Proceedings Volume 3259

Systems and Technologies for Clinical Diagnostics and Drug Discovery

Gerald E. Cohn, John C. Owicki
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Proceedings Volume 3259

Systems and Technologies for Clinical Diagnostics and Drug Discovery

Gerald E. Cohn, John C. Owicki
View the digital version of this volume at SPIE Digital Libarary.

Volume Details

Date Published: 10 April 1998
Contents: 6 Sessions, 27 Papers, 0 Presentations
Conference: BiOS '98 International Biomedical Optics Symposium 1998
Volume Number: 3259

Table of Contents

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Table of Contents

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  • Multispectral Imaging in Clinical Diagnostics
  • Fluorescence-based Diagnostics
  • Ultrasensitive Clinical Diagnostic Systems
  • Cell and Tissue Diagnostics
  • Fluorescence-based Diagnostics
  • Fluorometric Analysis of DNA and Proteins
  • Optical Methods in High-Throughput Screening for Drug Discovery
  • Cell and Tissue Diagnostics
Multispectral Imaging in Clinical Diagnostics
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Automated spectral imaging for clinical diagnostics
John Breneman, David M. Heffelfinger, Ken Pettipiece, et al.
Bio-Rad Laboratories supplies imaging equipment for many applications in the life sciences. As part of our effort to offer more flexibility to the investigator, we are developing a microscope-based imaging spectrometer for the automated detection and analysis of either conventionally or fluorescently labeled samples. Immediate applications will include the use of fluorescence in situ hybridization (FISH) technology. The field of cytogenetics has benefited greatly from the increased sensitivity of FISH producing simplified analysis of complex chromosomal rearrangements. FISH methods for identification lends itself to automation more easily than the current cytogenetics industry standard of G- banding, however, the methods are complementary. Several technologies have been demonstrated successfully for analyzing the signals from labeled samples, including filter exchanging and interferometry. The detection system lends itself to other fluorescent applications including the display of labeled tissue sections, DNA chips, capillary electrophoresis or any other system using color as an event marker. Enhanced displays of conventionally stained specimens will also be possible.
High-speed spectral imager for imaging transient fluorescence phenomena
We describe fluorescence spectral-imaging results with the microscope computed-tomography imaging spectrometer ((mu) CTIS). This imaging spectrometer is capable of recording spatial and spectral data simultaneously. Consequently, (mu) CTIS can be used to image dynamic phenomena. The results presented in this paper consist of imaging results using static targets consisting of 1 micrometers and 6 micrometers fluorescing microspheres. The emission spectra were sampled at a 10-nm interval between 430 nm and 710 nm. The smallest spatial sampling interval presented in this paper is 1.7 micrometers . Image data were collected in integration times of 16 msec.
Real-time in-vivo endoscopic imaging of fluorescence from human colonic adenomas
Thomas D. Wang, Jacques Van Dam, James M. Crawford, et al.
Previous in vitro studies showed that autofluorescence images of colonic mucosa collected endoscopically can be used to detect dysplasia with high sensitivity. This method is extended to collection of fluorescence images of adenomatous polyps in vivo. Fluorescence images were collected during colonoscopy in 30 patients. A total of 12 adenomatous and 6 hyperplastic polyps were identified. An optical fiber excitation probe, located in the instrument channel of the colonoscope, delivered 300 mW of near- ultraviolet light at (lambda) ex equals 351 and 364 nm. Mucosal fluorescence in the spectral bandwidth between 400 and 700 nm was imaged, processed, and displayed with various likelihoods of associated dysplasia. Adenomatous polyps exhibited decreased fluorescence intensity compared to adjacent mucosa with normal appearance. With the fluorescence threshold set to 80% of the average intensity of normal mucosa, a sensitivity of 83% for dysplasia detection was achieved. All hyperplastic polyps were correctly identified as being non-dysplastic. Optimal identification of dysplastic regions was obtained with the colonoscope oriented at near-normal incidence to the polyps. At higher angles of incidence, artifacts due to illumination shadows were introduced. The dysplasia associated with adenomatous polyps can be detected in vivo on fluorescence imaging with high sensitivity, thus demonstrating the potential to guide endoscopic biopsy.
Fluorescence-based Diagnostics
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Fluorescent capillary fill device: a quantitative whole-blood assay system suited to point-of-care use
Phelim B. Daniels, J. Philip Vessey, Janys E. Fletcher, et al.
The Fluorescence Capillary Fill Device (FCFD) has previously been shown to be a candidate test vehicle for the point of care market and instrumentation suitable for commercialization has now been developed. The FCFD instrumentation is a fluorimeter with custom optics to collect a given angular spread of light from the output edge of the device. Rigorous selection of fluorophore, light source, filters and detector (taking into account cost, availability in manufacturing quantity and the influence of inherent blood fluorescence) was undertaken when optimizing the system. The instrument utilizes allophycocyanin as a near-red fluorophore, an array of ultra-bright light emitting diodes and an inexpensive photomultiplier. Assay calibration is achieved by incorporating a light source movement mechanism that allows reference zones within the device to be interrogated individually. The resulting instrument has a footprint approximately 10 inches by 8 inches, requires no specialist operator training and is well suited to the point of care market. The capability of the system is illustrated by a whole blood assay for the cancer marker prostate specific antigen. This assay has a sensitivity of <0.5 ng/mL and precision of < 10% between 5 ng/mL and 50 ng/mL.
High-sensitivity determination of Zn(II) and Cu(II) in vitro by fluorescence polarization
Richard B. Thompson, Badri P. Maliwal, Vincent Feliccia, et al.
Recent work has suggested that free Cu(II) may play a role in syndromes such as Crohn's and Wilson's diseases, as well as being a pollutant toxic at low levels to shellfish and sheep. Similarly, Zn(II) has been implicated in some neural damage in the brain resulting from epilepsy and ischemia. Several high sensitivity methods exist for determining these ions in solution, including GFAAS, ICP-MS, ICP-ES, and electrochemical techniques. However, these techniques are generally slow and costly, require pretreatment of the sample, require complex instruments and skilled personnel, and are incapable of imaging at the cellular and subcellular level. To address these shortcomings we developed fluorescence polarization (anisotropy) biosensing methods for these ions which are very sensitivity, highly selective, require simple instrumentation and little pretreatment, and are inexpensive. Thus free Cu(II) or Zn(II) can be determined at picomolar levels by changes in fluorescence polarization, lifetime, or wavelength ratio using these methods; these techniques may be adapted to microscopy.
Spectral-selective fluorescence imaging for diagnostics of surface cancer
Alexander V. Vinogradov, Alexey I. Trushin, Pavel G. Leonov, et al.
We report on porphyrin autofluorescence imaging diagnostic of surface cancer. Digital computer fluorescent methods get real time, non-invasive, in vivo cancer diagnostic. Output information has clear picture form. Results may be placed in imaging data bases and sent via computer networks. We received our laboratory results without exogenous porphyrins or pharmaceutical stimulation of endogenous porphyrin production. The results were confirmed by histological tests. Autofluorescence was exited via He-Ne laser. Extra sensitive digital TV system was used for image capturing. Image was grabbed in the narrow spectral fluorescence band about 680 nm. The fluorescent image delivered quantity information on the porphyrin spatial distribution. Bright spots on the image were cancer suspected places. We combined usual color image with the fluorescent image and marked suspected places. Then these places were tested by standard diagnostic methods. Combination of image fluorescent and standard methods allowed us to find out invisible or very small cancer lesions. In our report we discuss the relation between traditional laser induced fluorescent spectroscopy diagnostic and fluorescent imaging. In our opinion the method is especially efficient at early disease stages, when lesion is very compact and clinical picture is not clear.
Noninvasive spatially resolved kinetic study of hydrolyzable camptothecin antitumor drugs in vitro and in vivo by confocal scanning microspectrofluorometry
Igor Chourpa, Serguei Charonov, Alexandre Kokota, et al.
Hydrolysis of the lactone ring of camptothecins (CPTs) leads to a loss of their antitumor activity. The non-hydrolyzable derivatives are also inactive. Thus, the state of the lactone ring during the drug interaction with biological partners is of a great interest. High performance liquid chromatography currently employed to study the lactone hydrolysis in free CPTs can not be applied to the drug- target complexes and in vivo measurements. We followed kinetics of the lactone hydrolysis in CPTs using hydrolysis- induced time-dependent evolution of their fluorescence spectra. Spectra were obtained from micro-volumes of the samples under the microscope of a computer-controlled confocal microspectrofluorometer (M51, DILOR, France). Spectral recording and treatment (filtering, decomposition into model spectra of the intact and hydrolyzed forms, etc.) were performed using a software package developed in our laboratory. Data obtained for a series of CPTs at very low concentrations, ca. 10-7 M, demonstrated a good reproducibility, even at basic pH, where the hydrolysis is fast. Then the kinetics studies were extended to CPTs in complexes with their potential biological targets, DNA and topoisomerase I, in vitro. The in vivo studies of the lactone status at the level of single living cancer cells treated with CPTs are actually in progress.
Comparison between time-correlated single-photon counting and fluorescence correlation spectroscopy in single-molecule identification
Joerg Enderlein, Malte Koellner
Currently, two methods of detection and identification of single molecules are widely used: fluorescence correlation spectroscopy (FCS) and time-correlated single photon counting (TCSPC). We present a thorough theoretical analysis of the error rates for identifying single molecules according to their diffusion coefficients (using FCS), and to their fluorescence lifetimes (using TCSPC). In most cases, the error rate using TCSPC is much lower. TCSPC is thus proven to be more versatile for analyzing single molecule events. The study is significant for a broad range of ultrasensitive fluorescence detection applications.
Ultrasensitive Clinical Diagnostic Systems
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LabCD: a centrifuge-based microfluidic platform for diagnostics
Marc J. Madou, Gregory J. Kellogg
Diagnostics for point-of-care (POC) and field use requires the integration of fluid processes with means of detection in a user-friendly, portable package. A drawback to the use of many current analyzers for POC and field applications is their reliance on expensive and fragile robotic technology for automation, lack of portability, and incomplete integration of sample processing into the device. As a result, a number of microfluidic technologies are being developed for diagnostics applications outside of central laboratories. We compare several of these technologies with our own preferred centrifugal flow system, the LabCDTM, with an emphasis on fluid propulsion. LabCDTM has been developed to perform a variety of fluidic processes necessary in diagnostics while dispensing with traditional pumps and valves. The use of the CD-ROM model provides a natural division of the system into an instrument and a disposable component, each with well-defined functions. The CD format also allows for the use of encoded information to integrate process control, data acquisition, and analysis. Finally, the `solid state' nature of the microfluidics and use of standard manufacturing techniques should yield a low- cost platform.
Quantitation of HIV-1 by real-time amplification and detection
Paul M. Jung, Naiquan Yang, Paul E. Kroeger
A model assay for HIV-1 using a non-competitive internal standard in quantitative RT-PCR was coupled with real-time detection of both analyte and internal standard (IS) signals in a closed system. Real-time detection by the PE-ABI Prism 7700 relied on TaqMan probes specific for HIV and IS. The exogenous, non-competitive IS RNA was added in the same, known amount to a series of HIV RNA standards. The threshold cycle ratio from this internal standard calibration curve was used in the quantitation of HIV. Two configurations of reporter labels were compared. The HEX-HIV:FAM-IS system was the most precise, with nearly half-log discrimination over a range of 102 through 105 copies HIV-1 RNA. The FAM- HIV:HEX-IS system was less precise, but more sensitive and resistant to sample inhibition. The analysis of these signals and their impact on the range and precision of HIV quantitation is discussed. The design and synthesis of the fluorescently-labelled probes is also described.
Single-molecule DNA detection in microfabricated capillary electrophoresis chips
Brian B. Haab, Richard A. Mathies
Single-molecule fluorescence burst counting is a highly sensitive method for detecting electrophoretic separations of ds-DNA fragments. In previous work we demonstrated the detection of single DNA fragments as small as 100 bp in capillary electrophoresis separations. To further enhance the applicability of this method to low level pathogen and mutation detection, we have now successfully performed single molecule detection of DNA separations in microfabricated glass capillary electrophoresis (CE) chips. By fabricating CE chips with a 200 micrometers thick top cover plate and by using a 40X, 1.3 NA immersion microscope objective, the S/N ratio for single molecule detection is enhanced by more than two-fold over conventional capillaries. By constricting the sample in the detection region to an approximately 10 micrometers wide by approximately 10 micrometers deep cross section, approximately 10% of the molecules passing through the channel are probed by the approximately 2 micrometers wide focused laser beam. This is a 1000-fold improvement over our previous work. We have now achieved an on-column detection limit of approximately 600 fM or 500 molecules for 500 bp DNA fragments (S/N equals 3).
Cell and Tissue Diagnostics
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New high-speed cell sorting methods for stem cell sorting and breast cancer cell purging
James F. Leary, Scott R. McLaughlin, James A. Hokanson, et al.
An important problem in clinical medicine is that of positively selecting hematopoietic stem cells or mobilized peripheral blood stem cells for autologous bone marrow transplantation while purging it of contaminating tumor cells. Since both the stem cells to be positively selected and the tumor cells to be purged are relatively rare cells, this poses special problems for their isolation in terms of purity and yield of stem cells, with a high penalty of misclassification for contaminating tumor cells. A model system of tumor cells spiked into bone marrow or blood cells was used to validate the system. Multiparameter data mixtures of human MCF-7 breast cancer cells and human peripheral blood or bone marrow cells were first analyzed by discriminant function analysis. Mathematical methods were developed to assess the relative probabilities of misclassification. 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. Then the classification system was implemented using lookup tables allowing for real-time (in this system approximately 625 microseconds) rapid separation of these cell types. Isolated cell types, purities and yields were assessed by single-cell PCR molecular characterizations.
Optical measurement of tissue pH for surgical and critical care monitoring
Babs R. Soller, Songbiao Zhang
Tissue pH is an important physiological parameter which indicates both blood flow and cell metabolic state. Continuous monitoring of tissue pH can provide an assessment of the level of anaerobic metabolism and a measure of whether organs or muscles are revivable or have died. A noninvasive, optical technique for deep tissue pH determination has been demonstrated in-vivo using near infrared (NIR) spectroscopy and partial least-squares (PLS) multivariate calibration. NIR reflectance spectra (700 - 1100 nm) were collected from skin covered muscle in a rabbit, canine myocardium, and swine bowel along with reference pH values measured in the same tissue using microelectrodes. Muscle and myocardial pH were varied by controlling the blood supply through vessel occlusion; bowel pH was altered through hemorrhagic shock. PLS cross- validation techniques and data preprocessing methods were used to relate the tissue pH to spectra. The standard error of prediction for each of the multivariate calibrations was less than 13% of the average pH change in each of the animal models. Optically measured tissue pH promises to provide a noninvasive monitor for ischemia during heart and plastic surgery and an early indicator of shock in the ICU patient.
Development of a new near-IR laser system for clinical monitoring of cerebral oxygenation in the newborn infant
The provision of sufficient oxygen to the brain is a major goal for neonatologists to prevent major neuro-developmental handicaps and to improve the chances for intact survival of sick newborn infants. In this paper the authors present the theory and design of a new noninvasive device for transcutaneous monitoring of cerebral blood and tissue oxygenation using a differential absorption laser system. The new technique depends upon illuminating the head of the neonate with radiation from six near infrared laser diodes. The choice of the used laser wavelengths (775, 780, 810, 825, 830, 850 nanometers) follows the principles of near infrared spectroscopy for obtaining accurate measurements. Different constructions of fiber optic probes arranged around the head of the neonate along the biparietal diameter guide the transmitted and received laser signals for signal processing. The detailed description and operating characteristics of this system are presented.
Application of a laser radar photogoniometer in the diagnosis of osteoporosis
Hans-Georg Eberle, Juergen Beuthan, Marc Dierolf, et al.
First results on angle dependent light scattering performed on normal and osteoporotic human bone tissue in vitro are presented. The scattering distributions are measured in cw mode as well as in time-resolved (time-gated) mode. Significant differences in the angular distribution of 675 nm laser radiation transmitted through normal or osteoporotic bones are found in cw-scattering measurements. For aspiring in situ results the transition in time-resolved measurements is needed to suppress the disturbing soft tissue signals. In this way, a characterization of the bone part is aimed by selectively recording the scattered signal in a suitable chosen time window. The measurement's principle is based on time-correlated single photon counting using a 10 ps laser diode (830 nm) and a micro channel plate- photomultiplier tube. First in vitro results on human lumbar vertebrae (L1) obtained with this equipment show a correlation between the thermal peak position and the FWHM of the scattered light pulses and the bone mineral densities determined by Dual X-ray absorptiometry. The angle dependent scattering curves give distinct indications on the bone status in transmission mode.
Fluorescence-based Diagnostics
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Rapid clinical diagnostics assays using injection-molded planar waveguides
James N. Herron, Hsu-Kun Wang, Allen H. Terry, et al.
The goal of our research program is to develop an evanescent wave immunoassay system that can be used in point-of-care and critical care settings. Several key attributes are required to accomplish this goal: (1) the assay system should be at least as sensitive as present day immunoassays; (2) assay time should be 5 minutes or less; (3) the assay protocol should be relatively simple; (4) the sensor should be capable of performing more than one assay on a single specimen; (5) the assay system should be able to accommodate specimens such as serum, plasma and whole blood; and (6) the sensor should be an inexpensive, disposable cartridge. Our laboratory has developed an injection-molded planar waveguide sensor that meets most, if not all, of these attributes. This sensor has been evaluated in a number of different immunoassays for analytes such as bovine serum albumin, human chorionic gonadotrophin, creatine phosphokinase MB and cardiac troponin I.
Fluorometric Analysis of DNA and Proteins
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Multicolor instrumentation for direct fluorescent detection of nucleic acids in a microchip format
Valery L. Bogdanov, Yu-Hui Rogers, Guang Lan, et al.
Deposition of nucleic acids on solid support in the form of high density arrays (a DNA microarray) creates a powerful nonelectrophoretic technology for highly parallel genetic analysis. Microarrays have applications in the areas of DNA sequencing, genetic mutation detection and gene expression monitoring. We report here the design and utility of an experimental instrument for microchip parallel hyperspectral fluorescent imaging. The instrument integrates in-line laser excitation of microarray, parallel fluorescent spectrometry with cooled CCD and dye-base spectral classification software. Instrument has been applied for imaging detection, spectral analysis and base classification of Genetic Bit Analysis (GBA) reactions in a microchip format on a glass support. GBA is a solid phase DNA sequence analysis method that provides single nucleotide resolution by specific extension of dye-labeled dideoxynucleotidetriphosphates (ddNTPs). GBA testing yields one or two different ddNTPs on any given microarray spot, so analysis must resolve any pair wise combination of all possible ddNTPs labeled with distinct fluorescent dyes.
Fabrication of multiple-antibody arrays using a hydrogel microstamp
Brett D. Martin, Bruce Paul Gaber, Charles H. Patterson, et al.
This work demonstrates a simple method for protein micropatterning. A new sugar poly(acrylate) hydrogel acts as a reusable `stamper' which deposits antibodies directly onto a silane modified capture surface. The antibodies are directly placed on the surface so a precise arrangement can be obtained with minimal cross-contamination among the antibody spots. Circular antibody spots have been created using this approach with diameters ranging from less than 20 to greater than 100 microns. Patterns were imaged using immunofluorescence microscopy wherein a fluorescently- labeled IgG antigen was selectively bound to the patterned (stamped) antibody. Signal to background ratios of greater than 25:1 have been routinely observed. Arrays with three antibodies were prepared and visualized by sequential exposure to fluorescently-labeled partner antigens. No cross-reactivity among the different antibody spots was observed. Engineered biosurfaces created using this new technology may be useful in the areas of multianalyte immunosensors, the assembly of protein ensembles, and cell patterning methods.
Hardware solution for continuous time-resolved burst detection of single molecules in flow
Michael Wahl, Rainer Erdmann, Kristian Lauritsen, et al.
Time Correlated Single Photon Counting (TCSPC) is a valuable tool for Single Molecule Detection (SMD). However, existing TCSPC systems did not support continuous data collection and processing as is desirable for applications such as SMD for e.g. DNA-sequencing in a liquid flow. First attempts at using existing instrumentation in this kind of operation mode required additional routing hardware to switch between several memory banks and were not truly continuous. We have designed a hard- and software system to perform continuous real-time TCSPC based upon a modern solid state Time to Digital Converter (TDC). Short dead times of the fully digital TDC design combined with fast Field Programmable Gay Array logic permit a continuous data throughput as high as 3 Mcounts/sec. The histogramming time may be set as short as 100 microsecond(s) . Every histogram or every single fluorescence photon can be real-time tagged at 200 ns resolution in addition to recording its arrival time relative to the excitation pulse. Continuous switching between memory banks permits concurrent histogramming and data read-out. The instrument provides a time resolution of 60 ps and up to 4096 histogram channels. The overall instrument response function in combination with a low cost picosecond diode laser and an inexpensive photomultiplier tube was found to be 180 ps and well sufficient to measure sub-nanosecond fluorescence lifetimes.
Parallelism in integrated fluidic circuits
Luc J. Bousse, Anne R. Kopf-Sill, J. Wallace Parce
Many research groups around the world are working on integrated microfluidics. The goal of these projects is to automate and integrate the handling of liquid samples and reagents for measurement and assay procedures in chemistry and biology. Ultimately, it is hoped that this will lead to a revolution in chemical and biological procedures similar to that caused in electronics by the invention of the integrated circuit. The optimal size scale of channels for liquid flow is determined by basic constraints to be somewhere between 10 and 100 micrometers . In larger channels, mixing by diffusion takes too long; in smaller channels, the number of molecules present is so low it makes detection difficult. At Caliper, we are making fluidic systems in glass chips with channels in this size range, based on electroosmotic flow, and fluorescence detection. One application of this technology is rapid assays for drug screening, such as enzyme assays and binding assays. A further challenge in this area is to perform multiple functions on a chip in parallel, without a large increase in the number of inputs and outputs. A first step in this direction is a fluidic serial-to-parallel converter. Fluidic circuits will be shown with the ability to distribute an incoming serial sample stream to multiple parallel channels.
Optical Methods in High-Throughput Screening for Drug Discovery
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Development of fluorescence-based high-throughput screening assays: choice of appropriate instrumentation
David J. Burns, Elisabeth Alder, Yi-Hong Fan, et al.
Fluorescence-based assays have become increasingly popular in high throughput screening for a variety of reasons (e.g. sensitivity). However, new screening technologies are pushing the limits of conventional fluorescence plate readers. For example, instruments that have optical sensitivities beyond most of the commercially available plate readers are required to reproducibly measure the fluorescence generated by the green fluorescent protein (GFP)--a novel reporter gene. Also, miniaturization of screening formats (with densities higher than the conventional 96-well plate) requires high resolution instrumentation to measure fluorescence. Several assays based on optical fluorescence measurements have been developed and screened in our Biological Screening group. These assays include various fluorescence-based protease assays (standard end-point and kinetic modes) and a functional cell-based screen using the green fluorescent protein as a reporter gene. The choice of instrumentation was the critical factor in the performance and success of each of these arrays. Data will be presented for the cell- based reporter assay including the type of instrumentation (fluorescence plate readers; fluorescence imaging systems) used for detection of GFP fluorescence.
Development of an ultrahigh-throughput screening system: plate design, liquid handling, and image analysis
Kevin R. Oldenburg
Recent advances in plate design, liquid handling, and imaging have made it possible, for the first time, to create truly ultra-high throughput screening systems. The key engineering challenges which needed to be overcome in order to develop this system as well as system limitations are discussed. Data from model systems is presented comparing the results obtained from the miniaturized format with those obtained from the standard 96-well format. These results demonstrate that the signal to noise ratio and robustness of an assay can be preserved upon ultra-miniaturization. The flexibility of this system should make it amenable for screening of not only pharmaceuticals but also for agrochemicals and applied materials.
Fluorescence-lifetime technologies for high-throughput screening
Todd E. French, John C. Owicki, Douglas N. Modlin, et al.
Fluorescence lifetime, the mean interval between absorption and emission, is as fundamental a characteristic of fluorescence as excitation and emission wavelengths and quantum yield. Yet, with the exception of time-resolved fluorescence assays utilizing lanthanide chelates, the analytical possibilities of methods based on fluorescence lifetime are virtually unexploited outside the academic research laboratory. We discuss the potential use of fluorescence-lifetime technologies in high-throughput screening from the standpoint of assay reagents and instrumentation. Among these applications are fluorescence- polarization assays based on long-lifetime probes and fluorescence-intensity assays using lifetime-resolved detection to reject background. We find that fluorescence- lifetime technologies offer significant practical advantages over existing methods.
Quantitative imaging of assays in miniaturized plate formats
Gang Sun, Peter Ramm
Miniaturized sample formats offer advantages in cost and throughput to screening laboratories. Imaging systems may be valuable tolls for assay miniaturization. We describe fluorescence performance of our TundraTM assay imaging system, which incorporates a thinned, back illuminated CCD area detector, a telecentric lens with internal excitation optics, and AssayVisionTM software. Using a flat field correction, Tundra imaged a difficult (faint) gel with performance similar to that of a high performance scanning laser. The accuracy of Tundra's dedicated well plate calibration functions was also evaluated in 384 (40 (mu) l sample volume) and 1536 (5 (mu) l) well plates, and across a realistic range of fluor concentrations. With both plate formats, and across the full range of fluor concentrations, Tundra shows coefficients of variation which are similar to those of a scanning laser, and to those reported for PMT devices. Tundra also exhibits highly linear response to fluor concentration.
Imaging proximity assays
Robert A. Jessop
Scintillation Proximity Assay is an established radioisotopic assay technique widely used in high throughput screening laboratories. The requirement for miniaturization of these arrays has led to the development of microplates containing 384, 864, 1536 or even higher numbers of wells. Imaging is the only practical detection method for quantifying the assays in such high density plates.
Light scattering from metal sol labels on high-density DNA probe arrays
Mark O. Trulson, Ian D. Walton, Audrey D. Suseno, et al.
We have been exploring the use of light scattering as a means to detect the binding of nucleic acids to high density DNA probe arrays. Initial work has concentrated on the use of 100 nanometer gold particles conjugated to monoclonal antibodies. A probe array scanner that utilizes an arc lamp source and a `photocopier grade' linear CCD detector has been developed. The optical configuration of the scanner maximizes dynamic range and minimizes optical backgrounds. Initial development of light scattering detection for the p53 cancer gene application shows that functional performance may be obtained that is essentially equivalent to existing fluorescence detection methodology.
Cell and Tissue Diagnostics
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Fluorescence detection of camptothecin anticancer drugs by two-photon excitation
Thomas G. Burke, Magda Malak, David Bom, et al.
Hycamtin is a camptothecin anticancer analogue containing a dimethylaminomethyl substituent at position 9 and a hydroxy functionality at position 10. Using an excitation wavelength of 800 nm we have compared the two-photon cross sections and excited-state lifetimes from several camptothecins in phosphate buffered saline solution with and without the presence of human serum albumin (HSA). Drug and HSA concentrations of 10 (mu) M and 46 (mu) M were employed in our studies. In phosphate buffered saline solution containing HSA the following excited-state lifetimes (ns) and two- photon cross-sections (10-50 cm4 s/photon), respectively, were determined: hycamtin (4.3 nm, 36); camptothecin (1.3 ns, 1); 7-t-butyldimethylsilyl-10- hydroxycamptothecin (1.7 ns, 3.7); 7-t-butyldimethylsilyl- camptothecin (1.9 ns, 1.9); 7-trimethylsilyl-10- aminocamptothecin (6.3 ns; 35); and 7-trimethylsilyl-10- hydroxycamptothecin (1.8 ns; 2.2). Our results indicate that Hycamtin exhibits a high cross-section relative to the parent camptothecin molecule and represents one of the best camptothecin analogues to detect using two-photon excitation. Hycamtin was detected at concentrations as low as 0.05 (mu) M and 1 (mu) M in plasma and whole blood, respectively. The newly synthesized analogue 7- trimethylsilyl-10-aminocamptothecin was found to display similar lifetime and two-photon cross section values relative to Hycamtin. Thus, fluorescence detection with two- photon excitation may prove to be of advantage in the development of this promising new experimental therapeutic.