Optics: measuring and testing – Velocity or velocity/height measuring – With light detector
Reexamination Certificate
2001-08-24
2003-03-11
Buczinski, Stephen C. (Department: 3662)
Optics: measuring and testing
Velocity or velocity/height measuring
With light detector
C356S028500, C356S039000
Reexamination Certificate
active
06532061
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally relates to a method and apparatus for measuring the velocity of an object, and more specifically, to sensing light from an object with a light sensitive detector, the amplitude of that light signal having been modulated by an optical grating, and measuring the velocity of the object by analysis of the modulated light signal.
BACKGROUND OF THE INVENTION
Cells and cell groupings are three-dimensional objects containing rich spatial information. The distribution of a tremendous variety of bio-molecules can be identified within a cell using an ever-increasing number of probes. In the post-genome era there is mounting interest in understanding the cell, not only as a static structure, but as a dynamic combination of numerous interacting feedback control systems. This understanding can lead to new drugs, better diagnostics, more effective therapies, and better health care management strategies. However, this understanding will require the ability to extract a far greater amount of information from cells than is currently possible.
The principal technologies for cellular analysis are automated microscopy and flow cytometry. The information generated by these mature technologies, although useful, is often not as detailed as desired. Automated microscopy allows two-dimensional (2D) imaging of from one to three colors of cells on slides. Typical video frame rates limit kinetic studies to time intervals of 30 ms.
Instruments known as flow cytometers currently provide vital information for clinical medicine and biomedical research by performing optical measurements on cells in liquid suspension. Whole blood, fractionated components of blood, suspensions of cells from biopsy specimens and from cell cultures, and suspensions of proteins and nucleic acid chains are some of the candidates suitable for analysis by flow cytometry. In flow cytometers specialized for routine blood sample analysis, cell type classification is performed by measuring the angular distribution of light scattered by the cells and the absorption of light by specially treated and stained cells. The approximate numbers of red blood cells, white blood cells of several types, and platelets are reported as the differential blood count. Some blood-related disorders can be detected as shifts in optical characteristics, as compared to baseline optical characteristics, such shifts being indicative of morphological and histochemical cell abnormalities. Flow cytometers have been adapted for use with fluorescent antibody probes, which attach themselves to specific protein targets, and for use with fluorescent nucleic acid probes, which bind to specific DNA and RNA base sequences by hybridization. Such probes find application in medicine for the detection and categorization of leukemia, for example, in biomedical research, and drug discovery. By employing such prior art techniques, flow cytometry can measure four to ten colors from living cells. However, such prior art flow cytometry offers little spatial resolution, and no ability to study a cell over time. There is clearly a motivation to address the limitations of existing cell analysis technologies with a novel platform for high speed, high sensitivity cell imaging.
A key issue that arises in cell analysis carried out with imaging systems is the measurement of the velocity of a cell or other object through the imaging system. In a conventional time-domain methodology, cell velocity is measured using time-of-flight (TOF). Two detectors are spaced a known distance apart and a clock measures the time it takes a cell to traverse the two detectors. The accuracy of a TOF measurement is enhanced by increasing detector spacing. However, this increases the likelihood that multiple cells will occupy the measurement region, requiring multiple timers to simultaneously track all cells in view. Initially, the region between the detectors is cleared before starting sample flow. As cells enter the measurement region, each entry signal is timed separately. The system is synchronized with the sample by noting the number of entry signals that occur before the first exit signal.
TOF velocity measurement systems are prone to desynchronization when the entry and exit signals are near threshold, noise is present, or expected waveform. characteristics change due to the presence of different cell types and orientations. Desynchronization causes errors in velocity measurement which can lead to degraded signals and misdiagnosed cells until the desynchronized condition is detected and corrected. Resynchronization may require that all cells be cleared from the region between the detectors before restarting sample flow, causing the loss of sample.
Significant advancements in the art of flow cytometry are described in commonly assigned U.S. Pat. No. 6,249,341, issued on Jun. 19, 2001, and entitled IMAGING AND ANALYZING PARAMETERS OF SMALL MOVING OBJECTS SUCH AS CELLS, as well as in commonly assigned U.S. Pat. No. 6,211,955, issued on Apr. 3, 2001, also entitled IMAGING AND ANALYZING PARAMETERS OF SMALL MOVING OBJECTS SUCH AS CELLS. The specifications and drawings of each of these patents are hereby specifically incorporated herein by reference.
The inventions disclosed in the above noted patents perform high resolution, high-sensitivity two-dimensional (2D) and three-dimensional (3D) imaging using time-delay-integration (TDI) electronic image acquisition with cells in flow. These instruments are designed to expand the analysis of biological specimens in fluid suspensions beyond the limits of conventional flow cytometers. TDI sensors utilize solid-state photon detectors such as charge-coupled device (CCD) arrays and shift lines of photon-induced charge in synchronization with the flow of the specimen. The method allows a long exposure time to increase a signal-to-noise ratio (SNR) in the image while avoiding blurring. However, precise synchronization of the TDI detector timing with the motion of the moving targets is required. For example, if a target is to traverse 100 lines of a TDI sensor to build an image, and the blurring is expected to be less than a single line width, then the velocity of the target must be known to less than one percent of its actual value. It would thus be desirable to provide method and apparatus capable of producing highly accurate flow velocity for such moving targets.
Several methods have been suggested in the prior art to address the limitations of TOF velocity measurements, and to achieve highly accurate flow velocity for moving targets such as cells. One such technique is laser Doppler anemometry (LDA), in which one or more laser beams are used to interrogate a moving target. The Doppler frequency shift is detected as modulation from the interference of multiple beams that have traversed different paths in the apparatus. An example of such an apparatus is disclosed in U.S. Pat. No. 3,832,059, issued on Aug. 27, 1974, and entitled FLOW VELOCITY MEASURING ARRANGEMENT UTILIZING LASER DOPPLER PROBE. That apparatus employs two laser beams that are directed toward a moving target at converging angles. The back-scattered light from both beams is collected and focused on a common photodetector. Coherent interference at the detector generates modulation with frequency equal to twice the Doppler shift frequency, allowing the detection of the target velocity. While LDA is a functional technique, the LDA systems described in U.S. Pat. No. 3,832,059 are elaborate, expensive, and prone to instability.
In an attempt to overcome the problem of instabilities in the laser wavelength, an improved LDA apparatus is disclosed in U.S. Pat. No. 5,229,830, issued on Jul. 20, 1993, and entitled DOPPLER VELOCIMETER. In this improved apparatus, a rotating grating is added for the purpose of extracting diffraction side-lobes for use in interrogating the target. This approach eliminates the wavelength dependency in the velocity measurement, but adds even more cost and complexity to the velocimeter.
An alternative approach to measuring object velocity b
Basiji David A.
Bauer Richard A.
Frost Keith L.
Ortyn William E.
Perry David J.
Amnis Corporation
Anderson Ronald M.
Buczinski Stephen C.
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