Proceedings Volume 2386

Ultrasensitive Instrumentation for DNA Sequencing and Biochemical Diagnostics

Gerald E. Cohn, Jeremy M. Lerner, Kevin J. Liddane, et al.
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Proceedings Volume 2386

Ultrasensitive Instrumentation for DNA Sequencing and Biochemical Diagnostics

Gerald E. Cohn, Jeremy M. Lerner, Kevin J. Liddane, et al.
View the digital version of this volume at SPIE Digital Libarary.

Volume Details

Date Published: 3 April 1995
Contents: 9 Sessions, 33 Papers, 0 Presentations
Conference: Photonics West '95 1995
Volume Number: 2386

Table of Contents

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Table of Contents

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  • Non-Gel-Based and Mass-Spectrometric DNA Sequencing Strategies
  • Gel-Based DNA Sequencing Technologies
  • Immunoassay Systems
  • Techniques of Ultracentrifugation
  • Ultrasensitive Systems
  • Chemometrics and Neural Networks in Data Analysis
  • Data Processing Software for Diagnostic Instruments
  • Advances in Instrumentation for Spectroscopy I
  • Advances in Instrumentation for Spectroscopy II
  • Immunoassay Systems
  • Gel-Based DNA Sequencing Technologies
  • Ultrasensitive Systems
Non-Gel-Based and Mass-Spectrometric DNA Sequencing Strategies
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Flow-based continuous DNA sequencing via single molecule detection of enzymatically cleaved fluorescent nucleotides
Jay A. Schecker, Peter M. Goodwin, Rhett L. Affleck, et al.
The development of our rapid, continuous DNA sequencing technology, based on single molecule detection of fluorescently-tagged nucleotides, has proceeded along separate research fronts, each with specific goals: the faithful replication of long sequences of template DNA using one or more fluorescent nucleotide analogues, the incorporation and stable mounting of a single DNA strand into a flow chamber, the enzymatic cleavage of labeled DNA by exonucleases, and the detection of single fluorescent nucleotides in a flow stream by the method of time-gated photon counting. Each individual goal of the sequencing technology has now been realized, and we have begun integrating these efforts in order to demonstrate the feasibility of flow-based sequencing. We are currently detecting photon bursts from TRITC labeled nucleotides which have been cleaved from DNA suspended in our flow cell. The sample size is estimated to be tens of DNA strands.
Laser desorption mass spectrometry for DNA analysis and sequencing
C. H. Winston Chen, N. I. Taranenko, K. Tang, et al.
Laser desorption mass spectrometry has been considered as a potential new method for fast DNA sequencing. Our approach is to use matrix-assisted laser desorption to produce parent ions of DNA segments and a time-of-flight mass spectrometer to identify the sizes of DNA segments. Thus, the approach is similar to gel electrophoresis sequencing using Sanger's enzymatic method. However, gel, radioactive tagging, and dye labeling are not required. In addition, the sequencing process can possibly be finished within a few hundred microseconds instead of hours and days. In order to use mass spectrometry for fast DNA sequencing, the following three criteria need to be satisfied. They are (1) detection of large DNA segments, (2) sensitivity reaching the femtomole region, and (3) mass resolution good enough to separate DNA segments of a single nucleotide difference. It has been very difficult to detect large DNA segments by mass spectrometry before due to the fragile chemical properties of DNA and low detection sensitivity of DNA ions. We discovered several new matrices to increase the production of DNA ions. By innovative design of a mass spectrometer, we can increase the ion energy up to 45 KeV to enhance the detection sensitivity. Recently, we succeeded in detecting a DNA segment with 500 nucleotides. The sensitivity was 100 femtomole. Thus, we have fulfilled two key criteria for using mass spectrometry for fast DNA sequencing. The major effort in the near future is to improve the resolution. Different approaches are being pursued. When high resolution of mass spectrometry can be achieved and automation of sample preparation is developed, the sequencing speed to reach 500 megabases per year can be feasible.
Mapping site-specific endonuclease binding to DNA by direct imaging with atomic force microscopy (AFM)
David P. Allison, Thomas G. Thundat, P. Modrich, et al.
Physical mapping of DNA can be accomplished by direct AFM imaging of site specific proteins bound to DNA molecules. Using Gln-111, a mutant of EcoRI endonuclease with a specific affinity for EcoRI sites 1000 times greater than wild type enzyme but with cleavage rate constants reduced by a factor of 104, we demonstrate site-specific mapping by direct AFM imaging. Images are presented showing specific-site binding of Gln-111 to plasmids having either one (pBS+) or two (pMP32) EcoRI sites. Identification of the Gln-111/DNA complex is greatly enhanced by biotinylation of the complex followed by reaction with streptavidin gold prior to imaging. Image enhancement coupled with improvements in our preparation techniques for imaging large DNA molecules, such as lambda DNA (47 kb), has the potential to contribute to direct AFM restriction mapping of cosmid-sized genomic DNAs.
Optical melting as a tool for optimizing sequencing by hybridization (SBH) analysis of DNA
Mitchel J. Doktycz, K. Bruce Jacobson, K. L. Beattie, et al.
Sequencing by hybridization is a technique that relies on the specific hybridization properties of nucleic acids to derive sequence information. Hybridization properties are highly dependent on the DNA sequence and the solution environment. Identification of the optimal SBH conditions can be obtained by optical melting. By optical melting of 128 octamer pairs that have the appropriate choice of nucleic acid structures, a useful model of stability has been obtained which will aid in the design and implementation of SBH assays.
Gel-Based DNA Sequencing Technologies
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Ultrahigh-speed DNA sequencing using capillary array electrophoresis chips
Adam T. Woolley, Richard A. Mathies
Capillary electrophoresis arrays have been fabricated on planar glass substrates using photolithographic masking and chemical etching. The photolithographically defined channel patterns were first etched in a glass substrate, and then capillaries were formed by thermally bonding the etched substrate to a second glass slide. High-speed separations of restriction fragment digests were performed on these chips in under 1 minute. Multiple separations in the same channel demonstrated excellent reproducibility. Rapid genetic typing of short tandem repeats on the HUMTHO1 locus have also been performed. Finally, separations with single- base resolution have been successful, indicating that these microfabricated devices will also be useful for high-speed DNA sequencing.
High-speed automated DNA sequencing utilizing from-the-side laser excitation
Michael S. Westphall, Robert L. Brumley Jr., Erin C. Buxton, et al.
The Human Genome Initiative is an ambitious international effort to map and sequence the three billion bases of DNA encoded in the human genome. If successfully completed, the resultant sequence database will be a tool of unparalleled power for biomedical research. One of the major challenges of this project is in the area of DNA sequencing technology. At this time, virtually all DNA sequencing is based upon the separation of DNA fragments in high resolution polyacrylamide gels. This method, as generally practiced, is one to two orders of magnitude too slow and expensive for the successful completion of the Human Genome projection. One reasonable approach is improved sequencing of DNA fragments is to increase the performance of such gel-based sequencing methods. Decreased sequencing times may be obtained by increasing the magnitude of the electric field employed. This is not possible with conventional sequencing, due to the fact that the additional heat associated with the increased electric field cannot be adequately dissipated. Recent developments in the use of thin gels have addressed this problem. Performing electrophoresis in ultrathin (50 to 100 microns) gels greatly increases the heat transfer efficiency, thus allowing the benefits of larger electric fields to be obtained. An increase in separation speed of about an order of magnitude is readily achieved. Thin gels have successfully been used in capillary and slab formats. A detection system has been designed for use with a multiple fluorophore sequencing strategy in horizontal ultrathin slab gels. The system employs laser through-the-side excitation and a cooled CCD detector; this allows for the parallel detection of up to 24 sets of four fluorescently labeled DNA sequencing reactions during their electrophoretic separation in ultrathin (115 micrometers ) denaturing polyacrylamide gels. Four hundred bases of sequence information is obtained from 100 ng of M13 template DNA in an hour, corresponding to an overall instrument throughput of over 9600 bases/hr. A detailed description and the operating characteristics of this systems are presented.
Single-lane single-fluor sequencing using dideoxy-labeled, heavy-atom-modified near-IR fluorescent dyes
Daryl C. Williams, James H. Flanagan Jr., Benjamin L. Legendre Jr., et al.
Using a near-IR (NIR) fluorescence detection system and labels synthesized in our laboratories, electropherograms of oligonucleotides separated by capillary gel electrophoresis and detected using NIR fluorescence will be presented. The sequence of nucleotide bases was determined using a single-lane, single-dye technique. The molar concentrations of the ddNTP's used during extension reactions were varied in order to achieve a ratio of 4:2:1:0 (A:C:G:T) which allowed the identification of each terminal base via fluorescence intensity measurements. Sequencing ladders were prepared from the template, M13mp18, using standard Sanger dideoxy chain termination techniques, the modified T7 DNA polymerase, and a NIR-labeled M13 primer. The data indicated reliable sequence determination up to 300 bases with a base-calling accuracy of 90%. In order to eliminate the need for dye-labeled primers and the T7 DNA polymerase enzyme, we have developed a sequencing strategy which utilizes dye-labeled dideoxy nucleotides in a single-lane, single-fluor approach. Base-calling is accomplished by measuring the fluorescence lifetime of intramolecular heavy-atom modified dyes.
Enhanced throughput for infrared automated DNA sequencing
Lyle Richard Middendorf, Bill O. Gartside, Pat G. Humphrey, et al.
Several enhancements have been developed and applied to infrared automated DNA sequencing resulting in significantly higher throughput. A 41 cm sequencing gel (31 cm well- to-read distance) combines high resolution of DNA sequencing fragments with optimized run times yielding two runs per day of 500 bases per sample. A 66 cm sequencing gel (56 cm well-to-read distance) produces sequence read lengths of up to 1000 bases for ds and ss templates using either T7 polymerase or cycle-sequencing protocols. Using a multichannel syringe to load 64 lanes allows 16 samples (compatible with 96-well format) to be visualized for each run. The 41 cm gel configuration allows 16,000 bases per day (16 samples X 500 bases/sample X 2 ten hour runs/day) to be sequenced with the advantages of infrared technology. Enhancements to internal labeling techniques using an infrared-labeled dATP molecule (Boehringer Mannheim GmbH, Penzberg, Germany) and Sequenase (U.S. Biochemical) have also been made. The inclusion of glycerol in the sequencing reactions yields greatly improved results for some primer and template combinations. The inclusion of (alpha) -Thio-dNTP's in the labeling reaction increases signal intensity two- to three-fold.
Single-molecule electrophoresis: applications to biomolecular detection
Alonso Castro, E. Brooks Shera
A new method for the detection and identification of biological molecules at the single molecule level of sensitivity has been developed. The technique involves measuring the electrophoretic velocity of each molecule present in a sample. Since different chemical species generally exhibit different electrophoretic velocities, identification is accomplished by classification according to electrophoretic velocity. The solution is contained in a capillary to which an external voltage is applied, and the velocity is determined by measuring the time taken by an individual molecule to travel the distance between two tightly focused laser beams due to the electrophoretic effect. The detection of the migration of individual molecules through each laser beam was accomplished by a modified version of our recently developed technique of single fluorescent molecule detection. Monte Carlo computer simulations of the process were performed beforehand in order to estimate the experimental feasibility of the method, and to determine the optimum values for the various experimental parameters. The method has been applied to the analysis of single-fluorophore molecules such as rhodamine 6G, and to biological macromolecules, such as mixtures of nucleic acids and of proteins. Applications of the technique to the detection of specific DNA sequences are discussed.
Immunoassay Systems
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Particle-enhanced total internal reflectance fluorescence
Victor P. Chu, Steven J. Zoha, Jim Davis, et al.
This paper presents work toward a retrofit of an acaTMIV Discrete Clinical Analyzer (E. I. du Pont de Nemours and Company, Wilmington, DE) with a Particle Enhanced Total Internal Reflection Fluorescence technology. The DuPont acaTM line of instruments was designed to perform highly accurate unit dose clinical chemistry assays. Introduction of a Total Reflectance Fluorescence optics into an acaTMIV could provide capability to perform highly sensitive homogeneous immunoassays. Sensitivity and precision depended upon uniform immobilization of immunoglobulins or immunogens, representing examples of specific binding partners, onto an ionomeric material, acting as both solid support and wall material of the disposable reagent units called the `pack'. This study examines the uniformity of antibody coating onto various types of tough and flexible optical polymeric films including those made from the DuPont SurlynTM and NucrelTM polymers. A number of coating processes will also be described. Bioanalytical tools were developed to determine uniformity, quantity, and activity of antibody coated onto the `pack' film, thus allowing for optimization of polymer film type and process of antibody coating.
Energy transfer of nucleic acid products
Paul M. Jung, Hsiang-Yun Hu, Omar S. Khalil
Fluorescence energy transfer was investigated as a homogeneous detection method for the gapped ligase chain reaction (G-LCR). Oligonucleotides of a Chlamydia trachomatic G-LCR probe set were labeled with fluorescein as the donor and Texas Red as the acceptor fluorophore. Amplification and detection of 10 molecules of synthetic target was demonstrated in spiked urine samples.
Techniques of Ultracentrifugation
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Methods for increasing the sensitivity of sedimentation velocity analysis: a signal averaging Rayleigh optical system for the Beckman Instruments Optima XL-A analytical ultracentrifuge
Walter F. Stafford III, Sen Liu
A large increase in the sensitivity of sedimentation velocity experiments can be achieved by combining time derivative analysis with signal averaging. Computation of the time derivative results in the complete elimination of time independent optical background. The ability to rapidly acquire sedimentation profiles using real-time Rayleigh interferometry has made it possible to average large numbers of profiles over relatively short periods of time. Currently available, inexpensive computers and video frame grabbers have made it possible to acquire and reduce Rayleigh interferograms at the rate of about 1 per second. The ability to achieve high sensitivity means that high affinity interacting systems that can not be studied by the conventional ultracentrifugal methods are now accessible to analysis by analytical ultracentrifugation. A Rayleigh optical system for the Beckman Instruments Optima Series XL-A Analytical Ultracentrifuge is described, and methods of data acquisition and reduction are presented.
Ultrasensitive Systems
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From in vitro to in vivo by dynamic multiwavelength imaging
Daniel L. Farkas, Byron T. Ballou, Gregory W. Fisher, et al.
There is a clear trend today towards non-invasive, dynamic, digital approaches to biomedical imaging, and a need for even higher resolution. Light is particularly well suited for such investigations, as its temporal, spatial and intensity range are unparalleled. A convergence of new capabilities from fields as diverse as electronics, optics, molecular biology, computer science and dye chemistry have transformed light microscopy from a traditional, static, 2D tool into a highly useful, dynamic, 3D research capability for biology and medicine. We believe that the understanding of certain fundamental biological functions by dynamic mapping of events in living systems is within reach, based on novel, interdisciplinary methods. For imaging molecular events with high resolution (live cells, in vitro), light microscopy has continued to improve in performance, and we survey here some of our recent progress. The same dynamic mapping can be extended to organs, whole animals and humans, by monitoring molecules labeled with the long-wavelength dyes that proved useful in microscopy. We report here results obtained by in vivo imaging of fluorescently labeled monoclonal antibodies, indicative of tumor location and evolution in nude mice.
Catheter-sized fiber optic absorbance sensor with compact instrumentation
David A. Landis, Carl J. Seliskar
Catheter-sized absorbance sensors have been constructed and tested using the medically significant dye indocyanine green as an analyte. Sensors were found to be equally usable with conventional wavelength scanning instrumentation or with a very compact solid state instrument. Statistical evaluation of the data acquired with these small sensors showed that accurate working curves provide high sensitivity and wide dynamic range of use. Potential uses of these sensors are suggested.
Transcutaneous analyte measuring method (TAMM): a reflective, noninvasive, near-infrared blood chemistry analyzer
Kenneth J. Schlager, Timothy L. Ruchti
TAMM for Transcutaneous Analyte Measuring Method is a near infrared spectroscopic technique for the noninvasive measurement of human blood chemistry. A near infrared indium gallium arsenide (InGaAs) photodiode array spectrometer has been developed and tested on over 1,000 patients as a part of an SBIR program sponsored by the Naval Medical Research and Development Command. Nine (9) blood analytes have been measured and evaluated during pre-clinical testing: sodium, chloride, calcium, potassium, bicarbonate, BUN, glucose, hematocrit and hemoglobin. A reflective rather than a transmissive invasive approach to measurement has been taken to avoid variations resulting from skin color and sensor positioning. The current status of the instrumentation, neural network pattern recognition algorithms and test results will be discussed.
Interaction of instrumental and process uncertainties in the overall precision of clinical diagnostics
Clinical biochemical analysis requires the interaction of the signal from an assay reaction product with a sequence of optical, mechanical, electronic and computational subassemblies. Each of these components can introduce an uncertainty into the final precision of measurement. Contributions from uncertainties in the concentration of sample chromophore differ from standard models of detector response in absorbance measurements. A Beer's Law absorbance model predicts that relative errors in concentration decrease momotonically with increasing absorbance. The product of molar absorptivity, path length and chromophore concentration uncertainty characterizes this effect. Precision of concentration must be controlled according to the particular molar absorptivity and detector response of the instrument. Assay chemistry factors dominate at low absorbance values, making high precision difficult at low absorbance.
Light scattering and fluorescence of single particles measured by a scanning flow cytometer
Valerii Pavlovich Maltsev, Andrej V. Chernyshev, Alexander A. Doroshkin, et al.
A novel type of Scanning Flow Cytometer (SFC) has been developed for improving particle analysis from light scattering data. The use of the SFC and the Flying Light Scattering Indicatrix (FLSI) method allows absolute measurement of the size and refractive index of spherical particles. The separation efficiency of the single particle analysis using FLSI method is verified measuring the size distribution of standard latex particles. It is shown that particles differed in size more than 1.2 micrometers can be separated by the FLSI method to different classes. In the second part of this paper the feasibility of a Pulsed Laser Flow Cytometer (PLFC) equipped by a pulsed nitrogen laser is tested for fluorometric analysis of human lymphocytes stained with FITC-labeled monoclonal antibodies. In addition the feasibility for the fluorescence measurement in time-resolved regime of the PLFC is analyzed from measurement of photon collection efficiency of scanning optical system.
Chemometrics and Neural Networks in Data Analysis
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Overview of chemometrics
Kenneth J. Schlager, Timothy L. Ruchti
Chemometrics is a broad field concerned with the application of mathematical and statistical methods to problems in chemistry. Biotronics Technologies has applied chemometrics to demanding chemical applications involving noninvasive medical diagnostic measurement instrumentation using advanced signal processing and calibration techniques. The chemometrics methods have also been extended to quantitative analysis in microbiology. Signal processing transforms data measurements to enhance the extraction of physically significant information. Examples include the Fourier Transform, first and second derivatives, and digital and adaptive filtering. Calibration is the process of relating data measurements to a chemical concentration for the purpose of estimation. Standard methods of calibration include linear regression, multiple-linear regression, partial linear regression, and principal components regression. For more demanding applications, novel techniques involving artificial neural networks, genetic algorithms, and rotated principal components have been developed. This paper summarizes the chemometric experience of Biotronics Technologies including relevant theoretical background.
Role of X-axis uncertainties on standard curves
Michael L. Johnson
The commonest method for the determination of standard curves is to use a least-squares technique to fit a function to a set of standard data. This fitted curve is then used to interpolate values from the standard curve. The problem addressed is that the standard data used for this process will usually contain experimental uncertainties in the X-axis (the independent variable) and in the Y-axis (dependent variable). When such X-axis uncertainties exist in the data it is statistically invalid to apply a least-squares procedure to evaluate the coefficients of the standard curve. This statistical invalidity generally cannot be corrected by the application of an `appropriate weighting factor.' However, a simple maximum likelihood procedure can be used to correctly consider the uncertainties in both the X-axis and Y-axis.
Novel approach to the use of neural networks to solve real-world analytical problems
Jeremy M. Lerner, Thomas Taiwei Lu, David T. Mintzer, et al.
This paper describes the general architecture of a hybrid neural network used to identify noisy and extremely complex spectra. A hybrid neural network has been built for environmental monitoring, medical diagnosis, and process control applications. The hybrid neural network consists of preprocessing algorithms to enhance the features of the spectra and an interconnect weight matrix for recognition. Results suggest that the hybrid neural network, through careful design of both the preprocessing algorithms and the neural network architecture, is capable of increasing the detection limit and speed of many analytical instruments.
Data Processing Software for Diagnostic Instruments
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Simple solution to the medical instrumentation software problem
Robert C. Leif, Suzanne B. Leif, Stephanie H. Leif, et al.
Medical devices now include a substantial software component, which is both difficult and expensive to produce and maintain. Medical software must be developed according to `Good Manufacturing Practices', GMP. Good Manufacturing Practices as specified by the FDA and ISO requires the definition and compliance to a software processes which ensures quality products by specifying a detailed method of software construction. The software process should be based on accepted standards. US Department of Defense software standards and technology can both facilitate the development and improve the quality of medical systems. We describe the advantages of employing Mil-Std-498, Software Development and Documentation, and the Ada programming language. Ada provides the very broad range of functionalities, from embedded real-time to management information systems required by many medical devices. It also includes advanced facilities for object oriented programming and software engineering.
Brief introduction to using asynchronous transfer mode (ATM) for communications in medical devices
Stephanie H. Leif
ATM (Asynchronous Transfer Mode) provides a new means of transferring data over a network. Medical device manufactures may move to ATM because it provides more bandwidth. However, there are safety concerns associated with moving to ATM. This paper provides a brief introduction of ATM and discusses some of the associated challenges.
Advances in Instrumentation for Spectroscopy I
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Hardware, software, brainware, noware
Developments in politics, communications, economics, and population have all had profound effects on the market for analytical chemical instrumentation. This essay examines the assumptions behind the current training of instrumentation scientists and marketing of instruments, and suggests changes in both. The market must be taken to be all of society, not just technically literate society. Cost tradeoffs between hardware and software are context- dependent. Chemometrics allows extraction of information from data that leaves the typical reductionist scientist queasy. And clever chemistry can sometimes obliterate entire markets. The implications of this evolution are explored.
Linear charge-coupled devices as multichannel detectors for optical spectroscopy
Linear charge coupled device (CCD) array detectors designed for use in document scanning applications have been proven to be useful as array spectroscopic detectors in some instances. The principles of operation, device architecture, and general characteristics of several common low-cost linear array CCD detectors as they pertain to use as spectroscopic detectors are described. In particular, the readout noise, quantum efficiency, low light level linearity and charge transfer characteristics, and charge capacity as it affects absorbance and fluorescence spectroscopic measurements are discussed.
Designing advanced instrumentation: real world, real time, real answers
Andrew T. Zander
In addressing the relationships among instrument design, applications and signal processing, it is necessary to consider the manner in which the hardware of a system is configured, i.e., how it is physically engineered and what considerations support that effort. Systems engineering concerns are in addition to those leading to a full understanding of all the system parameters associated with the physical measurement intended to be conducted. This multiplicity of considerations is even more important when the goal is the development of high performance instrumentation dedicated to applications in the real world, but which is also compact and inexpensive. A number of systems engineering considerations must be addressed when planning the development of advanced versions of physical property measurement equipment. A basic assumption is that the new equipment will not always be used in a high quality laboratory environment. It still will be required to meet its performance, size and cost specifications, however. Among these considerations are: the intended mobility of the equipment; the dependency on provided facilities; the expected system operational rates; the balance of operational flexibility and complexity; and the approach taken to contain cost.
Advances in Instrumentation for Spectroscopy II
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Integrated fiber optic backscatter system for photon correlation spectroscopy
Harbans S. Dhadwal, Romel R. Khan, Kwang Suh
Design and fabrication of an integrated backscatter fiber optic probe is described in this paper. A short section of a gradient index optical fiber is fusion spliced to two separate monomode optical fibers; the two fibers are mounted into a stainless steel face plate. One fiber is used to transmit either a focussed or collimated laser beam to the scattering region. The second fiber collects and guides the laser light scattered at a predefined scattering angle to a photomultiplier. Subsequent photon correlation and analysis yields size information of the scattering species.
Increased photon-counting efficiency for multispectral imaging using rotational spectrotomography
Paul A. Bernhardt
Multi-spectral imaging can be powerful tool for medical diagnostics. Many time resolved or low-light-level applications require large photon throughput to distinguish between areas of normal and diseased tissues. The throughput of a dispersive, imaging spectrometer is often much less than unity and consequently limits sensitivity. Common multi-spectral approaches use either (1) narrow band filters to isolate 2D spatial images for each spectral wavelength channel or (2) a slit spectrograph to image one spatial and one spectral dimension as the slit is scanned across the object. Both of these approaches are inefficient because photons outside the filter passband or the slit area are not detected. A new imaging technique called spectro- tomography collects all available photons and employs computer tomography to reconstruct the 3D data cube of the image. A rotational spectro-tomographic imager has been designed with a circular aperture, objective-grating camera that is rotated in steps around its optical axis. A sequence of images is obtained with fixed steps in camera angle by rotation and lens focal-length by zooming. These images provide a sufficient number of 2D projections of the 3D data cube for accurate reconstruction. Both direct Fourier transform and filter- backprojection algorithms have been developed for tomographic reconstructions. The data cube of a broad spectrum object with 64 spectral bands and 64 X 64 spatial resolution elements has been used as the test case for a numerical example of the technique.
Fluorescence microscopic spectral study of fluorescein and some amino acids
Narahari V. Joshi, Virginia Otero de Joshi, Nestor Rodrigues, et al.
A conventional collinear laser induced fluorescence detection system for capillary electrophoresis was modified by replacing a GaAs photomultiplier by a Charge coupled Device which was operated at liquid nitrogen temperature. This permits longer exposure time to collect the weak radiation and hence extends the limit of detection and spectral analysis of lower concentration of biological active molecules. Using the above technique, and passing the solution through a capillary of 20 micrometers inside diameter, we have recorded fluorescence spectra of biological important molecules such as Arginine, Glutamate and Glutamine; all of them were derivatized with Fluorescein Isothiocyanate (FITC) isomer I. Spectra of FITC as a function of the pH were examined to lower the detection limit. The concentration of the molecules was as low as 10-14 M. All the spectra recorded lied in the expected region (510 nm to 550 nm) and showed considerable similarity. The spectrum of low concentration of molecules provides significant information as the intensity of intra-molecular interaction is reduced. The recorded spectra gave information about the distribution of vibrational states near S0 and S1 levels.
Optical Multichannel Analyzer (OMA) computer system used in time-resolved spectra measurement
Tong Ye, Shuicai Wang, Bin Wu, et al.
Time-resolved spectroscopy techniques are the most powerful tools in the research of the ultrafast phenomena. Optical Multichannel Analyzer (OMA) is often used in such spectra's measurement. In the old product of OMA, OMA II, its console runs too slow to cope with the large quantity of data in the experiments, and its limited functions and unexpansibility lead to much inconvenience in using. A parallel interface is made to realize the communication between the controller and the lab computer so that the OMA console can be replaced by the computer. Clearly the computer has much higher rate to process the date and much larger media to store data. Furthermore, the computer can control the other devices such as a shutter, a step motor for delay line and so on. Also the method of data process can be changed according to the different experiments by program. In one word, it is the most important that the computer can be used to control the entire course of the experiments and do some necessary real-time process. A femtosecond time-resolved absorption apparatus based on OMA II and a 386 computer is established and a control software for DOS is developed.
Subpicosecond time-resolved fluorescence spectrometer using a Ti:sapphire laser
Tong Ye, Yaodong Chen, Jianming Tang, et al.
In this paper a subpicosecond time-resolved fluorescence spectra's measurement apparatus is presented. The principle of measurement is based on sum frequency in nonlinear crystal between the pump laser pulses and the fluorescence from sample stimulated by excitation laser pulses. The fluorescence decay can be obtained by varying time delay of the excitation laser pulses to the gating pulses. Adjusting the phasematching angle of nonlinear crystal records the fluorescence decay at various wavelengths. In this spectrometer, a CW mode-locked Ti:sapphire tunable laser is used, with approximately 100 fs pulse width and 82 MHz repetition. The IR light from the lasers is frequency doubled to excite the sample with the remained unconverted light used as gating pulses. A very thin BBO (500 micrometers ) is selected to be a sum frequency nonlinear crystal. The time resolution of the apparatus mainly depends on the pulse width of laser and the effects of group velocity in the crystal. The upconversion UV light is detected by a single photon counting system. The delay line driven by stepper motor is controlled by computer. This computer controls the course of experiments and accumulates data meanwhile. Some experimental results are also presented in study of dynamics of photosystem II reaction center which is isolated and purified from higher plants.
Immunoassay Systems
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Pseudointrinsic probes for generating spectrally enhanced proteins and nucleic acids
J. B. Alexander Ross, Carol A. Hasselbacher, Elena Rusinova, et al.
The convergence of methods and techniques in biological fluorescence spectroscopy and molecular biotechnology have resulted in improved strategies for labelling specific sites in proteins and nucleic acids. Extrinsic probes, such as dansyl or fluorescein, are commonly used for labelling of proteins and nucleic acids. Introduction of extrinsic probes by covalent modification, however, is always accompanied by the potential risk of altering structure and/or function of these macromolecules. As an alternative to the use of extrinsic probes, there has been a developing interest in the use of tryptophan or nucleic acid base analogs as pseudo intrinsic probes in proteins and nucleic acids. The objective is to generate spectrally enhanced proteins or nucleic acids that are labelled at specific sites and that retain most or all of the functional features of the non-enhanced parent macromolecule. Base analogs with desirable spectroscopic properties can be introduced by direct synthesis. Tryptophan analogs with desirable spectroscopic properties can be introduced into proteins by expression in vivo or in vitro, or by direct chemical synthesis.
Gel-Based DNA Sequencing Technologies
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Three-dimensional imaging of DNA fragments during electrophoresis using a confocal detector
Laurence R. Brewer, J. Courtney Davidson, Joseph W. Balch, et al.
We have measured the 3D distribution of DNA fragments within an electrophoretic band. The measurements were made using a confocal microscope and a photon counting photomultiplier detector. A DNA sequencing standard was loaded into glass microchannel plates containing polyacrylamide gel. The measurements were made by scanning the plates in three dimensions using a mechanical stage under computer control, while electrophoresis was taking place. We found that the distribution of DNA was the same for all the bands measured in the sequencing ladder with an approximate Gaussian distribution along all three axis. These measurements are important to understand what physical forces shape electrophoretic bands confined by a channel and also as an aid in the design of high throughput DNA sequencers.
Ultrasensitive Systems
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New technology for ultrasensitive detection and isolation of rare cells for clinical diagnostics and therapeutics
James F. Leary, Scott R. McLaughlin
A high-speed, 11-parameter, 6-color fluorescence, laser flow cytometer/cell sorter with a number of special and unique features has been built for ultrasensitive detection and isolation of rare cells for clinical diagnostics and therapeutics. The software for real-time data acquisition and sort control, written as C++ programming language modules with a WindowsTM graphical user interface, runs on a 66-MHz 80486 computer joined by an extended bus to 23 sophisticated multi-layered boards of special data acquisition and sorting electronics. Special features include: high-speed (> 100,000 cells/sec) real-time data classification module (U.S. Patent 5,204,884 (1993)); real-time principal component cell sorting; multi-queue signal-processing system with multiple hardware and software event buffers to reduce instrument dead time, LUT charge-pulse definition, high-resolution `flexible' sorting for optimal yield/purity sort strategies (U.S. Patent 5,199,576); pre-focusing optical wavelength correction for a second laser beam; and two trains of three fluorescence detectors-- each adjustable for spatial separation to interrogate only one of two laser beams, syringe- driven or pressure-driven fluidics, and time-windowed parameters. The system has been built to be both expandable and versatile through the use of LUT's and a modular hardware and software design. The instrument is especially useful at detection and isolation of rare cell subpopulations for which our laboratory is well-known. Cell subpopulations at frequencies as small as 10-7 have been successfully studied with this system. Current applications in clinical diagnostics and therapeutics include detection and isolation of (1) fetal cells from material blood for prenatal diagnosis of birth defects, (2) hematopoietic stem and precursor cells for autologous bone marrow transplantation, (3) metastatic breast cancer cells for molecular characterization, and (4) HIV-infected maternal cells in newborn blood to study mother-to-infant vertical transmission of AIDS.