We report the implementation of an optical laser trap that incorporates phase conjugate microscopy as one of its key elements. The laser trap consists of a primary trapping beam, and a counterpropagating self-pumped phase conjugate beam, the latter being derived from the primary signalbeam when it is transmitted through the sample and phase conjugated in a photorefractive crystal. Using a fundamental TEM₀₀ gaussian beam from an Ar-ion laser (514 nm), and a barium titanate (BaTiO₃) crystal in the CAT (total internal reflection) geometry, an optical laser trap was created using approximately 25 mW of laser power. Strong transverse optical confinement is reported for approximately 2.5 micrometers diameter polystyrene microspheres, using both high and low numerical aperture (e.g. 60 X, 0.85 N.A. and 10 X, 0.25 N.A.) objective lenses. By virtue of the phase conjugation process, the present geometry achieves self-alignment of the two counter-propagating beams, as well as photorefractive gain in the ratio of nearly 4:1. The system has the potential for implementing several novel image processing functions unique to the nonlinear phase conjugation process, on living cells confined to an optical laser trap, including aberration correction, contrast reversal, and novelty filtering, respectively.

We report the observation of two-photon fluorescence excitation and cell confinement, simultaneously, in a continuous-wave (cw) single-beam gradient force optical trap, and demonstrate its use as an in-situ probe to study the physiological state of an optically confined cell sample. At the wavelength of 1064 nm, a single focused gaussian laser beam is used to simultaneously confine, and excite visible fluorescence from, a human sperm cell that has been tagged with propidium iodide, a exogenous fluorescent dye that functions as a viability assay of cellular physiological state. The intensity at the dye peak emission wavelength of 620 nm exhibits a near-square-law dependence on incident trapping beam photon laser power, a behavior consistent with a two-photon absorption process. In addition, for a sperm cell held stationary in the optical tweezers for a period of several minutes at a constant trapping power, red fluorescence emission was observed to increase the time, indicating that the cell has gradually transitioned between a live and dead state. Two-photon excited fluorescence was also observed in Chinese hamster ovary cells that were confined by cw laser tweezers and stained with either propidium iodide or Snarf, a pH-sensitive dye probe. These results suggest that, for samples suitably tagged with fluorescent probes and vital stains, optical tweezers can be used to generate their own in-situ diagnostic optical probes of cellular viability or induced photodamage, via two-photon processes.

The stimulated emission process has been rarely exploited in spectroscopy and microscopy. Instead, a spectroscopy method based on the pump-probe principle has been frequently used to observe picosecond and femtosecond processes. This common approach has not been applied to microscopy due to the relatively slow acquisition time and the lack of 3D information. We have exploited an idea originally proposed by F. Lytle group²⁸ in which two pulsed lasers are simultaneously focused on the sample. One laser is used to excite a population of molecules and the second laser to induce stimulated emission. The stimulated radiation is carried away in the same direction of the stimulating laser beam. By collecting the fluorescence emission in other directions, we observe a modulation of the fluorescence signal as a function of the delay between the two laser pulses. The repetition rate of the two lasers is slightly different producing a frequency beating at the laser overlapping volume. We have extended this method to achieve very high spatial and temporal resolution in the microscope environment. By recording only the beating frequency, we obtain a 3D sectional effect similar to two-photon excitation. The harmonic content of the beating signal is limited by the laser pulse width and by the sample frequency response. Information of picosecond processes are extracted by standard frequency-domain methods. Using this principle, we built a stimulated emission microscope that has 3D and fluorescence lifetime capabilities.

The Automated Interactive Microscope is a robotic light microscope workstation that combines high performance light microscopy and computing to explore the chemical and molecular dynamics of cells and tissues.

A glioma produces some of the most rapidly growing, angiogenic, and invasive primary brain tumor cells known. A lack of understanding about the intricately coupled molecular mechanisms that result in cell transformation is responsible, in part, for the minimal progress made in treating this disease over the past century. To begin dissecting molecular interrelationships in time and space within living normal and transformed cells, a morphological assay is being developed wherein patient-derived tumor cells are allowed to attach to a substrate, spread, change shape, and locomote. During this process (approximately 6.5 h), low magnification phase contrast images of the cells are recorded at 1 min intervals. Quantitative image analyses of these time-lapse images are used to measure dynamic parameter such as projected cell area, shape, and displacement for each cell. Although there is considerable cellular heterogeneity, patterns of patient-specific tumor cell behavior are beginning to emerge. In addition, the assay is being used to test the effects of drugs that alter specific intracellular processes (e.g., cytochalasin, 2-deoxyglucose, and chemotherapeutic drugs). To dissect tumor cell physiology into its molecular components, I have focused on the actin-cytoskeleton because it is involved in the temporal and spatial orchestration of ions, metabolites, macromolecules, and organelles that underlies the interconnected processes of tumor cell growth, motility, and differentiation. I have used fluorescent analogs of actin and its associated proteins in conjunction with multimode-based light microscopy of living cells to measure the complex interrelationships responsible for motility in patient-derived normal and transformed glia. I have simultaneously measured the dynamics of actin assembly and focal contact formation using new fluorescent analogs of actin and vinculin in single migrating human glioblastoma cells and used this information to begin developing a molecular model of tumor cell migration. By engineering new protein-based reagents and fluorescence spectroscopic methodologies we will be able to measure and manipulate a greater number of molecular processes and therefore further refine the model. The ultimate goal of this work is to use living cells to diagnose and design treatment for primary brain tumors in a more patient-specific manner.

Chromosomes are often arranged into specific configurations. One example is the metaphase plate of the Drosophila embryo in which chromosomes are arranged into a parallel bundle. How is this configuration established and maintained? Quantitative analysis of chromosomes motion in vivo should help answer this question by providing a measure of the relevant mechanical properties of the chromosomes themselves. In addition, motion analysis will allow us to study interactions of chromosomes with the mitotic spindle. In order to analyze moving mitotic chromosomes, we acquire time-lapse 3D images of chromosomes in living Drosophila embryos, and then interactively model the chromosome configuration at each time point. A model-based motion estimation algorithm is then applied. From the motion estimate, we can visualize trajectories of different regions on the chromosomes, such as centromeres and telomeres, during metaphase and during prometaphase congression. In addition, quantitative estimates of mechanical properties such as mobility and flexibility can be computed. In this preliminary report we describe computational tools for tracking and visualizing 3D chromosome motion, and for detecting oscillations in position along the mitotic spindle.

Voltage sensitive dyes permit the measurement of biomembrane electrical activity where microelectrode measurements are unsuitable or inadequate. This laboratory has developed 2 class of such dyes as well as some new optical methods for mapping membrane potential in single cells. One class of compounds consists of amphiphilic membrane staining dyes containing a putative electrochromic chromophore aligned perpendicular to the membrane/aqueous interface. We have used these dyes to map the membrane potential along the surface of single cells with high resolution dual wavelength ratiometric imaging microscopy techniques. We have found intrinsic regional variations in the electrical properties of cell membranes. Adding chirality produces compounds that display remarkable efficiencies for second harmonic generation. Further, the second harmonic signal can be generated from a dye-stained membrane and is modulatable by changing the membrane voltage. This can form the basis for measurements of membrane electrophysiology at high 3D resolution with infrared light. A second class of dyes are membrane-permeant cations which act as Nernstian indicators. We have developed calibration methods which allow the use of these compounds to quantitate the membrane potential of individual mitochondria from confocal or widefield images of living cells.

Determination of the organization, sign and strength of neuronal circuitry has resisted conventional anatomical and electrophysiological methods; it has proved particularly difficult to discretely activate isolated single circuits within the network of connections in the mammalian brain slice. Traditional electrical stimulating electrodes are poor tools for a detailed investigation of the organization of functional connections within brain slices, as they lack good spatial resolution and activate multiple neuronal circuits simultaneously. Scanning laser photostimulation offers many advantages over this and other current approaches: spatial resolution is superb, fibers of passage are not activated, and thousands of presynaptic locations can be stimulated. With this approach, caged neurotransmitters are activated in a restricted region of the brain slice by photolysis with a UV argon laser. Combining computer-controlled positioning of the laser light and whole cell recording in brain slices allows the construction of detailed maps of the position, strength, sign and number of inputs converging on a single postsynaptic neuron. We describe the technique of photostimulation, outline the instrumentation necessary to implement it, and discuss the interpretation of photostimulation- derived data. Finally, we will present examples of mapping circuits in the mammalian visual cortex using this approach. Although only recently developed, scanning laser photostimulation offers neuroscientists a powerful tool for determining the organization and function of local brain circuits.

The intracellular concentration of ions such as H+, Mg2+, Ca2+ is known to monitor the activity of many intracellular enzymes. Furthermore these ions are considered as intracellular messengers involved in signal transducing. Moreover recent technological progresses gave rise to the feeling that accurate data are instantly accessible on microvolumes. So the determination of ionic intracellular concentrations has been achieved using fluorescent specific probes and different equipments (Microspectrofluorometer, Flow Cytometer, Numerical Image Analyzer with or without Confocal system), without taking care of the physico-chemical properties of the probe. Unfortunately fluorescent probes are supposed to fill up conflicting requirements in terms of ionic affinity, specificity, fluorescence quantum yield of the free and ion-bound probe, absence of fading and diffusibility out of the cell. Because most of the probes are not so specific than it is claimed, unexpected interactions may obscure the interpretation of results and even make it difficult to get an intracellular calibration curve. Such a situation generally precludes the use of the popular simplest methods of data acquisition and treatment. The scope of this presentation is to point out some underestimated difficulties, to discuss different ways for bypassing some of them and to rationale the use of Videomicrofluorometry.

T-cell contact with antigen-presenting B cells initiates an activation cascade which includes an increase in T-cell [Ca²⁺] and leads to T-cell differentiation and proliferation. We evaluated cell-cell contact requirements for T-cell activation by using an optical trap to control the orientation of T-cell/B-cell pairs and fluorescence microscopy to measure subsequent T-cell [Ca²⁺] responses. B cells were trapped with a titanium-sapphire laser tuned to 760 nm and placed at various locations along the T cells, which had a polarized appearance defined by shape and the direction of crawling. T-cell intracellular [Ca²⁺] was detected as an emission shift from the combination of fura-red and fluo-3, two cytoplasmic [Ca²⁺] indicators. T cells which were presented antigen at the leading edge had a higher probability of responding (84% vs. 31%) and a shorter latency of response (42 s vs. 143 s) than those contacting B cells with their trailing end. Similar results were obtained using beads coated with antibodies to the T-cell receptor. These findings demonstrate a role for initial T-cell/B-cell contact geometry in T-cell activation by showing that the T cell is a polarized antigen sensor.

There is growing evidence that generation of reactive oxygen molecules (e.g., hydrogen peroxide, superoxide, hydroxyl and nitric oxide radicals) plays an important role in cell death. In this report we evaluated the effectiveness of the membrane permeable probe carboxy-dichlorodihydrofluorescein diacetate acetoxymethyl ester (C-DCDHF-DA-AM) for imaging the production of H₂O₂ in cultured cells. We examined the properties of three derivatives of the ester in saline droplets and compared the results with responses recorded from cells loaded with the ester. Results indicated that fluorescence was generated in cells and droplets by a photo-oxidative process involving H₂O₂. Videomicroscopy demonstrated that the cellular responses originated in small vesicles (presumably peroxisomes), with large responses filling the cytosol and enveloping the nucleus. We interpreted these responses as due to light-induced activation of flavin-containing oxidases, which generate H₂O₂ in peroxisomes, followed by diffusion of H₂O₂ throughout the cell. Escape of H₂O₂ from peroxisomes into Fe²⁺-containing compartments could have dire consequences on cell viability due to the production of hydroxyl free radicals. Such a mechanism could underly the phototoxic effects of visible light on cultured cells.

We demonstrate multi-photon excitation in optically-trapped living cells. Intracellular non-resonant two-photon excitation of endogenous and exogenous chromophores was induced by CW near infrared (NIR) trapping beams of 105 mW power. In the case of fluorescent chromophores, detection of NIR-excited visible fluorescence was achieved by imaging and spectroscopy methods. Trap-induced, two-photon excited fluorescence was employed as a novel diagnostic method to monitor intracellular redox state and cell vitality of single motile spermatozoa and Chinese hamster ovary cells. We found, that nonlinear absorption of NIR photons <800 nm may lead to oxidative stress and severe cell damage. Biological response was amplified in multimode CW lasers due to longitudinal mode-beating and partial mode- locking. As a result, we recommend the use of longwavelength-NIR, single-frequency traps ("optical tweezers") for micromanipulation of vital cells.

The problems of fluorescence bleaching and fluorescence attenuation in thick specimens need to be solved for quantitative fluorescence imaging of thick specimens by confocal microscopy. Methods for fluorescence bleaching correction based on data collected from the same specimen as the one used for quantitative investigation, have been devised (Nagelhus et al. Cytometry, In press). The present work describes model systems for the estimation of degradation effects such as attenuation caused by scattering and absorption and decreasing resolution with increasing depth in thick specimens. Monodisperse, fluorescently stained particles with fluorescence spectrally separate from the tissue fluorescence to be investigated, were placed on top of and underneath the tissue specimen to be studied. From these particles imaging parameters such as the lateral and axial point spread function and attenuation coefficients for excitation and emission light, were estimated. By recording the particle fluorescence and specific tissue fluorescence on separate detectors, correction parameters may be estimated for localized areas throughout the specimen. If, based on detailed investigation, the change in imaging parameters with increasing imaging depth in the tissue can be modeled, correction factors for each depth and lateral location can be estimated from recordings of particle fluorescence from the top and bottom of the biological specimen. We have investigated such problems by means of 0.2 μm and 6 μm fluorescent particles imaged through frozen sections of various thicknesses of tumor tissue grown in athymic nude mice, using a Bio-Rad MRC-600 confocal microscope. Together with correction factors for bleaching of the specific tissue fluorescence, we hope to use the estimated imaging parameters to correct for image deterioration with imaging depth, and thus obtain quantitative 3D data on fluorescence intensity throughout thick specimens.

A major problem in the cancer chemotherapy is the development of resistance to a whole range of drugs not only similar to the drugs used for resistance induction but also to some functionally and structurally unrelated. It's one of the multifactorial causes of failure of chemotherapy. Thus it appears essential to evaluate the multi-drug resistance (MDR) in living cells populations to: detect the MDR phenotype, to discriminate between resistant and sensitive cells, to identify mechanisms which are involved in the induction or the reversion of resistance and to study the cytotoxic process. Such a challenge implies the use of multiparametric approach that has been possible using a protocol involving microfluorometry connected to numerical image analysis on single living cells. This protocol relays on the correlation existing between the decreased intracellular accumulation of some fluorescent probes such as Hoechst 33342 (Ho342) and Rhodamine 123 (R123) in resistant cells. The simultaneous estimation of the fluorescence intensities of these probes has required the use of a third probe, the Nile Red, for cell contour delineation. The analysis of parameters related to Ho342 and R123 allows the discrimination of sensitive and resistant cells. So the multiparametric approach using multi-wavelength image analysis, which appears to be a powerful technique, has allowed us to show on human lymphoblastoid CCRF-CEM cells lines that the cytotoxic effects could be different depending on the cell resistance or on the cytotoxic drug used: Adriamycine, Vinblastine and the different cell behavior could be used for cell differentiation.

A new epi-fluorescence microscope for analysis of cells stained with two fluorochromes which can be spectrally isolated is described. The system makes it possible to perform independent and specific spectral selection of each dye (e.g. DAPI and CY3) while perceiving the two specific images simultaneously by eye. The optics uses splitting of the primary excitation and emission light beams, independent modification of the separated beams, and their reunification. Modifications in the separated beams comprise: (1) isolation of specific wavelengths (365 nm and 546 nm in the excitation light path, 435-500 nm and 590- 750 nm in the emission light beams), (2) wavelength switching without image displacement and blur by means of a light chopper alternating between ultraviolet-excitation/blue-detection and green-excitation/red-detection at frequencies of up to 140 Hz for observation by eye without image flicker, and (3) the possible separate positioning of lenses for compensation of chromatic aberrations. The system demonstrates a good transmission of the chosen wavelengths. A high specificity of double fluorescence analysis with minimal effects of spectral overlap was attained with good temporal resolution. It has been shown that it is feasible to obtain separate chromatic compensations for the use of a microscope objective in spectral regions outside the range for which the objective is corrected. Quantitative and independent measurements of the two fluorescence images by a CCD camera synchronized with the light chopper are feasible. In conclusion, this imaging system is outlined for highly specific visual analysis and exact quantitative measurement of double fluorescence labeled specimens in cytology and histology.

Light microscopy has become a versatile tool for investigating biological phenomena as they unfold, using cells as living microcuvettes. The progress is based on improvements in a number of technological fields, including optics, electronics, reagent chemistry and computer science. The non-destructiveness, spatial resolution and speed of optical imaging can provide high versatility for investigating biological structure and function at the tissue or even whole organism level as well. Having previously reported on some new approaches to meeting the challenges posed by the fluorescence-based imaging of cells and tissues, we concentrate here on imaging with increased spectral content and resolution. We improved and applied two techniques of spectral selection: (1) acousto-optic tunable filtering, allowing for multiwavelength fluorescence microscopy with diffraction-limited spatial imaging and sub- millisecond temporal resolution, and (2) 2D Sagnac interferometry-based Fourier spectroscopy, yielding advanced spectral imaging capabilities. Enhanced implementations of existing optical technologies, coupled with image processing, were key in these approaches. We present an overview of the methods, and summarize some of our results in applying these advances to imaging biological specimens. The extension of spectral selection approaches to the mesoscopic domain, suitable for in vivo imaging is also illustrated, by fluorescence-based tumor visualization in the near infrared spectral region. Finally, some future directions are discussed.

The development of peripherally substituted europium(III)-macrocycles suitable as luminescent bio-markers was continued in three related areas. (1) Protocols were established for the coupling of NCS-substituted Eu-macrocycles to proteins and for the mounting on microscope slides of particles labeled with luminescent Eu-macrocycles. The emission/excitation spectra of the dried, slide-mounted particles were investigated. (2) A procedure was developed for the synthesis of lanthanide-macrocycles having a single peripheral functionality. The structure and properties of the mono-functionalized macrocyclic complexes were established. (3) A study was undertaken to explore whether the emission intensity of the Eu-macrocycles can be increased by energy transfer from yttrium(III) complexes. Preliminary results have shown that a considerable luminescence enhancement can be achieved by this method. The results obtained in these three areas are evaluated in the light of the research reported by other investigators.

A phase-sensitive flow cytometer has been developed to quantify fluorescence decay lifetimes on fluorochrome-labeled cells/particles. This instrument combines flow cytometry (FCM) and frequency-domain fluorescence spectroscopy measurement principles to provide unique capabilities for making phase-resolved lifetime measurements, while preserving conventional FCM capabilities. Cells are analyzed as they intersect a high-frequency, intensity-modulated (sine wave) laser excitation beam. Fluorescence signals are processed by conventional and phase-sensitive signal detection electronics and displayed as frequency distribution histograms. In this study we describe results of fluorescence intensity and lifetime measurements on fluorescently labeled particles, cells, and chromosomes. Examples of measurements on intrinsic cellular autofluorescence, cells labeled with immunofluorescence markers for cell-surface antigens, mitochondria stains, and on cellular DNA and protein binding fluorochromes will be presented to illustrate unique differences in measured lifetimes and changes caused by fluorescence quenching. This innovative technology will be used to probe fluorochrome/molecular interactions in the microenvironment of cells/chromosomes as a new parameter and thus expand the researchers' understanding of biochemical processes and structural features at the cellular and molecular level.

Two existing Ada tools AdaSAGE and AYACC were combined to produce a system that parses International Society for Analytical Cytology, ISAC, Flow Cytometry Standard 2.0 files and stores the data in AdaSAGE tables. There are significant differences in the way manufacturers interpret and conform to Flow Cytometry Standard 2.0. AdaSAGE is employed to analyze and plot the data from multiple experiments. This data is used to assess the stability of flow cytometers. The initial release will be for DOS. The utilization of AdaSAGE, which is a flexible database tool, will facilitate subsequent development of other products. The software engineer, whose previous professional experience was with C and C++, had very few problems with Ada syntax. The interface to the compiler and other tools was immature compared to those available for C++. The DOS text based user interface environment provided by AdaSAGE limited the functionality of the user interface. However, the present DOS 386 program can be directly ported to the newly released version of AdaSAGE for Microsoft Windows 95. Ada's strong type checking and package structure have significantly facilitated the development of the product.

New methods for flow cytometric detection, analysis and cell sorting of rare (< 0.001 percent) cell subpopulations have been developed. A 2-stage system of high-speed cell (and data) classification (U.S. Patent 5,204,884) allows first-stage cell classifications and error-Checking at rates in excess of 100 000 cells/sec. Multi-queuing, secondary-stage electronic processing on the basis of up to 11 parameters, including mathematical functions such as principal components calculated in real-time using lookup tables, allows for reduction of false-positive cells multiply-labeled with positive- and negative-selection fluorescent markers. This has allowed detection of rare cell subpopulations with frequencies below 10^-6. Special "flexible sorting" hardware and software (U.S. Patent 5,199,576) permits sorting of cell subpopulations on the basis of mathematical algorithms which are not limited to conventional rectilinear or bitmap sort boundaries. Both high-speed enrichment sorting of live cells and high-resolution sorting for single-cell molecular characterizations are supported. New sampling statistics software allows for prediction of required sorting times for rare cell subpopulations. Home-built "bridging" software facilitates analysis of rare cells by a number of home-built (e.g. PC/Biplot) and commercial software packages (e.g. S-Plus for Windows). 3D stereo visualization and interactive software provide viewing of three raw and/or mathematically transformed or constructed data parameters, to aid in subsequent selection of optimal sort criteria. Special software has been developed for improved data analysis and selection of sort boundaries through the use of cell classification-tagged listmode data mixtures. This permits comparison of different classification algorithms (e.g. cluster analysis, neural networks, or recursive partitioning) for rare cells.

Analysis of white blood cell flow cytometry light scatter data remains a challenging problem. Conventional methods for analyzing flow cytometry data use 2D scatterplots of multidimensional data. We have developed an automated method to locate and characterize clusters within WBC data. Our method uses the full dimensionality of the data and employs the recursive application of a two-step algorithm. Input to the first step is a dataset of cellular events divided into two assumed clusters. A clustering algorithm iteratively refines these initial clusters. This algorithm is based upon an extension of the k-means clustering algorithm. If two populations are confirmed, the data for each cluster are passed, in turn, to the second step, a splitting algorithm. This algorithm determines the potential for further subdivision of the data. When this potential exists, an approximate division of the data is made. These new subclusters are passed as input to step one and the process is repeated. The process terminates when either the clustering algorithm converges to a single population or when the splitting algorithm finds no potential for further subdivisions. Using the full dimensionality of the data, our method can characterize clusters within WBC data even in cases where these clusters overlap in the standard 2D scatterplots.

A benchtop optical flow cytometer utilizing microfabricated silicon V-groove flow channels (25 micron diameter) has been constructed and tested using diluted whole blood. A 1.2 mw diode laser probe beam focused onto the flow stream is used to generate scattered light from passing blood cells. Photodiode detectors are used to collect both small and large angle signals which are counted and analyzed for pulse peak intensities. Count rates as high as 1000/second have been obtained using pressure heads of about 0.5 psi. Optical modeling has also been carried out in order to determine the light scattering signature of blood cells passing down various channel flow lines. Results suggest that a significant amount of experimental signal variability may be due to variations in the positions of cells passing through the sampling region, but that this degree of signal variation should not prohibit the discrimination of different cell populations. Experimental and computational results are presented and discussed, as well as the possibility of developing a miniature portable flow cytometer based on microfabricated flow channels.

We determine the optical properties of whole blood samples in the near infrared spectral range from double integrating sphere measurements using an inverse Monte Carlo technique. The measured values included the diffuse reflectance, the total transmittance, and the collimated transmittance. From these data, the absorption coefficient, the scattering coefficient, and the anisotropy factor were derived. The spectral range investigated extended from 700 nm to 1200 nm. It was found that the optical properties of blood were substantially different from the respective data for other relevant human tissues known so far. In addition, we analyzed the effect of the scattering phase function approximation on the resulting estimates of the optical properties. The Henyev-Greenstein and the Gegenbauer kernel phase functions were considered. The calculated angular distributions of scattered light were compared with goniophotometric measurements performed at the wavelength of 633 nm. The data presented in this study prove that the variations of the employed scattering phase function approximation can cause large discrepancies in the derived optical properties. This leads to the conclusion that the exact knowledge of the scattering phase function is required for the precise determination of the optical constants from the double integrating sphere measurements.

The effects of pulsed light (2 Hz) with a 55% duty ratio and continuous light on the migration of human mononuclear leukocytes, MNLs (monocytes and lymphocytes) are reported for red light ((lambda) equals 660 nm) and green light ((lambda) equals 565 nm). The comparison of the relative value of the distance to blood cell migration under light to the control cell migration without light stimulus is recorded as cytokinetic index, K.I. K.I. is a measure of the cytokinesis which is the progress of the cell movement in which the migration is enhanced by substances in the cell environment irrespective of a concentration gradient. Red light stimulation produces K.I.'s for PMNs which are 30% grater than for MNLs. Green light stimulation produces K.I.'s for PMNs less than 1.0 indicative of inhibited migration, while for MNLs the K.I.'s are slightly greater than 1.0 indicative of enhanced migration.

Hemolysis as a consequence of open heart surgery is well investigated and explained by the oxidative and/or mechanical stress produced, e.g. by the heart lung machine. In Europe O₃ is widely used by physicians, dedicated to alternative medicine. They apply O₃ mostly by means of the Major Autohematotherapy (MAH, a process of removing 50-100 ml of blood, adding O₃ gas to it and returning it to the patient's body). No controlled studies on the efficacy of O₃ are available so far, but several anecdotal cases appear to confirm that MAH improves microcirculation, possibly due to increased RBC flexibility. Most methods established to estimate RBC deformability are hard to standardize and include high error of measurement. For our present investigation we used the method of laser diffraction in combination with image analysis. The variation coefficient of the measurement is less than 1%. Previous investigations of our group have shown, that mechanical stress decreases deformability, already at rather low levels of mechanical stress which do not include hemolysis. On the other hand exposure to O₂, H₂O₂ or O₃ does not alter the deformability of RBC and--except O₃--does not induce considerably hemolysis. However this only holds true if deformability (shear rates 36/s - 2620/s) is determined in isotonic solutions. In hypertonic solutions O₃ decreases RBC deformability, but improves it in hypotonic solutions. The results indicate that peroxidative stress dehydrates RBC and reduces their size. To explain the positive effect of O₃ on the mechanical fragility of RBC we tentatively assume, that the reduction of RBC size facilitates the feed through small pore filters. In consequence, the size reduction in combination with undisturbed deformability at iso-osmolarity may have a beneficial effect on microcirculation.

There are various optical methods to study the rheological properties of RBC's subjected to shear stress. The commonly used techniques are rheoscopy and ektacytometry, which use the diffraction pattern of light transmitted through the dilute suspension to study some changes in the shape and deformability of RBC's¹. But the conditions of light scattering in the dilute suspensions are different from that in the concentrated suspensions or whole blood. The aggregometry method based on the regisration of light scattered from the blood layer is used for the monitoring of aggregational properties of erythrocytes in stasis and under the shear stress^2'3. One of the methodological approaches in the development of aggregometry is based on the registration of light transmission through a thin blood layer in a cone-plate type viscometer^4'5. Another method is based on the registration of the backscattered light from a blood layer in a Couette flow viscometer^6'7. The differences in the flow geometry and in the modes of detection of the outcoming light have yielded different results in the study of hyperaggregational syndrome even in the in vitro experiments⁸. Comparative clinical approbation of the methods in three hemorheological centers have shown wide variation ofred blood cell aggregation characteristics⁹. We use the laser backscattering nephelometry technique to study the deformation and aggregation (or disaggregation induced by shear stress) of RBC's in whole blood in Couette flow. The estimated parameters of a blood sample are the characteristic times of aggregation T₁ and T₂, the index of hydrodynamic strength of aggregates β, and deformability of erythrocytes at high shear stress D. The measured parameters of the aggregation process in whole blood essentially depend on the degree of deformability of RBC's. The deformation and orientation of cells in a shear flow can be indirectly determined from the registration of the anisotropy in light scattering. Such measurements were carried out with a rotational viscometer¹⁰. These experiments with deformable erythrocytes showed the existence of an angular assymetry in the backscattering diagram. This assymetry was higher when the shear rate and the viscosity of the suspending medium increased. When performed with cells hardened with glutaraldehyde the backscattering experiments showed no changes in the backscattered diagram. In this paper we discuss a modified aggregometry technique which enables to register the anisotropy of light-scattering thus yielding additional information on deformation of RBC's under high shear stress and some parameters of the aggregation process in whole blood.

Static laser light scattering technique has been used to study the aggregation of red blood cells in plasma with dextran in it in different concentrations. The radius of gyration, the structure factor and the fractal dimension are determined for the clusters using the method given by Sorensen et al for the fractal clusters.

We are reporting on the results obtained in the development of our previous paper. We have implemented the previously described unified approach of particle image velocimetry and computer morphodensitometry trying to find correlation between morphofunctional and dynamic parameters of red blood cells of human donors who had undergone low-doze ionizing irradiation conventionally used in radiology. Specifically we examined the suspension of RBCs obtained from children who had accidentally or therapeutically undergone irradiation with different doses (0.03-0.04 Gy), and compared them to controls. Our experiments have shown the existence of correlation between the rheological and morphometric indexes obtained with the help of the computer image processing system Diamorph.

Bio-speckles obtained from the human retina are used to measure the retinal blood-flow velocity on the basis of the photon correlation analysis. The reproducibility of measurements was experimentally investigated by using a stable rotating ground-glass disk and for the normal human retina, and the error was estimated to be less than 20%. Using a glass capillary model, the reciprocal of correlation time was calibrated to the mean flow velocity by considering the influences of the vessel diameter and the background reflectance. The blood- flow volume rate in the normal human retina was estimated by using the calibrated velocity and the vessel diameter. The results were well compared with the previous data, and show the usefulness of the method for clinical diagnostic purposes.

Recently a blood velocimeter was developed, based on the principle of self-mixing in a semiconductor laser. This means that the intensity of the light is modulated by feedback from moving scattering particles, which contains the Doppler shift frequency. Upon feedback the characteristics of the laser diode will change. The threshold current will decrease and an instable region may become present just above the new threshold. It turns out that the amplitude of the Doppler signal is related to the difference in intensity between the situations with and without feedback. This amplitude is highest, but also most unstable, just above feedback. The suppression of reflection from the glass fiber facets is of paramount importance. Using an optical stabilization of the feedback, we are able to optimize the performance of the laser-fiber system and the Doppler modulation depth, and to clarify the behavior with a suitable physical model. The velocimeter has been used in vivo with the glass fiber inserted in normal catheters, but in upstream and in downstream situations. For the latter, the fiber facet in the liquid has been provided with a special side-reflecting device.

By irradiation of the skin with a diode laser the blood flow can be measured using the back scattered light. The majority of the back scattered light is scattered from fixed cells, but a small amount is scattered from moving erythrocytes and is Doppler shifted. The mixed light detected with a photodiode leads directly to a photocurrent proportional to the Doppler signal. A laser Doppler blood flow meter was developed, consisting of a PC-AT (66 MHz), a commercial A/D card (14 bit, 100 kHz) and a special head control card for two laser heads. The heads have a diameter of 35 mm and a height of 15 mm. The heads are fixed to the skin with tape. A DOS-Pascal software program provides the measurement, the calculation of a whole blood flow spectrum and shows the results on the screen with a time resolution of up to 50 Hz. The laser head is connected via 2 m cables with the head control card and contains a diode laser (5 mW, 670, 785 nm) a micro lens, two photodiodes and a pre-amplifier. On the head control card there is the current supply for the laser diodes, also the Doppler signals are band passed (400 Hz to 50 kHz), further amplified and fed into the A/D card. On the A/D card the analog signal is sampled with 100 kHz and digitized with 14 bit resolution. With 256 samples a frequency spectrum (128 channels, 0-50 kHz) is calculated by a FFT, but only the first 100 channels (0-39 kHz) are used to prevent ghosts. The measurements of the Doppler signal of the two laser heads need 5.2 ms and for all software calculations 15 ms are necessary. A quantitative recalculation of a velocity spectrum from the frequency spectrum is only possible if the velocity-, the irradiation-, and scattering-directions are known. In skin the small blood vessels have various directions and because of scattering in skin the irradiation of an erythrocyte can be assumed to be random. With this assumption of a random direction distribution a velocity spectrum can be calculated from the frequency spectrum. A flow spectrum is defined as the product of the velocity and the intensity at this velocity. The flow is defined as the integral over a given region of the flow spectrum. Four independent flow curves can be shown simultaneously on the screen and the frequency region for each flow can be set independently. The flows from low (1-3 mm/s) and high (7-9 mm/s) velocities show a different behavior and give the possibility to distinguish between the flow in the micro capillaries and larger vessels in the skin.

An approach for accelerated simulating of the Doppler frequency spectra histogram (DFSH) of a signal from the laser Doppler flowmeter (LDF) using a Monte-Carlo method is suggested. An extra averaging over permutations of the moving and stationary particles along the photon trajectories enables noticeably less trajectories to be simulated in order to obtain an estimate of the DFSH with a pre-set accuracy as compared with the traditional method. The convergence of the DFSH by this 'fast' method to that by the traditional method is demonstrated for different experimental conditions and the computational time advantage of the fast method is evaluated.

Laser Doppler Perfusion Imaging (LDPI) is a method for visualization of tissue blood perfusion. A low power laser beam is used to step-wise scan a tissue area of interest and a perfusion estimate based on the backscattered, partially Doppler broadened, light is generated. Although the basic operating principle of LDPI is the same as that of conventional Laser Doppler Perfusion Monitoring (LDPM), significant differences exist between the implementation of the methods which must be taken into account in order to generate high quality perfusion images. The purpose of this study is to investigate the relevance of a number of LDPI design parameters, such as: (1) The influence of artifact noise when using a continuously moving laser beam instead of a step-wise moving beam to scan the image. (2) The signal processor output's dependency on the distance between the measurement object and the scanner head when using collimated laser light. (3) The speed and mode of the scanning. The results show a substantial rise in the noise level when using a continuously moving beam as opposed to a step-wise. Skin measurements using a collimated laser beam demonstrated an amplification factor dependency on the distance between the skin surface and the scanner head not present when using a divergent laser beam. The scanning speed is limited by the trade-off between the Doppler signal lower cut-off frequency and the image quality.

Noninvasive registration and adequate analysis of response of living systems to low energy physical stimuli play an important role in contemporary biological and biomedical research. In this paper we discuss the possibilities of laser Doppler microscopy (LDM) application for the study of light irradiation and thermal effects on animal organisms. Fish embryos are shown to fit many requirements related to the performance of experiments in clearly defined conditions. LDM yielding quantitative data on the dynamic response of the embryos to temperature variations and light irradiation is proved to be an adequate diagnostic technique. A fringe- mode sign-sensitive LD microscope was used to monitor the alterations of blood flow in fish embryos. Measurements were performed with spatial resolution as small as 10 micrometers in real time scale. Experimental data on the biological effects of thermal and nonthermal light stimuli on different fish species are presented.

The theoretical and experimental investigations of the focused Gaussian beam (FGB) diffraction both in blood and lymph microvessels have been carried out. Speckle-interferometrical technique with the spectral analysis of scattered light intensity fluctuations has been applied for the investigation of lymph circulation in native microvessels. The measuring errors of bioflow velocity has been analyzed. Alterations caused by the drug influence on lymph vessels have been studied. Doppler method using FGB scattering is considered theoretically and experimentally.

Perfusion of tumor tissue is a necessary prerequisite for radiotherapy and Photodynamic Tumor Therapy (PDT). For PDT perfusion means oxygen supply to a light activated photosensitizing drug inside tumor cells to destroy these cells specifically by light activated singlet oxygen. These experiments are normally done in rodents, but can be much more easily performed on tumors transplanted to the extraembryonal painfree vessels developed during chick embryogenesis. We chose the yolk sac membrane (YSM) and the chorioallantoic membrane (CAM) for quantitative blood flow measurements. Near infrared light (830 nm) was used for measurements in vascularized xenotransplanted tumors on the extraembryonal membranes because at this wavelength blood flow can be detected also in vessels covered by tumor cells. We measured the influences on blood flow of different photosensitizers with and without therapeutic irradiation and single components of a polyphasic PDT System.

The ability of reliable, non-invasive, and quantitative characterization of biological fluids containing submicroscopic particles in a in-situ/in-vivo environment may open new possibilities to better understand these fluids. This paper focuses the use of a newly developed dynamic light scattering fiber optic probe to assist in this goal. A in-situ/on-line application to study protein aggregation and growth of protein crystals from solutions and one in-vivo application involving study of cataractogenesis in an animal model are presented.

This paper examines non-invasive methods for absolute determination of the hemoglobin content of arterial blood and the water content of skin. Both methods are based on diffuse reflectance spectrophotometry in the near-infrared band (800 - 1600 nm). Separation of blood and background tissue spectra is accomplished by a technique similar to pulse oximetry, with the added feature that the set of measurement wavelengths is chosen to be sensitive to both hemoglobin and water concentration in the blood. Regressions performed on a simulated tissue spectra suggest that {1060, 1160, 1200 and 1320 nm} is an optimal set of wavelengths for measurement of tissue hydration and {1040, 1120, 1140 and 1200 nm} is an optimal set of wavelengths for measurement of hemoglobin content under typical measurement conditions. A simple in vitro tissue phantom whose optical properties can be altered in a controlled manner was developed to test the feasibility of the methods. Measurements were made with a custom-designed NIR spectrophotometer.

A new technique based on the optogalvanic effect has been developed for the measurement of stable isotope ratios in the carbon dioxide of exhaled breath. Data obtained before and after ingestion of harmless stable isotope labeled compounds, metabolized to carbon dioxide, can be used for sensitive noninvasive diagnostics of various disease conditions. The technique uses the specificity of laser resonance spectroscopy and achieves sensitivity and accuracy typical of sophisticated isotope ratio mass spectrometers. Using fixed frequency carbon dioxide lasers, ¹³C/¹²C ratios can be determined with a precision of 2 ppm with 100 second averaging times. Multiple samples can be analyzed simultaneously providing real time continuous calibration. In a first application, analysis of ¹³C/¹²C ratios in exhaled human breath after ingestion of ¹³C labeled urea is being developed as a diagnostic for the bacterium H-pylori, known to be the causative agent for most peptic and duodenal ulcers.

Micro-endoscopes based on a fiber optic conduit and thin rod lens are investigated for applications in tissue diagnostics and studies of fluid transport in tissue-like matrices. The design, evaluation and implementation of these moderate to high resolution optical instruments is presented. The optical configuration employs a micro-endoscopic probe, an objective lens and a commercial color CCD. The flexible micro-endoscope is 2.65 meters in length, with an outer diameter of 1.5 mm and is employed to quantitatively measure EBT transport through a tissue-like substrate. The determined resolution is 20 line-pairs/mm (LP/mm). Calibration is accomplished by measuring absorbance of Eriochrome Black T dye against a reflective background. Fluorescence detection of fluorescein is evaluated using a thin rod lens micro- endoscope. The rod lens is 210 mm in length, 3 mm in outside diameter and can resolve 160 LP/mm. This resolution allows imaging of cellular organelles having a diameter of 2.8 mu;m. The limit of detection of the rod lens endoscopic system is approximately 20 pmol for fluorescein.

An non-invasive optical method for obtaining quantitative biochemical information is desirable. While quantitative methods for predicting fluorophore concentrations in dilute solutions are well known, human tissue is highly scattering and absorbing and cannot be accurately treated with these techniques. We have employed the method of partial least squares (PLS) to develop an empirical, linear model of sample fluorescence from a training set with optical properties and known concentrations representative of those to be predicted. This model can be applied to predict chromophore concentrations in the unknown samples.

A review of the optical methods designed for the study of intracellular protoplasmic streaming is presented. While in case of low streaming velocities (less than 10 mcm/s) the computer analysis of the images of moving protoplasm is effective, for higher velocities the laser Doppler microscopy has an advantage. With the help of the Doppler technique it is possible to determine the velocity profiles for nonstationary protoplasmic streamings in amoeboid cells. These data and also complex time changes of protoplasmic flow in response to external factors enable to determine and to specify parameters used for designing mathematical models of intracellular dynamics. As an example, results of experiments, modelling and simulations are presented for myxomycete plasmodium Physarum polycephalum which is a huge cell with typical amoeboid properties.

Under laser irradiation of different photosensitizers (PS) mixtures with pure albumin or without hemolysis blood serum the photodynamic effect (PE) is scarcely to be manifested. The coupling of PS with albumins prevents the interactions of dissolved oxygen molecules with PS molecules and formation of active oxygen forms. In order to promote the PE it is necessary to add the solution of hemoglobin. The PE is readily recognized in mixtures of PS with the blood. Such mixing leads to the erythrocytes' destruction and yields uncombined hemoglobin in blood plasma. The irradiation of hemoglobin mixtures with PS leads to the destruction of hemoglobin. In this case the direct combination of oxygen molecules with hemoglobin is important for PE performance (the deoxy hemoglobin can not promote PE otherwise).

The original optical diagnostic device for measuring the RBC membrane permeability and RBC charge is considered in this message. A blood microsample drips in the mixer filled with a solution of NaCl. The resultant RBC suspension trickles down the pipe into the drain vessel. The flat thin cell is fitted into the pipe. The optical channel consists of He-Ne laser whose beam goes through the flat cell perpendicularly to its sides and scatters by the RBC flowing through the thin cell. The scattered light falls on a frosted screen put in the focal plane of a lens. As the RBC concentration is more than 0.1% of suspension volume, RBC form two flows moving along slightly heparinized sides of the thin cell due to repel each other electrostatically. The two flows orient each other so that RBC round bases are perpendicular to the sides of the flat cell due to RBC dipole momentum. In this case RBC viewed from the side will form on the screen a visible diffraction ellipse with axes lengths related in the initial time as 4:1. The measurement of the rate of the changes of the lengths of the axes of the diffraction ellipse due to osmos made it possible to develop a number of original optical diagnostic techniques approved by clinical practice. The method of measuring the membrane permeability was approbated clinically by examining blood samples (50 mcl) of 30 patients suffering from heavy poisoning by alcohol and barbiturates before and after detoxifying treatment and allowed the use the method developed for diagnosis the degree of poisoning and choosing the appropriate detoxifying rehabilitation. Unlike the ectacytometer where the shear stress between two planes is constant, the device offered has an area in the center of the cell with zero shear stress. It is the area where RBC should go with the increasing shear stress in the cell. The electric charge of RBC prevent them from going to the central plane, loosing mutual orientation, and can be measured.

Hydrophobic binding sites of native and defatted human serum albumin (HSA) of individual donors were tested using a lipophilic fluorescent dye, Nile red (NR), over the pH interval of 6 to 10 covering the range of the neutral to base (N yields B) transition of HSA. A single NR-binding site of high affinity is present on the protein molecule, more hydrophobic (bound dye fluorescence maximum at (lambda)_max approximately 630 nm) as compared to low- affinity sites ((lambda)_max approximately 640 nm). A calculation procedure has been developed for monitoring the dissociation constant (K_d) of the dye complex with protein high-affinity binding site, based on the pH-profiles of dye fluorescence registered at two protein concentrations. A pH-profile of NR decoloration rate in the presence of HSA correlates with that of K_d and reflects pH-dependence of the protein affinity to the dye. N yields B transition of HSA results in an essential increase in the protein-NR affinity (lowering K_d from 4 - 7 μM for N-form down to 1 - 3 μM for B-form). Spectral features of protein-bound NR monitor an alkaline shift of the pH-range of N yields B transition upon deliganding HSA, midpoint pH increasing from 7.6 - 8.0 to 8.2 - 8.3. Numerous changes in the parameters of protein-bound NR fluorescence were revealed over the range of pH 6 - 10. The pH-profiles of the parameters varied for HSA of different donors and were not unified upon albumin deliganding. This variability is presumably caused by individual structural differences arising from both the ligand loading and covalent modification of the protein. The value of HSA-NR affinity is considered as possessive of a diagnostic potential.

By means of the new method of transmission-resonance EHF/SHF radiowave spectroscopy alteration of the resonance structure of water, whole blood, blood plasma, serum and erythrocyte hemolysate under the influence of He-Ne laser radiation was established. Fine molecular alterations was occurred after interaction of bioliquids with laser light.

Experimental studies were performed on 220 male rats of Wistar line to reveal optimal parameters of laser radiation causing positive changes in biotissues and to select methods of laser therapy. Irradiation of the ventral abdominal wall performed by arsenide-gallium injector (710 - 890 nm, exposure - 128 sec) in pulse rate: 3000 Hz, 1500 Hz, 80 Hz. Content of lymphoblasts, medium and small lymphocytes, plasmocytes, T-lymphocytes and T-helpers as well as the activity of chromatin and lysosomal enzymes were determined in the dynamics of thymus, spleen and lymph nodes. During irradiation with the rate of 3000 Hz prevailing inhibiting influence on the immumocytopoesis and functional activity of lymphocytes in all organs studied was state, the effect being manifested by the decrease in the number of all forms of lymphocytes particular on the 3rd-5th-7th day followed by normalization on the 15th-21st-30th day. Irradiation with the rate of 1500 Hz produced stimulating effect on the immune organs accompanied by reliable excess of control indices of lymphocyte content particularly of poorly differentiated forms (blasts and medium ones), as well as by the increase of the number of plasmocytes, T-lymphocytes, T-helpers with maximum manifestation on the 7th day. On the 15th day there is a decrease, and on the 21st-30th day--there is normalization. Irradiation with the rate of 80 Hz produced the smallest but most marked effect, particularly on the number of lymphoblasts. Peculiarities in kinetics of cellular elements studied were revealed in different lymphoid organs and in different functional zones of these organs.

Problem of studies of biological micro-ojects is actual one for ecology, medicine, biology. Holographic techniques are useful to solve the problem. The above microojects are transparent or semitransparent ones in a visible light rather often. The case of an optically soft particle, (that is of a particle whose substance has the refractive index close to that of the surrounding medium) is quite probable in biological water suspensions. Some peculiarities of holographing optically soft microparticles are analyzed in this paper. We propose a technique to calculate a light intensity distribution in the plane of a hologram and in the plane of a holographic image of a particle of an arbitrary shape at an arbitrary distance from the latter plane. The efficiency of the approach proposed is demonstrated by calculational results obtained analytically for some simple cases. In a more complicated cases the technique can make a basis for numerical computations. The method of determining of refractive index of transparent and semitransparent microparticles is proposed. We also present in this paper some experimental results on holographic detection of the water drops and such optically soft particles as ovums of helmints in human jaundice.

The possibility is shown to use optical methods of measurement of suspension transparency and holographic techniques of diagnostics of spatial particles ensembles simultaneously for nondestructive testing of microstructure parameters of water suspensions in biology, medicine, and environmental control. On the basis of calculated and experimental results, characteristics of the above methods are analyzed in the paper. We propose an optical scheme allowed us to realize both base method of measurement of suspension transparency and holographic technique with image transfer. An estimations have showed possibility of measurement of attenuation from about 10^-5. An allowable size of holographing microparticles is no less than 2 micrometers in a volume of several cm³ and more in dependence on the particle size and number density. These estimations demonstrate the possibility to use the above methods for investigation of natural media. We also propose in this paper some potential applications of these methods.

This work is the optical study of interinfluence between different physiological parameters of the human blood such as oxygenation rate and erythrocyte aggregation level. The evaluation of such influence upon optical properties of the blood has been also carried out in vitro. Optical parameters such as total and diaphragm transmission and diffuse reflection have been investigated by in vitro set up for the whole blood. Change of the blood oxygenation has been created by membrane oxygenator. According to the received data the coefficients of scattering and absorption and the mean cosine of scattering have been calculated for the whole blood by inverse Monte-Carlo simulations for following wavelengths: 633, 675, 820, 860 nm.

An Imaging Spectrometer has been developed which allows spectroscopy to be exploited in new and easier ways: as a result, new and useful information, not achievable so far with reasonable efforts, will be at the disposal of the researcher and the clinician. Chemical mapping of biological material with a spatial resolution at pixel level, will be performed under the microscope with high sensitivity and accuracy, and much faster than has been possible so far.

Differential interference contrast imaging is frequently used to give improved contrast in studies of thin unstained live samples. Similarly, confocal microscopy is used to study stained live samples, because of its ability to optically section thick cells or tissues. We present two fast laser scanning heterodyne differential phase confocal microscopes, which combine the advantages of both these systems. The output of both systems is proportional to the sine of the phase gradient, which gives increased sensitivity when compared to DC interference systems. In one system the differentiation is performed in the image plane, with a single probe beam and a split detector, in the other system, the differentiation is performed in the object plane, using a split beam and a single detector. A direct comparison between the imaging performance of the two systems is made, and an optimized design has been developed from them. Resolution of better than 0.3 μm is achieved for both systems, fast beam scanning gives frame rates of approximately one second. The optimized microscope will also contain a fluorescence detection channel, the fluorescence image being obtained simultaneously with the reflection/differential phase image to ensure precise spatial and temporal alignment. The images obtained from these two detection techniques can be superimposed, and may be used to enhance each other, the differential phase image showing general structure. A number of novel imaging techniques will be investigated using the microscope.

High resolution fluorescence imaging in intact tissues faces special challenges posed by scattering of excitation and fluorescence light and the need to avoid photodynamic damage. Significant improvements over conventional widefield and confocal imaging are provided when two-photon excitation is used. Applications to the functional imaging of the calcium dynamics in synaptic spines, small invertebrate neurites, and auditory hair cells are shown. Two-photon absorption induced photolysis can also be used for scanning photochemical microscopy and for high resolution measurements of diffusional coupling between cellular compartments.

FISH (fluorescence in situ hybridization) is a cytogenetic technique for locating and quantifying small genetic defects that cannot be detected by traditional karyotyping, the banding analysis of stained chromosomes. The selectivity of FISH holds the promise for the accurate, low cost and fast diagnosis of genetic disorders, birth defects, and various types of cancer that are not detectable by other means. To aid investigators with their FISH measurements, filter-based digital imaging systems are available to enhance and analyze faint FISH images. The accuracy and reliability of traditional FISH measurements is generally compromised when there is (1) sample autofluorescence, (2) image movement, e.g., due to lack of registration between images acquired through different filter sets, or (3) spectral overlap between fluorescent DNA probe emission spectra. SpectraCube^TM is an interferometric imaging method which is not subject to the limitations of filters-based systems. By measuring a definitive spectrum, simultaneously at all points in a sample, SpectraCube^TM has the potential to revolutionize FISH by enabling the detection and separation of a large number of spectrally and spatially overlapping fluorescent DNA probes, and by eliminating sample autofluorescence. Details of the Applied Spectral Imaging SD200 spectral bio-imaging system and the results of a six-color FISH measurement will be discussed.

Applications of fluorescence microscopy in biology range from macromolecular to multicellular, with living specimens in particular placing severe constraints on the resolution, precision, speed, and optical efficiency of the image-forming instrumentation. In most cases, a high-resolution optical system will produce images in which the axial position of a feature in the specimen is encoded through sharpness of focus, leading to inherently 3D data. Even in the case where quantitative analysis of only one particular plane of focus within the specimen is to be made, fluorescence from that stratum must be isolated from out-of-focus contributions. At the present time, (1) confocal scanning and (2) direct imaging used with computational deconvolution are the main methods by which object structure is extracted from serial-focus fluorescence image sets. In addition, fluorescence microscopes utilizing interferometric illumination, multi-photon excitation, composite apertures and image interferometry have been introduced specifically to improve 3D resolution. In this report, we look specifically at the constraints imposed by biological specimens on instrumentation used for 3D fluorescence imaging.

Two-photon excitation imaging is a recently described optical sectioning technique where fluorophore excitation is confined to--and therefore defines--the optical section being observed. This characteristic offers a significant advantage over laser-scanning confocal microscopy; the volume of fluorophore excited in the minimum necessary for imaging, thereby minimizing the destructive effects of fluorophore excitation in living tissues. In addition, a confocal pinhole is not required for optical scattering--thus further reducing the excitation needed for efficient photon collection. We have set up a two-photon excitation imaging system which uses an all-solid-state, short-pulse, long-wavelength laser as an excitation source. The source is a diode-pumped, mode-locked Nd:YLF laser operating in the infrared (1047nm). This laser is small, has modest power requirements, and has proven reliable and stable in operation. The short laser pulses from the laser are affected by the system optical path; this has been investigated with second harmonic generation derived from a nonlinear crystal. The system has been specifically designed for the study of live biological specimens. Two cell types especially sensitive to high-energy illumination, the developing Caenorhabditis elegans embryo and the crawling sperm of the nematode, Ascaris, were used to demonstrate the dramatic increase in viability when fluorescence is generated by two-photon excitation. The system has the capability of switching between two-photon and confocal imaging modes to facilitate direct comparison of theory of these two optical sectioning techniques on the same specimen. A heavily stained zebra fish embryo was used to demonstrate the increase in sectioning depth when fluorescence is generated by infrared two-photon excitation. Two-photon excitation with the 1047nm laser produces bright images with a variety of red emitting fluorophores, and some green emitting fluorophores, commonly used in biological research. Fortuitously, we have found that at least four blue emitting fluorophores normally excited by UV light are excited by the pulsed 1047nm laser, by what we believe to be three-photon excitation. Multi-photon excitation is demonstrated by a double labelled C. elegans embryo.

The measurement of fluorescence lifetimes offers the advantages of being independent of local intensity and concentration of the fluorophore, and can provide information regarding the molecular environment in a single living cell. Historically, measurements of fluorescence lifetimes have employed photomultipliers as detectors, providing high sensitivity but sacrificing spatial information. Fluorescence Lifetime Imaging Microscopy (FLIM) provides a 2- or 3D spatial map of the distribution of fluorescent lifetime(s) in the sample under observation. Picosecond laser pulses from a tunable dye laser are delivered to fluorophore containing living cells on the stage of a fluorescent microscope, and images of the fluorescence emission at various times during the decay of the fluorescence lifetime are collected using a high speed nanosecond-gated multichannel plate image intensifier. FLIM promises to substantially enhance the information obtainable from living cells and tissues, and will allow observations of the dynamic organization and interaction of cellular components on a spatial and temporal scale previously not possible using other microscopic techniques.

Optical coherent-domain tomography (OCT) uses low-coherence light interference to achieve on-axis optical sectioning and lateral scan for 3D optical imaging in scattering media. Owing to its exceptional resolution of approximately 10μm and high dynamic range in excess of 100dB, this technique is potential for the detection of the microstructures in biological tissues. Although not being able to resolve to the cell extent in most biological tissues because of multiple light scattering, it can still provide important diagnostic information for either low- scattering or superficial, high-scattering biological tissues according to our preliminary clinical experiments. In this paper, after showing the influences of multiple scattering effects on imaging contrast, we will present some 2D OCT images for evaluating the effects of laser thermal keratoplasty (LKT), then show the images of in vitro porcine bladder and human tongue. These results show that OCT can be developed into a promising means of noninvasive evaluation of laser-tissue effects, e.g., laser coagulation and ablation, in vivo location of superficial lesion and cancerous regions to aid minimum invasive surgery.