1. Field of the Invention
The present invention relates to apparatus and methods for high resolution imaging and classification 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
Imaging and classification of low concentrations of selected target particles, cells in particular, 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 cancerous cells, and 4) environmental monitoring. Cell imaging and classification systems developed to date usually suffer from 1) high cost, 2) unsatisfactory sensitivity, 3) slowness, 4) large size, 5) insufficient spectral and/or spatial resolution, and/or 6) labor-intensive preparation steps.
Direct detection may 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 (about 20 microns) 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 food-borne 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).
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.
U.S. Pat. No. 6,309,886,“High throughput analysis of samples in flowing liquid,” by Ambrose et al. discloses 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. It is also not an imaging technology, but rather a technology suitable for single-point photometric detection and characterization.
U.S. Pat. No. 6,473,176 (Baseji et al.), U.S. Pat. No. 6,249,341 (Baseji et al.), and U.S. Pat. No. 6,211,955 (Baseji et al.) describe a method to perform multi-spectral imaging of cells in a sample flowing in a flow cytometer using a technique called Time Delayed Integration or TDI. TDI is used with charge coupled device (CCD) detectors to produced enhanced signal-to-noise images of a moving scene (such as cells in a flow). The pixels of the CCD are arranged in rows and columns, and the signal is moved from row to row in synchrony with a moving image projected onto the device, allowing an extended integration time without blurring. Time Delayed Integration Multi-spectral flow cytometry, as described in the three above-mentioned patents, has advantages over previous flow cytometric techniques in that it recovers not only the fluorescence and/or scattering parameters from cells in the flow, but provides multi-spectral imaging as well. The latter allows for cell classification and differentiation based characteristics such as cell shape, overall size, nuclear size, nuclear shape, optical density, the detection and characterization of numerous fluorescent markers and FISH probes, the quantification of the total amount of DNA in the nucleus, and the detection of other cellular components at multiple wavelengths. The main disadvantage of this technique is that it requires TDI as well as conventional flow cytometry with all of its complexities including a hydrodynamically focused sheath flow.
A precursor invention, described in U.S. patent application Ser. No. 10/323,535 by the present inventor, is shown in FIGS. 2 and 3. This invention incorporates detection, but not high resolution imaging.
In FIG. 2 (Prior Art), a sample of fluorescently tagged cells 210 flows up the tube 206 toward the CCD camera and foreoptics 208. The cells are illuminated in the focal plane by a laser 228 through transparent end element 220. When the cell(s) pass through the CCD camera focal plane 234 they are imaged by the CCD camera 218 and lens assembly 212, through a transparent window and a filter 214 that isolates the wavelength of fluorescent emission. The fluid in which the cells are suspended then passes by the window and out the drain tube 230.
In FIG. 3 (Prior Art), a flow block 322 is used with a device like that shown in FIG. 1. FIG. 3A is a side schematic drawing of the aluminum flow block. FIG. 3B is a top plan view of the flow black. FIG. 3C shows a detail of the device flow and imaging. The sample enters the flow block through Tygon tube 312 and stainless steel tube 310 and exits through stainless steel tube 324 and Tygon tube 315. Two 2-mm holes have been drilled into the aluminum flow block 322, an entrance hole 302 and an exit hole 306. As the sample flows up the internal entrance hole 302, it passes through the focal plane of the CCD camera 326. This hole is generally painted black to reduce scattered light. Component 320 is a Teflon tape gasket. The gasket is sandwiched between the aluminum flow block and a circular window 220, and tightly held with a screw-on brass cap 318. The gasket is cut to form a channel 304 through which the fluid is diverted into the exit hole 306. FIG. 3D is a photograph of a working flow block with attached tubing. The block is mounted onto a black-anodized plate.
A need remains in the art for improved apparatus and methods for high throughput, high resolution imaging analysis of samples in a translucent flowing liquid.