Simultaneous acquisition of time- and space-information in time-domain single photon counting spectroscopy became feasible by a recent advance in microchannel-plate photomultiplier-tube technology: we present a novel MCP-PMT detector, featuring a space- sensitive delay-line anode. The detector is characterized by temporal and spatial instrument response functions of 75 ps and 100 micrometer FWHM, respectively, at 200 space channels and a dynamic range of 10⁵. By employing a two-dimensional multichannel analyzer with transputer, 70.000 cps through-put or higher is possible. No photons are lost at the exit slit of the monochromator, as in standard, one-channel time-correlated single photon counting spectroscopy, and sensitive biological samples can be studied at reduced excitation energies. We applied the novel detector to study the basic photophysics of DAPI and its interaction with DNA.

We report the first two-photon excited autofluorescence measurements in single cells induced by continuous wave NIR laser microbeams. In particular we demonstrate NIR-excited NAD(P)H fluorescence of motile spermatozoa in a single-beam gradient force optical trap ('optical tweezers'). In addition, two-photon NIR excited autofluorescence imaging and 'tau- mapping' of CHO cells was performed with laser scanning microradiation of a tunable femtosecond-Ti:sapphire laser. Cellular response on autofluorescence to two-photon absorption of NIR radiation was compared with exposure to UVA light (one-photon absorption).

A novel set-up for time-gated (nanosecond) detection of fluorescence spectra and images of microscopic samples was recently developed. The apparatus was now used to measure the fluorescence of mitochondrial coenzymes (NADH, flavins) and a marker molecule (rhodamine 123) in endothelial cells from calf aorta. In these cultivated cells the electron transport chain was inhibited at various sites of the inner mitochondrial membrane. It could be shown that the fluorescence intensity of the free coenzyme NADH depended on the site of inhibition. In addition, an increased energy transfer from mitochondrial coenzymes (NADH, flavins) to rhodamine 123 molecules was observed, if the inhibition occurred in complex I (NADH- coenzyme Q reductase) or complex III (coenzyme QH₂-cytochrome c reductase) of the respiratory chain. The diagnostic potential of these findings are discussed.

The intracellular concentration of ions such as H⁺, Mg²⁺, Ca²⁺, is known to monitor the activity of many fundamental enzymes. Furthermore these ions are generally considered as intracellular messengers involved in the transduction of extracellular signals. Recent technological progress, occulting the physicochemical properties of the probe, led to the feeling that accurate data on microvolumes are instantly accessible. Unfortunately fluorescent probes are supposed to fill up conflicting requirements for ionic affinity, absence of fading and intracellular calibration. Such a situation generally precludes the use of the simplest methods of data acquisition and treatment. This paper is based on the use of microspectrofluorometry, resolution of single cell complex fluorescence spectrum, and videomicrofluorometry. The methods of data handling allow us to demonstrate that most of the problems met in intracellular calibration come from the fighting of cells against the modification of the extracellular pH. Using these techniques allows us to restrict the need of comparison between results in cuvettes and intracellular results to the physiological pH range. A consequence of such an approach is that the effect with time of known concentrations of amiloride and nigericin on pHi became accessible. Data is presented allowing us to get information on the behavior of the ionic channels and/or cation/H⁺ exchangers involved in the pHi regulation. Such a method leads the way to direct investigations and monitoring of the different processes of regulation of the intracellular ionic concentrations in different cell lines at the level of single cells. Using different specific modifiers (activators or blocking agents) and convenient specific fluorescent probes, the efficiency of such pathways is expected to be checked at will. Compared to the patch clamp techniques, the method can be extended to the study of pathways located on the inner cell membranes.

Intracellular Ca²⁺ is a ubiquitous second messenger that regulates a wide variety of cellular functions including secretion, transepithelial solute and fluid transport. Laser confocal microspectrofluorometry (DILOR, Lille, France) was applied to visualize fluorescence emission spectra of the Indo-1 for measuring the intracellular free Ca²⁺ levels ([Ca²⁺]_i) in a human tracheal gland immortalized cell line (MM39 cell line). Under a 351 nm laser excitation (0.5 (mu) W), the intracellular spectrum was analyzed as a ratio of the emission intensities at 420 and 500 nm. Previously, the intracellular Ca²⁺ calibration has been performed to define the relation between the intensity ratio and [Ca²⁺]_i. Dynamic changes of single-cell [Ca²⁺]_i were measured either from one substrate-attached cell or from different adjacent cells in monolayer culture. Measurements of [Ca²⁺]_i are taken successively in different subcellular locations (up to 10 measurement points). Each measurement cycle was repeated 60 times. To do so, an (X,Y) motorized stage coupled with a computer allowed us to store the (X,Y) positions of several chosen points for the laser radiation. Cells were monitored for about 10 min. After agonist stimulation. Upon stimulating with calcium ionophore, 4BrA23187 (1 (mu) M), [Ca²⁺]_i increased immediately up to 10 fold from a resting value of 31 plus or minus 6 nM (n equals 36). Histamine (1 to 100 (mu) M) increased [Ca²⁺]_i in a concentration dependent manner with levels of up to 88 nM and 140 nM for 1 (mu) M and 100 (mu) M concentration, respectively, followed by a smooth decay back to baseline. Removal of extracellular Ca²⁺ did not abolish the histamine-stimulation [Ca²⁺]_i rise, suggesting that a part of Ca²⁺ mobilization comes from intracellular Ca²⁺ stores. These results show that the combined use of the UV microspectrofluorometry and Indo-1 is well adapted and straight forward for the measurement of rapid responses of substrate-attached cells during experiments of long duration.

Cell damage by low-power 365 nm radiation of a 50 W high-pressure mercury microscopy lamp was studied. UVA exposure to CHO cells resulted for radiant exposures greater than 10 kJ/m² in significant modifications of NADH-attributed autofluorescence and in inhibition of cell division. Single cell gel electrophoresis (comet assay) revealed UVA-induced single strand DNA breaks. According to these results, UVA excitation radiation in fluorescence microscopy may damage cells. This has to be considered in vital cell microscopy, e.g. in calcium measurements.

Two methods are proposed to improve microscopic fluorescence images. In the first method, the second derivative of the image is taken with respect to the focal depth. To obtain the derivative, the focus of the microscope objective is modulated piezoelectrically by 5 - 10 micrometer, and three images with focal positions z plus h, z and z minus h are taken. The resulting image is calculated according to the formula h² f'(z) approximately equals 2f(z)-f(z plus h)- f(z minus h) that follows immediately from Taylors formula. In the second method, a 2D- Fourier transform is applied to the image, followed by multiplication with a model function that reduces low spatial frequencies. Then, the image is transformed back into the spatial domain. The improvements are demonstrated on fluorescence microscopical images of bronchial tumor tissue.

By means of scanning and transmission electron microscopy the subcellular target sites of photodynamic therapy (PDT) with derivatives of sulfonated aluminum phthalocyanine (AlPcS₁, AlPcS₂, AlPcS₃, and AlPcS₄) were studied in a human melanoma LOX cell line. It was found that PDT with AlPcS₁ or AlPcS₂ damaged mainly the biomembraneous system of the cells, such as cytoplasmic membrane, mitochondria, endoplasmic reticulum, etc., while AlPcS₃- or AlPcS₄- induced PDT largely destroyed the lysosomes. However, none of the AlPcS_ns led to nuclear damage at an early stage after PDT. The subcellular photodynamic targets of the derivatives of AlPcS_n are related to the subcellular localization pattern of the dye in the LOX cells.

In vivo uptake of the natural porphyrins, uroporphyrin III (UP), coproporphyrin III (CP) and protoporphyrin IX (PP), was monitored by fluorescence microscopy. Experiments were performed using the chick chorioallantoic membrane (CAM) model, which allowed video documentation of fluorescence both in real time and after integration over a chosen time interval (usually 2 s). Sensitizers at a concentration of 50 (mu) M (100 (mu) L) were injected into a medium-sized vein (diameter approximately 40 micrometer) using an ultra-fine 10 micrometer diameter needle. Fluorescence images were quantitated by subtracting the fluorescence intensity of surrounding CAM tissue (F_matrix) from the intravascular fluorescence intensity (F_{intravascular}), after transformation of the video frames into digital form. The differential fluorescence intensity, F_{intravascular} - F_matrix, is a measure of the biodistribution. Real time measurements clearly showed that CP and UP fluorescence is associated with moving erythrocytes and not with endothelial cells of the vessel wall. Fluorescence intensity was monitored, up to 60 minutes after injection, by averaging the fluorescence over time intervals of 2 s and recording the integrated images. The fluorescence intensity reached its maximum in about 20 - 30 min after injection, presumably after monomerization inside erythrocyte membranes. The results are interpreted in terms of physical-chemical characteristics (e.g. hydrophilicity) and correlated with the photodynamically induced hemostasis in CAM blood vessels.

In in vitro illumination of HE stained frozen sections of healthy lymph nodes with different excitation wavelengths (365-546 nm) results, at least, in three different fluorescence emission peaks located at ca. 460, 545 and 592 nm. These might correspond to NADH, flavins, and protoporphyrin. Lymph nodes invaded by cancer cells resulted in the same spectra; they differ, however, in intensity. The HE stained cryosections can also be used for determining the spatial distribution of the endogenous fluorophores. In accordance with results obtained previously, cancer tissue of lymph nodes exhibit a small fluorescence intensity only. The adjacent healthy tissue shows a rather high intensity in comparison to normal values. Furthermore, necrotic tumor cells at different stages including its final degradation products (minute debris) can be detected by using a double illumination technique. HE stained frozen sections were illuminated concomitantly with a halogen lamp (bright-field transmitted light technique) and an Hg high-pressure lamp with an appropriate filter (395-440 nm) (incident light fluorescence technique). The minute debris can be seen in an area in which the histological image shows hardly anything. The fluorescence intensity observed in healthy tissue adjacent to the tumor, seems to decrease with progression of necrosis. Thus, autofluorescence studies on lymph nodes seem to be able to detect metastasis and the different stages of necrosis more clearly than the methods used conventionally.

The monitoring of the state of cellular energy metabolism and respiratory activity is a necessary procedure in cell biology and pharmacology. One method is the observation of the redox state by NADH and FAD autofluorescence measurements. Using this technique, investigations on endothelial cell cultures were done to study their behavior under pharmacologic influences. One application was the investigation of cytotoxicity of cyanides, blocking the mitochondrial respiratory chain. Further we studied the activation of energy metabolism as a step of the cellular reaction on extracellular impacts. The measurements have been performed with a fluorescence microscope Zei(beta) Axioplan, extended by a PMT and a CCD camera. During examination, the cell cultures were kept under nearly physiological conditions using a specialized perfusion chamber. The measurements took place on cellular monolayers. Different excitation geometries have been studied to overcome the difficulties, which arose from the very weak absorption of the cell monolayer, resulting in a low quantum yield and SNR. In classical cytotoxicity studies, only the statistical long-time effects (e.g. IC50) of cell damages are recorded. By redox microspectrofluorometry it is possible to observe the process of damage in its progress, shown by the presented results. In the second, more complex model, we studied the reaction of cells on ligands like PIA (Phenylisopropyladenosin). In this case, the intracellular reaction is connected with an increased production of cAMP. Again, this requires an increased production of ATP, which leads to an activation of the cellular energy metabolism. The spectroscopic results are interpreted by a first model.

Confluent L-929 mouse fibroblasts were stimulated after 11 days of nutritional deprivation by fresh medium with serum to resume proliferation in synchronized manner. At different time intervals after stimulation microscopic preparations of the cells were made with the Acriflavine-Feulgen-SITS method. The intensity distributions of the two fluorochromes were scanned across the cell nuclei in each 59 rows and columns. Data were gray level displayed and evaluated interactively. The fluorescence intensities in different cell cycle phases, monitored by flow cytometry, indicated a linear increase of nucleolar proteins from the stimulation until the next mitosis, whereas the DNA amount only increased during the S- phase. The width of the distributions revealed larger differences in fluorescence intensities shortly after the stimulation than during the S-phase with more homogeneously stained chromatin. With this procedure subpopulations of stimulated cells and of those, which did not proliferate, could be determined. The results confirm that scanning microfluorometry and evaluation of the intensity distributions yield interesting information about metabolic processes in cells and allow differentiation of subpopulations.

Laser microbeams and optical tweezers work by focusing lasers into a microscope. The energy density of a laser with a beam cross section of 1 cm² can be condensed by almost nine orders of magnitude and focused into a volume of less than 1 femtoliter. When a comparably soft nitrogen laser pulse with 1 (mu) Joule total energy is focused to the diffraction limit, intensities above 1 Terawatt per cm² and local temperatures above 100,000 Kelvin can be obtained. Probably a physical microplasma is generated where the laser pulse hits directly. This is the case even for comparably transparent biological objects, provided the plasma threshold can be reached. Since the heat is generated in a very small volume only, it can dissipate into the environment within a few tens of nanoseconds. This is faster than biological macromolecules can denature. Therefore, the laser microbeam interacts very locally with biological matter. In contrast to laser microbeams, optical tweezers use continuous infrared lasers of only moderate power at wavelengths with only small absorption by biological material. In such cases, the generation of heat is less prevalent and light pressure and gradient forces can be exploited to move microscopic particles. In the very inhomogeneous electric field of a highly focused laser, dielectric objects such as macromolecules, biological subcellular structures, cells or nonliving microspheres are, under suitable conditions, pulled towards the focus and are fixed there similarly as they would be fixed by micromechanical tweezers. This is true for particles with dimensions much smaller than the wavelength of the light used for trapping Rayleigh particles) as well as for particles much larger (Mie particles). Theoretical treatment of the Rayleigh particles assumes that they are linear dipoles. In contrast, many biological objects can be treated as Mie particles, where the basis for force generation is the interaction of the electromagnetic field of light with induced currents. Since Mie particles are large enough, ray optics can be used to explain the interplay of the different forces involved in optical trapping. Both, laser microbeams and optical tweezers (or synonymously 'single beam gradient laser traps') work most economically when the aperture of the microscope objective is just fully illuminated. Trapping effects are largest when the effective refractive index is between 1.1 and 1.6 -- a condition which is often satisfied with biological material.

The determination of scattering phase function as a microscopic basic tissue-optic parameter is very important for the theoretical description and practical realization of optical tomography in biomedical engineering. Right now there are only a few publications containing experimental data, measuring arrangements or even theoretical explanations for the realistic scattering phase functions of biological tissue. This is probably due to the very complex structure of biological specimen and some difficulties with the experimental setup. For the important field of radiation propagation simulations the analytical Henyey-Greenstein phase function has been assumed generally. But for the often used numerical modeling by the Monte-Carlo method an analytical representation of the angular scattering behavior is not required; sufficient are individual sample points. The determination of scattering phase functions of biological specimen is pretty difficult because of the several shapes, sizes, and concentrations of scatterers. But their distinguished consideration is necessary for the comparison with different theories of scattering, e.g. Mie theory. In particular the angular distribution of the scattered light intensity for a single scattering event has to be measured. Therefore the angular scattering distribution of tissues has to be evaluated as superpositioned individual scattering phase functions. The main problem with the instrumentation for investigating scattering phase functions is the need for generating and detecting the angular intensity distribution caused by single scattering events and its high dynamics. Because of the multiple scattering properties of tissues almost the entire object is illuminated often. But an intensity distribution caused by multiple scattering events could not represent the scattering phase function. Thus its contribution to the scattering distribution has to be suppressed. Therefore the detector's view field (i.e. the viewable solid angle with regard to the illuminated area) has to be limited to obtain a high angular resolution. This could be done for instance by controlling the detector's aperture, by introducing angular light wave guides or different techniques like polarization evaluation. Eventually the scattering location can be monitored by a built-in microscope.

The spatial resolution of a conventional light microscope or a confocal laser scanning microscope can be determined by calculating the point spread function for the objective used. Normally, ideal conditions are assumed for these calculations. Such conditions, however, are often not fulfilled in biological applications especially in those cases where biochemical requirements (e.g. buffer conditions) influence the specimen preparation on the microscope slide (i.e. 'practical' light microscopy). It has been shown that the problem of a reduced z- resolution in 3D-microscopy (optical sectioning) can be overcome by a capillary in a 2(pi) - tilting device that allows object rotation into an optimal perspective. The application of the glass capillary instead of a standard slide has an additional influence on the imaging properties of the microscope. Therefore, another 2(pi) -tilting device was developed, using a glass fiber for object fixation and rotation. Such a fiber could be covered by standard cover glasses. To estimate the resolution of this setup, point spread functions were measured under different conditions using fluorescent microspheres of subwavelength dimensions. Results obtained from standard slide setups were compared to the glass fiber setup. These results showed that in practice rotation leads to an overall 3D-resolution improvement.

The use of spontaneous and evoked otacoustic emissions is now a standard clinical tool for diagnosis of the function of the inner ear. However, it is not possible to extract this information over the entire, functionally relevant frequency range because of imperfect coupling of: (1) stapedial to ear-drum vibrations through the ossicular chain of the middle ear and (2) ear-drum vibrations to air in the external auditory meatus. The problem could be circumvented if it were possible to measure the vibration of the stapes and ear drum. The ear drum can be visualized non-invasively, whereas the stapes is only accessible intra-operatively. Therefore, we designed a laser-interferometric system to non-invasively measure the vibration of the human ear drum. Vibrations were measured with a laser Doppler velocimeter (Polytec OFV-302) coupled into the side arm of an operating microscope (Zeiss OPMI MDM). The wavelength was 633 nm and emitted power was less than 1 mW. Direct coupling through the optics of the operating microscope, instead of through glass fibers, enabled a larger signal-to- noise ratio (20 - 30 dB) due to collection of more reflected light. This coupling scheme avoids the problems associated with having to place a reflecting material on the ear drum. The developed vibration measurement system allows non-invasive, fast and reproducible characterization of the dynamics of the human ear drum and as such can be used for clinical diagnostics.

A new application of holographic interferometry in biomedicine and bioengineering is presented. Holographic interferometry techniques for non-destructive testing and biomechanical evaluation of prosthetic heart valves are developed, and experimental results obtained with tissue and mechanical values are demonstrated.

A depth-resolved imaging system is described for recording three dimensional images of objects embedded in diffuse media. Time-gated holographic imaging, employing rhodium- doped barium titanate as the recording medium, is used to obtain whole-field depth-resolved two dimensional images. Infra-red radiation has been used which corresponds to the medical imaging window, as well as blue radiation which may be suitable for undersea imaging.

Optical reflectometers are potentially useful tools for imaging internal structures of turbid media, particularly of biological media. To get a point by point image, an active imaging system has to distinguish light scattered from a sample volume and light scattered by other locations in the media. Operating this discrimination of light with reflectometers based on coherence can be realized in two ways: assuring a geometric selection or a temporal selection. In this paper we present both methods, showing in each case the influence of the different parameters on the size of the sample volume under the assumption of single scattering. We also study the influence on the detection efficiency of the coherence loss of the incident light resulting from multiple scattering. We adapt a model, first developed for atmospheric lidar in turbulent atmosphere, to get an analytical expression of this detection efficiency in the function of the optical coefficients of the media.

For evaluation of respiratory efficiency, data on shape changes of front body surface in various phases of breathing received by means of Moire technique are used in this paper. There is correlation between experimental data and pathological process course of patients with lungs pathology.

A fast and accurate measurement of corneal topography is an important task especially since laser induced corneal reshaping has been used for the correction of ametropia. The classical measuring system uses Placido rings for the measurement and calculation of the topography or local curvatures. Another approach is the projection of a known fringe map to be imaged onto the surface under a certain angle of incidence. We present a set-up using telecentric illumination and detection units. With a special grating we get a synthetic wavelength with a nearly sinusoidal profile. In combination with a very fast data acquisition the topography can be evaluated using as special selfnormalizing phase evaluation algorithm. It calculates local Fourier coefficients and corrects errors caused by imperfect illumination or inhomogeneous scattering by fringe normalization. The topography can be determined over 700 by 256 pixel. The set-up is suitable to measure optically rough silicon replica of the human cornea as well as the cornea in vivo over a field of 8 mm and more. The resolution is mainly limited by noise and is better than two micrometers. We discuss the principle benefits and the drawbacks compared with standard Placido technique.

The birefringence of the human eye with the use of the polarized light have been investigated. The experiments were conducted in an eye clinic on patients eyes in vivo. A video camera was focused on the iris of the non-dilated pupil. The polarized light beam that passed twice through the cornea and is scattered from the iris of the human eye, generates a polarized pattern on that iris. Such patterns were recorded and analyzed. The results which contribute to better understanding of the cornea behavior are discussed.

An anisotropy of optical elements of the human eye is examined. A theoretical model of the corneal birefringence is considered. Ray tracing through such a model depending on different approximation of the corneal topography is calculated. In the paper photographs of the polarization patterns of the cornea in vivo with linearly polarized light are presented. Numerical and experimental results are compared and discussed.

Investigations in performing an optical tomography of biological specimen have been carried out by using the phase-correlated single photon counting method. This method is capable of selecting non-interacted, on-beam-axis forward scattered and snake-line photons, i.e. the straight-forward propagating photons. The phase correlated single photon counting unit performs the mutual monitoring of a high-speed photomultiplier tube and adjacent gain by a broadband preamplifier. The detection occurs in phase correlation to the difference frequency of the optical modulation of the two arms of a Mach-Zehender interferometer arrangement. The object arm is crossing the scattering test medium. To prevent strong intensity ratios between the two interferometer arms the reference arm crosses an optically equivalent phantom medium, but without introduction of inhomogeneities. The correlation between the phase of the optical modulation and the electrically derived phase can be varied by 2(pi) phase shifting of the electrical signal. This allows the user to set a phase range comparable to the time window in time-resolved detection. Two different principle methods can be used for generating tomographic images: the confocal scanning and conventional (object rotation and translation) tomographic setups.

Optical imaging of obscure structures in biological tissue can be accomplished by means of time-gated or coherence gated technique which enables effective rejection of multiple scattering from outside the small tissue volume of interest. However the mechanisms of these modalities differ according to our recent theoretical model: unlike the time-resolved technique, coherence-gated method traces out the local variations of the pathlength-resolved backscattering. In this paper, measurements on tissue scattering phantoms which provide apparent evidences in support of our model prediction are presented. Speckle-related effects are exhibited and analyzed. Some in vitro and in vivo tissue measurements are presented Monte-Carlo simulation is incorporated to provide pathlength-resolved reflectance in order to analyze OCT measurements. The results show that OCT is a promising means of detecting optical heterogeneity due to tissue microstructural differences.

Early diagnosis of cutaneous melanoma followed by prompt surgical excision of the lesion is, at present, the most reliable means to assure the best prognosis of survival to the patient. Recent studies have shown that only 50% clinical accuracy in diagnosis of early melanoma is usually achieved by visual inspection, whereas accuracy can increase up to 90% when diagnosis is performed by experienced clinicians. For this reason, there is increasing interest in the possibility of new technologies to be used to assist the clinician in performing diagnosis of melanoma. Since 1976 several attempts have been made to set up methods to quantitatively estimate the clinical features which are subjectively evaluated in making clinical diagnosis. In this paper some of these techniques are reviewed including photographic photometry, integrating sphere spectrophotometry, multispectral image spectrophotometry and color CCD- camera based techniques. The two latter image acquisition systems seem to provide similar sensitivity and specificity in discriminating melanoma from benign nevi. In an attempt to improve the diagnostic capability of a computer-assisted diagnosis of melanoma, we are studying the potential of the computer image analysis of the two different image acquisition systems. By combining the information derived from the image analysis of both methods, the reliability of a computer-assisted diagnosis of melanoma might be enhanced.

A collimated transillumination system has been developed for the detection of small lesions in thick tissues. The test object is raster scanned by a collimated light beam and a synchronous in-line post collimated receiver. The images are recorded either in analog form to a film using a light emitting diode, or stored digitally in a microcomputer and displayed by a 256 gray shade monitor. Tests show that the system can achieve high resolution in detecting small details in images of whole teeth, live mice, breast sections, and bones in the regular operating mode. The resolution can be increased by using a newly developed high resolution method and/or by digitizing the image. In addition, different types of details can be separated from each other by using different wavelengths of light.

Skin temperatures on the chest and in the wrist area are interesting for continuous monitoring because they can be easily instrumented using an elastic belt or wristband which do not hamper movement in sports, for example. An infrared thermograph camera and NTC thermistors were used to take temperature profiles at these sensing points with a resolution of 0.1 degrees Celsius, and colored thermograms were used to analyze and compare the results. The effect of environmental changes on the skin temperature in the wrist area was studied by cooling and heating the fingers in water at 10 degrees Celsius and 40 degrees Celsius, and the effects of a loading situation on the chest area and wrist area were tested by means of a 30 min bicycle ergometer exercise. NTC thermistors were also used to measure wrist and chest temperatures in two environmental tests at minus 10 degrees Celsius and plus 60 degrees Celsius. Cooling of the fingers naturally reduces the skin temperature in the wrist area and heating increases it due to the venous circulation. The area of the radial artery in the wrist seems to be the most stable temperature point, altering by only about 2 degrees Celsius, whereas the temperature change at other points is up to 4 degrees Celsius. The bicycle ergometer exercise caused a decrease in skin temperature on the chest because of sweating. At the same time the skin temperature on the wrist decreased by about 1.5 degrees Celsius after the first 20 minutes and then returned to its previous level. The area of the radial artery in the wrist seems to be an attractive point for continuous temperature monitoring, especially under normal conditions, and also seems to reflect body temperature quite well upon loading and under different environmental conditions.

Photoacoustic (PA) techniques may be used for imaging of absorbing structures within a light scattering medium. By use of an array of detectors, the macroscopic structure of the absorbers in the medium may be determined, based on differences in light absorption. A physical explanation of the spherical PA profile is presented, from which the signals generated by other source geometries can be derived. The potentialities of PA imaging of blood perfused tissue have been investigated. Experiments were performed with a pulsed frequency-doubled Nd:YAG laser which delivered 10 ns pulses at 532 nm wavelength. Dilutions of India ink, dyed epoxy strands and dyed polystyrene spheres acted as PA sources. Characteristic source dimensions varied between ca. 10 and 250 micrometer. The PA signals were detected with wide band piezoelectric transducers made of 9 and 28 micrometer thick PVdF film with mm and sub-mm lateral dimensions. Detection distances were between 1 and 50 mm. Nonlinear effects have been observed for higher levels of absorbed energy. Dilutions of Liposyn were used as optically scattering media. The calculations suggest the applicability of the method. For imaging of dermal blood vessels up to a depth of 1 mm, piezoelectric signals in the (mu) V range may be expected. The experimental PA signals contain significant frequency components up to 75 MHz, depending on the source characteristics. In principle from such signals the detection distance can be determined with micrometer resolution. The detectors show a forward directivity caused by acoustic interference on the detector surface which depends on the lateral dimensions and the acoustic pulse shape and pulse duration. Optical attenuation coefficients of the Liposyn dilutions have been determined photoacoustically. A spatial resolution of ca. 5-50 micrometer was achieved in the reconstruction of the PA source locations.

A PC plug-in spectrometer has been used clinically to collect autofluorescence and diffuse reflectance spectral data from various skin lesions including skin cancer. To date, 82 patients with a total of 141 diseased skin sites have been measured. Our database on the spectroscopic properties of diseased skin has been enlarged. Analysis of these preliminary data confirmed our previous observations that certain types of skin lesions possess consistent characteristic autofluorescence and diffuse reflectance properties. These spectral characteristics can help the differential diagnosis in dermatology practice.

The method for RBC state control is based upon single cell viability control after illumination with laser pulse. Heat shock resulting from absorption of laser energy by a cell is considered as a cell load. This load acts from inside of the cell, is pulsed (10^-5) and can be delivered directly to the chosen cells. The result of each illumination as cell survival or damage is controlled optically by monitoring the cells' response on a pulse through photothermal technique. Under fixed laser parameters the percentage of damaged cells is viability index (VI) for a certain cell population. The testing procedure includes consequential illumination of each cell in population and calculation of VI. Experimental set up is based upon optical microscope. Dual laser thermal lens technique is used for cell illumination and monitoring. For cell loading 5 ns pulses, 400 divided by 600 nm with energies up to 20 (mu) J are generated by tunable dye laser. Cell monitoring is realized with cw He-Ne (632.8 nm) laser and photodetector. All data acquisition routines are automated. Up to 3 cell suspensions can be studied in a multisample chamber designed to secure cells. An amount of cell suspension required is 1 (mu) l. One population test at a fixed wavelength takes 2 divided by 3 min. In experiments with rats, treated with LPS E. Coli injection to stimulate fever and a septic stress we found that variation of RBC viability becomes apparent in 20 - 30 min after injection, while the clinical changes (blood pressure, body temperature, skin temperature) become detectable after 1 hour. The results obtained show that the method can reveal additional properties of the cells most abundant for monitoring and diagnostic tasks.

Up to now objective criteria for the oxygen supply of tissue only arose from global parameters, for example heart-rate, blood-pressure, blood gases. A judgement of the local oxygen supply -- as an early indicator of organ dysfunction -- is almost impossible. The development of the Erlanger microlightguide photometer (EMPHO) for the first time allows the registration of local parameters as intracapillary hemoglobinoxigenation (HbO₂). The instrument enables the measurement of hemoglobin spectra in tissue micro volumes and thus resolves the spatial heterogeneity of HbO₂. Measurements were performed in healthy human volunteers. HbO₂ was monitored at six points in the skin of the back of the hand. At each measuring site 2000 spectra were recorded. The measurements were performed during normoxia as well as during a period of hypoxia (FiO₂ equals 0.1). It could be shown that changes in the distribution of HbO₂ occur as an indicator of activated local regulatory mechanisms. This opens up the possibility of using the EMPHO for other organs, in order to investigate the relevance of these regulatory mechanisms under physiological and pathophysiological conditions.

In pulse oximetry, the red and infrared intensity fluctuations are often assumed to have the same form. However, we observed strong phase delays between these signals during measurements on the forehead of some of our subjects when no pressure was applied onto the probe, which practically disappeared when pressure was applied on the probe. The signals obtained at different distances from the light source were transformed into fluctuations in the absorption and reduced scattering coefficient by means of results from Monte Carlo simulations. The changes in the reduced scattering coefficient appeared to be inverted when no pressure was applied onto the probe. Although the calculated relation between the red and infrared fluctuations in the absorption coefficient was sometimes free of hysteresis, the ratio between the fluctuations still depend on pressure on the probe, and on the chosen optical properties of the medium.

The physiology and pathology of dissected blood vessels are studied by perfusion myography combined with video microscopy. Images of the vessels are formed under diffuse white light illumination and contrast is achieved by differential absorption with respect to the vessel wall. To obtain the vessel dimensional information in quasi real time an edge-tracking algorithm is used, allowing the edges to be found by applying common image processing tools to a very small number of pixels rather than the whole image. Employing a low order optical model of the light transmission properties of vessels with circular cross section, a relationship between the positions of edges found by a typical image processing algorithm and actual dimensions is derived. The dimensional analysis is demonstrated on rat mesenteric resistance arteries (internal diameter less than 300 micrometer) mounted in a perfusion arteriograph. Segments of vessels are secured on two glass cannulae using single strands of a nylon braided suture. The artery is perfused with physiological salt solution and the perfusion pressure maintained at 60 mmHg before starting the experiment. Changes in vascular diameter to the vasoconstrictor noradrenaline and the endothelium-dependent vasodilator acetylcholine were then observed.

Living cells investigation and cell response to external factors are of great interest for practical medicine and biology. The main advantages of computer aided phase microscope (CPM) Cytoscan which permits us to observe the cell surface and internal structure consists in superresolution and the possibility of the dynamic processes registering. We attempt to characterize some aspects of the morphofunctional status of human lymphocytes determining the dynamics of the selected points in normal or pathological cells. To evaluate the lymphocyte homeostasis donors and persons of autoimmune diseases were analyzed and the changes of optical and geometrical cell parameters registered by CPM Cytoscan. The dynamic process registration allowed us to perform the real-time quantitative analysis of the living lymphocyte activity in norm and pathology.

In this paper the heterodyning detection of the Doppler signal of dynamic speckles by spaced photodetectors is studied theoretically. The investigation is carried out on the assumption that the laser field scattered by the diffuse object is the Gaussian speckles and the signal processing is implemented by the maximum amplitude channel section. It is shown that the detection probability of Doppler signal by spaced photodetectors increases with the increase of detection probability P_D1 when only one photodetector is used. The spaced detection is practically effective for P_D1 approximately greater than 0.85. The detection probability value close to the theoretically attainable maximum value takes place when 4-5 spaced photodetectors are used.

Experimental data having no analogs is presented. Holograms and interferograms are recorded and momentarily photoprocessed in the presence of rather intense polychromatic light. Russian and Western silver halide media for holography are used. A scientific demonstration of the technique is planned with holograms momentarily produced on the site during the presentation. In other words totally daylight holographic technique avoiding darkrooms is presented de facto. The technique with its unique simplicity and speed, guaranteed quality of holographic reconstructions, and extreme cheapness seems to be very promising to biomedical applications.

The paper presents an analysis of the optical technique for low frequency biovibrations sensing. The suggested method makes use of dynamics of speckles induced by focused coherent fields diffraction from vibrating surface. Computer simulation of strongly focused coherent beams scattering from a vibrating skin surface has been carried out for cases of in- plane and angular vibrations. The dependence of output characteristics of the developed speckle-using vibration sensor on vibration type and relations between the amplitude of surface oscillations, illuminating beam waist size and the scale of the surface inhomogeneities has been analyzed. Results of biovibration monitoring using the proposed technique are presented.

The study of the mechanical behavior of the human jaw during chewing is helpful in several specific medical fields that cover the maxillo-facial area. In this work, electronic speckle pattern interferometry has been applied to study dead jaw bones under external stress which simulates the deformations induced during chewing. Fringes obtained after subtraction of two images of the jaw, the image of the relaxed jaw and that of the jaw under stress, give us information about the most stressed zones. The interferometric analysis proposed here is attractive as it can be done in real time with the jaw under progressive stress. Image processing can be applied for improving the quality of fringes. This research can be of help in orthognathic surgery, for example in diagnosis and treatment of fractured jaws, in oral surgery, and in orthodontics because it would help us to know the stress dispersion when we insert an osseointegrated implant or place an orthodontic appliance, respectively. Studying fragments of human jaw some results about its elasticity and flexibility were obtained.

The general concept of volumic view (VV) as a universal property of space is introduced. VV exists in every point of the universe where electromagnetic (EM) waves can reach and a point or a quasi-point receiver (detector) of EM waves can be placed. Classification of receivers is given for the first time. They are classified into three main categories: biological, man-made non-biological, and mathematically specified hypothetical receivers. The principally novel concept of volumic perception is introduced. It differs chiefly from the traditional concept which traces back to Euclid and pre-Euclidean times and much later to Leonardo da Vinci and Giovanni Battista della Porta's discoveries and practical stereoscopy as introduced by C. Wheatstone. The basic idea of novel concept is that humans and animals acquire volumic visual data flows in series rather than in parallel. In this case the brain is free from extremely sophisticated real time parallel processing of two volumic visual data flows in order to combine them. Such procedure seems hardly probable even for humans who are unable to combine two primitive static stereoscopic images in one quicker than in a few seconds. Some people are unable to perform this procedure at all.

There is great significance in comparing medical x-ray pictures, which are taken in different periods. For the orthopaedics surgeon comparing the density of bone is one important way of clinical diagnosis. Some persons think that if these orthopaedics pictures are processed by histogram normalization their gray level and pseudo color can be compared directly. But for one picture we know that the gray distribution is different between the original picture and the processed picture. Comparing these pictures is not authentic. In this paper we discuss that subject and describe the basis of histogram normalization. The distribution on original x-ray pictures and processed pictures is provided. The methods to compare different pictures are mentioned.

The methods available for testing the efficacy of topical sunscreens have improved considerably in recent years. Nevertheless, so far no simple and rapid test has been proposed to measure in vivo transmission spectra of sunscreens in the UVA region. Spectral changes that occur after sunscreen application were measured with a fluorescence spectrometer (LS 50B, Perkin Elmer, UK) equipped with a Y-shape quartz guide for in vivo measurements. Three sunscreens with different protection factors in the UVA range were tested. The excitation-emission maps of human collagen, skin, and sunscreens were analyzed. Visual demonstrations of the protective effects of sunscreens were also performed with photo- and video imaging techniques. As a consequence of the human skin and sunscreen's fluorescence map analysis, the optimal spectral regions (both for direct and indirect fluorescence measurements) were detected. In vivo fluorescence and remittance spectroscopy were used to investigate the time dependence in transmission spectra of epidermis with applied sunscreens. We also evaluate the feasibility of in vivo fluorescence measurements for the investigation of the sunscreen's water-resistance. The procedure is simple, and values obtained can be used to predict UVA protection on the basis of the mathematical algorithms.

Methodics of PDT control and fluorescent-spectroscopic diagnostic of head and neck tumors and mammary gland cancer (nodular) with the use of Kr, He-Ne and semiconductor lasers and photosensitizer (PS) -- Al phtalocyanin (Photosense) are discussed. The results show that applied diagnostic methods permit us not only to identify the topology and malignancy of a tumor but also to correct PDT process directly during irradiation.

The simultaneous recording of several photomovement parameters of algae as test-functions during biomonitoring is proposed. Green alga Dunaliella viridis Teod. was used as the test- object for the estimation of different heavy metals. The quantitative changes of photomovement parameters as a criterion of toxicity were determined by means of the vectorial method of biomonitoring.

The laser diode device (LDD) leasing in near infrared region (IR), synchronized with the cardiac pulse, is designed for laser therapy. The special pulse sensor was used for monitoring of the cardiac action. The unique future of this device allows to synchronize the laser action with the cardiac rhythm. This laser is a result of Polish-Belarussian cooperation. It was used in the clinics for treatment of 88 patients suffering from high blood pressure. The positive clinical effects were seen in 86% of cases. The further studies are continued.

Topographic analysis of the ophthalmic surfaces is an important task. Especially recently, when a laser assisted refractive surgery becomes more and more popular in a daily clinical praxis. Ophthalmologists need to know exact corneal parameters as a basis for proper operational approach, as well as for monitoring of the post-operative process. The fitting of the contact lenses can be more accurate when topography of both, cornea and contacts, can be precisely measured. We develop new coherent methods for measuring of the topography of curved optical surfaces. One of the proposed techniques is based on interferometry with a special distance measurement unit and spatial phase shifting interferogram evaluation. The other one uses deflectometry with spatial carrier frequency. The sensitivity of this method is adjustable and thus it closes the gap between the white light and interferometric measuring methods. The techniques proposed here can be suitable for measurement of the contact lenses or corneal surface.