has been developed for measurements of photosensitizer distributions in frozen tissue sections using a highly sensitive charge-coupled device camera. Studies have been carried out on sulphonated aluminum phthalocyanine and Protoporphyrin IX induced by administration of exogenous 5-aminolaevulinic acid. This technique has enabled microscopic pharmacokinetic studies to be performed in a range of normal tissues and tumors in order to improve our understanding of the photosensitization mechanism and enable optimal treatment times and dosimetry to be determined. Comparisons with chemical extraction data are reviewed and the feasibility of using biopsies from patients for optimizing dosimetry is discussed.

In this paper, demonstrating necessity to take into consideration the erythema as a factor, leading to distortion of skin autofluorescence spectra during in vivo investigations of skin by the method of fluorescence spectroscopy, are presented.

The advantages of optical spectroscopy in skin are discussed. The basis for fluorescence spectroscopy is briefly presented. The potential for in vitro and in vivo fluorescence spectroscopy of biotissues is significant, but not yet well developed. With the help of fluorescence spectroscopy and layer-by-layer skin surface strippings technique the important knowledge for autofluorescence spectra from different skin layers have been obtained. Resolution of autofluorescence spectra into discrete excitation-emission bands is presented, related to various skin fluorophores.

Possibility for definition of organism's sensitivity to specific allergen by means of luminescent analysis of peripheral blood lymphocytes was shown. The positive correlation dependence between luminescence intensity increase at 640 nm of acridine orange colored lymphocytes after simulation by specific allergene in vitro and the serum antiallergene antibodies level was detected.

To optimize phototherapy and photodetection of cancer, one of the important variables is the localization of the dye after injection. To study this in a clinical context, we have constructed an apparatus based on a non-invasive optical fiber that detects the dye by light induced fluorescence. The time course of the fluorescence signal can be used directly for optimizing photodetection. However, complementary information on the detailed localization of the drug by fluorescence microscopy, and a correlation of this data with tumor necrosis efficacy, are necessary to optimize PDT timing. This will be demonstrated for the case of Photofrin II and tetra (meta- hydroxyphenyl) chlorin (mTHPC) and the two sets of data will be compared.

Visualization of early stage bladder cancer is considerably improved using Delta-Aminolevulinic acid (ALA) as tumor marking agent. This is due to an intracellular accumulation of fluorescing Protoporphyrin IX in tissue following intravesical installation of ALA in a pH-neutral solution.

A time-gated fluorescence imaging technique was applied on tumor-bearing porphyrin-treated mice to study the sensitizer distribution in different organs and tissue types, and to establish whether false positives in the diagnosis of tumors (based on porphyrin fluorescence) could be generated by this localization in healthy tissues. Mice were administered 25 mg/kg body weight (b.w.) of HpD or 5 mg/kg b.w. of PII, and sacrificed 8 hr later. Time- gated fluorescence images were acquired from tumor, skin, muscle, fat, brain, heart, lung, lymph nodes, liver, bowel, spleen, and bone of both treated and untreated animals. Similar results were obtained with HpD and PII. The presence of porphyrins clearly helps the localization of the neoplastic area, which is characterized by the strongest fluorescence in delayed images. An appreciable long-living emission was observed also in bones. With the exception of the bowel, the fluorescence of other organs was weaker and, in untreated mice, short-living.

A set for fluorescence detection of laser-induced processes in biological tissues in the spectral range from 200 nm to 1000 nm was applied for fluorescence diagnostics of wound processes. The water-soluble photosensitizer and tumor-making agent--sulphonated aluminum phthalocyanine (AlPcS) were tested for vital decoration of wound processes. The spectral (optical multichannel analyzer) and image (slow-scan CCD camera) detection of wound process were performed. A high efficiency of laser-induced dye-related fluorescence for wound processes detection and monitoring was demonstrated. The distribution of dye between untreated tissues and different wounds, inflammations and experimental tumors is described. The extremely long uptake time (up to 8 weeks) for AlPcS in wounds and inflammations was observed.

An intensity ration of the photosensitizing agent to the natural fluorescence was used to determine the condition of lymph nodes (normal, inflammatory, or with metastases). Eighty- one Lobund Wistar rats were inoculated with Pollard rat adenocarcinoma cells. After 40 days the rats were injected with 0.75 mg/kg BPD-MA. A helium-cadmium lasers (442 nm) was used for both light delivery ad fluorescence acquisition. Fluorescence spectra were acquired and analyzed by an optical multichannel analyzer 9 EG&G, OMA III). Unbalanced repeated measures analysis of variance (ANOVA) was done on the mean intensity ratio according to location. Several locations recorded for each rat were modeled as the repeated variable. The fluorescence signal obtained from lymph nodes with metastases indicated high drug amounts, greater than in the skin, tumors, and all other tissues. The lowest fluorescence intensity ratios were observed in the skin. This leads us to the conclusion that the amount of Photofrin porfimer sodium and BPD-MA which localized in the metastatic lymph nodes is higher than in tumor and all other healthy tissues. Among normal lymph nodes there were significant differences according to the site of the node, with the left iliac lymph nodes having higher IR values than either the mesenteric (MC I or MC II) nodes. The geometric means of IR are consistently higher in normal as compared to inflammatory nodes for each location. The optical biopsy needle with laser-induced fluorescence detection and exogenic fluorochrome is a sensitive, novel techniques to localize small boundaries of metastatic neoplasm in lymph nodes.

Sensitivity of laser-induced fluorescence spectroscopy (LIFS) in detecting a change in tissue pH, and blood perfusion was determined. Rabbits were anesthetized, paralyzed, and mechanically ventilated. The arterial and venous blood supplies of the kidney were isolated and ligated to alter the perfusion. The femoral artery was cannulated to extract samples for blood gas analysis. A 308-nm XeCl was used as an excitation source. A 600 micrometers core diameter fiber was used for fluorescence acquisition, and the spectra analyzed by an optical multichannel analyzer (EG & G, OMA III). the corresponding intensity ratio R equals I^NADH / I^COLL was used as an index for respiratory acidosis. Blood perfusion was assessed using the following algorithm: (I^ELAS minus I^COLL) divided by (I^NADH minus I^COLL). The intensity ratio linearly decreased with the reduction of blood perfusion. When we totally occluded the artery the ratio decreased tenfold when compared to the ratio of a fully perfused kidney. Results of monitoring blood acidosis by laser-induced fluorescence spectroscopy shows a significant trend between pH and intensity ratio. Since all the slopes were negative, there is an obvious significant correlation between the pH and NADH.COLLAGEN RATIO. Blue-light-induced fluorescence measurements and ratio fluorometry is a sensitive method for monitoring blood perfusion and acidity or alkalinity of an organ.

Laser induced fluorescence has often been used as a diagnostic method. Unfortunately the fluorescence signal is modified during the photons migration towards the detector. The purpose of this study is to determine the alterations of the laser induced fluorescence spectra in white matter of adult brain due to the spectral variations of the optical coefficients (mu) _a((lambda) ), (mu) _s((lambda) ) and of the mean cosine of the scattering angle g((lambda) ).

A time-gated technique to enhance viewing through highly scattering media, such as tissue, is discussed. Experiments have been performed on tissue-like plastic phantoms in order to determine the possibilities and limitations of the technique. The effects of time-gate width, refractive index, localization and size of hidden objects have been studied. A computer model to simulate light propagation in tissue is also presented. The model is compared to experimental results.

The oxygenation of human muscle tissue can be investigated using near IR spectroscopy (NIRS). Oxy and deoxy hemoglobin changes can be quantified combining attenuation measurements with pathlength data obtained by time resolved spectroscopy. This study reports the application of NIRS to non- invasive measurements of quadriceps oxygenation on muscular dystrophy patients during treadmill exercise.

This paper will deal with a band-limited cw-transillumination and illustrates that the differences between scattering distributions of tissues determines the functional status of body parts and thus enables an adequate diagnosis with a minimal technical expenditure.

Spectrophotometry of in vivo skin pigmented lesions by means of an integrating sphere coupled to a conventional spectrophotometer has recently been suggested as a useful tool to discriminate cutaneous melanoma from other pigmented cutaneous lesions. To improve reflectance spectral analysis of moles, a new spectrophotometric procedure based on the use of a CCD camera provided with interferential filters has been developed. Preliminary results suggest that the new method, allowing a spatially resolved analysis of the spectral components from 420 to 1040 nm, would improve, when properly implemented with imaging data handling, the quality of a computer assisted diagnosis of malignant lesions.

A non-invasive diagnostic tool that could identify malignancy in situ and in real time would have a major impact on the detection and treatment of cancer. We have developed and are testing early prototypes of an optical biopsy system (OBS) for detection of cancer and other tissue pathologies. 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 microscopic structure of the tissue. In addition to the reduced invasiveness of this technique compared with current state-of-the-art methods (surgical biopsy and pathology analysis), the OBS offers the possibility of impressively faster diagnostic assessment.

Recent advancements in the technique of Raman spectroscopy now make it possible to achieve rapid, minimally invasive and non-destructive characterization of tissues. In order to evaluate the efficacy of this technique for diagnosis, the Raman spectra of normal and neoplastic human tissues (e.g., breast, kidney, liver and colon) were obtained utilizing visible and near-IR excitation. Normal breast tissue and colon adenocarcinoma showed major Raman features due to the presence of carotenoids and lipids. In breast carcinoma, the features due to lipids were attenuated and as fibrosis (desmoplasia) increased, new spectral features attributable to collagen were observed. Samples of normal and neoplastic liver and kidney show unique spectral differences sufficient to permit tissue differentiation.

In vivo measurement of NADH autofluorescence is strongly disturbed by changes in the Hb absorption of excitation and fluorescence light. An approach to this problem is based on the diffuse reflectance measurement of 805 nm (isobestic point of Hb/HbO₂). According a Kubelka-Munk calculation an increase in Hb/HbO₂ concentration leads to a decrease of diffuse reflectance. This gives correction terms for the measured autofluorescence data. An equipment to perform such measurements will be presented.

In investigations of optical parameters of human skin fluorescence and autofluorescence are very important. Since today some problems of experimental conditions are not solved yet. One of the main problems is the evaluation of different methods of sample preparing. Some different methods of sample preparing technique and the optical parameters for the presented cases will be presented. The significance of contribution of the fluorescent intensity of the glue into the total integrated transmittance and reflectance intensity of the sample will be analyze;d. As an excitation source, the nitrogen gas laser ((lambda) equals 337 nm) was used.

New method of intrasurgical diagnostics of metastasis in lymphatic nodes is based on the determination of investigated tissue autofluorescence spectra peculiarities. Statistical data demonstrate correlations between criteria of spectral analysis and morphological data. High sensitivity of spectroscopical technique, good choice of laser source for autofluorescence excitation (HeNe laser (lambda) equals 633 nm) and convenient non-invasive procedure of express diagnostics provide identification of metastasis presence in lymphatic nodes with a specifity 95%.

In this work the results of head and neck oncological diseases diagnostics with the use of autofluorescence spectra are adduced. On the grounds of the investigations of malignant tumors different types (more than 50 patients) the perspectives of autofluorescence diagnostics method using in clinic are discussed. The relation between autofluorescent properties of the tumor and conditions of the patient treatment is determined (chemicotherapy, radiotherapy).

In present work the temporal behavior of autofluorescence of upper (30 - 40 micrometers ) layer of human skin under continuous 337 nm laser exposure was investigated. The samples were prepared using epidermis stripping technique. Kinetic curves of autofluorescence decay at 420 nm, 440 nm, and 460 nm have been obtained. In double exponential approximation the parameters of autofluorescence decay have been evaluated. Under the assumption that different terms in double exponential approximation correspond to different individual fluorophores the contribution of fast bleaching, slow bleaching, and non-bleaching components to fluorescence spectrum of the sample at 420 nm, 440 nm, and 460 nm have been calculated.

Using subnanosecond laser spectrofluorometry the spectral and polarization time-resolved characteristics of 1-phenylnaphthylamine (1-AN) fluorescent probe in phospholipid bilayer and red blood cell (RBC) membranes have been studied. It is shown that the electronic spectra of probe in model membranes inhomogeneously broadened. Inhomogeneous broadening affect significantly the spectroscopic properties of probe in membranes, such as the time-dependent fluorescence Stokes shift, the dependence of the fluorescence instantaneous spectra and fluorescence kinetics on excitation wavelength. The obtained results prove the existence of the earlier unknown effect of the wavelength- dependent rotation of a probe in phospholipid bilayer and RBC membranes.

The autofluorescence of cancerous and normal human stomach tissues was measured in vitro, by fluorescence spectroscopy, within three hours of surgery ablation. A new fluorescence emission band, centered at about 380 nm for the cancerous stomach tissue is reported, for 340 nm excitation. This band is practically absent for the normal tissue when this is excited at the same wavelength. For UVB excitation (between 283 and 305 nm) the emission bands are centered around 350 nm and 470 nm, for both tissues, in agreement with the literature. The ratios of the fluorescence intensities for cancerous and normal tissues are measured at the center of the three bands, the intensity for the diseased tissue being always higher than for the normal one. The presence of a new band centered at 380 nm, combined with the intensity ratios, may prove of great relevance towards early in vivo detection of stomach cancer.

In spectroscopic investigations of biological samples the problem of separation and quantitative evaluation of spectra lines connected with various molecules is the most important one. The situation is especially significant for in vivo investigations. This paper presents the results of fluorescence spectra analysis of some photosensitizers measured in various tissues both in experimental and clinic conditions and autofluorescence spectra analysis of some human tissues. It is shown, that technique proposed allows one to evaluate the component concentrations including the in vivo measurements.

There are many problems before dinica surgery. which can be solved and part of them is being solved successfully in present time with the use of laser spectroscopy methods. In accordance with it the following general work directions have been determined: investigations of the functional state of intracavital organs. metabolic processes. operative and conservative treatment efficiency: - diagnostics of neopiasms including the malignant tumors of intracavital organs and intravascular formations: - control to the dose of laser irradiation during the laser diagnostics, therapy and surgery. Taking into account these directions the main efforts are concentrated on the solution of the following tasks: determination of microcirculating blood hemoglobin concentration and oxygenation degree in the mucous of stomach bowel tract, trachea bronchial tree and skIn; - determination of oxygen utilization degree by the mucous of i ntracavital organs and defi n ition of tiss ue destruction degree: - diagnostics of intracavital organs malignant new growths early forms and skin cancer on the base of spectra analysis of endogen tumor organic compounds luminescence 'nder 'aser influence: laser spectroscopic control to the completeness of different organs malignant sections ectomy during the surgical operation: - investigation of the influence of laser excitation on reproducing capacity of intracavital organs mucous as in the plan of stimulation as its growing attenuation; laser therapy of stomach and duodenat ulcer; - laser photodynamic therapy of malignant new growths with the use of phototocsic medical preparations with the parallel spectrcscopic control to the dose of irradiation and therapy efficiency; - laser coagulation of malignant and non-malignant neoplasms, stopping of the bleeding; - rechannalisation of occlusive magistral vessels and obturated sections of trachea bronchial tree with the parallel soectroscopic control to the dose of laser rd type of irradiated tissue with the purpose of prevention of bronchial or vascuiar walls perforation and timely influence stopping -spectroscopic express-analyse of blood and peritonitis exudation with the purpose of prevention of sepsis and peritonitis possibie evauatiori after operation.