Image analysis – Pattern recognition – Feature extraction
Patent
1993-07-23
1996-01-16
Couso, Yon J.
Image analysis
Pattern recognition
Feature extraction
382133, 2504591, G06K 900
Patent
active
054855306
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention generally relates to a method and apparatus for providing fluorescence lifetime images and more particularly to a method and apparatus for providing multi-dimensional fluorescence lifetime imaging, using the phase-shift and/or modulation of the fluorescence signal. Also described is a method to suppress the fluorescence signal due to background and/or autofluorescence from the sample, or to visualize regions of the sample with decay times greater or less than a desired value.
2. Description of the Background Art
Quantitative fluorescence image analysis has application to a wide variety of practical arts including cellular physiology and biological and clinical research, immunology, chromosome analysis, environmental science, forensic analysis, fingerprint imaging and the like. However, significant problems have been encountered in such applications.
The availability of a variety of quantitative techniques for the evaluation of the chemical composition of specimens is of significant importance to the conduct of basic biological research and to a wide variety of clinical applications. Research involving the study and analysis of cells, generally known as cytology, employs a variety of analytical techniques for identifying and enumerating the subpopulations of cells in a specimen under study. For example, cytological materials may be examined to detect the presence of cancerous or malignant cells, or to determine the chemical composition of cells within a specimen. For purposes of analysis, the cells may be labeled with a variety of fluorescent materials, commonly known as probes or fluorophores, which have an identified affinity for cells or cell components which are of interest to an analysis. The probes will emit a particular fluorescence when stimulated by light at a known wavelength. The emitted light may have distinguishing characteristics, particularly wavelength and intensity, which permit an analysis of a subpopulation of cells or region of a cell to be conducted. The wavelength and intensity are dependent on the concentration of the analyte or cell component but, due to background or autofluorescence, measurements based on intensity or wavelength are limited to detection of analytes or compounds that are present at the highest concentrations in a cell or sample.
The study of collections of multiple cells using fluorescence spectroscopy has obvious additional problems such that an accurate determination of the number of cells in a subpopulation which have a given characteristic usually cannot be made since it is difficult to separate the subpopulations for analysis.
In one conventional approach using fluorescence microscopy (FM), specimens are tagged with fluorescent agents which bind or react with particular components of a specimen or cell component, and which are responsive to light emitted by a non-modulated light source at a characteristic wavelength to which the agent is sensitive.
When the fluorescent agent, bound to or reacted with a specimen, is excited by light at the agent's absorption wavelength, the energy level of an electron of the agent is raised above the relaxed or ground state to an excited state. Following excitation, the agent's electrons return to their relaxed state and emit light having a characteristic wavelength. Multiple agents may be used, each sensitive to light at the same or different wavelengths and each responsive to the stimulating light by emitting light at a characteristic wavelength that can be detected and used for analysis. Each agent can be responsive to various chemicals or biological molecules within the cell.
In a conventional FM system, as a particular fluorophore emits light following its excitation, a two-dimensional intensity image may be produced that is proportional to the local concentration of the fluorescent species having the characteristic wavelength which is detected. The areas having the highest intensity light emissions are detectable and identifiable as areas having t
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patent: 5093866 (1992-03-01), Douglas-Hamilton
patent: 5159397 (1992-10-01), Kosaica
Gratton et al. "Parallel Acquisition of Fluoresence Decay Using Array Detector", Proceedings of the SPIE, vol. 1204, 1, Oct. 1990, pp. 21-23.
McGown et al. "Phase Resolved Luminesence Spectroscopy", Analytical Chemistry, vol. 56, No. 13, 1, Nov. 1984, pp. 1400A-1402A.
Berndt Klaus W.
Johnson Michael L.
Lakowicz Joseph R.
Nowaczyk Kazimierz
Szmacinski Henryk
Couso Yon J.
Lakowicz Joseph R.
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