We present a new technique, time domain opto-thermal FTIR spectroscopy, designed to perform non-contacting, remote sensing absorption spectrum measurements on opaque samples. Measurements on homogeneous and layered samples are presented to illustrate the capabilities of the technique and its potential for in-vivo skin research.

A light imaging system is being developed which can at this time detect and characterize small lesions on the surface and deep inside a tooth or tissue. It depends on the existence of light photons which can fully traverse a tooth in a straight path and therefore can make -- similar to x rays -- a clear shadow of a pathology on the receiver. To observe the very weak, transmitted, unscattered light rays a special patented system has been developed using collimated, narrow light beams and a highly sensitive, post collimated receiver which are rasterscanned over the entire sample. The image can be transferred in analog form to a film or observed after digitization on a 256 grayshade monitor. The light image can be observed in a regular or slower high sensitivity mode.

An automated image analysis system with two imaging regimes is described. Photothermal (PT) effect is used for imaging of a temperature field or absorption structure of the sample (the cell) with high sensitivity and spatial resolution. In a cell study PT-technique enables imaging of live non-stained cells, and the monitoring of the cell shape/structure. The system includes a dual laser illumination unit coupled to a conventional optical microscope. A sample chamber provides automated or manual loading of up to 3 samples and cell positioning. For image detection a 256 X 256 10-bit CCD-camera is used. The lasers, scanning stage, and camera are controlled by PC. The system provides optical (transmitted light) image, probe laser optical image, and PT-image acquisition. Operation rate is 1 - 1.5 sec per cell for a cycle: cell positioning -- 3 images acquisition -- image parameters calculation. A special database provides image/parameters storage, presentation, and cell diagnostic according to quantitative image parameters. The described system has been tested during live and stained blood cell studies. PT-images of the cells have been used for cell differentiation. In experiments with the red blood cells (RBC) that originate from normal and anaemia blood parameters for disease differentiation have been found. For white blood cells in PT-images the details of cell structure have found that absent in their optical images.

The cornea is the most refractive element in the eye. Its refractive power is about 70% of the power of the whole eye. The shape of the cornea is aspheric, and almost always has no rotational symmetry. Even small surface irregularities can cause a perceptible reduction in visual acuity. Standard methods for evaluation of the corneal topography used in clinical practice include keratometry, photokeratoscopy, and computer assisted videokeratography. All of these methods used the principles of geometrical optics, and their accuracy is about 0.25 D. An application of interference phenomenon's to examine the corneal contour map significantly increase the accuracy. Using the interferometric inspection of the corneal shape one can easily observe the fine corneal topography, the fast, dynamic changes of the corneal surface, and the topology of the tear film and its irregularities. The paper presents the Twyman Green interferometer, used in experiments, an example of sequence of interferograms and their 3D presentations.

Among the new possibilities offered by an endoholographic method, the vision in turbid media could facilitate the surgical interventions, in particular in vessels, where blood masks the vascular wall. The holographic process allows us to select the coherent light, used to form the image of an object embedded in a turbid medium. An in situ holographic technique based on the use of a flexible miniaturized endoscope (diameter less than 1 mm) coupled to a CCD camera, to record the hologram, was developed for medical applications. The hologram is formed, by reflection, on the tip of a multicore optical fiber (MCF), sampled, and then treated electronically. The image is reconstructed numerically, providing more flexibility to the holographic process. We present here the first experimental results obtained with this imaging system, tested in vitro with conditions matching the typical situations encountered in endoscopy. The possibility of extracting an image out of the ambient noise, produced by the diffusers present in the turbid medium, is described and analyzed.

Electronic speckle pattern interferometry (ESPI) is a laser based technique developed for the study of stresses and strains in complex structures. This method consists of the projection of two laser beams onto the object of study. This produces a visible pattern that is recorded by a video camera. When the structure is stressed in a jig, the pattern changes depending on the in- plane and out-of-plane deformation and movement of the surface observed. This allows us to qualify and quantify strain in the whole area under study, in real time. We describe the method of application of ESPI to the study of the biomechanics of the proximal femur, before and after the implantation of the femoral component of a total hip replacement in cadaveric femora, under physiological loads and tensions, and report our preliminary results. The information obtained with this technique should provide a better understanding of the biomechanics of bone after joint replacement.

Recent advances in optical techniques for vibration measurement have made it feasible to purchase commercially available laser Doppler velocimeters that, in combination with a light microscope, are capable of detecting velocities of poorly reflecting biological materials down to fractions of a micrometers s^-1. Thus, in our particular application, the aim is to understand the micromechanics of the inner ear, particularly in the nanometer region where the ear is first able to detect sound. With currently available devices, it is now possible to make the necessary vibration measurements without need of introducing reflecting materials onto the object of interest. This not only avoids inertial loading, but also allows focusing through cellular layers onto otherwise inaccessible structures. Here we present the results of micromechanical experiments for the in vitro inner ear of the guinea pig, the most commonly used model in auditory physiology. Our experiments concentrate on the third and fourth cochlear turns because in this region the different cellular structures of the organ of Corti are optically accessible with present technology. The mechanical responses of all examined structures, including the basilar membrane, were equally frequency selective, but linear below 90 dB SPL, suggesting that active mechanical tuning is not as pronounced in the apex as in the base of the cochlea. This has important consequences for signal processing in the inner ear.

It is generally considered that in coupling the motion of the tympanic membrane to the incus, the malleus behaves as a rigid body. To investigate the bending of the manubrium, its vibrations were measured with an interferometer in response to sound applied to the ear at a large number of observation points along its center line. Measurements at an arbitrary position are now possible due to high interferometer sensitivity. The x, y, and z coordinates of the observation points were measured with optical scales incorporated in the positioning system. The displacements are plotted as a function of position along the manubrium at seven phases (30 degree(s) apart throughout a half cycle). At low frequencies the bending is moderate and near the tip. In portions in the mid and high frequencies the bending is quite strong and extends to the middle region of the manubrium or is absent and the extreme lower edge of the malleus tip does not seem to vibrate solidly with the rest of the manubrium.

Basically, other information becomes available when, instead of local slope, local height is measured. In contradistinction to the data obtained from Placido based systems, ambiguity can be avoided in the height data obtained from oblique projected grids on a diffusely reflecting surface, e.g., with an adapted set-up, discrimination between convex and concave surfaces is possible. We made a corneal topographer based on sodium-fluorescein installation in the precorneal tearfilm for obtaining a diffusely radiating surface. The local information available using a height measuring system, however, sometimes deviates from global information a.o. due to tearfilm breakup. This breakup may be controlled by applying artificial tear products. These products however, may influence the tearfilm thickness. With in vitro measurements we also obtained information about the thickness of a natural tearfilm that turned out to be at least several tens of micrometers s rather than 7 to 10 micrometers as is given in physiological handbooks. In this paper also a microslit-projection and observation method for direct tearlayer thickness measurements is described. The aim of this research is to investigate the maximum obtainable accuracy of measurements done in vivo and to optimize the sodium-fluorescein installation with respect to absorption of the excitation light and the fluorescent yield with minimum distortion of the natural corneal tearlayer.

With the use of corneal measuring devices there has been the need to describe the cornea in a simple fashion. The most commonly used method is the radius of curvature whereas the corneal surface is modeled, based on spherical assumptions. Basically two types of radii of curvature are used, axial and instantaneous. Both have their own advantages and drawbacks. Another method of corneal description is to use true topographical shape. From this topography, parameters are calculated using shape-matching in the form of best-fit sphere, B- spline approximation, or other general 3D approximation functions. Accuracy depends highly on the number of points used in matching and the degree of match-function. Using 3D approximation in a clinical environment is only possible if one knows what a normal cornea looks like. Since this is not known exactly, it is impossible to specify the correct matching- function at present. To proof clinical relevance in any measurement, results in 3D mapping have to be developed starting with thorough research in what does the cornea look like and what abnormalities do we want to detect for clinical use.

The new interferometric method for investigation of the corneal surface is presented. Fundamentals of the proposed method, based on dual-wavefront illumination, are explained and the experimental set-up for practical realization is described. This technique can be suitable for measurement of the corneal surface in vivo.

The results are presented on investigations to determine the optimum and most sensitive to a change in the surface blood flow informative parameters of the spectrum of the dynamic speckle-field formed by the laser radiation scattered from the skin surface. It has been found that along with the value of change in the ratio of the spectral power density at high and low frequencies, the medium frequency of the spectrum, the spectrum asymmetry about the medium frequency and the ratio of the medium frequency to asymmetry strongly depend on the functional state of the skin and, consequently, on the state of microhaemodynamics in a given portion of the skin. Maps of the distribution of micro-circulations in the investigated portion of the skin are given.

The analysis of statistical and correlation properties of speckle patterns formed during different skin tissue scanning by the sharply focused probing laser beam has been carried out. The influences of the biotissues' structural features on the speckle patterns formation under Gaussian beam illumination have been investigated. The relationships between the structural characteristics of the sample under study, Rayleigh range of the probing beam and normalized statistical moments of the speckle intensity (contrast and asymmetry coefficient) are discussed for the different scatterer models. A phenomenological model of speckle pattern formation for the large-scale scatterers allows us to explain the dependence of speckle contrast and the coefficient of asymmetry on the generalized structure parameters and illumination conditions for the samples under study. The experimental investigations of the human skin structure features have been carried out using two types of the tissue samples by means of coherent scanning microscopy (CSM). Firstly, D-SQUAME discs (CuDerm Corporation, Texas, USA) have been used for the evaluation of skin dryness level. Secondly, the samples under study were the thin layers of normal and psoriatic epidermis (skin strippings). The dependencies of contrast and coefficient of asymmetry on the beam defocusing parameter and 2D correlation functions of speckle pattern intensity have been analyzed for different zones on the biotissue's surface. Particularly, promising results in skin dryness studies (using D-SQUAME discs) have been obtained. Our results and conventional 5-pattern kit scale are in good agreement. So, the presented method is accurate and objective and may be useful in novel cosmetic research and development.

The aggregational properties of erythrocytes in whole blood are studied by backscattering nephelometry during their spontaneous aggregation and disaggregation in vitro. As the process of aggregate formation develops from pair via linear to 3-D aggregates the parameters of the aggregation kinetics are uniquely related to the aggregate morphology although the mechanisms of aggregation under pathologies are not quite clear. Different manifestations of disaggregation processes registered by the backscattering technique show that the same level of light scattering can correspond to different morphological states of aggregates and their behavior in shear flow. The variety of disaggregation kinetics obtained from different samples reflects different features of alteration of aggregational properties of erythrocytes and hydrodynamic behavior of aggregates under shear stress in the Couette flow. In case of some pathologies we observe different disaggregation effects such as the increase of aggregation at small shear rates (2.5 sec^-1 in our set-up), the transitional processes of disaggregation at stepwise increase of shear rate, aggregate destruction with possible increase of durability of 3-D aggregates or that of small aggregates, the hysteresis loops at direct and reverse change of shear rate. These effects are sensitive to the volume concentration of erythrocytes.

Based on the study of speckles polarization for multi-effect simulated biotissue, a new phenomenon called speckle-moire is created within a single diffraction halo of a laser specklegram. This is made by controlling the correlation between selected speckle pairs within a multiexposure specklegram via the polarization properties of laser speckles. This phenomenon can be used to measure the difference between two displacements or deformations processes, such as, in the field of speckle metrology for biomedical science.

The possibility of using strongly focused coherent fields diffraction for cardiovibration measurements is considered on the basis of a numerical modeling of light scattering by vibrating rough surface. The optical scheme for cardiovibration measurements is suggested. The forming of the output signal of the measuring system has been studied. The region of parameters when the system considered operates in the linear regime has been determined. The influence of profile statistics on investigated system output characteristics has been analyzed.

There are many methods of testing blood parameters. The light scattered by blood cells forms the diffraction picture which contains all the information about number, size, and form of blood cells. Measuring the parameters of the spatial spectrum gives one the possibility to get the necessary data. To record the spectrum a computer aided CCD-camera is used. The scattering spectrum can be observed as a process of interference of beams refracted and reflected on the blood cell surfaces, interference of waves scattered by cells themselves many times. A typical spectrum looks like a system of bright and dark circles. The total area under the curve plotted from scanning the spectrum gives us an idea of the number of blood cells on the illuminated area of preparation. The diameter of first and following circles gives us information about size distribution of the cells. We have compared the spectrum of the blood preparations of healthy people and patients who passed through stress and shock. We propose that this method can be one of the quickest and easiest methods of blood-cell analysis.

For the first time the method of the second harmonic generation was used to study the photo- and electrically induced nonlinear optical transformations in thin oriented films of purple membranes (PM). Variations of the film nonlinear susceptibility were investigated as the bacteriorhodopsin (bR) molecule underwent the cycle of photoinduced transformations for both dry electrically oriented films and bR molecules embedded into poly(vinyl alcohol) matrix. The electrically induced changes of the nonlinear optical properties were studied for the electrostatic field strength up to the values 4 (DOT) 10⁴ V/cm. Nonlinear susceptibilities of oriented and nonoriented dried PM films are compared.

The modulation polarimeter with an accuracy of 0.05% for single measurements has been created. It is made by using control electro-optical wave plate as a polarization converter in probing and receiving channels. This makes it possible to fully automate processes of measurement and calibration of a polarimeter. It also makes available the operational elasticity of the polarimeter. Measurement time reached a few microseconds. The exception of an optical field background component on measurement results without use of an interference filter is a distinction of this polarimeter. It is possible due to intensity modulation of optical radiation in the polarimeter probing channel with simultaneous modulation of polarization.

The special laser diode device (LDD) leasing in the near infrared region (IR) with two wavelengths: (lambda) ₁ equals 850 nm and (lambda) ₂ equals 1000 nm, designed for laser therapy, is presented. This device is characterized by a unique feature, namely a separate built-in illuminator, operating in 670 nm. The special construction of LDD and the illuminator enables the user to visualize exactly the surface irradiated by IR radiation. The exposure time and the output of laser power are also controlled and can be displayed on the LED monitor at the front panel. This new device, described here, is compact, low cost, and user friendly.

We proposed the usage of two-projection moire technique for contactless non-invasive investigations of the spine's shape in norm and pathology. The results of investigations for healthy people and for patients having vertebrogenic pathology are presented in the paper. The quantitative interpretation of the obtained moire topograms has been performed. The three- dimensional profiles of the spine surface have been drawn. It is shown, that the suggested method may be used both for screening investigations of various population groups, and to estimate the treatment efficiency of patients with vertebrogenic pathology.

A novel analytical spectral-imaging system and its results in the examination of biological specimens are presented. The SpectraCube 1000 system measures the transmission, absorbance, or fluorescence spectra of images studied by light microscopy. The system is based on an interferometer combined with a CCD camera, enabling measurement of the interferogram for each pixel constructing the image. Fourier transformation of the interferograms derives pixel by pixel spectra for 170 X 170 pixels of the image. A special `similarity mapping' program has been developed, enabling comparisons of spectral algorithms of all the spatial and spectral information measured by the system in the image. By comparing the spectrum of each pixel in the specimen with a selected reference spectrum (similarity mapping), there is a depiction of the spatial distribution of macromolecules possessing the characteristics of the reference spectrum. The system has been applied to analyses of bone marrow blood cells as well as fluorescent specimens, and has revealed information which could not be unveiled by other techniques. Similarity mapping has enabled visualization of fine details of chromatin packing in the nucleus of cells and other cytoplasmic compartments. Fluorescence analysis by the system has enabled the determination of porphyrin concentrations and distribution in cytoplasmic organelles of living cells.

NADH is an indispensable mediator of energy metabolism in cells. By laser fluorescence spectroscopy with an experimental setup we studied the influence of different concentrations of NaCN solutions on the NADH fluorescence intensity of endothelial cell cultures of the calf aorta (BKz-7). The results obtained are discussed against the background of published cytotoxicity studies of cyanides with endothelial cell cultures.

Photoinduced modifications of NAD(P)H attributed autofluorescence of CHO cells in a single- beam gradient force optical trap (optical tweezers) were studied. Fluorescence spectra of single cells in the optical trap were measured using a modified microscope with an IR microbeam at 1064 and 760 nm for trapping, UVA radiation at 365 nm for fluorescence excitation, and an optical multichannel analyzer for spectral recording. No strong effect of the 1064 nm trapping beam on fluorescence intensity and spectral characteristics was found, even for power densities up to 70 MW/cm². In contrast, 760 nm microirradiation resulted in a significant fluorescence increase, probably indicating cell damage due to absorption by heme- containing molecules. UVA exposure (1 W/cm²) of the trapped cells generated within seconds an initial fluorescence decrease, followed by a significant increase up to 5X of the value prior to irradiation. The UVA-induced modifications reflect NAD(P)H auto-oxidation and irreversible cell damage due to oxidative stress.

The basic principle of this approach relies on microspectrofluorometric observations of upheavals in the cell's energy metabolism and cell-to-cell metabolic communication in human and mouse melanoma cells. A striking feature is the definition of a highly active nuclear energy metabolism in M8255 human melanoma cells which is characterized by an intense fluorescence response associated with NAD(P) reduction by substrates of glycolysis or the hexose monophosphate shunt. Changes are also expected in the steady state levels of reduced/oxidized NAD(P) in the nuclear, cytoplasmic and mitochondrial compartments, which are probably dependent on ATP levels and distribution (as determined by cell metabolism and eventually the presence of ATP traps). A topographic scanning of skin lesions, either under metabolic steady state conditions or in the presence of permeating substrates, can lead to the recognition of characteristic patterns associated with pigmented and nonpigmented, malignant and nonmalignant skin lesions. The method is, in a way, an extension of microscopic transillumination techniques which have led to the identification of specific patterns associated with such lesions. However, here, a new dimension is added by introduction of fluorescence evaluations. This can represent the first step in a multiparameter approach to the non-invasive in situ fluorescence scan of dermatological lesions by inclusion of: (1) fluorescence excitation and emission spectra; (2) new fluorescence probes of cytoplasmic organelles and nuclear components. Primary emphasis should be placed on the highly active nuclear energy metabolism, which can be triggered to maximum levels when the role of mitochondria as the `cells's policeman' with regard to metabolic control is suppressed by use of topically cytotoxic agents such as the `antipsoriatic' anthralin and dicarboxylic acids used in the local treatment of melanoma. Fluorescence excitation spectroscopy may be of particular advantage in studies with the new highly sensitive cyanine nucleic acid dyes and their dimers, but caution should be exerted in the use of such compounds because of cytotoxicity (e.g., limiting it to cellular studies used in the interpretation of dermatological findings). Parallel cellular and non-invasive dermatological studies will help to define the most specific set of parameters to be used in diagnostic and prognostic evaluations of skin lesions.

A dye pair characterized by favorable spectral properties allows a simplified analytical procedure, based on the measurements of both donor and acceptor emission in double-stained cytological samples, to be applied to evaluate both the relative efficiency of the energy transfer (FRET) process and its topological distribution. Propidium Iodide, a DNA intercalating agent, has been used in combination with Hoechst 33258, a non-intercalating dye specific for A-T sequences of DNA, to assess the chromatin arrangement in human fibroblasts in both quiescent (G0) and cycling (G1) phases. The results indicate that the cells in the two phases, that cannot be distinguished on the basis of the DNA content, exhibit differences of the FRET efficiency relative value that can be ascribed to chromatin structure modification related to gene activation processes.

Intracellular modifications of anionic photosensitizers during PDT were observed, dependent on the growth phase of the incubated cells. In addition to steady-state spectroscopy, time- resolved fluorescence spectra between different time gates were measured. Comparative results were obtained for the sensitizers meso-tetra(4-sulfonatophenyl)porphyrine (TPPS₄) and aluminumsulfophthalocyanine (AlS_2-3Pc). In both cases relocalization of the sensitizer could be observed exclusively for cells in the growing phase, whereas no relocalization was detected for cells in the stationary phase. Moreover, time-gated spectra were different for the two cell types. The formation of a fluorescent band around 615 nm during PDT for the sensitizer TPPS₄ was observed mainly for cells in the growing phase. This species was correlated with a short fluorescence decay time (< 5 ns). Similar observations were done with AlS_2-3Pc. A fluorescence band blue-shifted in the same order of magnitude (about 30 nm from the original band) and measured predominantly between an `early' time-gate (0-5 ns) was detected for cells in the growing phase.

Phosphorescent metalloporphyrins have evoked interest in the field of bioaffinity assays as sensitive labels because the time-resolved detection of phosphorescence is a very efficient method for background rejection. In this paper, the photoluminescence properties of the two major classes of metalloporphyrins are described, namely Pd- and Pt-porphyrin. Both dyes feature high quantum efficiency, low non-specific adsorption on different surfaces, and sufficient photochemical stability. The measurement of phosphorescence of Pt- and Pd- coproporphyrin labeled compounds from the surface of microparticles can be performed in wet or dry state. The phosphorescence of the label was determined using a microscope equipped with a CCD camera with time-resolved phosphorometric detection principle. Different mounting media, particularly those of non-aqueous mountants forming polymerized matrix, are suitable for phosphorescence microscopy analysis. It is possible to measure the phosphorescence signal in the presence of oxygen.

Ion concentrations in biological cells are widely studied with fluorescent probes. The probes have a high selectivity for specific ions and exhibit marked changes in their photophysical properties upon binding ions. The fluorescence decay behavior of the probes in the presence of ions can now be used as a contrast mechanism for imaging purposes. This technique can be further exploited for the quantitative determination of ion concentrations within living cells. Here we describe the fluorescence lifetime properties of the free calcium probe CalciumGreen and the pH probe carboxy SNAFL-1. The potential of fluorescence lifetime imaging is illustrated by the imaging of Ca²⁺ concentrations and pH in single cells. In the case of the emission ratio probe c.SNAFL-1, it was possible to determine the pH in the same cell using both the ratio and the fluorescence lifetime method. It turns out that no cumbersome in vivo calibration procedure is required when c.SNAFL-1 is used for quantitative fluorescence lifetime imaging of pH in single cells.

The intracellular accumulation of a variety of photosensitizers in human (non-nucleated) and chicken (nucleated) erythrocytes, as well as the photodynamically induced hemolysis were studied using 488 nm laser microirradiation (15 (mu) W, 100X) and confocal laser scanning fluorescence microscopy. Cells incubated with the negatively charged hydrophilic compounds TPPS₄ and Pd-TPPS₄ exhibited no significant fluorescence before irradiation, but developed strong fluorescence in the cellular and nuclear membranes following photoinduced membrane damage. In contrast, microirradiation of Photofrin-incubated erythrocytes showed instantaneous fluorescence which decreased due to photodegradation. For the cationic, hydrophilic dye Methylene Blue, significant fluorescence was detected in the nucleus only. Following ALA incubation, large intercellular differences were observed in fluorescence in the red spectral region. These differences are probably due to the differential ability of individual erythrocytes to biosynthesize protoporphyrin IX. Photofrin was the most efficient photosensitizer to induce hemolysis. Higher radiant exposures were required for lysis of nucleated than of human red blood cells, except in the case of Methylene Blue. Irradiation was more efficient for unwashed cell suspensions than for washed suspensions, indicating the non-negligible role of extracellular photosensitizing molecules.

The differentiation between discocytic and stomatocytic red blood cell (RBC) shape using conventional microscopic imaging and image analysis tools is still on a very poor level. A procedure to differentiate the degree of stomatocytic shape changes was developed. We obtained multiple microscopic images of the same RBCs settled on a human albumin coated cover slip. The images were acquired when the microscope objective was subsequently focused through the cell layer. At equidistant horizontal planes (z-axis) below, within, and above the microscopic focal plane the light intensity distribution was considered. Using a model based on light refraction, we calculated the intensity distribution of the planes which are out of focus. Using this tool we are able to differentiate RBC shapes precisely. On the other hand, using this model from and the light intensity distributions of different focal planes, we are able to reconstruct the shape of one single RBC located in the optical axis of the microscope.

Fourier transform infrared spectroscopy (FTIRS) is used in cancer research, involving normal and malignant cell cultures and tissue samples. Preliminary studies have been made by some of us using ATR technique with the aid of silver halide MIR fibers. In the present work parallel preliminary results are presented concerning microreflectance technique. We studied bladder tissue in three patients. Cell clusters were obtained by mechanical apposition on an AgCl window of surgical samples of two patients. Cancer tissue and normal mucosa specimens obtained from the third patient were sliced by a scalpel and immediately relied upon ZnSe window. Diagnosis was checked on a sample counterpart. A Bio-Rad FTS 40 spectrometer and attached microscope were used. We discuss only phosphate stretching bands (PSB) which have been proposed to be `cancer sensitive.' Our results show: (1) apposition samples give fairly reproducible spectra shape and PSB peak frequencies (symmetrical: 1084.6 +/- 1.9(SD) vs. 1085.1 +/- 0.6; asymmetrical: 1240.6 +/- 1.4 vs. 1242.2 +/- 1.0; all cm^-1 units); (2) spectra of thick tissue slices are more complex and irregular due to the presence of occasionally strong confounding bands (in normal mucosa), and apparently shifted in frequency; (3) tissue slices exhibit a significant (p < 0.02) difference in the distribution of energy within the window (running Wilkoxon test) -- from roughly 1080 cm^-1 to 1170 cm^-1 it is greater in malignant samples and vice versa from 1230 cm^-1 to 1310 cm^-1.

Laser Doppler microscopy is an efficient method of in vivo measurements of flow velocities in different biological objects. It is based on the registration of frequency shifts in light quasielastically scattered from particles moving in the flows. To study the embryonic development of the cardiac-vascular system in embryos of warm water fishes, embryos of Macropodus opercularis have been used. Doppler spectra from pulsatile blood flows in selected vessels and their changes in the process of ontogenesis have been registered. The recording of the successive spectra and their computer processing yield the varying dynamics of blood flows. Typical age dependencies of velocity patterns in the embryos are presented.

Recent advances in video microscopy in the field of cytopathology need to define in detail requirements for rigorous technical quality control of DNA analysis. This study deals with the influence of both quality and calibration of instrumentation on DNA content measurements. Quality control of instrumentation in stability, linearity, and shading estimations has been achieved for two SAMBA^TM2005 (ALCATEL-TITN-Answare/France) image analysis systems by using prototype reference standard slide and Feulgen-stained rat liver imprints. The accuracy of the various conditions of work has been expressed as the CV of integrated optical density (IOD) and varied from 1.5% to 5.4%. These results demonstrate the dependence of the accuracy of measurements on the conditions for calibration of the instrument. Moreover, this work shows that each system can be characterized by its specific answer and all the systems don't really offer acceptable conditions for nuclear DNA content determination.

The aim of this study is to demonstrate the technical requirements and performance of fluorescent quantization using image analysis. The quality of fluorescent measurements is largely dependent on the characteristics of instrumentation: fluorescence microscope, low light level camera, fluorescence standard reference, and protocols for image acquisition and measurements calibration. This study is focused on the problem of the efficiency of microscope and low light level cameras. Various types of cameras are tested (SIT C2400-08, intensified CCD C2400-80, and cooled CCD C4880 from Hamamatsu) with respect to stability, linearity, and shading properties. Results show that evaluation of fluorescent instrumentation cannot be reduced to the evaluation of camera properties, the intrinsic evaluation of the fluorescent sample has to be considered as well. Moreover, this work demonstrates the deficiency of usual corrective protocols to lead to reliable quantitative fluorescence analysis.

The main advantages of the computer-aided phase microscope (CPM) Airyscan which permits the user to observe the cell surface and internal structure consists in superresolution and the possibility of the dynamic processes registering. Immunocompetent cells in process of activation with different agents were studied. Several criterions for evaluation of the functional status of immunocompetent cells based on the shape changes were suggested on the basis of real-time immunology reaction monitoring. The perspectives of the proposed method for immunology testing are discussed.

The complex study of bone structures has been done by microscopy and x-ray diffraction methods. The x-ray diffraction pictures were fixed in the angle range from 8 min. to 150 degrees. The results of measurements show essential differences between Mn (normal moose) and Md (moose suffering from growth disturbances) bone structures in the initial state. These differences become considerable after the compressing deformation of bone tissue cuts. X-ray data add microscopy patterns essentially because they allow us to fix fine changes of bone structures.

Stress factors can be visualized reliably by measurements of motion dynamics. Spatial portraits and temporal diagrams of impulses from two different kinds of cells, Physarum and Dictyostelium discoideum, are considered in the present work. Large numbers of biological applications require development of quantitative measurements in the dimensional range of classical optics. In order to apply this, we used a 3D RM 600 autofocus profilometer with spatial resolution of 1 micrometers ². It allows measurement at the dynamical bands of 0.001 - 120 Hz. Perspectives and measured results as well as applications of another computer-aided microscopical technique are discussed.

A simple and easy to use holographic microscope interferometer (HMI) for biological and material science applications is described. The unit is based on an ordinary microscope accomplished by He-Ne laser, several optical elements, a photothermoplastic (PTP) recorder, and a CCD-camera. Blood and plant cells, as well as internal solid bodies defect images, are demonstrated. Characteristics and application of the unit are discussed.

In recent years, there has been an increasing interest for applications of fluorescence measurements to studies on many physiological mechanisms in living cells. However, few studies have taken advantage of DNA quantification by fluorometry for dynamic assessment of chromatin organization. This type of approach involves both optimal conditions for DNA staining and the use of several investigation methods such as flow cytometry, image cytometry, laser scanning confocal microscopy and spectral imaging. In this context, this report describes a stoichiometric method for nuclear DNA specific staining, using bisbenzimidazole Hoechst 33342 associated with verapamil, a calcium membrane channel blocker. This method makes it possible to follow variations of nuclear DNA content in cells that are maintained alive.

X-ray microscopy makes it possible to obtain images at a higher spatial resolution (about 20 nm) as compared to optical microscopy. Moreover, x-ray microscopy permits direct acquisition from the specimen in 2D or 3D mode, without any preparation step (staining, fixation, ...), which is not possible in electron microscopy. Here we present deblurring methods to restore images after the acquisition process. An additive Poisson noise is generated by the use of x rays and also contributes to image degradation. Our purpose is to analyze such noise and to restore images. Due to the optical properties of the Fresnel zone plate, we presently associate it to an optical circular lens as is done for the optical microscope. Here we use the Richardson Lucy algorithm to deconvolute. A first step is to observe results of restoration obtained on an image grating (a star pattern) with inner zones of dimensions near the resolution. The next step involves the suppression of noise effects arising from the deconvolution process. The characteristics of the noise after deconvolution are evaluated by Fourier analysis. These effects are eliminated by a filtering process in the Fourier spectrum. This filtering is applied on images with different signal to noise ratio (obtained after different time exposures), in order to compare results obtained on noisy images with long time exposure images.

Investigation of the inner ear is still the subject of basic research. The concept of an active filtering process in the mechano-electrical transfer mechanism in the inner ear (cochlea) was introduced. In this paper an optical approach detecting microvibrations is discussed. A fiber optic heterodyne interferometer with a miniaturized sensorhead was built. The fibers ensure flexibility and easy handling of the interferometer whereas the miniaturized sensorhead allows a non-invasive approach to the organ of Corti. In heterodyne interferometry as compared to classical interferometry two slightly different light frequencies for the reference and object beams are used. The vibration of the object is detected as a modulation of the phase of the detector signal. To obtain the information on the vibration two methods were analyzed and applied: namely measuring the amplitude of the sidebands in the spectrum of the modulated signal and secondly signal demodulation. It is possible to detect amplitudes down to 0.3 angstrom in a frequency range of 500 Hz to 50 kHz with a simulated object reflectivity of 0.02%.

The ultrastructural changes that occurred in CaD2 mouse mammary carcinoma at 1, 24, and 96 h after photodynamic therapy (PDT) with meso-tetra(m-hydroxyphenyl)porphyrin (m- THPP, 5 mg/kg b.w.) or its corresponding chlorin (m-THPC, 1 mg/kg b.w.) were investigated. The destruction of the tumors occurred at a faster rate and was more severe by m-THPC-PDT than m-THPP-PDT, although the dose of m-THPC used was 5 times lower than that of m-THPP. The ultrastructral alterations of the tumors after PDT with m-THPP or m-THPC were qualitatively similar: direct damage to both microvasculature and neoplastic cells of the tumors.

Mechanisms of ligand binding by monoclonal anti-coproporphyrin antibodies are studied by steady-state and time-resolved fluorescence spectroscopy by use of a picosecond laser system. The antibodies quench the coproporphyrin (CP) fluorescence, but the CP fluorescence spectra show a strong shift of maxima at high concentrations of antibodies (Ab) or their Fab fragment. This can be explained by a special type of Ab or Fab dimerization. Fluorescence decays of CP are measured at different concentrations of Ab and different pH values. The following deconvolution procedure based on the non-linear least squares method reveals a two- exponential character of the fluorescence decay. Data obtained by polarized fluorescence spectroscopy allows us to attribute the slow component (16 ns) to the free CP and the fast one (2.5 ns) to the CP-Ab complex. The obtained dependence of the ratio of amplitudes of the fast and slow components on the ratio of concentrations of CP and Ab at different pH values makes it possible to register separately the binding at different centers within the Ab and investigation of the effects of cooperation in the Ab molecule. The following experiments with Pt-coproporphyrin reveal the ability of Ab to reduce quenching of long-lived phosphorescence of the ligand.