We describe a new optical system for the detection of laser- induced fluorescence of surface bound molecules. The system consists of two optical elements: a paraboloid glass segment, and a spherical long focal lens. The function of the paraboloid glass segment is twofold: First, due to jump in refractive index at the water-glass interface, surface bound molecules emit the major part of their fluorescence into the paraboloid. Second, the parabolic shape of the segment services as a collecting mirror. We demonstrate that with this simple system an extremely high light collection efficiency can be achieved, without using sophisticated and expensive objectives with high numerical aperture. Ray- tracing calculations show that the system is able of confocal imaging, which is very important for efficient suppression of scattered excitation light. Experimental result showing the exquisite performance and sensitivity are presented.

Previously, we had shown that the zinc-dependent binding of certain fluorescent aryl sulfonamide inhibitors could be used with apo-carbonic anhydrase II to transduce the level of free zinc as a change in the fluorescence of the inhibitor. While inhibitors such as dansylamide, ABD-M, and ABD-N made possible quantitation of free zinc in the picomolar range with high selectivity, they have only modest absorbance which limits their utility. We describe here the synthesis and properties of two new probes, Dapoxyl sulfonamide and BTCS, and their use in zinc biosensing. Dapoxyl sulfonamide exhibits a dramatic increase and blue shift in its emission upon binding to holo-carbonic anhydrase II, as well as a twenty-fold increase in lifetime: it is thus well suited for quantitating free Zn(II) down to picomolar ranges. The anisotropy of BTCS increases five-fold binding to the holoprotein making this probe well suited for anisotropy-based determination of zinc.

Kromoscopic analysis is the real-time simultaneous detection of radiation in different but overlapping spectral regions and their correlations. It is analogous to human color vision, which uses a set of overlapping spectral response to distinguish between up to 20 million colors. Kromoscopic data in the spectral region of 800-1300 nm, which is optimum for pulsatile in vivo measurement, is presented for glucose, and urea. The superiority of Kromoscopic analysis in this spectral region is clearly seen in the raw in vitro data we present here.

A mid-IR gas sensor using difference frequency generation was developed to measure trace levels of biological carbon monoxide (CO). A periodically-poled lithium niobate crystal is pumped by a continuous wave Ti:Sapphire laser and a compact diode pumped Nd:YAG laser operating at 864.86 and 1064.6 nm, respectively. The strong IR transition R(6) at 2169.2 cm^-1 is chosen for convenient CO detection without interference from other gas species. Carbon monoxide is collected and flowed into a multipass cell with an effective optical path length of 18.3 m. Using such an experimental arrangement, we detected the generation of CO at levels of 30 ppb during a 30 min period from living vascular smooth muscle cells in basal state.

Stable CO₂ isotope breath test are established as a valuable tool in diagnostic and investigate medicine. The instrument conventionally used is an Isotope Ratio Mass Spectrometer, however, the expensive and complexity of such an instrument severely restricts the widespread and routine use of isotope tests. To realize their full potential an alternative instrument which is reliable, uncomplicated, insensitive to environmental and component fluctuations and affordable is required.

Nowadays when the computation ability of the computers is rapidly increased, the existence of image processing system may be an essential tool for the pathology researcher who will benefit from the image processing facilities that such system may supply. This system may be implemented in areas as immunocytochemistry, DNA ploidy analysis, chromosomal analysis and 3D tissue reconstruction. The problem with similar system existing nowadays in the market is their high price, inflexibility and low speed of operation. In this paper we suggest a very low cost processing system. The suggested system does not require most of the expensive elements used in the systems commonly sold in the market. In addition to the low cost, in this paper, many novel image processing algorithms, to be used for pathology image processing, are introduced. Some of these algorithms may be implemented using a low cost optical data processor which speeds the processing procedure to much higher rates and increases the flexibility of the system.

Our initial studies indicate that children who develop post- operative capillary leak syndrome (CLS) following cardiac surgery with cardiopulmonary bypass (CPB) can be distinguished based on their pre-operative level of circulating cytokines an adhesion molecules. We tested flow cytometric analysis of surface antigen expression as a potential assay for risk assessment of CLS. 24th preoperative blood samples were stained with monoclonal antibodies for the adhesion molecules ICAM-1, LFA1, MAC1, (beta) -integrin, activation markers CD25, CD54, CD69, HLA- DR, CD14 or CD4. Cells were measured on a dual-laser flow cytometer calibrated with microbeads. Antigen expression was detected as mean fluorescence intensity. The data indicate, that neutrophils of CLS patients express preoperatively higher levels of LFA1 and monocytes higher levels of HLA-DR and activation markers thus are in a state of activation. This could in combination with surgical trauma and CPB lead to their additional stimulation and migration into sites of inflammation and induce postoperative CLS. It is planned to set up a Flow-Classification program for individual risk assessment. By discriminate analysis over 80 percent of the patients were correctly classified. Our preliminary study indicates that flow cytometry with its low samples requirements and rapid access of the results could be a powerful tool to perform risk assessment prior to pediatric open heart surgery.

In this communication we are reporting the recent developments on a system/method of automated analysis of electrophoreses gels' by image digitalization and processing. The image digitalization system is formed by a non-intensified color CCD camera interfaced to a microcomputer through a frame grabber that allows image acquisition to be performed in real time. The illumination and image acquisition conditions will be discussed. A few different macro routines for image processing and data treatment were built. They cope with the different requirements, specifically for each of the most frequently used types of electrophoreses gels. After selection of the proper routine the automated evaluation process begins with the images' digitalization and processing in order to enhance image's contrast and performing the detection of relevant features. The image processing techniques employed include: histogram equalization and contrast enhancement, noise reducing by low pass Fourier filtering; edge enhancement and location by the application of differential operators, posterization or binarization; blob analysis, matching and feature extraction. the obtained images are also compressed and stored on the patient's files for future reference. The process take up to just a couple of minutes to be completed.

The common usage of flow cytometry (FCM) in research and clinical diagnostic is limited by the lack visualizing the fluorescence labelled cells. The Laser Scanning Cytometer (LSC) enables multicolor cytometric measurements on a slide featuring relocation of single cells for further investigation via brightfield and fluorescence microscopy. Additionally, it is possible to capture these images for documentation. In a FISH application, the LSC was successfully used for automated scoring techniqeus for evaluating the frequency of aneuploid sperm in humans and mice. In just 30 minutes, we were able to acquire more than 15,000 sperms, a task which normally takes more than a day. After relocation, genetic defects were identified and confirmed via fluorescence microscopy. In an on going study, we investigate via the LSC the remain of a new radiopaque material for high resolution echocardiography in the blood circulation. At first the result exhibited that the radiopaque material is endocysed by leukocytes just after application but is still detectable via echocardiography for up to 40 minutes. In conclusion, with the additional data acquisition by the LSC, it is possible to perform further detailed information from very small samples. Therefore, we are working up to now on developing new methods to introduce the LSC in our clinical diagnostic of neonates undergoing cardiac surgery.

One of the problems in treating breast cancer patients is discovering the gene rearrangements that are occurring while the patient is in apparent remission. Spontaneous mutations in DNA sequences, particularly in tumor suppressor genes, can lead to the evolution of new clones of tumor cells that may be able to evade both clinical treatments and the patient's immune surveillance system. Isolation of these tumor clones is extremely difficult. Rare-event analysis and single-cell sorting techniques must be used to successfully detect and isolate these tumor clones. PCR amplification of selected gene sequences followed by TA cloning techniques can then be used to perform single-cell DNA sequencing in those gene regions. In this paper we present preliminary data showing successful detection and single-cell sorting of rare tumor clones from defined cell mixtures. Using TA cloning techniques and PCR we have been able to detect a single base-pair mutation in the PTEN tumor suppressor gene in single cells from a breast cancer cell line. Thus, while extremely difficult, it should in the future be possible to isolate tumor clones form a patient for subsequent molecular analyses of DNA mutations in critical gene regions.

Multispectral image data has been a key data type for land observational remote sensing from aircraft and spacecraft since the 1960's. Sensor technology was a primary limiting factor for many years for this method, as sensors such as Landsat could only collect data in four to seven spectral bands at once. In the last few years, advances in sensor technology have mae possible the collection of such image data in as many as several hundred spectral bands at once. In this paper, some results obtained in the study of data analysis methods for such high dimensional data will be overviewed. They show that such data have substantially increased potential for deriving more detailed and more accurate information, but to achieve it, the primary limiting factor has become the precision with which a user can specify the analysis classes of interest. Some methods and procedures for mitigating this limitation in practical circumstances will be described.

Functional imaging of the retina and associated structures may provide information for early assessment of risks of developing retinopathy in diabetic patients. Here we show results of retinal oximetry performed using multi-spectral reflectance imaging techniques to assess hemoglobin (Hb) oxygen saturation (OS) in blood vessels of the inner retina and oxygen utilization at the optic nerve in diabetic patients without retinopathy and early disease during experimental hyperglycemia. Retinal images were obtained through a fundus camera and simultaneously recorded at up to four wavelengths using image-splitting modules coupled to a digital camera. Changes in OS in large retinal vessels, in average OS in disk tissue, and in the reduced state of cytochrome oxidase (CO) at the disk were determined from changes in reflectance associated with the oxidation/reduction states of Hb and CO. Step to high sugar lowered venous oxygen saturation to a degree dependent on disease duration. Moderate increase in sugar produced higher levels of reduced CO in both the disk and surrounding tissue without a detectable change in average tissue OS. Results suggest that regulation of retinal blood supply and oxygen consumption are altered by hyperglycemia and that such functional changes are present before clinical signs of retinopathy.

This research discusses the use of a new multispectral imaging system in fluorescence microscopy applications. We present the result of experiments designed to demonstrate the utility of this instrument for biological investigations. A phantom object was constructed using fluorescent microspheres and imaged to demonstrate the ability of the system to differentiate microscopic structures based on spectral properties. Excellent spectral isolation of subcellular structures, differentially labeled with fluorescent probes, was also obtained in images of neurons and rat sinusoidal epithelial cells demonstrating that separation of subcellular structures in biological imaging applications is possible with high spatial resolution and excellent signal to noise ratio. A final imaging experiment using this device with a confocal micro- endoscope shows the flexibility of the developed instrument when used with different imaging systems. In addition, the results indicate that multispectral investigations of tissue in-vivo may be possible.

The ISIS approach to spectral imaging seeks to bridge the gap between tuned multispectral and fixed hyperspectral imaging sensor. By allowing the definition of completely general spectral filter functions, truly optimal measurement can be made for a given task. These optimal measurements significantly improve signal to noise ratio and speed, minimize data volume and data rate, while preserving classification accuracy. This paper investigates the application of the ISIS sensing approach in two sample biomedical applications: prostate and colon cancer screening. It is shown that is these applications, two to three optimal measurements are sufficient to capture the majority of classification information for critical samples constituents. In the prostate cancer example, the optimal measurements allow 8 percent relative improvement in classification accuracy of critical cell constituents over a red, green, blue (RGB) sensor. In the colon cancer example, use of optimal measurements boost the classification accuracy of critical cell constituents by 28 percent relative to the RGB sensor. In both cases, optimal measurements match the performance achieved by the entire hyperspectral data set. The paper concludes that an ISIS style spectral imager can acquire these optimal spectral images directly, allowing improved classification accuracy imager can acquire these optimal spectral images directly, allowing improved classification accuracy over an RGB sensor. Compared to a hyperspectral sensor, the ISIS approach can achieve similar classification accuracy using a significantly lower number of spectral samples, thus minimizing overall sample classification time and cost.

Spectral unmixing algorithms tend to make the simplifying assumptions that each type of material in a spectral library may be represented by a single reference spectrum and that the mixing process is linear. While these assumptions are convenient in that they allow techniques of linear algebra to be used, they lack realism as each material type in a spectral image will in general emit a distribution of spectra while the mixing itself need not be linear. We describe a 'common sense' spectral unmixing algorithm for the general case where endmembers are described by arbitrary D-dimensional probability distribution and the mixing can be non-linear. As an application we outline an unsupervised procedure for deriving the fractional material content of every pixel in an image and identifying anomalies given no a priori knowledge. Accurate endmember distribution are obtained by first masking out impure pixels using locally normalized Sobel and Laplacian filters and then performing single-link hierarchical clustering on the pure pixels which remain. The most probable endmember decomposition for a given target spectrum is found by selecting an appropriate set of endmembers based on the target's immediate neighborhood, and performing a constrained maximum likelihood search over the space of fractional abundances. We also explain how the procedure may be applied to subpixel and anomaly detection. To illustrate our ideas the techniques described are applied to biomedical images throughout.

The development of a biosensor with adequate sensitivity generally requires a biospecific interaction with high binding affinity. The affinity constants for most antibody- antigen interactions are determined largely by the dissociation constants, k_d, with little variation observed in rates of associated. additionally, surface immobilization typically results in a reduced k_d. In this case, the sensor binds analyte kinetically irreversibly preventing response to changes in analytic concentration or reuse. Regeneration of the sensor surface is difficult, at best. On the other hand, a higher dissociation rate which would lend itself to a linear and reusable sensor, results in lower affinity and poor sensitivity. Consequently, most biosensors are disposable devices and quantitation is obtained using multiple single-use sensors. In this work, a new reusable biosensor platform which provides simultaneous fluorescence detection and electrochemical control of biospecific binding has been developed. Biotin was covalently attached to a transparent indium tin oxide electrode, which also served as an integral part of a total internal reflection fluorescence (TIRF) flow cell. TIRF was used to monitor biospecific interactions while electrochemical polarization was employed to control the interactions. Two possible mechanisms of the electric field effect involving interactions with induced and permanent dipoles of proteins will be discussed.

A planar optical waveguide was used to simultaneously excite fluorescence due to antigen binding in three separate areas of immobilized antibody. Biotin labeled, polyclonal antibodies to goat, human, and rabbit IgG were immobilized through surface bound, photo-activated MeNPOC-biotin-bSA and avidin. Exposing the MeNPOC to UV light effectively uncaged the biotin molecule attached to the bSA and allowed avidin, followed by the biotin labeled antibody, to bind to the waveguide surface. Whereas a time intensive, non-specific binding prone step-and-repeat method is normally used to form the individual capture layers, we chose to pursue a combined deposition method involving sample wells and photo- activated crosslinkers. The result was a covalently linked multi-component capture layer formed in a short period of the time. Specific and cross-reactive activities of this antibody array were gauged by sequentially injecting analyte specific to one antibody area at a time. Results suggested that the binding of each analyte occurred predominately in the correct area and, depending on the particular antibody, generated varying levels of cross reactivity. A comparison of result with previously acquired, physically adsorbed capture layer data did not infer one deposition technique was better than the other.

A total internal reflection fluorescence (TIRF) flow system was used to detect DNA adsorption and hybridization of a single stranded target DNA with a surface immobilized DNA probe. A transparent SnO₂ or indium tin oxide (ITO) film served as a spectroscopic surface in the TIRF flow system and simultaneously as a working electrode for electrochemical (EC) control. The SnO₂ and ITO electrodes were chemically modified to provide hydrophilic or hydrophobic surfaces with different functional groups. Charge of the sensor surface was modulated by external electrochemical polarization. Results indicate that DNA oligonucleotides exhibit higher adsorption affinity to positively charged aminated sensor surfaces, while negative polarization stimulates desorption of DNA molecules. To investigate DNA hybridization at the TIRF EC sensor surface, a thiolated probe oligonucleotide, with the complementary sequence to that of the target DNA, was covalently attached to the aminated sensor surface using a heterobifunctional crosslinker. A solution phase 27-base target DNA labeled by fluorescein was exposed to the sensor surface. The kinetics of heterogeneous hybridization between target DNA and surface immobilized probe was studied at different electrode potentials. The results show that cathodic polarization can accelerate hybridization and, at the same time, suppress nonspecific DNA adsorption.

Millennium Predictive Medicine (MPMx), a subsidiary of Millennium Pharmaceuticals, is focusing on the discovery and clinical validation of Diagnomic and Pharmacogenomic Tests which will replace many of the subjective elements of clinical decision making. Diagnomics are molecular diagnostic markers with prognostic and economic impact. While the majority of currently available diagnostics represent data points, Diagnomics allow patients and physicians to make scientifically based, individualized decisions about their disease and its therapy. Pharmacogenomics are diagnostics that specify the use or avoidance of specific therapeutics based on an individual genotype and/or disease subtype. MPMx uses the broad Millennium genomics, proteomics, and bioinformatics technologies in the analysis of human disease and drug response. These technologies permit global and unbiased approaches towards the elucidation of disease pathways and mechanisms at the molecular level. Germline or somatic mutations, RNA levels, or protein levels comprising these pathways and mechanisms are currently being evaluated as markers for disease predisposition, stage, aggressiveness, and likely drug response or drug toxicity. Diagnomic and Pharmacogenomic Tests are part of the new molecular medicine that is transforming clinical practice forma symptom/pathology-based art into a pre-symptom, mechanism- based science.

Microfluidic control in microfabricated glass channels enables miniaturized, fast, and multianalyte assays. We are applying this technology in several areas, including real- time environmental monitoring for airborne biological agents. Two complementary approaches are being used in parallel. the first is assaying for the presence of nucleotide sequences that are markers for specific hazardous, engineered bacteria. The second is an assay that monitors the functionality of an in vitro biochemical pathway, in which the pathway that is chosen is sensitive to the presence of the class of toxins to be detected. The first approach is discussed here. The detected signal from the nucleic-acid-based assay is from fluorescently labeled probes that hybridize to bead-bound amplified DNA sequences. Detection approaches and their benefits will be discussed.

In virtually everyapplicationofbio-medical technology there is at least on step in the process where detection oflight is employed for measurement or quantification. Some cases involve light which is emitted at the molecular level (e.g. chemiluminescence), while others require an external light source and measure changes in the illumination (e.g. fluorescence, absorption, etc.). There are a wide variety of light sources (LEDs, lasers, filament or arc lamps, etc.) available for illumination, as well as a number of photon detectors (photo-multiplier tubes, photodiodes, vidicons, charge coupled devices, etc.). Many bio-medical assays are evolving toward ever greater numbers of active sites per test platform (often hundreds or even thousands), as well as miniaturization ofboth the individual features and the over-all array size. We will present here arguments for using charge coupled devices (CCDs), perhaps cooled or intensified, as "area detectors' and bulbs (filament or arc lamps) as "area illuminators" for such applications. Because of time restrictions, we will limit much of the presentation to simply itemizing features, advantages, and disadvantages. Anyone who has a potential application is encouraged to contact the author for more details and supporting documentation.

T-Sensors enable the optical detection of clinically significant analytes directly in whole blood. In microfluidic channels, fluids usually show laminar behavior. This allows the movement of different fluidic layers in a channel without mixing other than by diffusion. A sample solution, a detection solution, and a reference solution are introduce din a common channel. Smaller particles such as ions or small proteins diffuse rapidly across the fluid boundaries, whereas larger molecules diffuse more slowly. Large particles show no significant diffusion within the time the flow streams are in contact. Two interface zones are formed between the fluidic layers. The ratio of a property of the two interface zones is a function of the concentration of the analyte. In this paper, we introduce a novel optical geometry to determine absorbance or fluorescence at a number of distinct wavelength ranges, and to perform spectroscopic measurements with 1D or 2D spatial. Resolution in a flow channel. A flow channel is coupled to an optical filter with variable transmission in one or two dimensions, a light source, and a CCD detector. Such a device allows, for example, the absorption- or fluorescence- based detection of a variety of analytes in a T-sensor using a non-color-sensitive spatial detector, while retaining diffusion and reference information provided by the T-sensor principle.

We describe fluorescence spectral-imaging results with the computed-tomography imaging spectrometer (CTIS). This imaging spectrometer is capable of recording spatial and spectral data simultaneously. Consequently, the CTIS can be used to image dynamic phenomena involving multiple, spectrally overlapping fluorescence probes. This system is also optimal for simultaneously monitoring changes in spectral characteristics of multiple probes from different locations within the same sample. This advantage will provide additional information about the physiological changes in function form populations of cells which respond in a heterogeneous manner. The results presented in this paper consist of proof-of-concept imaging results from the CTIS in combination with two different systems of fore- optics. In the first configuration, raw image data were collected using the CTIS coupled to an inverted fluorescence microscope. The second configuration combined the CTIS with a confocal microscope equipped with a fiber-optic imaging bundle, previously for in vivo imaging. Image data were collected at frame rates of 15 frame per second and emission spectra were sample at 10-nm intervals with a minimum of 29 spectral bands. The smallest spatial sampling interval presented in this paper is 0.7 micrometers .

The new medical developments, e.g. immune therapy, patient oriented chemotherapy or even gene therapy, create a questionable doubt to the further requirement of animal test. Instead the call for humanitarian reproductive in vitro models becomes increasingly louder. Pharmaceutical usage of in vitro has a long proven history. In cancer research and therapy, the effect of chemostatica in vitro in the so-called oncobiogram is being tested; but the assays do not always correlate with in vivo-like drug resistance and sensitivity. We developed a drug test system in vitro, feasible for therapeutic drug monitoring by the combination of tissue cultivation in hollow fiber bioreactors and fiber optic sensors for monitoring the pharmaceutical effect. Using two fiber optic sensors - an optical oxygen sensor and a metabolism detecting Laserfluoroscope, we were able to successfully monitor the biological status of tissue culture and the drug or toxic effects of in vitro pharmaceutical testing. Furthermore, we developed and patented a system for monitoring the effect of minor toxic compounds which can induce Sick Building Syndrome.

Surface-Enhanced Raman Scattering (SERS) spectroscopy and Flow Linear Dichroism (FLD) technique have been employed to study the anticancer agent fagaronine and its derivative ethoxidine - double inhibitors of DNA topoisomerases I and II. Cooperative use of two methods permitted (i) to determine the molecular determinants of the drug-DNA interactions; (ii) to monitor in real time the process of topo I inhibition by these anticancer agents. FLD technique allowed us to identify the mode of drug interactions with the DNA as a 'major groove intercalation' and to determine orientation of the drugs chromophores within the complexes. Using SERS spectroscopy we have determined the drugs molecular determinants interacting with the DNA. FLD was also used for real time monitoring of the process of sc DNA relaxation by topo I and of inhibition of relaxation with the pharmaceuticals. Ethoxidine was found to exhibit the same activity of inhibition of sc DNA relaxation as fagaronine at the 10-fold less concentration. The proposed SERS-FLD combined approach demonstrates the new perspectives for screening new pharmaceuticals due to its relative simplicity and low expense, high sensitivity and selectivity, and, finally, possibility of real-time monitoring of the structure-function correlation within the series of drug derivatives.

A fluorescence polarization assay was developed as an alternative to the radiolabeled SPA assays currently used to monitor the activity of tyrosine kinases in drug discovery. The assay can be used with enzymes having substrate specificity similar to that of the insulin receptor, the EGF receptor and the Src kinase receptor enzymes. The assay is easy to configure in 96, 384 and 1536-well microplates in assay volumes ranging from (mu) L with minimal efforts. The reconstituted reagents are stable for up to 24 hr at ambient temperatures, thereby minimizing the need for replenishing the stock solutions during the course of a high-throughput screen. Because of the stability and equilibrium kinetics, the assay allows the user the luxury of scheduling the reading of plates any time up to 24 hr after the completion of the assay without substantial deterioration in the assay signal. The antibody and the tracer solutions can also be premixed and added as a preformed complex in a single step. The performance of the assay with the insulin receptor kinase is described. In addition, given the diversity of the substrates used in measuring the activity of different tyrosine kinases, LJL's on-going efforts to provide different antibodies of wide ranging specificity and sensitivity are described.

We report the implementation of intensity modulated diode lasers in frequency-domain pump-probe studies, diode lasers are compact, stable, and economical units that require little maintenance. In our study, a 365 nm diode laser is used as the excitation source and the output of a 680 nm unit induces stimulated emission from excited state fluorophores. By modulating the intensities of the two diode lasers at slightly different frequencies, and detecting the fluorescence signal at the cross-correlation frequency, both time-resolved and high spatial resolution imaging can be achieved. The laser diodes are modulated in the 100 MHz cross-correlation signal has been used for time-resolved imaging of fluorescent microspheres and mouse fibroblasts labeled with nucleic acid stains TOTO-3. These results demonstrate and feasibility of using intensity modulated diode lasers for frequency-domain, pump-probe studies.

Fluorescence instrumentation for high-throughput screening (HTS) must be sensitive, accurate and reliable. An instrument must be capable of robust measurement as well. HTS assays are often complicated by interfering signals from background fluorescence, scattering, absorption and quenching. Traditional fluorescence intensity methods do not remove the effect of interferences well. Fluorescence lifetime methods, however, have the ability to retrieve the true assay signal from a signal plagued with interferences. We have developed fluorescence-lifetime methods specifically for high-throughput screening with a high-frequency time- resolved fluorometer created from the optics of a high- throughput screening instrument, a high-speed LED or laser diode light source and the phase and modulation method of fluorescence lifetime measurement. The prototype instrument is capable of measuring the lifetime of samples from 1-1000 ns, able to measure a single frequency in less than one second and able to distinguish nanomolar concentrations of fluorophores in small volumes. Furthermore, the often complex and troublesome fluorescence lifetime measurement is made simple and reliable with this prototype. To demonstrate robustness and reliability, we used lifetime-based methods to measure a model system with background fluorescence form biological membranes. Specifically, methods based upon long- lifetime fluorophores can significantly improve immunity to assay interferences.

Cell membrane remnants represent a probable nucleation site for calcium deposition in bioprosthetic heart valves. Calcification is a primary failure mode of both bovine pericardial and porcine aortic heterograft bioprosthesis but the nonuniform pattern of calcium distribution within the tissue remains unexplained. Searching for a likely cellular source, we considered the possibility of a previously overlooked small blood vessel network. Using a videomicroscopy technique, we examined 5 matched pairs of porcine aortic and pulmonary valves and 14 samples from 6 bovine pericardia. Tissue was placed on a Leitz Metallux microscope and transilluminated with a 75 watt mercury lamp. Video images were obtained using a silicon intensified target camera equipped with a 431 nm interference filter to maximize contrast of red cells trapped in a capillary microvasculature. Video images were recorded for analysis on a Silicon Graphics Image Analysis work station equipped with a video frame grabber. For porcine valves, the technique demonstrated a vascular bed in the central spongiosa at cusp bases with vessel sizes from 6-80 micrometers . Bovine pericardium differed with a more uniform distribution of 7-100 micrometers vessels residing centrally. Thus, small blood vessel endothelial cells provide a potential explanation patterns of bioprosthetic calcification.

In studying the molecular mechanics of stimulation of a receptor and mechanisms of the receptor's interaction with ligands, the widely sued approach is to characterize dose- dependent functional responses of stimulation or inhibition of the receptor. Many GPCRs respond to the stimulation by transient changes in cytoplasmic calcium. A time trace of the ligand-evoked 'calcium signal' visualizes the sequence of signaling events taking place after the initial receptor stimulation. It is more important to know how those events depend on the ligand concentration. This type of data provides ligand affinity profiles together with information about mechanisms of the receptor/ligand interaction - competitive, non-competitive antagonism, full or partial stimulation. We have developed an automated system, HT-PS 100, for registering continuous concentration-dependent functional responses in real time at the rate of 2 min per dose-response curve. The flow-through fluidics prepares a concentration gradient of the compound and sequentially mixes it with another reagent, agonist or antagonist, and finally with cells. The resulting 'real time' concentration dependent signal is registered with a fluorescence detector. By monitoring calcium mobilization with Fura-2, we have functionally and mechanistically characterized a variety of G protein-coupled receptors, cholinergic, histaminergic, purinergic, endothelin, and bradykinin, endogenously expressed in different cell lines, SK-N-MC, TE671 and DDT₁MF-2.

High-throughput screening (HTS) efforts at Abbott Laboratories have been greatly facilitated by the use of a Fluorometric Imaging Plate Reader. The FLIPR consists of an incubated cabinet with integrated 96-channel pipettor and fluorometer. An argon laser is used to excite fluorophores in a 96-well microtiter plate and the emitted fluorometer. An argon laser is used to excite fluorophores in a 96-well microtiter plate and the emitted fluorescence is imaged by a cooled CCD camera. The image data is downloaded from the camera and processed to average the signal form each well of the microtiter pate for each time point. The data is presented in real time on the computer screen, facilitating interpretation and trouble-shooting. In addition to fluorescence, the camera can also detect luminescence form firefly luciferase.

Several different types of proximity assays have been developed using various isotopes in miniaturized format using 384-well microplates and have been quantified using the Leadseekers homogeneous imaging system. Further miniaturization to 1536-well plates is feasible, with the ultimate goal being an attomated high throughput instrument capable of screening up to 100,000 compounds per day.

A surface plasmon resonance fiber optic system is presented for the simultaneous analysis of ninety-six micro-well plates for purposes of high throughput biochemical screening analysis. The sensing element is compared of ninety-six discrete fiber optic sensor housed in a containment plate. A white light source is used to introduce light to the sensor via a multiplexed fiber optic bundle. The transmitted spectral intensity distribution of each sensor is detected via a multiplexed fiber optic bundle. The transmitted spectral intensity distribution of each sensor is detected simultaneously using a lens-based holographic imaging spectrograph and a 2D CCD detector. Experimental results confirm the feasibility of the application of this label free and real-time transduction mechanism of surface plasmon resonance towards high throughput biochemical analysis.