Coordination of cell migration and adhesion is essential for movement of tissues during morphogenesis. During Drosophila oogenesis so called border cells (BCs) break from an anterior epithelium of egg chamber, acquire a mesenchymal-like morphology, and migrate posteriorly between nurse cells to oocyte. The confocal microscopic observation of BCs has revealed well-developed forepart lamellipodium stained with Drosophila E-cadherin (DE-cadherin), PS2 integrin, cytoplasmic myosin and F-actin. To investigate mechanism of BC migration in vivo we have constructed a DE-cadherin-GFP and a GFP-actin fusion proteins and induced their expression BCs utilizing the UAS/GAL4 system. The DE-cadherin-GFP signal as well as immunostaining of PS2 integrin visualized a track of migrating BCs providing an evidence that adhesive molecules are pulled out and left behind on the surface of nurse cells. Our data suggest that two distinct adhesive systems, DE-cadherins and PS2 integrins simultaneously mediate the migration of BCs. Release of adhesive contacts in the tail region is a rate- limited event in BC migration. The spatial-temporal sequence of actin-based events visualized by the GFP-actin suggest a treadmilling model for actin behavior in BC lamellipodium. BC migration can be considered as simultaneous reiterating processes of lamellipodium extension and adhesive attachment, cytoskeletal contraction, and rear detachment.

Lymphocytes are the central players in the human adaptive immune response. In the body, individual T helper lymphocytes need to be activated first by physical contact with antigen- presenting cells (APC). T-cell contact with APCs initiated an activation cascade, which includes an increase in T-cell intracellular calcium, leading to T-cell proliferation, differentiation and lymphokine production. Calcium imaging are combined with optical manipulation to investigate the physical properties of T-cell activation. We study cell-cell contact requirements for T-cell activation using optical tweezers to control the orientation of T-cell/APC pairs and fluorescence microscopy to measure the subsequent T-cell intracellular calcium level [(Ca²⁺)_i] response. APCs or beads coated with antibodies to the T-cell receptor (TCR) are trapped with a near-infrared titanium-sapphire laser and placed at different locations along the T-cell, which has a polarized appearance defined by the shape and direction of crawling. T cells which are presented with antigen at the leading edge have a higher probability of responding and a shorter latency of response than those contacting APCs or beads with their trailing end. Alterations in antibody density and bead size are used to determine the spatial requirements for T cell activation and the minimum number of receptors which must be engaged in order to transmit a positive signal.

Stroke has been shown to cause exitotoxic injury, two of its manifestations being cellular and mitochondrial swelling. In vitro models of stroke attempt to reproduce the effects of stroke by treating brain tissue with excitotoxins or hypotonic solutions. To further resolve the mechanism of stroke injury, we have designed a dual-angle scatter imaging (DASI) system sensitive to particle size. The DASI system has been used with a hippocampal slice preparation to contrast cellular swelling, induced by hypotonicity, and combined cellular and mitochondrial swelling caused by excitotoxicity. We found that both hypotonic end excitotoxic treatments caused changes in light scatter. However, only excitotoxic treatment caused a significant change in DASI.

Advances in infrared spectroscopic methodology permit excellent infrared spectra to be collected from objects as small as single human cells. These advances have lead to an increased interest of the use of infrared spectroscopy as a medical diagnostic tool. Infrared spectra of myeloid leukemia (ML-1) cells are reported for cells derived from an asynchronous, exponentially-growing culture, as well as for cells that were fractionated according to their stage within the cell division cycle. The observed results suggest that the cells' DNA is detectable by infrared spectroscopy mainly when the cell is in the S phase, during the replication of DNA. In the G1 and G2 phases, the DNA is so tightly packed in the nucleus that it appears opaque to infrared radiation. Consequently, the nucleic acid spectral contributions in the G1 and G2 phases would be mostly that of cytoplasmic RNA. These results suggest that infrared spectral changes observed earlier between normal and abnormal cells may have been due to different distributions of cells within the stages of the cell division cycle.

The influence of the peak power, laser wavelength and the pulse duration of near infrared (NIR) ultrashort laser pulses on the reproduction behavior of Chinese hamster ovary (CHO) cells has been studied. In particular we determined the cloning efficiency of single cell pairs after exposure to ultrashort laser pulses with an intensity in the range of GW/cm² and TW/cm². A total of more than 3500 non- labeled cells were exposed to a highly focused scanning beam of a multiphoton laser microscope with 60 microsecond pixel dwell time per scan. The beam was provided by a tunable argon ion laser pumped mode-locked 76 MHz Titanium:Sapphire laser as well as by a compact solid-state laser based system (Vitesse) at a fixed wavelength of 800 nm. Pulse duration (tau) was varied in the range of 100 fs to 4 ps by out-of-cavity pulse- stretching units consisting of SF14 prisms and blazed gratings. Within an optical (laser power) window CHO cells could be scanned for hours without severe impact on reproduction behavior, morphology and vitality. Ultrastructural studies reveal that mitochondria are the major targets of intense destructive laser pulses. Above certain laser power P thresholds, CHO cells started to delay or failed to undergo cell division and, in part, to develop uncontrolled cell growth (giant cell formation). The damage followed a P²/(tau) relation which is typical for a two-photon excitation process. Therefore, cell damage was found to be more pronounced at shorter pulses. Due to the same P²/(tau) relation for the efficiency of fluorescence excitation, two- photon microscopy of living cells does not require extremely short femtosecond laser pulses nor pulse compression units. Picosecond as well as femtosecond layers can be used as efficient light sources in safe two photon fluorescence microscopy. Only in three photon fluorescence microscopy, femtosecond laser pulses are advantageous over picosecond pulses.

The use of the two-photon excitation (TPE) is believed to be prominent for fluorometric studies with cells. We evaluated the advantages and limitations of the two-photon technique compared to the single photon one when it used to detect potent anticancer drugs, camptothecins (CPTs), within single living cancer cells. The technique we used was confocal microspectrofluorometry amplified with possibility of the spectral imaging analysis. We have previously reported the use of the florescence emission of CPTs to study them qualitatively and quantitatively, namely, to follow the status of their hydrolyzable lactone moiety. However, the intracellular investigation of CPTs using microspectrofluorometry with single photon UV excitation (SPE) is hindered by significant interference of their fluorescence emission with cellular autofluorescence. We attempted to overcome these problems using the two-photon excitation. The intracellular single-photon- and two-photon-excited emission spectra from treated and control cells (HCT-116 line) were recorded using a spectral imaging approach. The obtained data demonstrate that, apart from intrinsically increased three- dimensional resolution, the two-photon approach was advantageous over the single-photon method with respect to selective fluorometric detection of intracellular CPTs. Nevertheless, much attention should be paid to avoid any excessive irradiation of the cells with UV and even NIR light.

Non-invasive optical diagnosis of cellular and extracellular structure and biochemistry in thick tissue is becoming a reality with the maturation of the two-photon imaging. Today, the slow imaging speed of typical two-photon microscopes is a major hurdle in realizing their clinical potential. We have developed a high-speed two-photon microscope optimized for acquiring 3-D tissue images in real time. The scanning speed improvement of this system is obtained by the use of an air bearing polygonal mirror. The maximum achievable scanning rate is 40 microseconds per line, which is about 100 times faster than conventional scanning microscopes. High-resolution fluorescence images were recorded in real-time by an intensified CCD camera. Using this instrument, we have monitored the movements of protozoas and mapped the collagen/elastin fiber structures in excised human skin.

Two-photon microscopy (TPM) is a non-invasive biological imaging technique that can be used to selectively image cellular activity and photosensitizer (PS) localization within highly scattering epithelial tissues at depths of approximately 200 micrometer with submicron resolution. The principal objective of this study was to develop a model system for understanding the impact of photodynamic therapy on cellular and extracellular matrix remodeling in biological tissues. An artificial tissue model (RAFT) composed of collagen, embedded fibroblasts, and macrophage cells has been developed for this purpose. TPM is utilized to monitor extracellular matrix remodeling following PDT by imaging collagen/elastin autofluorescence. Selective uptake of photosensitizers by specific cellular components in the matrix can also be visualized by TPM.

We describe a new method of optical microscopy which allows simultaneously intensity and quantitative phase contrast imaging. The method is based on a new holographic imaging technique which involves a CCD camera as recording device and a numerical method for the reconstruction. An application to phase contrast imaging of living astrocytes is presented.

It is shown here that an optical tweezer can successfully be employed to study interactions between coated microbeads and either biological surfaces, viz. membranes of neuronal cells, or artificial surfaces derivatized with amino acids. For biological applications, polystyrene beads of a diameter of 3 micrometer were coated with different proteins of the Extracellular Matrix (ECM) and brought into contact with glial cells and neurons. By comparing interaction forces, effects of bacterial phospholipase C, formation of membrane threads and surfing distances of bound ECM protein coated beads on cells we demonstrate a different anchorage of the tenascin-C receptor in primary neurons and glia. To study interactions at artificial surfaces with optical tweezers, polystyrene beads with a diameter of 10 micrometer were coated with different amino acids. Friction coefficients were measured between these beads and glass surfaces comprising amino acids coupled to well-defined layers of cellulose derivatives produced by the Langmuir-Blodgett technique. The technique provides an easy method for friction measurements and the characterization and quality control of surfaces. We conclude that optical tweezers are a powerful tool for the characterization of biological and artificial surfaces.

An ultramicroscope coupled to a square-aspect-ratio sensor was used to image the dynamic geometry of live muscle cells. Skeletal muscle cells, dissected from frogs, were suspended in the optical axis and illuminated from one side by a focused slit of white light. The sensor detected light scattered at 90 degrees to the incident beam. Serial cross-sections were acquired as a motorized stage moved the cell through the slit of light. The axial force at right angles to the cross- sections was recorded simultaneously. Cross-sections were aligned by a least-squares fit of their centroids to a straight line, to correct for misalignments between the axes of the microscope, the stage, and the sensor. Three- dimensional volumes were reconstructed from each series and viewed from all directions to locate regions that remained at matching axial positions. The angle of the principal axis and the cross-sectional area were calculated and associated with force recorded concurrently. The cells adjusted their profile and volume to remain stable against turning as contractile force rose and fell, as predicted by the law of conservation of angular momentum.

Planar fibrous connective tissues are composed of a dense extra-cellular network of collagen and elastin fibers embedded in a ground matrix. Thus, quantification of fiber architecture an important step in developing an understanding of the mechanics of planar tissues. We have extensively used small angle light scattering (SALS) to map the gross fiber orientation of several soft membrane connective tissues using a custom built high speed mapping instrument. However, the current technique is limited to total through-thickness tissue structural analysis. The current study was undertaken to determine the feasibility of obtaining transmural tissue structural information from 2D SALS data. Methods: The basic approach is to utilize precisely aligned serial histological sections cut en-face through a tissue block and obtain 2D fiber structure from each section using SALS. Transumural fiber structure information is then derived by integration of 2D data to form a single data set containing the complete transmural fiber structure. To demonstrate the feasibility of the method, both explanted bioprosthetic heart valve (BHV) and native bovine pericardium (BP) tissues were evaluated. Results: The transmural SALS technique revealed for explanted BHV preferential damage in the fibrosa layer, while for BP variations in transmural fiber architecture were found consistent with optical histology. Conclusions: The transmural SALS technique successfully demonstrated quantitative transmural variations in fiber architecture in two dense collagenous tissues in a rapid, cost-effective approach.

The diffusive transport characteristics of a unique class of small fluorescent molecular probes in an interstitial tissue model are investigated using micro-endoscopy. The probes employed in the present work are organo-metallic complexes of polyazamacrocycles chelated to Terbium. These particular molecules have large Stoke's shifts, making them amendable to tissue analysis. The delocalized electronic structure of the organic chelate absorbs ultra-violate light (approximately 270 nm) and, after inter-molecular transfer, the lanthanide cation fluoresces in the visible region (550 nm). The diffusive transport properties of the probe molecules are related to their chemical structure, which governs their affinity toward the components of the interstitial model. The basic polyazamacrocycle is functionalized with three phosphate groups. Presently, methyl, ethyl, propyl and butyl alkyl chains are added to the phosphate groups on the polyazamacrocycle to modify the affinity of the probes toward the components of the interstitial model. Micro-endoscopy coupled with digital imaging allows remote, quantitative analysis of the transport process in near real time. Cross sectional analysis of the images yields the concentration profile of the probe as it diffuses through the gel. The concentration profile is fit to Fick's second law of diffusion to determine the diffusion coefficient, D, for each of the problem molecules. Presently the measured D values for each of the compounds are typical for small molecules in water (approximately 10^-6 cm²/sec), however, D is observed to increase with decreasing hydrocarbon chain length which demonstrates interstitial transport is structurally dependent.

There is a current trend to design innovative mitral valve replacements that mimic the native mitral valve (MV). A prerequisite for these new designs is the characterization of MV structure. This study was conducted to determine the distribution of MV collagen and glycosaminoglycan (GAGs) in MV anterior leaflets. Methods: Specimens from the mid-line of eight sheep MV anterior leaflets were stained with aniline blue (collagen) and alcian blue (GAGs). These specimens were analyzed using an image analysis system running Optimas software. Based on the luminance of stains within individual valve layers, the distribution of valvular collagen and GAGs from leaflet annulus to free-edge were determined. Results: Near the annulus, 100% of MV thickness is fibrosa (collagen dominated layer). Moving towards the free-edge, fibrosa prominence decreases and there is a transition to spongiosa (GAG dominated layer). Near the free-edge 100% of MV thickness is dominated by the spongiosa. Conclusions: Valvular collagen dominates MV structure near the annulus to support the stresses of bending and pressurization. Valvular GAGs dominate the MV near the free-edge to absorb the impact of leaflet coaptation. Image analysis has proven to be an effective tool to evaluate MV structure and facilitate the design of valve replacements.

Multivariate statistics can be used for visualization of cell subpopulations in multidimensional data space and for classification of cells within that data space. New data mining techniques we have developed, such as subtractive clustering, can be used to find the differences between test and control multiparameter flow cytometric data, e.g. in the problem of human stem cell isolation with tumor purging. They also can provide training data for subsequent multivariate statistical classification techniques such as discriminant function or logistic regression analyses. Using lookup tables, these multivariate statistical calculations can be performed in real-time, and can even include probabilities of misclassification. Thus, the only distinction between off-line classification of cells in data analysis and real-time statistical decision-making for cell sorting is the time limit in which a classification decision must be made. For real-time cell sorting we presently are able to perform these classifications in less than 625 microseconds, corresponding to the time that it takes the cell to travel from the laser intersection point to the sort decision point in a flow cytometer/cell sorter. Statistical decision making and the ability to include the costs of misclassification into that decision process will become important as flow cytometry/cell sorting moves from diagnostics to therapeutics.

Light scatter is often a source of interference in dual-laser excitation experiments by flow cytometry. If the two laser beams are not adequately separated or optical masking is not used, and if gated sequential fluorescence signal detection is not employed, light scatter from particles/cells as they pass across one beam can interfere in the fluorescence measurement channel of the other laser excitation beam. In this study we discuss the problem and present the theory for improving fluorescence measurements in dual-laser flow cytometry by use of modulated laser excitation and synchronous (phase- sensitive) detection of the resulting fluorescence emission to eliminate light scatter interference from particles/cells. Fluorescence emission from particles/cells generated by the first laser beam (unmodulated cw) is measured using conventional means (detection channel no. 1). Light scatter interference from particles/cells as they pass across the first laser excitation beam, whose wavelength lies in the fluorescence measurement region of detection channel no. 2, is rejected by the synchronously tuned phase-sensitive channel no. 2 detector, whereas the fluorescence emission corresponding to laser no. 2 excitation is detected and measured. The efficacy of the technology in relation to analytical cytology measurements is demonstrated using simulated fluorescence and light scatter signals.

Inhalable particulate dusts are involved in the genesis of several lung diseases. Besides the well-known toxic dusts, i.e. asbestos and quartz, heavy metal-containing pollutants are considered as possible harmful substances. In the present study we compared the effect of silica chemically coated with certain metal oxides on several cell physiological parameters of bovine alveolar macrophages (BAM). The cytosolic free calcium concentration [(Ca²⁺)_i], the intracellular pH (pH_i), and the plasma membrane potential (MP) of BAM were measured by flow cytometry whereas the dust- induced secretion of reactive oxygen species (ROS) was measured enzymatically. Compared to control incubations with pure silica the dust-induced secretion of ROS by BAM was not affected when the particles were coated with Cr₂O₃, NiO, and Fe₃O₄, whereas VO₂-coated dust induced a marked increase in ROS release. This effect was not correlated to changes in (Ca²⁺)_i, pH_i, or MP. On the other hand Cr₂O₃-coated silica caused alterations in all of the three latter parameters. The same pattern of changes has been reported previously for quartz dusts (Tarnok et al., Anal. Cell Pathol., 15:61-72, 1997). We conclude that cell physiological measurements by flow cytometry could extend the pallet of tools to evaluate possible toxic effects of environmental dust samples.

Cardiac surgery with cardiopulmonary bypass (CPB) can induce severe post-operative immune responses. During CPB loss of activated lymphocytes from the peripheral blood (PBL) was observed. We investigated if PBL get lost by binding to the CPB or by migration into the peripheral tissue and if the cells adhere selectively to different filter types. PBL were collected before, during and after surgery of pediatric patients and from the filters of the CPB by washing. Immunophenotype was determined by four color flow cytometry (FCM). In addition, PBL adhesion to CPB was analyzed in vitro. During surgery, B-cell counts decreased by greater than 50% due to the loss of CD69⁺ cells. The fraction of CD25⁺ and CD54⁺ T-lymphocytes decreased by 70%, that of CD69⁺ natural killer cells by 40%. In vivo in the CPB the proportion of CD69⁺ cells increased by up to 50%. These findings were supported by in vitro filtration studies. In contrast, the proportion of T-lymphocytes CD25⁺ or CD54⁺ were lower in the CPB. CD69⁺ cells adhere selectively to CPB filters. Loss of activated CD25⁺ or CD54⁺ T-lymphocytes could be due to their selective migration into the peripheral tissue. This FCM technique could be applied to test various filter types used in CPB in order to test their biocompatibility.

We have studied the kinetics of binding of dissolved antigens to antibody-receptors on the surface of the intact living cells. We have combined the Scanning Flow Cytometry with fluorescent immunoassay. Measurements of the kinetics were carried out for rabbit anti-mouse immunoglobulins that bind to receptors of the mouse hybridomas. Anti-mouse immunoglobulins were labeled with fluorescein isothiocyanate molecules (FITC). The individual cells were identified from the native Scanning Flow Cytometer records in our experiment. The kinetics of the formation of antigen-antibody complexes was measured from the fluorescence from the individual cells. A kinetic scheme for the heterogeneous reactions between ligands and receptors has been suggested. This scheme utilizes the concept of a distribution function of an amount of active receptors on the cell surface. The analytical solution for the system of the kinetic equations has been found in the reaction-limited approximation. The experimental kinetics were fitted by this mathematical model to determine the rate constant for binding reaction of the used antigen-antibody pair.

Fluorescent In-Situ Hybridization (FISH) is becoming an accepted technique for identification of aneuploidies in interphase fetal cells obtained by either CVS (chorionic villus sampling) or amniocentesis. Currently the analysis is done manually by a skilled operator and is a lengthy and fatiguing process. Applied Imaging is developing an automated procedure for counting FISH spots in these samples. Spot counting involves slide preparation, probe hybridization, filter selection, FISH image acquisition, image analysis, operator verification, and analysis of count distributions. We concentrate on the tasks starting with image acquisition. The following topics are covered: selection of appropriate cells, acquisition and processing of Z-stacks of FISH images for presentation and spot counting, background removal, formation of segmentation tree and selection of spot markers, growing of spot markers by means of constrained watershed, detection of irregular spots and flagging them for the user, time and accuracy compared with manual method, and applicability to a clinical research setting.

Numerical aberrations involving parts of or entire chromosomes have detrimental effects on mammalian embryonic, and perinatal development. Only few fetuses with chromosomal imbalances survive to term, and their abnormalities lead to stillbirth or cause severely altered phenotypes in the offspring (such as trisomies involving chromosomes 13, 18, 21, and anomalies of X, and Y). Because aneuploidy of any of the 24 chromosomes will have significant consequences, an optimized preimplantation and prenatal genetic diagnosis (PGD) test will score all the chromosomes. Since most cells to be analyzed will be in interphase rather than metaphase, we developed a rapid procedure for the analysis of interphase cells such as lymphocytes, amniocytes, or early embryonic cells (blastomeres). Our approach was based on in situ hybridization of chromosome-specific non-isotopically labeled DNA probes and Spectral Imaging. The Spectral Imaging system uses an interferometer instead of standard emission filters in a fluorescence microscope to record high resolution spectra from fluorescently stained specimens. This bio-imaging system combines the techniques of fluorescence optical microscopy, charged coupled device imaging, Fourier spectroscopy, light microscopy, and powerful analysis software. The probe set used here allowed simultaneous detection of 10 chromosomes (9, 13, 14, 15, 16, 18, 21, 22, X, Y) in interphase nuclei. Probes were obtained commercially or prepared in-house. Following 16 - 40 h hybridization to interphase cells and removal of unbound probes, image spectra (range 450 - 850 nm, resolution 10 nm) were recorded and analyzed using an SD200 Spectral Imaging system (ASI, Carlsbad, CA). Initially some amniocytes were unscoreable due to their thickness, and fixation protocols had to be modified to achieve satisfactory results. In summary, this study shows the simultaneous detection of at least 10 different chromosomes in interphase cells using a novel approach for multi-chromosome analysis.

The chromatin organization of interphase cell nuclei, albeit an object of intense investigation, is only poorly understood. In the past, this has hampered the cytogenetic analysis of tissues derived from specimens where only few cells were actively proliferating or a significant number of metaphase cells could be obtained by induction of growth. Typical examples of such hard to analyze cell systems are solid tumors, germ cells and, to a certain extent, fetal cells such as amniocytes, blastomeres or cytotrophoblasts. Balanced reciprocal translocations that do not disrupt essential genes and thus do not led to disease symptoms exit in less than one percent of the general population. Since the presence of translocations interferes with homologue pairing in meiosis, many of these individuals experience problems in their reproduction, such as reduced fertility, infertility or a history of spontaneous abortions. The majority of translocation carriers enrolled in our in vitro fertilization (IVF) programs carry simple translocations involving only two autosomes. While most translocations are relatively easy to spot in metaphase cells, the majority of cells biopsied from embryos produced by IVF are in interphase and thus unsuitable for analysis by chromosome banding or FISH-painting. We therefore set out to analyze single interphase cells for presence or absence of specific translocations. Our assay, based on fluorescence in situ hybridization (FISH) of breakpoint-spanning DNA probes, detects translocations in interphase by visual microscopic inspection of hybridization domains. Probes are prepared so that they span a breakpoint and cover several hundred kb of DNA adjacent to the breakpoint. On normal chromosomes, such probes label a contiguous stretch of DNA and produce a single hybridization domain per chromosome in interphase cells. The translocation disrupts the hybridization domain and the resulting two fragments appear as physically separated hybridization domains in the nucleus. To facilitate the detection, DNA probes for breakpoints on different chromosomes are labeled in different colors, so the translocation event can be detected as a fusion of red and green hybridization domains. We applied this scheme successfully for the analysis of somatic and germ cells from more than 20 translocation patients, each with individual breakpoints, and provide summaries of our experience as well as strategies, cost and time frames to prepare case-specific translocation probes.

Recent advances in autofocus, image segmentation, and fluorescence lamp stabilization have resulted in a fully automated high-speed image cytometer. This system performs computer-controlled autofocus, incremental stage scanning, image processing and data storage at a combined 1 Hz field rate. DAPI stained 3T3 cells that were cultured on microscope cover slips were scanned using the scanning cytometer. The integrated intensity for each detected cell object was computed and stored as well as its area and information about its location. Performance of the system was evaluated by repeatedly scanning cellular objects and comparing the results of each object over several hundred scans. This method of performance evaluation has the advantage of being independent of variations in stain homogeneity of the slide being scanned. For several hundred repeat scans, coefficients of variation (CVs) of significantly less than 1% were consistently obtained for the integrated intensity of each cellular object in the experiment.

With the typically narrow depth-of-field microscope optics, biological specimens do not lie in a single focal plane across the slide and this complicates automated scanning for image cytometry. An on-the-fly autofocus system for high-resolution image cytometry is presented which keeps the image sharply focused during continuous stage travel. To track possible foci, an image volume is acquired by concurrent optical sectioning of the specimen with a dedicated imaging array. This volume scanning camera was designed for adjustment of the optical path lengths to allow simple adaptation to objectives with different depths-of-field and magnifications. The computational demand of calculating and adjusting focus dynamically is absorbed by an array of parallel autofocus circuits that measure the 3D image in real time. In conventional optical sectioning microcopy, where the image data is acquired by sequential sectioning, a priori knowledge of the specimen and its boundaries exists. In continuous volume scanning, this is usually not the case and variations in specimen thickness and information content are routine. This makes implementation of fully automatic, high-speed, high-resolution image cytometry a challenge. A model of the combined specimen and optical system was developed to evaluate strategies for tracking focus. Data from this model is described along with the volume scanning camera array design.

There are many different modes of microscopy, and all of these modes deliver light in a controlled fashion to the object to be examined and collect as much of the light containing the desired information about the object as possible. The system being presented replaces the simple irises of a conventional microscope with digital micromirror devices (DMDs, made by Texas Instruments) to produce a digital microscope. The DMDs are placed in the optical path at positions corresponding to the field and aperture diaphragms of a conventional microscope. This allows for more precise and flexible control over the spatial location, amount, and angles of the illumination light, and the light to be collected. This digital microscope will improve existing modes of microscopy, specifically in quantitative microscopy. Using the intensity modulation feature of the DMDs, the system can correct for inhomogeneous illumination sources to achieve uniform distributions. In various configurations, one can perform brightfield, darkfield, confocal and fluorescence microscopy. In addition, new microscopy modes will be possible, such as reconstruction microscopy. Utilizing the fast switching times of the mirrors (under 20 microseconds), one can switch between modes efficiently.

At present, the microscopic visualization of luminescent labels containing lanthanide(III) ions, primarily europium(III), as light-emitting centers is best performed with time-gated instrumentation, which by virtually eliminating the background fluorescence results in an improved signal to noise ratio. However, the use of the europium(III) macrocycle, Quantum Dye^TM, in conjunction with the strong luminescence enhancing effect (cofluorescence) of yttrium(III) or gadolinium(III), can eliminate the need for such specialized instrumentation. In the presence of Gd(III), the luminescence of the Eu-macrocycles can be conveniently observed with conventional fluorescence instrumentation at previously unattainable low levels. The Eu(III) ⁵D_O yields ⁷F₂ emission of the Eu-macrocycles was observed as an extremely sharp band with a maximum at 619 nm and a clearly resolved characteristic pattern. At very low Eu- macrocycle concentrations, another sharp emission was detected at 614 nm, arising from traces of Eu(III) present in even the purest commercially available gadolinium products. Discrimination of the resolved emissions of the Eu-macrocycle and Eu(III) contaminant should provide a means to further lower the limit of detection of the Eu-macrocycle.

Observing dynamic reorganization and molecular interactions of cellular components on a precise spatial and temporal scale is not possible using existing microscopic techniques. However, fluorescence lifetimes occur on a nanosecond time scale, are independent of local signal intensity and concentration of the fluorophore, and provide sensitive discrimination of the molecular environment. We designed and implemented a fluorescence lifetime imaging microscope (FLIM) using a picosecond-gated multi-channel plate image intensifier, providing two-dimensional time-resolved images of single cell specimen. BHK21 cells were transfected with vectors for green fluorescent protein (GFP) and placed on an infinity-corrected Olympus epi-fluorescence microscope, coupled to a Coherent tunable femtosecond ti-sapphire pulsed laser and a frequency doubler to select an appropriate excitation wave length. After synchronizing the high-speed gated image intensifier to the excitation laser pulses, time-resolved nanosecond images of fluorescent emission were acquired. These images were processed pixel-by-pixel for single exponential decay to obtain an image based on fluorescence lifetime. Although the nucleus appeared brighter than the cytoplasm by fluorescence intensity measurement, FILM showed a uniform lifetime of the GFP fluorescence in both compartments, indicating that the GFP was in similar molecular environments. This technology also has important applications in fluorescence resonance energy transfer (FRET) imaging.

Modern research in molecular, cellular, and developmental biology requires the precise measurement of cellular or subcellular activity in two- or three-dimensions. Many available fluorescence microscope techniques are yielding limited detail about the organization and dynamics of complex cellular structures. But the multi-photon (two- or more photon) excitation fluorescence imaging microscopy (MEFIM) system proposed here will provide a unique, state-of-the-art opportunity to integrate advances in optical microscopy, low- light video detection, and two- or three-dimensional image analysis in measuring the fluorescent signals from living cells. The MEFIM system uses longer excitation wavelength, which increases the penetration of the excitation of the sample, yet essentially reduces photobleaching and autofluorescence. But, more importantly, the infrared light excitation in the MEFIM techniques allows maintaining cell viability for longer periods of time and thus acquisition of more images. In our current work, we integrated the MEFIM system with one-photon laser scanning confocal microscope coupled to a verdi pumped tunable femtosecond pulsed ti- sapphire laser. Appropriate wavelength was tuned to excite the fluorescently labeled specimens and MEFIM images were acquired from living biological specimens at different optical sections. The importance of the MEFIM image acquisition and processing for biomedical sciences are discussed.

This paper describes results obtained with a novel imaging system based on a fast CCD device. Sensor's output was digitized at 12 bit/pixel by customized electronics, reaching acquisition rates as high as 800 frames/s with a full-frame resolution of 128 X 128 pixels. The software developed for the project permitted the sequential capture of thousands of images directly to host PC RAM without frame loss even at the maximum readout rate (16 MHz). It is shown that the high spatio-temporal resolution of this apparatus is of value when investigating the time-course of rapid intracellular Ca²⁺ fluorescence transients, particularly those associated with neuronal action potentials near physiological temperature.