For the study of biological systems such as living cells, access to oxygen concentrations in various organelles is important. In living cells, the lifetime of pyrene fluorescence can be used to measure local oxygen concentrations. We designed and synthesized a new probe to measure the oxygen concentration in mitochondria ([1'- pyrene butyl]-2-rhodamine ester, PRE). The localization of the probe was studied by videomicrofluometry in 3T3 cells and confirmed to label the mitochondria. We compared the lifetime of PRE with the well-known cytosol specific probe Pyrene Butyric Acid in (1) in living cells (2) in liposomes and (3) in solution. Liposomes were used to investigate the effect of phospholipid bilayer organization on the fluorescence lifetimes. Depending on the oxygen concentration we observed lifetime variation ranges of (1) 60 ns (hyperoxygenation) - 110 ns (anoxy) in cells (2) 60 - 220 ns in liposomes and (3) 6 - 220 ns in ethanolic solution. These results indicate that, under hyperoxygenation, quenching is less efficient in organized environments than in solution. Without oxygen and in cellular medium, the quenching depends on the composition of the probe environment. Accordingly, these probes can be used to measure the intracellular oxygen concentrations as well as changes in the environment.

Confocal and Two-photon excitation laser scanning microscopy allow gathering three-dimensional and temporal information from biological systems exploiting fluorescence labeling and autofluorescence properties. In this work we study biological events linked to functionality in Paramecium primaurelia. The internalization of material in ciliated one-celled organisms (protozoa) occurs via different mechanisms, even if most of nutrients, particulate or not, is taken up by food vacuoles formed at the bottom of the oral cavity. The endocytosis of small-sized molecules occurs at the parasomal sacs, located next the ciliar basal bodies. Vital fluorescent dyes (BSA-FITC, WGA-FITC, dextran-Texas Red, cholesteryl-Bodipy) and autofluorescence were used to study formation, movement, and fusion of vesicles during endocytosis and phagocytosis of Paramecium primaurelia. By immobilizing living cells pulsed with food vacuole and endosome markers at successive times after chasing in unlabeled medium, the intracellular movement and fusion of food vacuoles and of endosomes were visualized. A temporal analysis of fluorescence images and the false-color technique were used. Starting from time series or 3D data sets composite images were generated by associating with each originally acquired image a different color corresponding to each sampling point in time and along the z-axis. Second Harmonic Generation Imaging attempts are also outlined.

A mathematical model was developed to predict the bi- directional transport rate of fluorescent proteins across the nuclear membrane during a fluorescence recovery after photobleaching (FRAP) experiment. The model assumes that the total amount of fluorescent protein remains the same in the cell (i.e. no production, loss or exchange with the outside of the cell) and that the cell is in a state of equilibrium; i.e. proteins are leaving and entering the nucleus at an equal rate. The latter assumption has the advantage of not needing to take into account the method of protein transport (e.g. active or passive). The model includes correction for the photobleaching that happens during image acquisition following the deliberate photobleach. In this study, the green fluorescent protein (GFP) was transfected into cells in order to study its free behavior. In the FRAP experiments, either the entire nucleus and part of the cytoplasm or only part of the cytoplasm was photobleached followed by time-series imaging of the fluorescence redistribution. The model was fitted to the curves of intensity loss or recovery after photobleaching using numerical, non-linear methods. In addition, the mobile fractions of free GFP in the cytoplasm and the nucleus could be determined.

In this paper a comparison of suitability for different segmentation schemes applied to test images of different complexity with intracellular fluorescent dye concentrations of living cells is presented. The analysis of experimental results is done for the segmented images using color segmentation method, adaptive intensity histogram segmentation method and edge threshold segmentation method. The statistical analysis of the data obtained from images using different segmentation methods is also discussed. Two different segmentations methods can be applied to the same image before the statistical analysis is carried out. Our system implemented in Visual C++ allows analysis of statistic information for part of an image, for the whole image and for the group of images. All the operations have multi-step undo and redo functions and are performed fast even for complicated images.

In our system developed in Visual C++ for images with intracellular fluorescent dye concentrations of living cells after the boundaries and labels are drawn for all living cells, the new image is pushed into the stack pointer for the purpose of redo function and the window is updated. For the user interface, different buttons and their control algorithms have been tested. In our program we used some special control algorithms to ensure that the user gets less chance to make an error operation. Drawing line can split joined objects and Regions-of-Interest can be selected or removed by drawing a rectangle or an ellipse. Once we select any part of an image by drawing rectangle or ellipse, the cutting button and the file generation button are enabled together with the clear button. Such an approach allows the user not to be concerned so much which operation is suitable at any time and the continuous undo and redo operation encourage the user to perform whatever operation is suitable at any time without being afraid of losing information. Finally the reminding messages will guide the user how to perform operation of the system correctly and to retrieve any removed data.

Using Monte-Carlo methods, we have investigated the signal- to-noise ratio obtainable for different fluorescence lifetime imaging methods. Quantum noise limited performance and mono-exponential decays were assumed. We have also investigated the importance of parameter choice and implementation for the different methods. In addition, our simulations were in many cases compared with analytical theoretical investigations. The results from the simulations proved to be in good agreement with the theoretical results. It was found that all the investigated lifetime imaging methods have the potential to produce a high signal-to-noise ratio, but careful attention must be paid to implementation method and parameter choice in order to get optimal results.

Cardiac surgery with cardiopulmonary bypass (CPB) alters the leukocyte composition of the peripheral blood (PB). This response contributes to the sometimes adverse outcome with capillary leakage. Migration of activated cells to sites of inflammation, driven by chemokines is part of this response. In order to determine the chemotactic activity of patients serum during and after surgery we established an assay for PB leukocytes (PBL). PBL from healthy donors were isolated and 250,000 cells were placed into a migration chamber separated by a filter from a second lower chamber filled with patient serum. After incubation cells from top and bottom chamber were removed and stained with a cocktail of monoclonal antibodies for leukocyte subsets and analyzed on a flow cytometer (FCM). Cells at the bottom of the filter belong to the migrating compartment and were quantified by LSC after staining of nucleated cells. Increased chemotactic activity started at onset of anaesthesia followed by a phase of low activity immediately after surgery and a second phase of a high post-operative activity. The in vitro results correlated with results obtained by immunopenotyping of circulating PBL. Manipulation of the chemokine pattern might prove beneficial to prevent extravasation of cells leading to tissue damage. In chemotaxis assays with low amount of available serum the combined use of FCM and Laser Scanning LSC proved as an appropriate analytical tool.

The development of therapeutics for the control of tumor angiogenesis requires a simple, reliable in vivo assay for tumor-induced vascularization. For this purpose, we have adapted the orthotopic implantation model of angiogenesis by using human and rodent tumors genetically tagged with Aequorea victoria green fluorescent protein (GFP) for grafting into nude mice. Genetically-fluorescent tumors can be readily imaged in vivo. The non-luminous induced capillaries are clearly visible against the bright tumor fluorescence examined either intravitally or by whole-body luminance in real time. Fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. High-level GFP-expressing tumor cell lines made it possible to acquire the high-resolution real-time fluorescent optical images of angiogenesis in both primary tumors and their metastatic lesions in various human and rodent tumor models by means of a light-based imaging system. Intravital images of angiogenesis onset and development were acquired and quantified from a GFP- expressing orthotopically-growing human prostate tumor over a 19-day period. Whole-body optical imaging visualized vessel density increasing linearly over a 20-week period in orthotopically-growing, GFP-expressing human breast tumor MDA-MB-435. Vessels in an orthotopically-growing GFP- expressing Lewis lung carcinoma tumor were visualized through the chest wall via a reversible skin flap. These clinically-relevant angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumor angiogenesis in physiological micro- environments.

The results of experimentally investigated laser heating of optically absorbing inhomogeneities inside the biological objects accompanied with monitoring of internal temperature by acoustical brightness thermometry (ABT) have been presented. One of the urgent problems of modern medicine is to provide organism safety during photodynamic therapy of various neoplasms including malignant ones. In the case when neoplasm differs from normal tissue mainly in optical absorption it seems to be effective to use laser heating for this purpose. In our experiments we used the NIR emission of CW and pulse-periodic Nd:YAG lasers (1064 nm) as well as CW semiconductor laser (800 nm) for heating of tissue- simulating phantom. Optically transparent gelatine with absorbing inhomogeneity inside was used as a phantom. Internal temperature was measured non-invasively by means of multi-channel ABT after long heating of an object by laser radiation. Temperature was also measured independently by contact electronic thermometer. The results of experiments demonstrated high efficiency of ABT application for internal temperature monitoring during PDT and other hyperthermia procedures. Besides that laser radiation can be used for backlighting followed by ABT investigation of internal structure of temperature distribution inside biological tissues. This work was supported by Russian Foundation for Basic Research (Projects # 00-02-16600; 01-02-06417; 01-02- 17645) and 6th competition-expertise of young scientists of Russian Academy of Sciences (Project #399).

We report here on bacterial biofilm detection with an optical fiber probe and a compact detection system. This probe was tested on cells of the Pseudomonas aeruginosa and other species of bacteria in planktonic and sessile forms. Optical signal changes corresponding to the number density of the bacterial cells were measured.

Multiphoton microscopy is a powerful tool for imaging sub- cellular distribution of luminescent compounds present in living cells. We have used this tool to study the distribution and pharmacology of photosensitizers in tissue and tissue culture. Murine hepatoma tumor cells dosed with a photosensitizer were briefly photoactivated, then imaged for periods up to several hours. Using the photosensitizer Rose Bengal with green light activation, nearly immediate photolytic release of lysosomal enzymes resulted in catastrophic cell destruction within 5 - 30 minutes. The magnitude and rapidity of this response is markedly different than that observed with other photosensitizer agents, and is consistent with in vivo studies illustrating that Rose Bengal is capable of causing extremely rapid destruction of treated tumors.

Bacterial adhesion is a primary cause of failure in implanted medical devices. Bacteria commonly found in device-related infections, such as S. aureus, have multiple cell surface adhesins which mediate specific adhesion to molecules found in extracellular matrix and blood plasma. Adhesins recognizing fibrinogen, fibronectin, collagen, and elastin molecules have been isolated in S. aureus. We have used optical tweezers to measure the adhesive force between a single bacterium and a protein-coated surface. Various concentrations of fibronectin, fibrinogen, or whole plasma were immobilized onto 10-micrometers diameter polystyrene microspheres. We optically trapped a bacterium with a titanium-sapphire laser tuned to 830 nm and contacted the cell with a coated bead. We determined the minimum force necessary to separate the cell and bead. For beads coated with fibronectin and fibrinogen, detachment force values occurred as approximate integer multiples of an estimated single bond detachment force. With plasma-coated beads, only cells lacking the fibrinogen adhesin could be detached; therefore, we believe that either this adhesin is prevalent on wilde-type cells, or it is preferentially adsorbed onto the beads. Additionally, the whole plasma detachment forces appeared random; therefore, we believe that many adhesins participate in boding to plasma.

Thrombus formation occurs when a platelet membrane receptor, glycoprotein (GP) Ib-IX-V complex, binds to its ligand, von Willebrand factor (vWf), in the subendothelium or plasma. To determine which GP Ib-IX-V amino acid sequences are critical for bond formation, we have used optical tweezers to measure forces involved in the binding of vWf to GP Ib-IX-V variants. Inasmuch as GP Ib(alpha) subunit is the primary component in human GP Ib-IX-V complex that binds to vWf, and that canine GP Ib(alpha) , on the other hand, does not bind to human vWf, we progressively replaced human GP Ib(alpha) amino acid sequences with canine GP Ib(alpha) sequences to determine the sequences essential for vWf/GP Ib(alpha) binding. After measuring the adhesive forces between optically trapped, vWf-coated beads and GP Ib(alpha) variants expressed on mammalian cells, we determined that leucine- rich repeat 2 of GP Ib(alpha) was necessary for vWf/GP Ib-IX- V bond formation. We also found that deletion of the N- terminal flanking sequence and leucine-rich repeat 1 reduced adhesion strength to vWf but did not abolish binding. While divalent cations are known to influence binding of vWf, addition of 1mM CaCl₂ had no effect on measured vWf/GP Ib(alpha) bond strengths.

Laser manipulation system combining microfluidic and microimaging devices was developed, in which a cell sorting in a transparent microchip was successfully demonstrated. The microchip containing two microchambers was prepared with laser microfabrication, in which a solution containing yeast cells was injected as a sample. In the microchip, a cell transfer from one chamber to another one was performed by using single, fixed trapping laser beam. Furthermore, to realize an efficient cell sorting, the trapping laser bam was split into two by a polarizing beam splitter and each beam was modulated independently; one trapping beam was used to trap individual cells and to move them, which is freely controlled by a mouse pointer, and another was used to store the selected yeast cells with its liner scanning. In this method, the cells on a locus of the scanned beam were isolated to transfer in the microchip. From these results, it is concluded that shortening of the cell sorting time in microchip by a few time was realized by using dual-beam laser manipulation.

Confocal microscopy and optical tweezers were combined to develop a minimally invasive instrument capable of making hydrodynamic measurements more rapidly than is possible with other devices. This result leads to the possibility of making scanning images of the viscosity distribution of materials around bipolymer producing cells. An image of the viscosity distribution around a pullulan producing cell of Aureobasidium pullulans is shown as an example. We present results from experiments supporting a linearized model for the motion of a trapped bead in an oscillating harmonic potential. Fluid velocity measurements are tested by comparing to an independent video based measurement. We apply the technique to obtain a 2-D map of the flow past a microscopic wedge and compare to a theoretical solution for the stream lines assuming potential flow. Since the velocity is measured simultaneously with the trap relaxation time, it requires practically no calibration and is independent of the trap stiffness and the particle size.

We have developed applications of Captivate^TM ferrofluids, paramagnetic particles (approximately 200 nm diameter), for isolating and analyzing cell populations in combination with fluorescence-based techniques. Using a microscope-mounted magnetic yoke and sample insertion chamber, fluorescent images of magnetically captured cells were obtained in culture media, buffer, or whole blood, while non-magnetically labeled cells sedimented to the bottom of the chamber. We combined this immunomagnetic cell separation and imaging technique with fluorescent staining, spectroscopy, and analysis to evaluate cell surface receptor-containing subpopulations, live/dead cell ratios, apoptotic/dead cell ratios, etc. The acquired images were analyzed using multi-color parameters, as produced by nucleic acid staining, esterase activity, or antibody labeling. In addition, the immunomagnetically separated cell fractions were assessed through microplate analysis using the CyQUANT Cell Proliferation Assay. These methods should provide an inexpensive alternative to some flow cytometric measurements. The binding capacities of the streptavidin- labled Captivate ferrofluid (SA-FF) particles were determined to be 8.8 nmol biotin/mg SA-FF, using biotin-4- fluorescein, and > 10⁶ cells/mg SA-FF, using several cell types labeled with biotinylated probes. For goat anti- mouse IgG-labeled ferrofluids (GAM-FF), binding capacities were established to be approximately 0.2 - 7.5 nmol protein/mg GAM-FF using fluorescent conjugates of antibodies, protein G, and protein A.

Ongoing efforts to engineer a system capable of selecting and labeling single cells using optical micromanipulation tools and performing electrophoretic separation on the contents of a single cell using the 'lab-on-a-chip' format are presented. At the heart of this design, are channels with 10micrometers diameter cross-sections, etched using a molecular fluorine laser. Individual cells are moved on the microchip using optical tweezers. These single cells are brought into contact with a liposome containing fluorescent tags. The liposome and cell are fused using optical scissors; resulting in a cell with labeled components. This cell is lysed using the optical scissors, and high voltage is applied to separate the contents. This design will allow us to directly look at protein and mRNA expression from a single cell without amplifying the contents of interest, as well as to obtain the population averages and their variations from the analysis of a sufficient number of individual cells.

The goal of this study was to develop a new approach to manipulating biological and nonbiological objects in liquid or gas, with a focus on living cells in liquid. This approach is based on laser-generated thermal and pressure gradients in the medium surrounding an object, which create forces of different origins acting on the object. In general, depending on the spatial geometry of these forces, particles can be trapped (symmetrical forces) or moved in desired directions (asymmetrical forces). Comparison of the different mechanisms for creating photothermal (PT) and photoacoustic (PA) gradients and their roles in manipulating particles are considered, including heating through absorption, optical breakdown, and plasma and bubble formation. The PA and PT gradients lead in turn to the formation of many physical phenomena, some of which may be important for particle movement, separation, sorting, and trapping. Among these phenomena are fast thermal expansion, acoustic radiation pressure, directed thermal convection, acoustic streamers, laser-induced jets, laser-generated high-frequency focused ultrasound, radiometric forces, nonisotropic Brownian motion, and asymmetry in thermal effects of irradiated particles, including thermal expansion, evaporation, ablation, and infrared (IR) radiation. Different phenomena are shown to play dominant roles in the manipulation of particles, depending on the particle or cell type, parameters of the surrounding medium, laser energy, and the beam's shape or geometry. The optical systems for particle manipulation using different laser spot shapes and geometry (i.e., circular, linear, crescent, or multispot and ring configurations) are presented. The advantages and limitations of this new member of the family of different 'tweezers' are discussed.

In this paper, we study optical trapping and manipulation of single electrically charged colloidal particles. We measured the dynamic motion of a charged particle captured in a linearly polarized optical trap. We found that the position's fluctuation of a trapped particle in the parallel direction to the lase polarization is larger than that in the normal direction to the polarization, which suggests that there exists an additional electric force parallel to the laser polarization direction exerting on the charged particle beside the known radiation forces on the dielectric particle. This asymmetry in dynamic motion is significant when the particle size is less than the wavelength of the trapping laser. We present both the theoretical and experimental results.

A three-fiber optical trapping/detection system has been molded in poly(dimethyl siloxane) (PDMS) using anisotropically etched Si V-grooves as the primary or master mold. The process of reverse molding in PDMS maintains the benefits of fiber optic self-alignment previously used in Si V-grooves. Two, pigtailed laser diodes emitting at 830 nm and 980 nm are connected to cleaved, single-mode (SM), counter-propagating fibers, used for trapping polystyrene beads. Orthogonal to the trapping fibers is a multi mode detection fiber coupled to a spectrometer. Chemically treated beads trapped by the 830 and 980 nm diode lasers were excited using a 660 nm diode laser. By utilizing the optical clarity of PDMS, the fourth excitation source fiber is mounted below the PDMS trap and used to excite the trapped beads. Changes in the relative intensity of the trapping light are used to indicate the capture and position of a bead in the trap. Additionally, detection of the excitation source and bead fluorescence is monitored.

Many applications of laser tweezers rely on the accurate measurement of the transverse or axial trapping force. We have concentrated on the transverse trapping force and the most common method used to measure it, applying a viscous drag force. A trapped sphere was subjected to a viscous drag force via a Stokesian flow. The flow was achieved by oscillating the sample stage at a constant speed of 750 microns/second. A Zeiss oil-immersion (N.A. equals 1.3) objective was used to focus a 1064 nm Nd:YVO₄ laser beam in order to trap 6 microns diameter polystyrene spheres suspended in distilled water. The minimum power needed to hold the particle in the trap at a particular viscous drag force was then measured. The influence of trap depth, oscillation amplitude used and particle concentration have been investigated, in particular the effects caused by the characteristics of the function used to create the oscillation. The minimum laser power needed to trap a sphere was found to increase with a rise in oscillation amplitude. The velocity profile through the fluid, the rotation of the trapped particle and the effect of interactions with other particles is considered when explaining these effects.

While many flow cytometric data analysis and 'discovery' methods have been developed, few of these have been applied to the problem of separating out purified cell subpopulations by cell sorting. The fundamental problem is that the data analysis techniques have been performed using relatively slow computational methods that take far more time than is allowed by the sort decision on a cell sorter (typically less than a millisecond). Thus cell sorting, which is really a form of 'real-time data classification,' is usually done with few, if any, multivariate statistical tools used either in the sort decision or in the evaluation of the correctness of the classification. We have developed new multivariate data analysis and 'data discovery' methods that can be implemented for real-time data classification for cell sorting using linked lookup tables. One multivariate 'data discovery' method, 'subtractive clustering,' has been used to find which clusters of cells are different between two or more files (cell samples) and to help guide analysis or sort boundaries for these cell subpopulations. Multivariate statistical methods (e.g. principal component analysis or discriminant function analysis) were implemented in linked lookup tables to establish analysis/sort boundaries that include 'costs (or penalties) of misclassification. Costs of misclassification provided a measure of the quality of the analysis/sort boundary and were expressed in simple terms that describe the tradeoff between yield and purity.

Hormone receptor expression in human breast and prostate tumors is of diagnostic and therapeutic importance. With the availability of anti-estrogen, androgen and progesterone antibodies, immunohistochemistry has become a standard tool for determination of receptor expression in human tumor biopsies. However, this method is dependent on examination of a small number of cells under a microscope and the data obtained in most cases is not quantitative. As most of the commercially used anti-hormone antibodies have nuclear specificity, we have developed methods for isolation and antigen unmasking of nuclei from formalin fixed/paraffin embedded archival human tumors. After immunostaining with the antibodies and propidium iodide (for DNA content and cell cycle analysis), nuclei are analyzed by multiparametric laser flow cytometry for hormone receptor expression, DNA content, aneuploidy and cell cycle determination. These multiparametric methods are especially important for retrospective studies seeking to correlate hormone receptor expression with clinical response to anti-hormonal therapy of human breast and prostate tumors.

Quantitative immunophenotypes varied widely among > 100 healthy young males but were maintained at characteristic levels within individuals. The initial results (SPIE Proceedings 4260:226) that examined cell numbers and the quantitative expression of adhesion and lineage-specific molecules, e.g., CD2 and CD14, have now been confirmed and extended to include the quantitative expression of inducible molecules such as HLA-DR and perforin (Pf). Some properties, such as the ratio of T helper (Th) to T cytotoxic/suppressor (Tc/s) cells, are known to be genetically determined. Other properties, e.g., the T:B cell ratio, the amount of CD19 per B cell, etc., behaved similarly and may also be inherited traits. Since some patterns observed in these healthy individuals resembled those found in pathological situations we tested whether the patterns could be associated with the occurrence of disease. The current studies shows that there were associations between quantitative immunophenotypes and the subsequent incidence and severity of disease. For example, individuals with characteristically low levels of HLA-DR or B cells or reduced numbers of Pf+ Tc/s cells had more frequent and/or more severe upper respiratory infections. Quantitative immunophenotypes will be more widely measured if the necessary standards are available and if appropriate procedures are made more accessible.

In lymphatic organs the quantitative analysis of the spatial distribution of leukocytes would give relevant information about alterations during diseases (leukemia, HIV, AIDS) and their therapeutic regimen. Analysis of them in solid tissues is difficult to perform but would yield important data in a variety of clinical and experimental settings. We have developed an automated analysis method for LSC suitable for archived or fresh biopsy material of human lymph nodes and tonsils. Sections are stained with PI for DNA and up to three antigens using direct or indirect immunofluorescence staining. Measurement is triggered on DNA-fluorescence (Argon Laser). Due to the heterogeneity in cell density measurements are repeatedly performed at different threshold levels (low threshold: regions of low cellular density, germinal centers; high threshold: dense regions, mantle zone). Data are acquired by single- (Ar) or dual-laser excitation (Ar-HeNe) in order to determine data from single- (FITC), up to triple-staining (FITC/PE-Cy5/APC). Percentage and cellular density of cell-subsets is quantified in different structural regions of the specimen. Comparison with manual analysis of identical specimens showed very good correlation. With LSC a semi-automated operator-independent and immunophenotyping of lymphatic tissues with simultaneously up to four antibodies is possible. This technique should yield new insight into processes during diseases and should help to quantify the success of therapeutic interventions.

LSC is a microscope-based technology. The principle of the instrument is that any specimen is immobilized on a microscope slide. Therefore the cells are not lost in a fluid stream but are kept on the slide and minimal specimens as low as 1.000 cells can be analyzed. Additionally cells are available for further analyses such as staining for another set of specific markers and re-analysis or cytological staining (H&E). This approach multiplies the information gained from a given sample. We have established an assay for immunophenotyping of peripheral blood leukocytes by LSC. Cells are prepared according to routine flow cytometry protocols with a first set of CD-antibodies and are fixed on microscope slides. As a stable trigger signal the nuclear DNA is stained by 7-aminoactinomycin-D. This guarantees that all nucleated cells and that only nucleated cells are included in the analysis, and many differentiate between lymphocytes and neutrophiles by staining intensity. After analysis cells are stained with a second set of CD-antibodies and analyzed again. This step can be repeated with a third set of CD-antigens. Since the location of the cells on the slide is fixed data from the analyses can be attributed to the same cell.

A Eu(III)-macrocycle-mono-isothiocyanate, Quantum Dye®, has been coupled to a monoclonal antibody against 5BrdU. Since Quantum Dyes do not undergo concentration quenching, the coupling conditions were optimized to achieve the maximum number of Eu(III) macrocycles bound to the antiBrdU, without decrease in solubility or loss of antigen-binding ability. In order to optimize the coupling conditions, a colorimetric method for the quantitation of the Eu(III)- macrocycle-mono-isothio-cyanate has been developed. A simple mixture composed of an ethanolic solution and a Gd(III)- containing aqueous solution is now used to provide lanthanide enhanced luminescence, LEL. Under LEL conditions, the specific binding of Eu(III) macrocycles to apoptotic cells has been observed in both aqueous and mounted slide preparations. A comparison between measurements of the same LEL model system, obtained in both time-gated luminescence and standard fluorescence modes, has demonstrated that time- gating significantly improves the signal to noise ratio.

Delayed Luminescence is a well established technique based on the illumination of biological sample and on the subsequent count of the number of photons re-emitted by the sample after the light source has been switched off together with their spectral distribution. Investigations have been performed on yeast cells and algae so that correlations can be established between biological activities and physical parameters of the samples. Moreover nonlinear mechanisms of interaction between optical fields and cells can be figured out. Further investigations will be reported on yeast cell samples deposited on paper filters after irradiation by soft X-rays. The results will be discussed by cross correlating the experimental evidence from Delayed Luminescence with those obtained by metabolic activity recording. Luminescence and Delayed Luminescence are strictly correlated with early insurgence of morphological alterations of normal or pathological nature in cells and tissues; a novel technique for morphological analysis has been developed by means of Focused Ion Beam machines. A straightforward approach to morphology at the nanoscale both of membranes and cellular inner structures is then made possible. The final aim is an experimental set up for an early and reliable detection technique for neoplastic cells and tissues sorting.

With recent advances in high-speed confocal imaging, data storage, and computational power, practical high-speed 3D cytometry instrumentation is on the horizon. For 3D cytometry to become practical for example from the perspective of a pathologist, speed attained in part by walk-away automation is fundamentally important. This level of automation can only be obtained with fully automated segmentation of image objects from background. Accuracy of this first image analysis task is crucial since it determines the results of all subsequent quantitative analyses. Confocal cell images often have low contrast due to both inherently low signal-to-noise ratios and high cell- cell contrast ratios that can occupy much of the available imaging dynamic range. A contrast-enhancing technique previously developed for 2D images of fluorescent cell nuclei was extended for 3D confocal images (stacks of 2D image slices). Edge sharpening and contrast-enhancement necessary for automatic thresholding are achieved by filtering with a finite impulse response (FIR) filter. These optimal FIR filters range in size from 3 X3X3X to 13X13X13 and were designed by utilizing the perceptron criterion and nonlinear least squares on confocal training datasets derived from fluorescent microspheres. By utilizing fluorescent beads of known shapes and sizes, the ideal (or standard) segmented image is known a priori. The contrast-enhancing performance of these filters on 3D confocal images of DAPI stained cell nuclei demonstrates that they should lead to accurate, fully automated 3D image segmentation.

Three-dimensional cytometry, whereby large volumes of tissue would be measured automatically, requires a computerized method for detecting the upper and lower tissue boundaries. In conventional confocal microscopy, the user interactively sets limits for axial scanning for each field-of-view. Biological specimens vary in section thickness, thereby driving the requirement for setting vertical scan limits. Limits could be set arbitrarily large to ensure the entire tissue is scanned, but automatic surface identification would eliminate storing undue numbers of empty optical sections and forms the basis for incorporating lateral microscope stage motion to collect unlimited numbers of stacks. This walk-away automation of 3D confocal scanning for biological imaging is the first sep towards practical, computerized statistical sampling from arbitrarily large tissue volumes. Preliminary results for automatic tissue surface tracking were obtained for phase-contrast microscopy by measuring focus sharpness (previously used for high-speed autofocus by our group). Measurements were taken from 5X5 fields-of-view from hamster liver sections, varying from five to twenty microns in thickness, then smoothed to lessen variations of in-focus information at each axial position. Because image sharpness (as the power of high spatial frequency components) drops across the axial boundaries of a tissue section, mathematical quantities including the full-width at half-maximum, extrema in the first derivative, and second derivative were used to locate the proximal and distal surfaces of a tissue. Results from these tests were evaluated against manual (i.e., visual) determination of section boundaries.

Since the 1970s, extensive research has been devoted to the development of a standard procedure for the isolation of fetal nucleated red cells (fnRBCs) from maternal blood. Since these cells are sources of fetal DNA, cytogenetic analysis would lead to a minimally-invasive method for the prenatal diagnosis of chromosomal and genetic disorders early in gestation. FnRBCs constitute a significant portion of the fetal blood, have a short and finite life span, and are rare in peripheral adult blood. They have been reported to exist in the maternal circulation at frequencies as low as 1:10⁵ - 1:10⁹ maternal nucleated cells. Due to these ultra-rare frequencies, isolation with minimal loss has been a time and labor-intensive process. To overcome this problem, a fully automated scanning cytometer that incorporates high-performance autofocus and image segmentation has been built and shown higher rate, quantity, sensitivity (true positive rate) and specificity (true negative rate) in a model cell preparation. For detecting fnRBCs, two discriminating characteristics may suffice: (1) the presence of fetal hemoglobin, which is the major intracytoplasmic protein found in fetal red cells from 5 to 35 weeks gestation, and (2) the presence of a nucleus. In clinical trials, the fetal origin of the isolated cells will be confirmed by fluorescence in situ hybridization (FISH) on the X and Y chromosomes in male pregnancies. The aim of the present study was to develop a reliable and reproducible staining method for combined immunofluorescence and FISH analysis for these clinical trials. This staining technique was developed using fnRBCs extracted from fetal liver blood and a human erythroleukemia cell line (HEL) that expresses fetal hemoglobin. The resulting method for four-color X- and Y-FISH , anti-(gamma) -Hb fluorescence and DAPI staining was consistent and bright.

Since hyperglycaemia changes fluidity of erythrocyte cell membrane and impair cell deformability, our goal was to characterize light refractive properties of haemoglobin and red blood cells (RBC) in diabetes patients. Microscopic investigation was carried out on intact and in methanol fixed RBCs of diabetes patients with long-term hyperglycaemia (glycosylated haemoglobin > 7,5%). Interference microscopy was used for refractive index (RI) measurements at 18 different pH levels in range of 2 - 13. Results showed that the curves of RI in intact and fixed cells of diabetes patients and fixed cells for control group were of similar configuration, with one branch in alkaline portion, one branch in acidic portion, and two minima and a maximum in neutral portion of pH scale. The curves of RI of intact cells of the control group were with one minimum in the neutral portion of pH scale. The curves of the individuals from the control group overlapped each other (maximum for fixed cells is at pH equals 6,3; minimum for intact cells at pH equals 7,2). On the contrary, curves of RI in RBC of diabetes patients were not uniform in the neutral portion and alkaline portion (maximum for fixed and intact cells at pH equals 6,9). The curves of RI in RBC of diabetes patients in the neutral zone was shifted towards the alkaline end of the pH scale, and the curves of RI of RBC were lower compared to the control curves.

Well-characterized phantom objects are necessary for investigating the performances of optical imaging systems based on time-resolved transmittance and fluorescence. For this purposes we have prepared inhomogeneous phantoms made of gelatinous objects placed in aqueous solutions of 10% Intralipid with different concentrations. The gelatinous objects have been prepared using a mixture of 10% Intralipid with agar at which absorbing ink have been added for transmittance based optical imaging system. For fluorescence measurements proper fluorescent dyes (rhodamine6G and IR125) have been added. Conventional optical characterization by spectrophotometric and spectrofluorimetric measurements have been performed. In addition, time-resolved transmittance and fluorescence measurements have been carried out. In particular, time-correlated single photon counting system has been used for time-resolved transmittance measurements. For time-resolved fluorescence measurements an optical imaging system based on a Ti:Sa laser and streak camera has been employed.

Partial thickness articular cartilage lesions are commonly encountered in orthopedic surgery. These lesions do not have the ability to heal by themselves, due to lack of vascular supply. Several types of treatment have addressed this problem, including mechanical debridement and thermal chondroplasty. The goal of these treatments is to provide a smooth cartilage surface and prevent propagation of the lesions. Early thermal chondroplasty was performed using lasers, and yielded very mixed results, including severe damage to the cartilage, due to poor control of the induced thermal effects. This led to the development (including commercial) of probes using radiofrequency to generate the thermal effects desired for chondroplasty. Similar concerns over the quantitative aspects and control ability of the induced thermal effects in these treatments led us to test the whole range of complex issues and parameters involved. Our investigations are designed to simultaneously evaluate clinical conditions, instrument variables for existing radiofrequency probes (pressure, speed, distance, dose) as well as the associated basic science issues such as damage temperature and controllability (down to the subcellular level), damage geometry, and effects of surrounding conditions (medium, temperature, flow, pressure). The overall goals of this work are (1) to establish whether thermal chondroplasty can be used in a safe and efficacious manner, and (2) provide a prescription for multi-variable optimization of the way treatments are delivered, based on quantitative analysis. The methods used form an interdisciplinary set, to include precise mechanical actuation, high accuracy temperature and temperature gradient control and measurement, advanced imaging approaches and mathematical modeling.