Optics: measuring and testing – Plural test
Reexamination Certificate
2002-12-18
2004-07-20
Stafira, Michael P. (Department: 2877)
Optics: measuring and testing
Plural test
C356S338000
Reexamination Certificate
active
06765656
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to apparatus and methods for detection and enumeration of sample particles in translucent or transparent flowing liquid. In particular the present invention relates to high throughput analysis of imaged particles in a translucent flow.
2. Description of the Prior Art
Detection and enumeration of low concentrations of selected microorganisms in large volumes of fluid has a number of applications including: 1.) bioterrorism and biowarfare defense, 2.) food and water quality control, 3.) clinical detection of pathogens, and 4.) environmental monitoring. Biodetection systems developed to date usually suffer from 1.) high cost, 2.) unsatisfactory sensitivity, 3.) slowness 4.) large size and/or 5.) labor-intensive preparation steps.
Common biodetection techniques include culture-based techniques, molecular microbiological techniques (such as Polymerase Chain Reaction or PCR), direct detection using epifluorescent microscopy, conventional flow cytometry and solid phase cytometry. All of these techniques suffer drawbacks. Culture-based techniques are slow (typically taking more than 24 hours) and usually require labor intensive steps. Microbiological techniques are not strictly quantitative and are limited by interference from background material. PCR, in particular, doesn't differentiate between live and dead cells. Direct detection of single cells without the requirement for growth is desirable, as it is more rapid. In addition, detection is quantitative, as the number of cells is directly determined.
A conventional technique for direct enumeration of microorganisms in water is filtration and microscopy. A known water sample volume is filtered so that cells in the size range of the microorganisms of interest are captured. These are then stained and examined by microscopy. Typically, staining of specific microorganisms is obtained through use of immunofluorescent antibodies (IFAs). IFAs are fluorescent molecules chemically bonded to antibodies. The antibodies are produced to have binding specificity for particular target microorganisms or particular cell surface receptors. Hence, staining a target cell with an IFA specific for it causes the cell to fluoresce and “stand out” from background, unstained material when illuminated. Filters are examined under epifluorescence microscopy. When the organism(s) of interest are specifically stained, they fluoresce against the background. In order to retain organisms as small as bacteria, the filter used for capture must have a small pore size, typically 0.22-0.45 microns. The volume filtered is limited to several liters maximum as membranes quickly clog with particulates. Microscopic scanning for bacteria is labor intensive, requiring a trained microscopist and a large non-portable epifluorescence-equipped microscope.
Direct detection may also be accomplished using flow cytometry. Flow cytometry is a commonly used technique to measure the chemical or physical properties of cells. Cells flow by a measuring apparatus in single file while suspended in a fluid, usually air or water. In immunofluorescence flow cytometry, cells can be identified by attaching fluorescent antibodies to each cell:
An antibody specific to the cell of interest is labeled with a fluorescent molecule or fluorochrome.
The labeled antibody is mixed in solution with the cell of interest. The antibodies attach to specific sites on the cells (called antigens).
The cells are passed in single file in a stream of liquid past a laser(s), which illuminates the fluorochromes and causes them to fluoresce at a different wavelength.
A photomultiplier or photodiode is used to detect a burst of fluorescence emission each time a marked cell passes in front of the detector.
The number of marked cells can then be counted. Antibodies can be chosen that are highly-specific to the cell(s) of interest.
Flow cytometry is currently used for a wide variety of applications including: measuring helper T-lymphocyte counts to monitor HIV treatment, measuring tumor cell DNA content in determining cancer treatment, and separating X- and Y-chromosome bearing sperm for animal breeding.
FIG. 1
(prior art) shows a typical flow cytometry system (from Shapiro, Practical Flow Cytometry, 2nd Edition). Putting flow cytometry into practice involves using two concentric cylindrical streams of fluid. The inner flow or core flow contains the cells to be sampled. The purpose of the outer stream or sheath flow is to reduce the diameter of the core flow. As the core and sheath fluids reach the tapered region of the flow, the cross-sectional area of the core flow is reduced. A small bore core flow (~20 &mgr;m) allows for precision photometric measurements of cells in the flow, illuminated by a small diameter laser beam; all of the cells will pass through nearly the same part of the beam and will be equally illuminated. Why not just pass the cells through a small-bore transparent tube? Small diameter orifices are generally unworkable because they experience frequent clogging. All commercial flow cytometers now use a sheath/core flow arrangement.
Laser-induced fluorescence of fluorescent labels in a flow cytometer is a uniquely powerful method of making fast, reliable, and relatively unambiguous detections of specific microorganisms, such as foodborne pathogens. Several monographs describe the methods and applications of flow cytometry (e.g., Flow Cytometry: First Principles by A. L. Givan, 1992, and references therein). The successful detection of single cells relies on several critical factors. First, the laser power must be sufficient to generate a large enough number of fluorescence photons during the brief passage of the labeled microbial cell through the irradiated region. Specifically, it is essential that the number of photons generated be large enough so that the fluorescence burst can be reliably differentiated from random fluctuations in the number of background photons. Second, reducing the background is important, i.e., minimizing the number of unwanted photons that strike the detector which arise from scattering and fluorescence of the apparatus and of impurities and unbound dye in the flowing liquid.
Historically, flow cytometers have been very large, expensive laboratory-based instruments. They consume large amounts of power, and use complex electronics. They are not typically considered within the realm of portable devices. The size (desktop at the smallest), power requirements, and susceptibility to clogging (requiring operator intervention) of conventional flow cytometers precludes their use for many applications, such as field monitoring of water biocontamination.
A technique called solid-phase cytometry is more rapid than flow cytometry. In this method, the fluid matrix is concentrated and then filtered. Fluorescently labeled microorganisms are then imaged on a filter membrane with a laser scanning system, detected, and enumerated. Using fluorescently tagged antibodies, this technique can detect specific microorganisms, and is highly suited for low concentrations of microorganisms. However, the high cost of this system (>$100,000) makes its use problematic for many applications, especially those requiring continuous on-site monitoring.
U.S. Pat. No. 6,309,886, “High throughput analysis of samples in flowing liquid,” by Ambrose et al. is an invention for the high throughput analysis of fluorescently labeled DNA in a transparent medium. This invention is a device that detects cells in a flow moving toward an imaging device. The flow is in a transparent tube illuminated in the focal plane from the side by a laser with a highly elongated beam. Although this invention does not suffer from the drawbacks listed above for alternative technologies, it is not suitable for applications where the flow medium is not transparent.
A need remains in the art for improved apparatus and methods for high throughput analysis of samples in a translucent flowing liquid.
SUMMARY OF THE INVENTION
The present invention has the adv
Bales Jennifer L.
Macheledt Bales & Heidmiller LLP
Stafira Michael P.
University of Wyoming
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