We developed a near-field scanning optical microscope (NSOM) which uses an optically trapped metallic Rayleigh particle as a near-field probe. This microscope is suitable for observations of biological specimen under aqueous condition because the probe particle is held by non-contact force from radiation pressure. The spring constant of trapping force is so weak (several mN/m order typically) that induced damage on a sample rarely occurs by this force. We used a gold colloidal particle which had a diameter of 40 nm as a probe. The scattering efficiency of the particle (at 488-nm wavelength, n_Au equals 0.916, K_Au equals 1.840) is 348 times as great as that of a glass particle (n_glass equals 1.5) in the same size under water. We investigated a surface of a cover glass with the gold colloidal particle and confirmed the ability to observe small irregularities of the surface in around 10 nm. We also observed aggregated gold colloidal particles (d equals 40 nm) on a cover glass and fluorescent beads on glass. The investigation of DNA stained with YOYO-1 iodide on glass under water conditions was also performed.

We have developed a new assay in which two mesoscale particles are caused to collide using two independently controlled optical tweezers. This assay involves the measurement of the adhesion probability following a collision. Since the relative orientation, impact parameter (i.e., distance of closest approach), and collision velocity of the particles, as well as the components of the solution, are all under the user's control, this assay can mimic a wide range of biologically relevant collisions. We illustrate the utility of our assay by evaluating the adhesion probability of a single erythrocyte (red blood cell) to an influenza virus-coated microsphere, in the presence of sialic acid-bearing inhibitors of adhesion. This probability as a function of inhibitor concentration yields a measure of the effectiveness of the inhibitor for blocking viral adhesion. Most of the inhibition constants obtained using the tweezers agree well with those obtained from other techniques, although the inhibition constants for the best of the inhibitors were beyond the limited resolution of conventional assays. They were readily evaluated using our tweezers-based assay, however, and prove to be the most potent inhibitors of adhesion between influenza virus and erythrocytes ever measured. Further studies are underway to investigate the effect of collision velocity on the adhesion probability, with the eventual goal of understanding the various mechanisms of inhibition (direct competition for viral binding sites versus steric stabilization). Analysis of these data also provide evidence that the density of binding sites may be a crucial parameter in the application of this assay and polymeric inhibition in general.

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 initiates an activation cascade which includes an increase in T-cell intracellular calcium and leads to T-cell proliferation and differentiation. We have combined fluroescence spectroscopy and imaging 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 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.

Laser tweezers, used as novel sterile micromanipulation tools of living cells, are employed in laser-assisted in vitro fertilization (IVF). For example, controlled spermatozoa transport with 1064 nm tweezers to human egg cells has been performed in European clinics in cases of male infertility. The interaction of approximately 100 mW near infrared (NIR) trapping beams at MW/cm² intensity with human gametes results in low mean less than 2 K temperature increases and less than 100 pN trapping forces. Therefore, photothermal or photomechanical induced destructive effects appear unlikely. However, the high photon flux densities may induce simultaneous absorption of two NIR photons resulting in nonlinear interactions. These nonlinear interactions imply non-resonant two-photon excitation of endogenous cellular chromophores. In the case of less than 800 nm tweezers, UV- like damage effects may occur. The destructive effect is amplified when multimode cw lasers are used as tweezer sources due to longitudinal mode-beating effects and partial mode- locking. Spermatozoa damage within seconds using 760 nm traps due to formation of unstable ps pulses in a cw Ti:Sa ring laser is demonstrated. We recommend the use of greater than or equal to 800 nm traps for optical gamete micromanipulation. To our opinion, further basic studies on the influence of nonlinear effects of laser tweezers on human gamete are necessary.

Highly focused pulsed laser microbeams can be used to precisely dissect, inactivate, or perturb cells and subcellular targets. Here we introduce a new technique which employs pulsed microbeams to transiently permeabilize the plasma cell membrane and affect the delivery of molecules from the extracellular environment into the cell. This optically produced cell permeabilization can be applied using non- specific or specific modalities. In the non-specific modality, which we term 'optoporation,' the pulsed microbeam is focused onto the glass coverslip on which the cells are plated. The generation of mechanical transients in connection with irradiation of the glass achieves molecular delivery to a number of cells proximal to the irradiation site. In the specific modality, termed 'optoinjection,' the microbeam is focused directly onto the plasma cell membrane and achieves molecular delivery into that cell alone. To quantify the irradiation geometry involved in these and other microbeam processes, as well as examine the possibility of certain non- linear effects, we have developed a system using photochromic polymer films to characterize microbeam propagation and its effects within microirradiated targets. These photochromic polymers confirm that the laser microbeam are indeed focused to submicron dimensions within the targets in our systems. In addition the behavior of such polymers at higher pulse energies and irradiances indicate that multiphoton absorption and/or plasma formation may mediate some laser microirradiation processes.

The technology of two-photon excitation has opened a window of opportunity for developing non-invasive medical diagnostic tools capable of monitoring thick tissue biochemical states. Using cellular endogenous chromophores, (beta) -nicotinamide- adenine dinucleotide phosphate [NAD(P)H], the cellular metabolic rates in living human skin were determined. Although important functional information can be obtained from the fluorescence spectroscopy of endogenous chromophores, these chromophores are rather poor contrast enhancing agent for mapping cellular morphology. First, most endogenous chromophores are confined to the cellular cytoplasm which prevents the visualization of other cellular organelles. Second, there is significant variability in the distribution and the quantum yield of endogenous chromophores which depends on tissue biochemistry but prevents consistent comparison of cellular morphology. On the other hand, the deep tissue cellular morphology has been imaged with excellent resolution using reflected light confocal microscopy. In reflected light microscopy, the image contrast originates from the index of refraction differences of the cellular structures. The organelle boundaries with significant index differences such as the plasma membrane and the nucleus envelope can be consistently visualized. A combination of morphological and functional information is required for a thorough tissue study. This presentation describes the development of a new microscope which is capable of simultaneously collecting both two-photon fluorescence and confocal reflected light signals. Promising biomedical applications include the non-invasive diagnosis of skin cancer and the study of wound healing.

Fluorescence microscopy is a ubiquitous and powerful tool for the biologist mainly due to the availability of a wealth of highly specific fluorescent probes. Multiphoton (two or more photon) excitation fluorescence microscopy is an optical sectioning technique that offers significant advantages over other optical sectioning techniques in terms of improved viability of living material and the ability to penetrate deeper into specimens. The use of a longer excitation wavelength (typically twice that of the excitation peak of the fluorophore) increases the penetration of the excitation into the sample, yet essentially eliminates single-photon excitation in the bulk of the sample. In order to attain the high peak-power densities necessary for the production of multiphoton events while keeping mean power levels below damaging levels, ultrashort-pulsed excitation sources are used. Some sources, such as mode-locked, Ti:sapphire lasers, can produce pulses less than 100 fs. Pulses this short need to be pre-chirped in order to compensate for the group velocity dispersion of the microscope optics so that the pulse width is maintained at the sample. Without such pre-compensation we show that the average power required to produce a fixed level of two-photon excited signal, using typical microscope optics, is fairly constant from 60fs to 250fs. We argue that the choice of pulse width is an important consideration for a biological imaging system since varying the source pulse width may be used to change the relative amounts of two- and three- photon excitation. With a pre-chirped (compensated) system, if the pulse length is quadrupled then twice the power will be required to attain the previous level of two-photon excited fluorescence, but only half the three-photon excitation (or absorption) will be produced. Pulse widths may be varied on compensated systems by adjusting the pre-compensation. This may be used to favor three-photon excitation of UV-excited fluorophores, or, on the other hand, it may be desirable to reduce levels of three-photon excitation during two-photon imaging of live samples using 700 nm - 800 nm radiation as deleterious excitation of endogenous fluorophores or absorption by (and therefore damage to) proteins and nucleic acids could occur. Variable pulse widths may therefore prove to be an important parameter for live cell studies. Alternatively, for a given range of applications, a simpler and cheaper fixed-pulse length source with the desired characteristics may be chosen.

Highly reproducible and stable surface gold island films exhibiting long-range enhancement have been investigated and characterized as compatible for biological systems. These surface-enhanced Raman scattering (SERS) substrates allowed the non-invasive detection of micromolar concentrations of antitumor drugs using red and near-infrared excitations. Thus, good quality SERS spectra of dimethylcrocetin (DMCRT) in a single living HL60 cell have been recorded on these substrates using red excitation, without any noticeable perturbation of the cell integrity. Comparison of these spectra with FT-Raman data obtained in HL60 cells on one hand, and with FT-SERS data of the DMCRT-retinoic acid receptor (RAR) complex on the other, shows practically the same spectral profiles. However, it should be noted that with the red laser the spectrum gives additional information on the cellular components. Similarity between the signal of DMCRT-treated K562 cells and the free drug is explained by either an absence of RAR in this cell line or a lack of binding.

We have developed a novel approach to study luminescent light emission during migration of living cells by low-light imaging techniques. The equipment consists in an anti-vibration table with a hole for a direct output under the frame of an inverted microscope. The image is directly captured by an ultra low- light level photon-counting camera equipped with an image intensifier coupled by an optical fiber to a CCD sensor. This installation is dedicated to measure in a dynamic manner the effect of SF/HGF (Scatter Factor/Hepatocyte Growth Factor) both on activation of gene promoter elements and on cell motility. Epithelial cells were stably transfected with promoter elements containing Ets transcription factor-binding sites driving a luciferase reporter gene. Luminescent light emitted by individual cells was measured by image analysis. Images of luminescent spots were acquired with a high aperture objective and time exposure of 10 - 30 min in photon-counting mode. The sensitivity of the camera was adjusted to a high value which required the use of a segmentation algorithm dedicated to eliminate the background noise. Hence, image segmentation and treatments by mathematical morphology were particularly indicated in these experimental conditions. In order to estimate the orientation of cells during their migration, we used a dedicated skeleton algorithm applied to the oblong spots of variable intensities emitted by the cells. Kinetic changes of luminescent sources, distance and speed of migration were recorded and then correlated with cellular morphological changes for each spot. Our results highlight the usefulness of the mathematical morphology to quantify kinetic changes in luminescence microscopy.

Two-dimensional fluorescence depolarization measurement was achieved by a fluorescence microscope equipped with polarizing devices and an image intensified CCD camera. Anisotropy images were acquired using living cells stained with a membrane specific binding dye. It was found that the bound dye is spatially distinguishable from the free dye which does not bind to the membrane of a cell, due to the difference in rotational Brownian motion. In addition, we succeeded in obtaining fluorescence depolarization images by means of time- resolved measurement and two-photon excitation measurement. Two-photon excitation images showed a superior signal-to-noise ratio compared to one-photon excitation images. Fluorescence depolarization imaging will therefore prove a powerful tool for studying molecular functions in cells.

Cytosolic acidification and mitochondrial dysfunction have been reported to be concomitant with apoptosis. In our study, we report on the use of carboxy-SNARF-1-AM and JC1 probes in association with confocal scanning microspectrofluorometry to examine respectively cytosolic pH and mitochondrial membrane potential during camptothecin (CPT) and daunorubicin (DNR) induced apoptosis in human leukemic HL60 cells. Emission intensity ratios (I_{580 nm}/I_{640 nm}) of carboxy-SNARF-1 spectra lead to determine cytosolic pH. Whereas, JC1 was able to form J-aggregates respectively from green (530 nm) to yellow-orange (590 nm) as mitochondrial membrane becomes more polarized. Our results show that control cells presented a neutral cytosolic pH (7.26 plus or minus 0.08). The cells induced in apoptosis in presence of 1 (mu) M CPT or DNR during 6hrs, undergo significant acidification of the cytosol (7.00 plus or minus 0.06 and 7.02 plus or minus 0.05 respectively). Decrease of I_{590 nm}/I_{530 nm} emission ratio of JC1 spectra (64 plus or minus 5% compared to control) was observed only with CPT. We conclude that cytosolic acidification and mitochondrial dysfunction are early events in apoptosis and may be essential for genome destruction. Confocal scanning microspectrofluorometry and dual ratiometric fluorescent probes appear to be a powerful approach for the study of ion response in living cells.

The spatiotemporal dynamics of dimerization of the transcription factor Pit-1 in the living cell nucleus have been visualized and monitored by expression of genetic vectors encoding green (GFP) and blue (BFP) fluorescent protein fusions and fluorescence resonance energy transfer (FRET) imaging techniques. However, in wide-field FRET imaging microscopy the FRET signals are a combination of signals from all focal planes and the fine image details are obscured by 'out-of-focus' light. Digital deconvolution FRET imaging microscopy is used here to remove the 'out-of-focus' light to improve the resolution of the protein localization in the optical axis. Cells expressing fluorescent Pit-1 fusion proteins were imaged with a high speed, high sensitivity CCD camera and a water immersion objective lens. The point spread function (PSF) of the system was used to deconvolve the donor and acceptor images which then were ratioed to obtain the FRET signal at different optical sections. These signals were used to create three-dimensional visualization of the distribution of Pit-1 protein dimers. Digitally deconvolved data with a water immersion lens has a better signal-to-noise ratio than data obtained with an oil immersion lens. The 3-D energy transfer imaging of Pit-1 protein in the nucleus of living cells offers the possibility of studying domains within the nucleus.

The vesicular transport of lipids from the endoplasmic reticulum via the Golgi apparatus affects the composition of the plasma membrane. The purpose of our study was to develop an in vitro test system for characterization of vesicular lipid transport kinetics by using confocal microscopy and fluorescence recovery after photobleaching (FRAP). Fibroblasts from two patients homozygous for the hypercatabolic HDL deficiency syndrome Tangier disease and 4 control subjects were pulsed with the C₆-NBD-ceramide for 30 minutes. Chase incubation at room temperature resulted in the metabolic accumulation of fluorescent C₆-NBD-sphingolyelin and C₆-NBD-glycosylceramides in the medial- and trans-Golgi region. Cells were analyzed with an inverted Leica TCS microscope. Calibration was performed through the analysis of diffusion of 50 nm microparticles embedded in media of different viscosity. An acousto optical tunable filter (AOTF) was used for the selective bleaching of the medial- and trans- Golgi region followed by analysis of the fluorescence recovery for 4 minutes. Post-bleach fluorescence recovery through the trans-Golgi-oriented transport of NBD-sphingomyelin was calculated from 2-dimensional scans. Tangier fibroblasts displayed a retarded recovery of fluorescence in the trans- Golgi region. This suggests that the vesicular transport of sphingomyelin and cholesterol is disturbed in Tangier disease confirming data from our laboratory generated with radiometabolites on whole cells. Our data suggest that FRAP analysis allows a sensitive kinetic and spatially resolved analysis of disturbances of vesicular lipid transport.

Rat adrenal glands fluoresce intensely after systemic application of meso-tetra(hydroxyphenyl)chlorin (mTHPC). We investigated which parts of the adrenal gland accumulate mTHPC. Furthermore we examined the time course of adrenal mTHPC-accumulation. Ten male Wistar rats each were given 0.5 or 0.7 mg mTHPC kg^-1 iv. Each two animals were perfused with normal saline and Zamboni fixative 6, 12, 24, 48 and 72 hours after photosensitization. Untreated animals served as controls. Fluorescence was quantified on 20 micrometer frozen sections with CCD-camera and appropriate software. Immunohistochemistry identified specific cell types with antibodies to steroid-synthesizing enzymes. The cortex exhibited an intense fluorescence, with weaker fluorescence of corticocytes in the zona glomerulosa compared to the other zones. Besides intensely fluorescing singly lying scattered cells, the medulla showed a faint mTHPC-induced fluorescence. Immunohistochemistry revealed that intramedullary cells with intense fluorescence were corticocytes, showing a positive reaction to the 21-(beta) -hydroxylase antibody. Peak accumulation of mTHPC was always observed after 24 hours. Our results indicate for the first time that only steroid synthesizing cells of the adrenal gland exhibit an intense photosensitizer-induced fluorescence. Thus mTHPC-application is an uncomplicated method to identify steroid-synthesizing cells, possibly also in other organs.

The molecular and structural analysis of cells within their tissue context helps us understand disease mechanisms, such as carcinogenesis. Standard analysis of cutting specimens into thin (4 micrometer) sections, followed by labeling and visual microscopic analysis, has the limitation that tissue properties can only be studied within the section plane, and not perpendicular to the plane. We solved these limitations by building a system for registering images of adjacent sections. In addition, the system enables analysis of many molecular markers in a specific tissue volume, by labeling different sections with different markers, followed by using the system to locate the relevant tissue volume in each section. The system has three stages. First, it automatically images each entire section and two fiducial markers per slide. After this stage, the slides can be removed from the microscope. In stage two, pairs of images of adjacent sections are registered. This is done by interactively marking several points that are common to both images, which are used to calculate the translation and rotation of the images relative to each other. Different registrations can be performed on different parts of the images to account for differential stretching, tearing and folding of sections. In stage three, a slide is placed on the microscope stage and the analyst can bring a specific location into the field of view by referring to it in the previously acquired image. Accuracy is approximately equal to 10 micrometers.

The detection and genetic analysis of fetal cells in maternal blood will permit noninvasive prenatal screening for genetic defects. Applied Imaging has developed and is currently evaluating a system for semiautomatic detection of fetal nucleated red blood cells on slides and acquisition of their DNA probe FISH images. The specimens are blood smears from pregnant women (9 - 16 weeks gestation) enriched for nucleated red blood cells (NRBC). The cells are identified by using labeled monoclonal antibodies directed to different types of hemoglobin chains (gamma, epsilon); the nuclei are stained with DAPI. The Applied Imaging system has been implemented with both Olympus BX and Nikon Eclipse series microscopes which were equipped with transmission and fluorescence optics. The system includes the following motorized components: stage, focus, transmission, and fluorescence filter wheels. A video camera with light integration (COHU 4910) permits low light imaging. The software capabilities include scanning, relocation, autofocusing, feature extraction, facilities for operator review, and data analysis. Detection of fetal NRBCs is achieved by employing a combination of brightfield and fluorescence images of nuclear and cytoplasmic markers. The brightfield and fluorescence images are all obtained with a single multi-bandpass dichroic mirror. A Z-stack of DNA probe FISH images is acquired by moving focus and switching excitation filters. This stack is combined to produce an enhanced image for presentation and spot counting.

There has been great interest in automated image cytometry because of the increased information afforded by a native 2D data set (as opposed to 1D in flow cytometry). High resolution scanning cytometry, however, has been severely hampered by cell analysis rates three orders of magnitude slower than that of flow cytometry. Thus far, a two-pass strategy of scanning at very low resolution followed by high resolution imaging of a few selected fields has been most commonly employed to avoid this problem and achieve higher rates of analysis. Fast scanning can be performed at high resolution using a time- delay-and-integrate (TDI) CCD camera synchronized to constant- velocity stage movement, but images collected by this method are degraded by inability to track focus during continuous stage movement. Here we introduce simultaneous multiplanar image acquisition and focus measurement for tracking focus. This volume-scanning, autofocus system combines multiplanar fiberoptic image coupling, an array of TDI CCD cameras, and an array of focus measurement circuits. This design will also enable future high speed 3D scanning cytometry applications.

There are numerous modes of microscopy such as brightfield, darkfield, phase contrast, fluorescence, reflected light, confocal, etc. All of these forms of microscopy deliver illumination light in a controlled fashion to the object to be examined and collect as much light containing the desired information as possible. The majority of these methods use appropriately placed and formed diaphragms (iris, pin hole, annulus, etc.) and lenses to control both the incident angles of the illumination light and its intensity as well as the size and location of the illuminated area in the sample. Usually these diaphragms are a simple iris or annulus and are almost always static. The novel aspect of the system being presented is to replace these simple mechanical diaphragms with digital micro mirror devices (¹DMDs made by Texas Instruments) to allow for more precise, flexible control over the transmission behavior of these optical planes. By placing DMDs in the same plane (actual or conjugate) as that of the field iris, illumination aperture iris (condenser diaphragm), objective lens aperture stop, and field stop, one has the ability to rapidly switch between brightfield, darkfield, confocal and reconstruction microscopy. In addition because of the intensity modulating features of DMDs, one can create a uniform illumination distributions in the sample or a non- uniform distribution.

Given the demographics of current and ex-smoking populations in North America, lung cancer will be a major problem in the foreseeable future. Early detection and treatment of lung cancer holds great promise for the management of this disease. Unlike cervical cancer, the physical, complete removal/destruction of all dysplastic lesions in the bronchial tree is not possible; however, treatment of the lesions using a chemopreventive agent is. Intermediate biomarkers have been used to screen promising chemopreventive agents for larger population studies. We have examined the natural history of lung cancer development by following a group of subjects at high risk of developing lung cancer using fluorescence endoscopy to identify the areas of abnormality for biopsy. Approximately 900 biopsies have been collected in this fashion and graded by at least two experienced, expert pathologists. Using an interactive version of the Cyto-Savant (Oncometrics Imaging Corp.), cytometric and tissue architectural data were collected from these biopsies. Using only the data from the normal and invasive cancer biopsies, quantitative morphometric and architectural indices were generated and calculated for all the collected biopsies. These indices were compared with Loss of Heterozygosity (LOH) of ten sites commonly associated with cancer. These results and the application of these quantitative measures to two small chemoprevention studies will be reported.

The study of in vitro cancerous cell behavior is a very important topic. We describe here a tool which we have developed and which enables cell motility, cell proliferation and cell death rate to be determined semi-automatically. The tool is based on the processing of images obtained by observing cultures over several days. Numerous experiments have already been performed. These experiments are described only briefly in this paper and must be considered as examples of applications of the device that we present.

The blood flow in capillary vessels plays an important role in sustaining the vital activity of the human organism. The computerized capillaroscope is used for the investigations of nailfold (eponychium) capillary blood flow. An important advantage of the instrument is the possibility of performing non-invasive investigations, i.e., without damage to skin or vessels and causing no pain or unpleasant sensations. The high-class equipment and software allow direct observation of capillary blood flow dynamics on a computer screen at a 700 - 1300 times magnification. For the first time in the clinical practice, it has become possible to precisely measure the speed of capillary blood flow, as well as the frequency of aggregate formation (glued together in clots of blood particles). In addition, provision is made for automatic measurement of capillary size and wall thickness and automatic recording of blood aggregate images for further visual study, documentation, and electronic database management.

A new technique of Focused Ion Beam (FIB) microscopy- nanomachining is proposed for life sciences. Its performances are compared with those of currently available ultramicroscopy apparatuses. Ultra-high resolution tridimensional tomography can be performed on whole cells without preparation. This can be achieved by sequentially etching layers of material and subsequently viewing the result of the operation under a different perspective. Very fast imaging times (minutes) allow quasi real time microscopy. The complementary technique of nano-biology can be performed on the same apparatus. The use of the ion beam allows to imaging both the surface and the inner part of the sample along any desired plane that can be chosen while the observation is on.

A phase-sensitive flow cytometer has been developed to quantify fluorescence lifetimes directly as a parameter in real time on particles and cells labeled with fluorophores. This instrument combines flow cytometry and frequency-domain fluorescence lifetime spectroscopy measurement principles to provide unique features for making excited-state lifetime measurements. Cells are analyzed as they intersect a high- frequency, intensity-modulated (sine wave) laser excitation beam. Fluorescence signals are processed by analog phase comparator measurement electronics to provide output signals proportional to the sine and cosine of the phase difference between the modulated signal input and a steady state reference signal. The ratios, which are proportional to the lifetimes, are displayed as frequency distributions histograms. In this study we present the underlying theory to measure fluorescence lifetimes by both phase shift and amplitude demodulation. Prototype analog amplitude demodulation electronics have been constructed and are being tested using simulated signals, fluorescent microspheres, and cells labeled with fluorescent probes.

We report the first demonstration of flow cytometry using upconverting phosphor microsphere reporters. These unique materials are excited by near infrared light from a diode laser and emit visible light in narrow emission bands in the 500 to 700 nm spectral region. Advantages of upconverting phosphors over conventional fluorescent reporters include greater multiplexing, no autofluorescence background, no photobleaching, and excitation by a compact laser source. We modified and optimized a commercial flow cytometer for upconversion detection, and we evaluated system performance in a sandwich assay format, using magnetic bead capture surfaces for small antigen detection. Preliminary results using mouse IgG targets show sensitivities at the ng/mL level using a 25 (mu) L sample volume and a total analysis time of several minutes. Efforts to improve the sensitivity to a variety of small antigen targets and to demonstrate multiplexing with different phosphor compositions will be discussed along with efforts to develop a compact upconverting flow cytometer for field applications.

An altered cellular membrane fluidity secondary to changes of cholesterol metabolism is a potentially important mechanism in the pathogenesis of atherosclerosis. Especially in blood platelets an increased sensitivity for stimulation dependent aggregation which is a risk factor for thrombosis has been experimentally linked to disorders of lipid and lipoprotein metabolism. The goal of this study was the development of a flow cytometric assay for the direct analysis of cellular membrane microviscosity in correlation to activation associated phenotypic changes of platelets in vitro. The analysis of fluorescence polarization following the staining of hydrophobic lipid regions of cell membranes with the fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) is a well established method for the analysis of membrane fluidity. The extent of fluorescence anisotropy dependent on the rotational mobility of this fluorochrome is indirectly proportional to the microviscosity of the stained membrane subcompartment. In this study, an alternative and more simple method based on the diffusion dependent excimer formation of pyrenedecanoic acid (PDA) (J. Immunol. Methods 96:225-31, 1987) was characterized in comparison to the DPH method as a reference. Human platelets showed a rapid uptake of both DPH and PDA resulting in the staining primarily of the plasma membrane after up to 30 min of incubation. Staining analyzed at 351 nm excitation resulted in a saturation of the depolarization coefficient of DPH at 20 (mu) M but an increase of the excimer to monomer ratio of PDA with increasing dye concentration. A 'membrane fluidity coefficient' which saturated at 5 (mu) M PDA was calculated as the excimer fluorescence divided through the square of monomer fluorescence thereby correcting for the influence of dye concentration on excimer formation. The temperature dependent changes of membrane viscosity were further used as a model for the comparison of both methods. Cells analyzed at temperatures between 12 degrees Celsius and 33 degrees Celsius showed a linear increase of the microviscosity values which were derived from the method by a factor of 3.1. The microviscosity calculated from the DPH method, in contrast, decreased only 2.2-fold with relatively smaller changes occurring above 24 degrees Celsius. Cholesterol depletion of platelets using cholesterol-poor phosphatidylcholine-cholesterol liposomes resulted in significant changes of the PDA fluorescence coefficient similar to the DPH polarization coefficient indicating similar specificity of both methods. A high sensitivity of the PDA method was further confirmed through the analysis of patient blood samples where the membrane viscosity of platelets as determined with PDA showed a good correlation to serum HDL cholesterol. In conclusion, the analysis of the excimer fluorescence of PDA is a technically simple, sensitive, and highly reproducible method for the flow cytometric analysis of an altered membrane fluidity of platelets.

Flow cytometric (FCM) measurement of intracellular free calcium [Ca²⁺]_i, transients is usually done by two methods: (a) after a short prerun period to assess the baseline the measurement is stopped, stimulus is added and the measurement continued or (b) stimulus is injected during measurement and the sample pressure briefly increased to deliver cells rapidly to the detection point. In (a) measurement of very short transients [Ca²⁺]_i is impeded by the lag time between stimulus addition and restart of acquisition. In (b) response of pressure sensitive cells is hard to analyze. Furthermore, (a) and (b) do not allow to quantify and sort rare responders. A simple Fixed- Time device has been developed. Ca²⁺ sensitive fluorescent dye labeled cells and a stimulus are placed in different vials. Both fluids are forced by the same pressure through tubing that merges into a T-junction where they mix and are delivered through a connecting tube to the FCM: [Ca²⁺]_i is measured at a certain time after stimulation that is adjusted by sample flow rate and length of the connecting tube. With Fixed-Time, the pressure sensitive neuronal NH15-CA2 cell was analyzed. Furthermore, rare neurotransmitter responsive fibroblast from normal and transfected cultures were sorted and cloned and their dose response characterized. The results demonstrate that fixed- time FCM is an important tool for the analysis of the cells physiology and the preparation of responders.

Data analysis and cell sorting are both fundamentally the same except in terms of the time available to make classification decisions. In the case of cell sorting the cell classification decisions must be made in real-time (in the case of cell sorting, real-time means in about 625 microseconds on this system). This dictates an approach to classification which can be implemented at memory speeds or in pre-programmed hardware. We have been developing new high-speed lookup table transformation methods, suitable for real-time data classification or cell sorting based on statistical classifiers. Multiparameter data mixtures of human MCF-7 breast cancer cells and human bone marrow were analyzed by discriminant function analysis. Cell identification tags, implemented as additional correlated listmode parameters not used for these analyses, were used to uniquely identify each cell type and to compare classifier results. The performance of classifier systems was also assessed using ROC ('receiver operating characteristics') analysis. The effectiveness of the classification system for cell sorting can be evaluated using molecular characterizations of sorted cells, either in small numbers or at single-cell level.

The addition of a list mode data type to the Digital Imaging and Communications in Medicine standard, DICOM will enhance the storage and transmission of digital microscopy data and extend DICOM to include flow cytometry data. This would permit the present International Society for analytical Cytology Flow Cytometry Standard to be retired. DICOM includes: image graphics objects, specifications for describing: studies, reports, the acquisition of the data, work list management, and the individuals involved (physician, patient, etc.) The glossary of terms (objects) suitable for use with DICOM has been extended to include the collaborative effort of Logical Observation Identifier Names and Codes (LOINC) and Systematized Nomenclature of Human and Veterinary Medicine (SNOMED) to create a consistent, unambiguous clinical reference terminology. It also appears that DICOM will be a significant part of the Common Object Request Broker Architecture, CORBA.

In order to enhance the quality and safety of bottled natural mineral waters, new methodologies besides classical bacteriology have been evaluated. Multi laser flow cytometry has been used to identify bacterial populations based on their DNA content, physiological activity and phylogeny from in situ hybridization with rRNA targeted DNA probes. Due to the low content of organic material in these waters, the bacterial population are under conditions (low ribosome content, low activity, etc.) which makes it hard to detect them flow cytometrically. The numbers of bacteria are in the range between 1000 and 100,000 per ml (for uncarbonated waters). Filtration techniques to enrich the bacterial population have been developed in combination with specific staining and hybridization protocols. First results on some selected brands show, that most bacteria belong to the beta subclass of proteobacteria. If the DNA containing cells (DAPI staining) are counted as 100%, 84% could be stained with a eubacteria probe. From these 84% 68% belong to the beta subclass, 8.2% to the alpha and 0.3% to the gamma subclass of roteobacteria. 8.5% could be identified as cytophaga flexibacter. By optimizing DNA staining with cyanine dyes and enhancing the sensitivity of light scatter detection, the detection limit could be considerably lowered.

A simple and inexpensive quantitative detection of protein in one droplet solution was achieved by a portable diode-laser- based-optical-fiber absorption spectrometry. A system consisted of a visible diode laser, a photodiode and a pair of optical fibers. The conventional Lowry method was used to measure the blue-colored protein solutions. In this study, one droplet of sample, which filled up the void between the end surfaces of two optical fibers, served as a sample cell. The volume of a droplet was 50 (mu) L, which is 1/100 of the cuvette of UV/VIS spectrometer cell used in Lowry method. Although the required sample volume decreased up to 100 times, the detection range was comparable to that of conventional Lowry method in the range of g/L - mg/L.

One of the principle ways that cells interact with their environment is by exerting mechanical forces. In order to understand the generation and application of forces by cells, the location, direction and magnitude of forces applied to a surface must be known and these related to dynamic cellular structures. Light microscopy is the method of choice for simultaneous measurements of these quantities, and therefore the surface must have suitable optical properties. These challenges have been approached by imaging cellular and cytoskeletal dynamics in living cells along with strain produced by traction forces in transparent silicone rubber substrata. The silicone has an index of refraction very similar to glass, facilitating many types of optical microscopy. Fluorescence microscopy was used to study cytoskeletal dynamics in fibroblasts by microinjecting a fluorescent analog of the motor protein myosin II. Cell- substratum contacts were studied by interference contrast microscopy (IRM), and cell morphology was monitored by Nomarski Differential Interference Contrast (DIC). Changes in traction force at cell-substratum contacts correlated strongly with assembly of myosin into the cytoskeleton. An important optical property of the silicone rubber used here allows its mechanical properties to be matched to the cells under study: illumination by UV light increases compliance, permitting measurement of forces in the range of nanonewtons to micronewtons during cell locomotion, contraction, and division.

Immune responses are strongly influenced by the cytokines following antigenic stimulation. Distinct cytokine-producing T cell subsets are well known to play a major role in immune responses and to be differentially regulated during immunological disorders, although the characterization and quantification of the TH-1/TH-2 cytokine pattern in T cells remained not clearly defined. Expression of cytokines by T lymphocytes is a highly balanced process, involving stimulatory and inhibitory intracellular signaling pathways. The aim of this study was (1) to quantify the cytokine expression in T cells at the single cell level using optical imaging, (2) and to analyze the influence of cyclic AMP- dependent signal transduction pathway in the balance between the TH-1 and TH-2 cytokine profile. We attempted to study several cytokines (IL-2, IFN-(gamma) , IL-4, IL-10 and IL-13) in peripheral blood mononuclear cells. Cells were prestimulated in vitro using phytohemagglutinin and phorbol ester for 36h, and then further cultured for 8h in the presence of monensin. Cells were permeabilized and then simple-, double- or triple-labeled with the corresponding specific fluorescent monoclonal antibodies. The cell phenotype was also determined by analyzing the expression of each of CD4, CD8, CD45RO and CD45RA with the cytokine expression. Conventional images of cells were recorded with a Peltier- cooled CCD camera (B/W C5985, Hamamatsu photonics) through an inverted microscope equipped with epi-fluorescence (Diaphot 300, Nikon). Images were digitalized using an acquisition video interface (Oculus TCX Coreco) in 762 by 570 pixels coded in 8 bits (256 gray levels), and analyzed thereafter in an IBM PC computer based on an intel pentium processor with an adequate software (Visilog 4, Noesis). The first image processing step is the extraction of cell areas using an edge detection and a binary thresholding method. In order to reduce the background noise of fluorescence, we performed an opening procedure of the original image using a structuring element. The opened image was therefore subtracted from the original one, and the gray intensities were subsequently measured. Fluorescence intensities are mapped in MD representation using Matlab software. Consequently, quantitative comparative expression of intracellular cytokines and cell membrane markers was achieved. Using this technique, we showed that CD4+ and CD8+T lymphocytes expressed a large panel of cytokines, and that protein kinase A (PKA) activation pathway induced a polarization of activated human T cells to the TH-2 type profile. Data also showed different sensitivities of CD45 RO/CD45RA lymphocytes to the activation of PKA, thus suggesting the implication of memory CD4+- and CD8+-T cells in the T cell specific immune and inflammatory processes and their control by PKA activation pathway. Finally, this method represents a powerful tool for the detection and quantification of intracellular cytokine expression and the analysis of the functional properties of T lymphocytes during immune responses.

We demonstrate the potential of dual-wavelength optical coherence-domain tomography (OCT) for noninvasive imaging of biological tissue in vivo. A prototype mono-mode fiber-optic coherence tomographic scanner is described, capable of providing rapid 2-D and 3-D images of biological tissues. The advantages of employing a dual-wavelength method for enhancing OCT image contrast, particularly in highly scattering tissue, are discussed. In the current OCT system, a long-wavelength superluminescence diode (SLD) emitting at 1.3-micrometer and a shorter-wavelength SLD emitting at 0.83-micrometer are used as light sources. Because of the optical configuration of dual-wavelength system, we can image the biological tissue at these two wavelengths simultaneously while avoiding the 'mode hopping' effects in the optical fibers. The experimental results demonstrate good correlation between tissue microstructures at these two wavelengths and show that image registration can effectively reduce speckle effects, suggesting that the dual-wavelength OCT has the potential for image contrast enhancement in highly scattering tissue.

Autofocus functions based on the measurement of the high spatial frequency content of the image have proven most precise and robust for microscopy. In some situations, however, the through-focus response of the highpass autofocus functions exhibit local maxima, or side peaks. These violations of unimodality can produce spurious autofocus results. Through-focus phase-contrast images of a stage micrometer and their spectra were studied in order to identify the conditions under which these side peaks are generated. The through-focus spectra suggest the side peaks occur when the bandpass of the filter includes the frequencies that undergo contrast reversals. These contrast reversals occur as a fundamental characteristic imposed by the optical system. Contrast reversals, however, are not seen in the frequency range near the optical cutoff. Thus, it was possible to design narrow bandpass filters that isolated only these frequencies and produced unimodal autofocus functions. Based on unimodality, improved sharpness, and consideration of the conditions present in scanning applications, sharply defined highpass filters should prove best for general microscopy autofocus.