Fluorescence imaging is a promising technique for the early detection of neoplastic changes in the human body. Investigation of the microscopic origin of tissue fluorescence can provide important information about the histology of tissue. In this study we report the synthesis and use of two novel Rhodamine B derivatives in an efficient microscopic technique for colon cancer diagnosis. Another important objective is the examination of the morphological structures on which the fluorescence analogs bind and the correlation of the fluorescence images with their underlying microstructural origin. Rh B-Leucine amide and Rh B-phenyl boronic acid distinguish clearly healthy from neoplastic colon tissue sections. Both derivatives accumulate mainly in the of neoplastic crypts. This finding can be exploited for colon cancer diagnosis using fluorescence microscopy as well as for a fluorescence-based optical biopsy method.

Changes in autofluorescence intensities at 550, 580, 680 and 720 nm as functions of incident intensity and exposure time were measured in normal mucosa and adenocarcinomatous of human colonic tissues when excited by the wavelengths of 457.9, 488, 514.5 and 632.8 nm, respectively. The obtained results show that the photobleaching process of the autofluorescence follows a double-exponential function. The slower decay rates of the photobleaching at 550 and 580 nm emissions could be found in normal tissues compared to those in tumor tissues, however, the faster decay times at 680 and 720 nm emissions were also found in normal tissues. It appears that the quantitative measurements of photobleaching processes may provide a method to evaluate the fractional contribution of the autofluorescence from different layers in the colon tissues. The evaluation of temporal behavior of photobleaching processes of autofluorescence emissions may also reveal the different accumulated concentrations of endogenous fluorophores between normal and tumor tissues.

Determination of the tumor's penetration into the tissue is actual task of optical biopsy. For this aim the depth resolved fluorescence of the second generation sensitizer's molecules has been studied in tissue's phantom at different excitation wavelength. Fluorescence spectra from the superficially and deeply (up to 10 mm) located Chlorin e₆ sensitizer's molecules at 355, 532, 660 nm laser excitation were registered. The comparison of the fluorescence signals registered at the different values of penetration into tissues of the probing radiation gives opportunity to define the presence of the sensitizer's molecules in different depth of tissue and to reveal such important clinical parameter as tumor's penetration into depth.

We have applied elastic-scattering spectroscopy (ESS) for noninvasive, real-time in vivo measurement of the concentrations of certain drugs in tissue, utilizing a simple fiber-optic-probe spectroscopic system. The system uses a broadband light source, enabling the detection of compounds with absorption bands in most regions of the visible, as well as the NIR to 1700 nm. Subcutaneous tumors were grown in 4 Nude mice; the mice were treated with one of two chemotherapy agents, and the ESS system was used to perform pharmacokinetics measurements on the tumors following drug administration. Time histories of the drug concentrations in the tumors agreed with the known pharmacokinetics of the two drugs, and HPLC assays following sacrifice showed good correlation with the ESS values. Most photodynamic therapy agents and many chemotherapy drugs, including any that are not fluorescent, are ideal candidates for the ESS system. The measurement can be calibrated absolutely, and is not susceptible to problems associated with fluorescence assay methods.

Topical application of acetic acid solution is routinely used as a marker for the clinical diagnosis of cervical cancer and more specifically to direct biopsy sampling. Abnormal areas are visualized as transient white patches and there are several evidences that acetic acid-tissue interaction kinetics is correlated with the malignancy grade. The visual assessment of this interaction can not be effective and therefore the obtained diagnostic information is limited. In this paper a new method and a multi-spectral imaging system are presented, capable to enhance the contrast between normal and acetic acid responsive tissue areas and to assess quantitatively the kinetics of acetic acid-tissue interaction. The former is achieved by cutting-off the regular reflection, in combination with the selection of the appropriate imaging spectral band. The second is achieved with the dynamic measurement of the intensity of the back-scattered light in any spatial point of the image, after acetic acid application. The obtained quantitative data provide a means for the early detection, and more accurate grading and mapping of the lesion. This method was experimentally implemented to colposcopy and a remarkable improvement of the sensitivity specificity and reproducibility was demonstrated during in initial clinical trials.

The infrared spectrum of biological tissue is due to the 'microheterogenous' composition as a whole and is based on complex vibrational modes. In detail, the spectrum represents the biochemical status resulting from a combination of the structural framework of tissues together with the biological active compounds of metabolism. According to the literature, the main IR-spectroscopic differences are to be expected by the characteristic spectral pattern which is located at the 'fingerprint' region (1500 - 1000 cm^-1). In order to design and develop an endoscopic system for the in vivo identification of healthy and malignant tissue FTIR- measurements were carried out using a fiberoptic device. The source of IR-radiation can be either the FTIR-spectrometer or tunable IR-diode lasers. Fiberoptic IR-spectra obtained using the attenuated total reflectance (ATR) and reflection mode were compared to spectra resulting from the FTIR- microspectroscopic measurements.

The accuracy of conventional and emerging methods for the dimensional analysis of optically imaged arterial vessels, isolated in a pressure myograph, is investigated. The pressure myograph is a device used to study the structure and function of isolated sections of small resistance arteries, as a function of chemical, mechanical and electrical stimuli. The arterial wall and lumen dimensions are particularly important indicators of anatomy and pathology. The conventional method of dimensional analysis uses edge detection, however the accuracy of this approach is questionable when the vessel is in a contracted state since contrast deteriorates or is lost between lumen and vessel wall. The conventional and emerging methods are examined experimentally with vessel phantoms, to provide known characteristics. A novel algorithm, based on a measurement of the vessel extinction coefficient, is also examined theoretically and experimentally. A discussion centers on the possibility for realistic lumen size measurement when edge detection can not be applied and when the accuracy of edge detection is questionable.

The spectral peculiarities of normal and tumor human tissues of different localization after sensitization by sodium fluorescein has been studied with help of multifrequency YAG:Nd laser. Fluorescence spectra of normal human tissue and breast, stomach, intestine, skin cancer at 355, 440 nm laser excitation were registered. The pharmacokinetic behavior of sodium fluorescein in normal and different tumor tissues has been investigated. The optimal time for diagnosis (12 - 18 hours after drug injection) was determined. The intensification of tumor tissue autofluorescence decreasing after sensitization of organisms by tumor seeking dye was observed. The electron excitation energy transfer from NAD(P)H to endogen chromophores or dyes localized in tumor has been discussed as possible reason of the observed phenomenon.

An apparatus for the measurement of the radial dependence of the continuous-wave diffuse reflectance of a tissue is described. It consists in a probe, which is applied on the tissue, and a detection unit. By employing an array of incoherent semiconductor light sources (LED's) and continuous- wave detection, probe structure is considerably simpler than other devices described in literature, allowing moreover an efficient coupling of the emitted light towards the tissue. The high responsivity so obtained permits fast and accurate measurements. Measurement speed, probe compactness and accuracy are potentially sufficient for the in-vivo application of the method to surgically exposed tissue. A preliminary set of data, measured on a scattering phantom and on non-exposed in-vivo tissue, is presented. Even though available models fitted to the measured data give the correct order of magnitude for light transport coefficients, in order to extract reliable absolute values they should be corrected for probe nonidealities. The availability of extensive high- quality in-vivo data is to this regard stimulating for further theoretical investigations.

When spectrofluorimetry is applied to the problem of diagnosis, correlation techniques relating spectral features to biotissue status should be used. But the coefficients of correlation equation should account for the relationship of spectral parameters with biochemical and morphological changes associated with the pathology. As a search for these dependencies in actual biotissue is difficult, we offer to employ tissue phantoms -- the physical models mimicking, under conditions of measurement, optical characteristics of the natural object. To model human cervix tissue, we used a three- layer planar structure, with upper 0.3 - 1.0 mm layer simulating epithelium, middle 0.03 - 0.1 mm layer representing basal membrane, and greater than 1.0 mm lower layer modeling subepithelial tissue. As a mechanical base of the structure we used 10% (per weight) gelatin gel. To simulate light scattering by biotissue, the nonabsorbing and nonluminiscent scatters were added to the upper and lower layers. NADH, FAD, and Protoporphyrin IX were added to upper layer. Collagen, as dried thin gelatin film, modeled basal membrane. To reproduce modulation of autofluorescence spectrum by reabsorption within the tissue, we added solution of human hemoglobin to the lower layer. Spectrofluorimetric measurement was performed using various excitation wavelengths (337 nm, 365 plus or minus 20 nm, and 405 plus or minus 20 nm).

There exists a number of fluorophores whose lifetimes that vary with the properties of the surrounding medium, such as pH and calcium ion concentration. Lifetime information can be gathered in the form of lifetime images where each pixel value represents the fluorophore lifetime. By using confocal microscopy it is possible to record such lifetime information in three dimensions, thus recording 3-D lifetime images. We have built a confocal microscope that allows simultaneous lifetime imaging of two fluorophores. Two intensity-modulated laser beams are used as excitation sources, and fluorescence is detected using phase-sensitive lock-in technique. We have previously verified that this instrument can simultaneously record lifetime images of two fluorophores with good channel separation. Excitation wavelengths of 488 and 568 nm were used, and the fluorophores tested had lifetimes in the range 2.3 - 6.5 nanoseconds. With the present study we have started work on biomedical applications of lifetime imaging with the IMS technique. In this first stage we have concentrated on one parameter, namely pH. Using the pH sensitive fluorophore SNAFL-2, we found that the lifetime varied between 1 and 3.7 ns for a pH range of 5.0 - 10.0. Experiments with SNAFL-2 were also carried out on living cells. The signal quality in confocal lifetime imaging is also discussed.

Confocal laser scanning fluorescence microscopy is presently being used widely in biomedical research. A severe limitation for its use is its often still insufficient resolution. In situ measurements in 3D conserved human cell nuclei showed that distance measurements between fluorescent targets located in the interior of such objects are limited a resolution regime of appr. greater than or equal to 0.3 micrometer in lateral and appr. greater than or equal to 0.7 micrometer in axial direction. A technique to overcome these restrictions is the recently developed Spectral Precision Distance Microscopy (SPM). This approach allows the determination of distances between targets which carry different spectral signatures with high precision. In situ measurements revealed that the SPM approach allows the determination of distances in 3D intact cell nuclei with a 'Resolution Equivalent' better than 50 nm. Here we present an improved chromatic shift calibration method for Spectral Precision Distance Microscopy. Furthermore, micro axial tomography allows the tilting of objects perpendicular to the optical axis; thus two objects can always be tilted in such a way that they can be recorded in the same focal plane. Therefore the error in distance determination is minimized. Here we present some preliminary data for the applicability of spectral precision distance microscopy (SPM) to micro axial microscopy.

The reliable and widely used pH-sensitive indicator BCECF (2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein) can also be employed in a classical confocal laser scanning microscopes using 488 nm argon laser excitation line. The intracellular pH maps based on the fluorescence emission ratio tally with those obtained by other techniques.

A dual scanning system forms the essential part of a conventional confocal microscope. Hence, its imaging mode is on principle serial (sequential). Parallel-mode confocal imaging is possible on the basis of a broad-source image-plane holography. We adapted this classical-holography technique for a real-time reflected-light microscopy.

In this contribution we use intravital microscopy to study the dynamics of extravasation into normal and tumor tissue of several hydrophilic cyanine dyes used as near-infrared (NIR) contrast agents. The technique provides information about the angiographic properties of the dyes and about their interaction with tumor tissue under dynamic conditions in vivo. In our previous work we demonstrated that several NIR- absorbing fluorescent dyes enable in vivo fluorescence detection of tumors in mice and rats. However, the mechanism leading to dye accumulation and enhanced fluorescence in tumors is not fully understood. Increased extravasation of dyes into tumor tissue due to pathologically altered tumor vessels may be an important factor in this process. Indocyanine green (ICG) displayed predominantly intravascular distribution and rapid elimination resulting in enhanced fluorescence signal of vessels during the first 15 min after administration only. No elevated extravasation into tumor tissue was observed with ICG. A hydrophilic indotricarbocyanine derivative with a high molecular weight displayed prolonged intravascular distribution and increased fluorescence signal of the vasculature compared to surrounding tissue for up to five hours. Rapid extravasation and accumulation in tumor areas, yielding elevated contrast of tumors up to 15 min after administration, was observed with hydrophilic, low molecular weight indotricarbocyanine derivatives.

Fluorescence in situ hybridization is used to study the arrangement of chromosomes in interphase nuclei of unsynchronized human lymphocytes. DNA probes specific for telomeric DNA, centromeric (alpha) -satellite DNA and whole chromosomes 2, 7, 9 and X are employed. It is demonstrated that the shape of the chromosome territories is variable in cycling cells, for example, close to the metaphase chromosome homologues are arranged pairwise. Furthermore, the relative arrangement of chromosome homologues to each other is not spatially defined. Also, the relative orientation of centromeres and telomeres within a chromosome domain is variable.

The evanescent wave (EW) elicited by total internal reflection of light provides a means to selectively excite fluorophores in an optical slice above a reflecting dielectric interface. EW excitation eliminates out-of-focus fluorescence present in epi-illumination microscopy, and can offer a 5-fold enhancement of axial resolution compared to confocal and two- photon microscopy. The decay length of the evanescent field is a function of the refractive indices at the interface, the wavelength of the light, and is modulated by the beam-angle. EW microscopy has been used to study the distribution and concentration of fluorophores at or near the interface in the presence of high concentrations in bulk solution on top of the interface. We modified an upright microscope to accommodate the condenser optics needed for EW excitation. Systematic variations of the angle of incidence were attained using an acousto-optical deflector, telecentric optics, and a hemicylindrical prism. 3-D reconstruction of image stacks by an inverse Laplace transform results in topographical information with an axial resolution of 10's of nanometers. We have labeled subcelluar storage organelles ('vesicles') of approximately equal 300 nm diameter and visualized the trajectories of single vesicles in the 'footprint' region of living neuroendocrine cells, grown on the interface.

To establish optical in situ detection of mitochondrial malfunction, non-radiative energy transfer from the coenzyme NADH to the mitochondrial marker rhodamine 123 (R123) was examined. Dual excitation of R123 via energy transfer from excited NADH molecules as well as by direct absorption of light results in two fluorescence signals whose ratio is a measure of mitochondrial NADH. These signals are detected simultaneously using a time-gated (nanosecond) technique for energy transfer measurements and a frequency selective technique for direct excitation and fluorescence monitoring of R123. Optical and electronic components of the experimental setup are described and compared with a previously established microscopic system.

Cellular uptake of transparent Latex particles by J774A.1 mouse macrophages has been studied: First, single beads were kept within an optical light trap and located in close vicinity to individual cells. Uptake of the beads was visualized, and intrinsic fluorescence was detected in the spectral range of 420 - 530 nm. Second, time-gated fluorescence spectra of single cells were recorded at pre- selected times during one hour after cellular uptake. A rapid increase of autofluorescence and a subsequent decrease to the level of control cells within about 10 min. was measured within a time gate of 0 - 5 ns after the exciting laser pulses, and attributed to the 'free' coenzyme NAD(P)H. In contrast, fluorescence increase of NAD(P)H bound to proteins (measured within time gates of 5 - 10 ns or 10 - 15 ns) was less pronounced, and the subsequent decrease occurred within a longer period (about one hour).

Methods for analysis, especially 2D reconstruction, of cyanobacterial S-layers were improved. The locally normalized correlation was used in the course of correlation averaging. For elimination of image distortion, the function of the distortion was approximated step by step by the linear fractional function or by the Lagrange interpolation polynomials. The modification of the methods is illustrated on figures of S-layers of Synechocystis aquatilis Kovacik 1990/8 and Microcystis aeruginosa Hindak 1971/1 (negatively stained), and Microcystis cf. wesenbergii Bitov 1994 (metal shadow).

A new panoramic microscope design is offered. An object is illuminated by two interfering laser beams of different close frequency. The angle between the beams and their direction is varied. An energy of scattered light is detected. The detected signal is filtered at the difference frequency at the fixed beam directions and further the image using two-dimensional Fourier transform algorithm of filtered signal is built. The intensity distribution in the image is shown to have a speckled structure, typical of coherent images, with the envelope dependent on the mean slant of the object's roughness and the curvature of its surface. It is also shown that the larger the size of scattered light detecting aperture, the less the effect of the speckled structure on the image quality. Various designs of the microscope are given. The function of the system that uses a thin Fresnel lens as the light source is described in detail. Two illuminating beams move, one continuously and the other discretely, in perpendicular directions along the lens diameter. The imaging of an object with various periodical structure is simulated. The panoramic microscope designs presented in the paper allows the imaging of objects as large as 10 cm located as long as 10 cm away from the light source and the detector with the Rayleigh resolution of about (lambda) /2.

Interferometric radar technique with low-coherent illumination provides comparatively large range of measurement definition by interference fringes visibility maxima location. The problem is that the interference fringes are often distorted by noise of speckles. Noise influence can be decreased by new method based on Markov theory of stochastic processes. Interferometric data are assumed to be a realization of random series of phase-shifting samples of vector signal with known stochastic properties. The processing algorithm gives the optimal assessment of vector of interference signal parameters, including visibility maximum position, on each point of phase shifting by the recurrence procedure of data processing.

High spatial frequencies in the illuminating light of microscopes lead to a shift of the object spatial frequencies detectable through the objective lens. If a suitable procedure is found for evaluation of the measured data, a microscopic image with a higher resolution than under flat illumination can be obtained. A simple method for generation of a laterally modulated illumination pattern is discussed here. A specially constructed diffraction grating was inserted in the illumination beam path at the conjugate object plane (position of the adjustable aperture) and projected through the objective into the object. Microscopic beads were imaged with this method and evaluated with an algorithm based on the structure of the Fourier space. The results indicate an improvement of resolution.

To obtain three-dimensional refractive index or density spatial distribution inside optically transparent living cells, it is necessary to combine computed tomography and interference microscopy. These methods use the oblique illumination and the sample viewing angles scanning for recording a number of the projections. A new application Linnik microscope reflected type for obtaining a high quality phase projections with spatially incoherent laser illumination is described. Phase-shifting technique for automated interferogram analysis is used. Interferometric computed- tomography Linnik laser microscope are fully automated integrated systems that incorporate CCD camera, frame grabber, computed operated PZT mirror as well as intelligible software for interferogram processing, projections preprocessing and tomographic reconstruction. Microscope measurement system and performance characterization are presented. There are some features in angle scanning, projections proceeding, and reconstruction for tomographic investigation of the phase samples that are placed near the mirror surface. For plane mirror the projections viewing angle range may be limited to 90-degree and the investigation object may be represented as a sum of the original object and its mirror reflection. The iterative algorithms for limited-angle tomographic reconstruction were used. Three-dimensional images of living blood cells (lymphocytes) were obtained.

Microscopy investigations of S-layers of plague microbes have been carried out. A protein forming S-layer was isolated, purified and its biochemical properties were studied. Images of plague cells with and without S-layer and images of isolated S-layer protein arrays on solid supports were obtained by transmission electron microscopy. An isolated protein forming the layer is self-assembled into crystalline structure with hexagonal pores are of regular size about 4 - 8 nm, which was observed by transmission electron micrographs. The STM images of plague microbes with S-layer and without one and the STM and AFM images of isolated S-layer protein arrays on mica surface were obtained.

The COMET assay, a single cell electrophoresis technique which allows to separate electrophoretically fractionated DNA according to size has been combined with fluorescence in situ hybridization (FISH) which allows to localize specific genes or gene regions. This combination (COMET FISH) allows the detection of DNA single strand breaks in specific regions of the genome of human lymphocytes at the single cell level. Various types of DNA probes, e.g. centromere-, (alpha) - satellite-, telomere-, whole chromosome-, single copy- and region specific DNA probes have been used to investigate whether the UV-A induced DNA single strand breaks are distributed randomly all over the human genome or induced at specific sites ('hot spots'). In the investigated human peripheral blood lymphocytes all but one centromere reveal low sensitivity for UV-A irradiation (500 kJ/m²), while telomeres are randomly distributed over COMET heads and tails. The human chromosome 1 is fractionated by irradiation, but remains in the COMET head, indicating an only moderate degree of fractionation. Among three tested single copy probes, c- myc, p53 and p58, the p53 gene located on chromosome 17p13.1 and the p58 gene (1p36) appear to be located in UV-A stable regions of the human genome in 95% of 65 investigated lymphocytes. In contrast, the c-myc proto-oncogene (8q24) is found in the COMET tail in 90% of the 27 investigated lymphocytes and thus appears to be more sensitive to UV-A irradiation.

We report on the results of using the optical coherence tomography (OCT) as one of the diagnostic methods at the Department of Gynecology of the Nizhny Novgorod Regional Hospital. An endoscopic OCT device adjusted for gynecological examinations with colposcopy, hysteroscopy and laparoscopy has been developed at the Institute of Applied Physics. It provides clinicians with sharp (up to 15 - 20 micron resolution) images of 1.5 mm thick superficial mucosa layers in the female genital tract, that are recorded at the 0.83 micron wavelength with approximately 1 frame/second rate for a 200 X 200 pixel image. Data obtained during examination of more than 100 patients demonstrate the capability of OCT in estimation of structural alterations in organs, connected with different types of pathologies and functional states of the female genital system. We present first results of OCT application to assess the adequacy of cervical pathologies treatment (electro-, laser surgery and cryotherapy) and to control the healing process.