It is well known that fluorescence spectroscopy can provide information about the differences in the concentration of chromophores in healthy and cancerous tissues. The tumor detection potential can be enhanced by using exogenous fluorescent agents with selective accumulation in cancerous tissue. In this study healthy and cancerous human colon tissue samples were obtained after colon surgery. Excitation-emission matrices were collected using a fluorescence spectrometer. The optimum excitation wavelength lied at 340 nm. After the acquisition of autofluorescence spectra, the samples were incubated in a solution of 4 (mu) g/ml of Rhodamine analogs. Rhodamine B, Rhodamine 6G and three recently synthesized analogs, were used. For the acquisition of fluorescence images, an endoscopic imaging system was developed. Fluorescence imaging with the concomitant use of Rhodamine analogs revealed a remarkable differentiation of cancerous from healthy colonic mucosa.

This paper describes an innovative approach to tissue fluorescence as a phenomenon of inelastic scattering of electromagnetic radiation. A general formulation of the polarization vector of the medium is used in order to describe the fluorescence inelastic scattering process. The general method described, is applied to two different tissue models. In the first model, tissue is represented as a single infinitely thick, homogeneous layer, under plane wave excitation. The second model used is based on a picture of tissue as a single dielectric layer, under pulse excitation. In both cases, fluorophores and absorbing species are assumed to be homogeneously distributed inside tissue. The mathematical techniques used together with the fully evaluated theoretical results are presented. Additionally, experimental measurements were performed on collagen gels which contained various dyes in order to evaluate and validate our mathematical modeling.

In order to investigate the effects of conservation in liquid nitrogen and formalin on peripheral vascular tissue (abdominal aortic, femoral and flank artery tissue) laser-induced fluorescence spectra were recorded during the exposure of these tissues to He-Cd radiation (442 nm). The spectral distribution of tissue fluorescence allowed the development of simple algorithms based on the intensity difference in order to discriminate the tissue samples when they were fresh and after they were stored for 24 and 48 hours in liquid nitrogen or formalin.

Laser-induced fluorescence (LIF) was studied in vivo from premalignant and benign lesions in the female genital tract, in particular the cervix. The aim of the study was to investigate the possibilities to differentiate cervical intraepithelial neoplasia (CIN) from normal tissue by means of two different fluorescence modalities. Most of the patients were given a low dose (5 mg/kg bw) of (delta) -amino levulinic acid (ALA). The ALA was orally administered 2 - 4 hours prior to the investigation. During this time, the ALA is transformed to the strongly fluorescent protoporphyrin IX (PpIX) via the haem cycle. Excitation light with a wavelength of 405 nm was used to excite the PpIX fluorescence. Excess amounts of PpIX were accumulated preferentially in diseased tissue. However, the variability in the PpIX accumulation from patient to patient was large. By using excitation light at 337 nm, the endogenous fluorophores are more efficiently excited. Therefore, this excitation modality was exploited for studying spectral characteristics of the autofluorescence in different tissue types. The spectra obtained were evaluated by forming fluorescence intensity ratios. The tissue types were grouped according to the histopathological examination. A correlation with the fluorescence ratios was performed. Some problems with the classification remain, mostly due to the difficulties in obtaining histopathologic evaluation of the biopsies at the exact location of the LIF measurements.

Autofluorescence spectra of normal and pathological mucosa of the bladder were measured in vivo in five patients using 410 nm excitation light and ex vivo in five samples, within 30 minutes after removal, using both 364 and 400 nm excitation lights. Inflammatory and angiodysplasia post-radiotherapy lesions, and papilloma were differentiated from normal mucosa by the low autofluorescence intensity and the shape of spectra. The autofluorescence intensity ratio, at a common emission peak (515 - 520 nm) induced by 410 nm and 1 mW excitation light, between normal mucosa and pathological tissues for each patient was variable. A minimum of 5 and a maximum of 22.5 were observed. The autofluorescence intensity for certain inflammatory post-radiotherapy lesions was null. Furthermore, due to high absorption of hemoglobin, spectra of angiodysplasia post-radiotherapy lesions were characterized by two peaks at about 555 and 600 nm. The measured autofluorescence spectra in samples caused by two excitation wavelengths (360 & 400 nm) and 1 mW light power showed similar structures to in vivo results. Though, a NADH peak appeared in variable intensities in spectra of both normal and pathological tissues at the excitation light of 364 nm. In certain lesions, this emission was highly attenuated.

Carcinoma in situ (CIS) of the bladder is a confounding disease that is difficult to recognize endoscopically since it is a flat cancer. Many studies have suggested its relationship with subsequent invasive disease. Early recognition of CIS therefore is essential in order to offer the patients the most appropriate treatment and the highest cure rate. Since white light cystoscopic examination is not sufficient to reveal areas of dysplasia or carcinoma in situ random biopsies are recommended. We wanted to evaluate whether amino levulinic acid (ALA) fluorescence detection could be helpful in diagnosing carcinoma in situ and if the specificity could be enhanced by reducing the ALA dose. Sixteen patients with papillary bladder cancer and carcinoma in situ and dysplasia were instilled with low dose ALA. Fluorescence detection of the metabolized ALA was performed three hours later, with the naked eye, after blue light illumination. CIS or dysplasia was found in 50 biopsies. The sensitivity for detecting CIS was 94% with a specificity of 89%. Carcinoma in situ can be diagnosed with a very high accuracy through fluorescence detection after ALA instillation. Fluorescence detection can be achieved with the naked eye and does not necessitate complex equipment neither specifically trained personnel.

Preliminary results of clinical experiments using UV induced autofluorescence spectroscopy in 23 patients with Barrett's esophagus are reported in this paper. Excitation wavelengths of 351 nm and 330 nm were used to induce Barrett's mucosa autofluorescence. Autofluorescence acquisition and signal processing were performed using a CP200 Jobin-Yvon system coupled to a flexible three optical fiber sensor. Three distinct emission bands were observed in the measured spectra after normalization according to the backscattered light power. These emissions were attributed to collagen, elastin, NADH and flavin. Fluorescence intensities and ratios between the emission bands were used to discriminate high grade dysplasia and early stage cancer in Barrett's esophagus from normal surrounding tissues. A significant decrease of the overall fluorescence intensity was observed for the Barrett's mucosa compared to the normal esophageal one. Autofluorescence spectral shape was modified. Collagen and elastin contribution was found to decrease going from normal to tumoral tissue.

In medical diagnosis of superficial lesions at inner or outer surfaces of the human body fluorescence imaging techniques are able to deliver additional information on the metabolic and structural state of the observed tissue. To subtract background fluorescence and to achieve a differential diagnosis a multispectral analysis in several wavelength windows is needed. Additionally, special image algorithms have to be applied which depend on the examined malignancy. For this purpose a multispectral fluorescence imaging device was developed. It can be used both endoscopically and in combination with a standard operational microscope from Carl Zeiss, Germany. In this paper, the device and first clinical results are presented. The device was built to detect superficial lesions like tumors, inflammations, etc. Target chromophores are NADH, Protoporphyrin IX, collagen and other. The measured optical bands are (405 plus or minus 5) nm, (442 plus or minus 5) nm, (458 plus or minus 5) nm, (550 plus or minus 5) nm, (630 plus or minus 5) nm and (690 plus or minus 5) nm. A special UV-source with a liquid light guide is used as the illumination source in two excitation bands of (365 plus or minus 10) nm and (420 plus or minus 20) nm. First clinical investigations of superficial malignancies like squamous cell carcinoma and basalioma are presented.

After the very promising clinical results for the detection of bladder cancer in urology, preclinical and clinical studies on aminolevulinic acid (5-ALA) induced protoporphyrin IX (PPIX) are preformed in various disciplines now. This paper provides a brief overview of the progress on 5-ALA assisted fluorescence diagnosis in urology, pulmonology, neurosurgery, gynecology and ENT performed in collaboration with the Laser Research Laboratory at the Department of Urology of the Ludwig-Maximilians-University in Munich. Five-ALA can be applied either topically or systemically to induce an intracellular accumulation of fluorescing PPIX. With appropriate dosage of 5-ALA, malignant tissue can be stained selectively, and irradiation with violet light excites a bright red fluorescence of the tumor. Optical properties of the tissue tend to hamper the precise identification and demarcation of suspect areas in fluorescence images. Multicolor remission and fluorescence imaging, therefore, seems to be indispensable for a reliable tumor localization.

We present a scheme by which tissue phantom systems can be designed rapidly and systematically according to individual demands. For the optical biopsies, organ structures of biological tissues have to be modeled which requires a solid host material determining the modeling potentialities. Complex structures of biological tissues can be modeled by phantom systems based on a solid host material which determines the modeling potentialities. Mie theory shows that scattering and absorption of particles depends strongly on their material constants and size distribution. According to these predictions particles can be selected or produced. Particles are not only useful to induce scattering but can also be an interesting alternative to absorbing and fluorescent dyes. We present organic, metallic and mineralic particles, their relevant properties and outlines of their characterization. We discuss how the predictions of theory and mutual interactions between components can be checked and report on problems frequently encountered with dyes and particles as components of tissue phantom systems.

FT-IR-microspectroscopic mapping technique has been used in combination with imaging methods for characterizing thin tissue sections of human adenocarcinomas of the colon and rectum as well as carcinomas of the breast. This paper presents results of microspectroscopic measurements in vitro of 10 micrometer cryosections of healthy and tumor tissue samples of gastro-intestinal and gastro-oesophageal origin by using a minimal spatial resolution of 100 micrometers squared. This technique is not only able to detect the amount of collagen, lipids and tissue related features as well as different substructures of the tissue samples, it could also be used for the differentiation between healthy and tumor tissue. The IR-maps based on the ratio of intensities of selected wavenumbers were compared with parallel cut and HE stained cryosections which were judged by a pathologist. The method is based on differences in IR-spectra of tissues which have been already described in the literature. Several papers have shown that the main differences are to be expected in the so-called 'fingerprint region' (1500 - 1000 cm^-1). Additional spectroscopic changes arise from carbonyl/amide vibrational modes. Measurements were carried out using transmission, attenuated total reflection and spatial reflectance infrared spectroscopy. IR-maps of healthy and tumor tissue specimen are presented and discussed. Different modes of spectra acquisition (transmission, ATR, diffuse reflectance) are compared. The aim of the investigations is the determination of suitable wavelengths to distinguish between healthy and tumor epithelia tissue for tumor diagnostic with an endoscopic approach in vivo.

Elastic-scattering spectroscopy examines the wavelength dependence of light that has passed through a small volume of tissue. Measurements are typically made by placing two optical fibers on the surface of the tissue to be examined. The analysis of these measurements to obtain quantitative information about scattering and absorption is important to many biomedical applications such as cancer diagnosis and measurement of bilirubin concentrations. For fiber separations large enough for the diffusion approximation to be valid, this is straight forward. However, for clinical applications such as those listed above, the separation is too small for the diffusion approximation to be applicable. To obtain insight into the question of how scattering and absorption changes affect the wavelength dependence of the elastic-scatter signal Monte Carlo simulations have been used. First, it is shown that the Monte Carlo simulations and elastic-scatter measurements of polystyrene spheres agree quite well. Monte Carlo simulations are then used to investigate how particle size and concentration affect the elastic-scatter signal. It is found that the concentration has very little effect of the wavelength dependence, but that the size of the scattering particles does affect the wavelength dependence. In general, the signal decreases more rapidly as a function of wavelength for smaller particles.

Each tissue type has a unique spectral signature (e.g. liver looks distinct from bowel due to differences in both absorbance and in the way the tissue scatters light). While differentiation between normal tissues and tumors is not trivial, automated discrimination among normal tissue types (e.g. nerve, artery, vein, muscle) is feasible and clinically important, as many medical errors in medicine involve the misidentification of normal tissues. In this study, we have found that spectroscopic differentiation of tissues can be successfully applied to tissue samples (kidney and uterus) and model systems (fruit). Such optical techniques may usher in use of optical tissue diagnosis, leading to automated and portable diagnostic devices which can identify tissues, and guide use of medical instruments, such as during ablation or biopsy.

It is shown that the amniotic fluid has intensive ultra-violet luminescence caused by proteins. Along with it amniotic fluid radiated in the field of 380 - 650 nm with maxima at 430 - 450 nm and 520 - 560 nm. The first peak of luminescence ((lambda) _exc equals 350 nm; (lambda) _em equals 430 - 440 nm) is caused (most probably) by the presence in amniotic fluid of some hormones, NADH₂ and NADPH₂. A more long-wave component ((lambda) _exc equals 460 nm; (lambda) _em equals 520 - 560 nm) is most likely connected with the presence in amniotic fluid pigments (bilirubin connected with protein and other). It is shown that intensity and maximum of ultra-violet luminescence spectra of amniotic fluid in normality and at pathology are identical. However both emission spectra and excitation spectra of long-wave ((lambda) greater than 450 nm) luminescence of amniotic fluid from pregnant women with such prenatal abnormal developments of a fetus as anencephaly and spina bifida are too long-wave region in comparison with the norm. Results of research testify that spectral luminescent analysis of amniotic fluid can be used for screening of malformations of the neural tube. It is very difficult for a practical obstetrician to reveal pregnant women with a high risk of congenital malformations of the fetus. Apart from ultrasonic examination, cytogenetic examination of amniotic fluid and defumination of concentrations of alpha-fetoprotein and acetylcholin-esterases in the amniotic fluid and blood plasma are the most widely used diagnostic approaches. However, biochemical and cytogenetic diagnostic methods are time-consuming. In the present work spectral luminescence properties of the amniotic fluid are investigated to determine spectral parameters that can be used to reveal pregnant women with a high risk of congenital malformations of their offsprings.

The spectral fluorescence analysis is a promising method for differential tissue diagnostic. Usually the UV and visible light is used for fluorescence excitation with emission registration in the visible wavelength range. The light penetration length in this wavelength range is very small allowing one to analyze only the surface region of the tissue. Here we present the tissue fluorescent spectra in vivo excited in the red wavelength region. As excitation light source we used compact He-Ne laser (632.8 nm) and observed the fluorescence in 650 - 800 nm spectral range. The various tissues including normal skin, psoriasis, tumors, necrosis as well as photosensitized tissues have been measured.

Video epifluorescence microscopy and image analysis are used for studying anthracycline resistance in breast cancer cells. In order to perform a semi-quantitative image analysis, several deconvolution algorithms are tested and validated on model beads. The most performant algorithm is applied to fluorescent biological specimens. We show that deconvolution makes image segmentation easier. Semi-quantitative measurements on resulting images are correlated with results obtained by cytometry.

First results with enzyme catalyzed reactions show that examination of reactions of single molecules with classical (non-scanning) fluorescence microscopy is possible. Two picoliters of enzyme solution (lactate dehydrogenase, LDH-1) are injected into an area with an excess of substrate (Lactate, NA⁺). From the dilution of the enzyme solution one can calculate that only few enzyme molecules (in the order of 10²) are injected. After a minute discrete zones with increasing fluorescence intensity from the reaction's product NADH can be observed in a microscope. The variation of the fluorescence intensity and the size of these zones is used to monitor the reaction kinetics and to estimate the number of individual enzyme molecules which catalyzed the reaction in every zone. Three reaction zones are observed which may be caused by 1, 1 and 2 LDH-1 molecules up to 5, 5 and 10 molecules.

Autofluorescence measurements on human tissue have revealed a decrease in intensity in malignant tumors and an increase in the healthy region adjacent to the tumor. This latter event might serve as a protective wall against the invasive tumor cells. The composition of this wall is still unknown. Antioxidants such as NADH might be involved. In the case of seborrheic keratosis (wart), the intensity is increased in the pigmented spots. Care must be taken, therefore, when warts are attached to malignant tumors. The resulting value is, then, not indicative for the condition of the system.

The application possibilities of fluorescent probes have increased dramatically in the last few years. The main areas are as follows (Slavik, 1994, 1996, 1998). Intracellular ionic cell composition: There are selective ion-sensitive dyes for H⁺, Ca²⁺, Mg²⁺, K⁺, Na⁺, Fe³⁺, Cl^-, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺, Ba²⁺, La³⁺. Membrane potential: Using the so-called slow (Nernstian dyes) or electrochromic dyes one can assess the value of the transmembrane potential. Membrane fluidity: Fluorescent probes inform about the freedom of rotational and translational movement of membrane proteins and lipids. Selective labeling: Almost any object of interest inside the cell or on its surface can be selectively fluorescently labeled. There are dyes specific for DNA, RNA, oligonucleotides (FISH), Golgi, endoplasmic reticulum, mitochondria, vacuoles, cytoskeleton, etc. Using fluorescent dyes specific receptors may be localized, their conformational changes followed and the polarity of corresponding binding sites accessed. The endocytic pathway may be followed, enzymes and their local enzymatic activity localized. For really selective labeling fluorescent labeled antibodies exist. Imaging: One of the main advantages of fluorescence imaging is its versatility. It allow choice among ratio imaging in excitation, ratio imaging in emission and lifetime imaging. These approaches can be applied to both the classical wide-field fluorescence microscopy and to the laser confocal fluorescence microscopy, one day possibly to the scanning near field optical microscopy. Simultaneous application of several fluorescent dyes: The technical progress in both excitation sources and in detectors allows to extend the excitation deeper in the blue and ultraviolet side and the detection further in the NIR and IR. Consequently, up to 6 peaks in excitation and up to 6 peaks in emission can be followed without any substantial difficulties. Application of dyes such with longer fluorescence lifetimes such as rare earth dyes gives chance for the separated detection of another six peak pairs. The literature data on simultaneous applications of several fluorescent dyes are rare, usually it is only pH and calcium, pH and membrane potential or pH and cytoskeleton changes that are mentioned. Nevertheless, I am sure that in the near future it will be quite common to employ several fluorescent dyes simultaneously. So, in a few years, you may expect to be comfortably seated in an armchair in front of the monitor screen, sip your coffee and follow simultaneously several physiological parameters trying to find out new relations among them. In this respect the potential of fluorescent probes is unsurpassed if you just recall only the discovery of calcium waves and calcium spikes during the past years.

Energy transfer from NADH to the mitochondrial marker rhodamine 123 (R123) was used to probe mitochondrial malfunction of cultivated endothelial cells incubated with various inhibitors of specific enzyme complexes of the respiratory chain. Pronounced differences of 'energy transfer efficacy' of incubated cells as compared to controls were deduced from the ratio of fluorescence intensity and intracellular amount of the acceptor. A combination of cw and time-gated (nanosecond) fluorescence spectroscopy appeared to be an appropriate tool for probing mitochondrial malfunction in various kinds of diseases.

The coordination of excitation in a biological system of cells such as cardiac myocytes in heart tissue has crucial influence on the function of the entire organ. This coordinated behavior can be visualized in a small group of embryonic cardiac myocytes derived from the hearts of unborn chicken. Loaded with a calcium sensitive dye the excitation can be imaged via the occurring transient rise in cytosolic calcium concentration. It can be shown that in regions with physiological or morphological restrictions the transient rise in cytosolic calcium occurs with a temporal delay compared to the ordinary array of coupled myocytes. The height of the transient rise of cytosolic calcium is related to the ability of the individual cell to participate in the coordinated contraction. The free cytosolic calcium concentration is decreased with the UV-labile calcium, chelator diazo-2. Our setup allows to decrease the free cytosolic calcium in a single cell of the contracting array of cells. This allows us to introduce mismatches in selected regions of the coordinated contraction and to visualize the effects simultaneously.

Analytical methods on individual cells are essential for studying both the functions of normal cells and the possibility to monitor pathological cells. The development of some polyvalent techniques like flow cytometry and fluorescence imaging spectroscopy has led to more and more accurate methods for analyzing heterogeneous cell populations. Unfortunately, most of the quantitative morphometric or biological parameters extracted from fluorescence measurements rely on the accuracy of the determination of the surface of the biological objects (whole cell, nucleus, . . .). That determination is a difficult task for each molecule of the fluorescent marker act as a secondary light source emitting in a solid angle of 360 degrees. As a result the spot of any fluorescent object appears larger than the shadow that it displays on an image obtained with absorption techniques. Furthermore the limits of the fluorescent spot are more difficult to determine automatically due to a lack of sharp change in the intensity profile of the spot. Of course this problem is more important when images are recorded with a low magnifying objective, which is generally the case when people want to harvest data on a large number of cells. In that case it is necessary to have an accurate but also automatic protocol allowing to standardize the way that the surface of fluorescent objects are measured. The problem has been previously addressed by suggesting different mathematical protocols allowing an easier determination of the convenient thresholding level. In this paper are suggested two empirical solutions, one usable for the nuclei and the other one for the whole cell delineation. Of course these protocols are usable only when the fluorescent images have been corrected from all the potential distortions introduced by the equipment.

Bioluminescence microscopy can be used to measure intracellular cofactors and ionic concentrations (Ca²⁺, K⁺, ATP, NADH), as an alternative to micro- spectrophotometry and micro-fluorimetry, due to the development of sensitive detectors (cooled photomultipliers tubes and CCD). The main limitation comes from the very small and brief intensity of the emitted light. Our instrumentation based on an inverted microscope, equipped with high aperture immersion lenses is presented. Light intensity measurements are carried out through a photomultiplier sorted for low dark current and cooled at -5 degree(s)C to reduce thermal noise. Our first aim is to quantify ATP on single living cells using the firefly luciferin-luciferase couple. Experimental and kinetic aspects are presented to emphasize the potentialities of the technique.

The last 20 years have seen an unexpected great renaissance and a partial revolution in light microscopy. This recent progress is due to new design in optics and instrumentation as well as improvement of optical contrast enhancement techniques. Recent progress in fluorescence microscopy is achieved by multiparameter fluorescence techniques, by improvement of conventional photomicrography as well as by optoelectronic imaging, confocal laser scanning microscopy, image processing and analysis. Due to the increase in number of fluorescence dyes, double and triple bandpass filter sets permit a rapid changeover between different fluorochromes simultaneously.

To study the 3D-organization and 3D-pathology of the genome in intact cell nuclei, precise and accurate 3D-object localizations and 3D-distance measurements of fluorescent labelled chromatin-nanostructures are required. For this purpose, a high precision fluorescence microscope setup with spatially modulated excitation (SME) has been built up combining advantages of an epifluorescence microscope with interferometric laser illumination in the direction of the optical axis and optical sectioning. The SME allows high precision localization in the nanometer range resulting in a considerable increase of the axial resolution equivalent. This is shown for a configuration of five fluorescent microspheres of 100 nm diameter. Since the optical sections are acquired in an epifluorescent mode, image analysis procedures of high precision spectral distance microscopy were applied to determine the lateral particle localization by the position of the bary center of intensity. From these data, relative axial distances as well as relative 3D-distances were calculated. The results indicate that distance measurements between fluorescent objects can be performed with an accuracy of more than one order of magnitude better than the lateral epifluorescent optical resolution given the full width half maximum of the central peak of the effective point spread function. Since the definition of the optical resolution in refraction limited optical systems is based on distance measurement, the measure for the accuracy of our results in precision distance calculation is called resolution equivalent.

The intention and first results of a research project called DELAS (diagnostic endo laser scanner) are presented. This paper is focused on the technology development of a scanning imaging method for endoscopy. This work is granted by the Bundesministerium fur Bildung, Wissenschaft, Forschung und Technologie (BMBF, FKZ 13 N 6796). The current endoscopic imaging technique is restricted to the visible spectral range. An extension of the usable wavelength range leads to new application fields of endoscopes, such as the use of fluorescence or other spectroscopic methods, e.g. to perform diagnostic investigations. The aim of the project is to investigate the feasibility of a scanning imaging technique on the basis of microsystem technology. The optical components are realized by using lithographic methods in addition to single fibers and microscanning mirrors being applied to avoid the spectral limitations of conventional image guides. The integration of the microscanning device into an imaging setup of an endoscope is planned in a further project part. Initially, an experimental setup using conventional components has to be realized in order to simulate a situation considering the requirements of an endoscopic utilization. Using this setup the verification of the imaging method regarding potential applications and an improvement of the imaging parameters are performed. These investigations lead to specific requirements of a microscanning device for a future prototype manufactured by using silicon technologies. First results of the investigations are discussed in terms of lateral and depth resolution, image distortion, and non linear behavior of the scanning mirrors. They are compared to the results obtained by conventional microscopy and evaluated with regard to the requirements of diagnostic applications.

In comparison with optical tweezers using near infrared lasers, red emitting high power laser diodes ((lambda) equals 670 - 680 nm) may offer several advantages, such as easier adjustment, smaller beam diameter in the focal plane and low absorption of optical radiation. One-photon absorption by water or two-photon absorption by various cellular components may induce cell killing by heat or by photochemical reactions. To test cell viability, cultivated CHO cells were exposed to various light doses of this wavelength as well as to a moderate light dose of a conventional Nd:YAG laser. Loss of clonogenicity of about 50% as well as a reduction of cell growth were measured at 680 nm when using a maximum light dose of 2.4 GJ/cm². In contrast, no cell damage was found at 670 nm as well as at 1064 nm when using a light dose of 340 MJ/cm², which revealed to be sufficient for several applications.

Rat adrenal glands exhibit an intense mTHPC-induced fluorescence. The objective of our study was the identification of adrenal cells exhibiting mTHPC-induced fluorescence under normal conditions and under stimulation of adrenal proliferation by reserpine. Furthermore mTHPC-uptake of rat pheochromocytoma (PC 12) cells was investigated. Four male Wistar rats received 0.5 mg mTHPC/kg iv 48 hours before perfusion. Furthermore four rats received reserpine (2 mg/kg im od), bromo-deoxy-uridine (BrdU; 50 mg/kg ip od) each for one week and mTHPC (0.5 mg/kg) 48 hours before perfusion. BrdU was detected immunohistochemically. PC 12-cells were incubated with 0.5 mg mTHPC/l culture medium for 24 or 48 hours. Cells and tissues were examined by fluorescence microscopy. The adrenal cortex exhibited an intense mTHPC-induced fluorescence. The adrenal medulla fluoresced faintly. Reserpine increased fluorescence of intramedullary cells, not coinciding with adrenal proliferation. Cortical fluorescence remained unchanged. PC 12-cells lying singly or in small groups and differentiating cells showed a more intense mTHPC- induced fluorescence than confluent cells. Differences of cortical and medullary uptake of mTHPC are independent of proliferation and may be explained by lipophilia of mTHPC, since adrenocytes have an uptake mechanism for cholesterol. The difference of mTHPC-uptake between PC 12-cells and chromaffin cells implicate the possibility of photodynamic applications for medullary neoplasia.

A Fourier transform spectrometer is used to simultaneously measure thickness, dispersion and absorption spectrum of a sample. It is shown that short coherence interferometry has the potential to measure the three dimensional distribution of the structure, the spectral absorption and dispersion of a sample.

The FT-IR-microspectroscopic mapping technique in combination with image construction methods has been used to characterize thin tissue sections from human melanoma. IR mapping spectroscopy as a spatially two-dimensional working technique is a non-invasive analytical method. Up to now IR-imaging has been based on distinct spectral parameters such as frequency, intensity or fullwidth of half maximum, ratio of single wavelengths and so on. We decided to compare this technique with other chemometric methods and to secure that these parameters will not give unsatisfactory results. For this purpose pattern recognition analysis (PRA) e.g. principal component analysis (PCA) or artificial neural networks (ANN) of IR-data, confirmed with standard histopathological techniques, has turned out to be helpful to discriminate also between different tissues.

Investigation of intracellular dynamics of Allium cepa inner epidermal cells are described. The applicability of the method for quantitative estimation of spatio-temporal phase fluctuations and the effect due to external factors is discussed. The analysis of time-sampled series allows one to detect the regions of various motility in cytoplasm. The intense Fourier-spectra harmonics in 0.2 - 8 Hz interval were observed inside a cell wall and cytoplasm. Regularly spaced 2- to 4-s long batches of 100-ms pulses at cell-wall sites are recorded. The phase-fluctuation intensity decreased and the frequencies of certain harmonics were shifted with lowering temperature. The advantages and specific features of the method are discussed.

In our biophysical laboratory has been developed a new scanned probe microscope (SPM) for biological application. The SPM allows to investigate a biological samples' surface by means of three different near field microscopes: scanning tunneling microscope (STM), atomic force microscope (AFM) and near field scanning optical microscope (NSOM). The SPM is very rigid and can be operated in ordinary laboratory without any vibration isolation. The scanning area of the microscope is about 10 by 10 micrometers. Some different biological objects were visualized by means of the SPM viz. bacteria (E. Coli, plague, cholera, staphylococcus), macromolecules (DNA, plague proteins) and phage (T2).

A new near field light microscope with SEM light generation (NFLEM) has been developed on the basis of modified SEM (Hitachi HU-12A). The scanning electron beam of the SEM passed through thin luminescent film coating a sample generates a small light spot. The light transmitted through the sample is registered by PMT. The wavelength of the light can be easily modified (from infrared to x-ray) by changing the luminescent film. The images of test objects (latex 0.1 micrometer, thin metal films) are obtained by the microscope.

Whole bacteria as well as thin sections were investigated in our laboratory by means of near field scanning optical microscope (NSOM). The main problem in NSOM operation is a control of distance between microscopy tip and sample surface. The bacteria thin section is a more preferable sample for NSOM investigation because of its flat surface. For increasing of thin sections' image contrast were used different light microscopy stainers (Eosin, Hematoxylin, etc.). We obtained images of thin sections of plague (Y. Pestis EV) and cholera (V. Cholerae). Lateral resolution in the investigation is about 300 angstroms.

Applications of scanning near field optical microscopy to the imaging and the structuring of biomolecules is presented. The paper introduces the technique and presents images of a test grating, showing the potential of the technique for subwavelength imaging in air as well as in water. A sub- micrometer size fiber tip characterized by its high transmission is presented as an interesting probe for near field fluorescence microscopy. The potential of the technique is exploited to detect bioactive molecules that have been marked with a fluorescent probe and photoimmobilized onto a glass substrate using near field UV-activation of a photolinker.

A tamoxifen resistant cell line (MCF7^TAM) was established from tamoxifen sensitive MCF-7 human adenocarcinoma cells expressing estrogen receptors. The resistant cell line was found to express estrogen receptors to similar level as the parent cell line but the receptors were found to be altered, having lost their ability to bind estradiol or tamoxifen. The fluorescence of eosin-tamoxifen ionic association was used to investigate intracellular location of tamoxifen in both sensitive and resistant cell lines. Fluorescence emission spectra of eosin, tamoxifen and eosin-tamoxifen complex ((lambda) _exc equals 480 nm) were analyzed and showed that maximal fluorescence intensity of the complex ((lambda) _em equals 540 nm) was four times higher than that of eosin alone while tamoxifen alone did not emit any fluorescence in this spectral range. In MCF-7 cells, tamoxifen was found to be diffusively located in the cytoplasm and nuclear fluorescence intensity was significantly lower. No difference was observed in fluorescence intensity or location in tamoxifen resistant cells, although it has been previously correlated with clinical responsiveness. Improvement of this fluorescence microscopy methodology appears necessary to provide accurate results taking into account the complexity of tamoxifen resistance molecular pathways.

We have applied Raman spectroscopy to the study of the distribution within cells of photosensitizing compounds designed for use in photodynamic therapy. A human endothelial hybridoma cell-line was incubated in the presence of substituted zinc phthalocyanines, with the cells subsequently fixed in formalin. Microscopic Raman analysis was performed using 782 nm excitation in order to avoid photosensitizer absorption, thereby minimizing both the background fluorescence signal and the generation of cytotoxic species. A method of Raman mapping is described and we demonstrate that it is possible to identify the distribution of photosensitizer within the cell. The results indicate that sub-cellular photosensitizer localization is dependent upon both molecular structure and incubation time.

Near-infrared Raman microscopy was used to study normal, adenomatous and carcinomatous colon tissue in vitro. Samples were fixed in formalin and it was shown that this procedure did not alter the observed Raman peaks of the tissue, and is therefore a suitable method of fixation. The samples were observed visually to be of varying shades ranging from cream to dark brown; this was independent of whether the samples were cancerous or normal. Differences were seen between the Raman spectra of the different colored tissue with the spectra of brown tissue displaying a greater number of resolved peaks. We believe these differences may be due to differing amounts of blood present in the tissue. This must be considered when using Raman spectra for diagnostic work to ensure that differences ascribed to cancerous changes are not in fact due to differing amounts of blood within the sample.

Three-day-old chick embryos were exposed intra-amniotically to bromodeoxyuridine within the range of teratogenic doses. Using comet assay, a significant damage of DNA was demonstrated in blood cells 3 h after the treatment. While the damage seemed to be partially repaired within 12 h, new peak of DNA fragmentation detected on incubation day 4 implied an apoptotic elimination of impaired cells. More frequent occurrence of macrophages in blood samples from BrdU treated embryos supports this assumption. The differentiating blood cells, however, did not exhibit any remarkable injury of cytoskeleton biogenesis. Nevertheless, an improved experimental procedure revealed the existence of intermediate 'wreath' stage preceding the consolidation of tubulin bundles into marginal band of chicken erythroblasts already within the course of embryonic period. The more, even the mature cells of primitive erhthroid series retained the visible bundles of radial microtubules attached to MTOC. Actin labeling disclosed in many primitive erythroblasts the special lace arrangement of microfilaments growing from nucleus surface while the rest of cells exhibited only a diffuse staining through cytoplasm, concentrated sometimes in area of marginal band. Such distribution was characteristic for mature form of primitive and definitive erythrocytes. The expression of vimentin in erythroid cells was very weak and quite different from patterns of adult definitive erythrocytes. The labeling was noticed only around the nucleus till incubation day 10 when implication of fiber growth through cytoplasm was detected. Conventional hematological analysis performed on incubation day 10 revealed in blood of BrdU treated embryos the lower incidence of definitive erythrocytes in favor of immature forms resulting probably from death of cells in consequence of primary DNA damage. Such effect could be associated with development of myelodysplastic syndrome in later life.