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US5936731A
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Method for simultaneous detection of multiple fluorophores for in situ hybridization and chromosome painting
Assignee: Applied Spectral Imaging Ltd
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Filing: 1995-12-20
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Granted: 1999-08-10
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Expires: 2015-12-20
Abstract
Abstract A fluorescent in situ hybridization method comprising the steps of (a) providing a cell nuclei having chromosomes hybridized with at least one nucleic acid probe including at least one nucleic acid molecule labeled with at least one fluorophore; (b) viewing the cell nuclei through a fluorescence microscope optically connected to an imaging spectrometer for obtaining a spectrum of each pixel of the cell nuclei by (i) collecting incident collimated light simultaneously from all pixels of the cell nuclei; (ii) passing the incident collimated light through an interferometer system so that the light is first split into two coherent beams and then recombine to interfere and form an exiting light beam; (iii) focusing the exiting light beam on a detector having an array of detector elements, so that at each instant each of the elements is the image of one and always the same pixel for the entire duration of the measurement and so that each of the elements produces a signal which is a particular linear combination of light intensity emitted by the pixel at different wavelengths; (iv) rotating or translating one or more of the elements of the interferometer, so that the optical path difference is scanned simultaneously for all the pixels of the cell nuclei; and (v) recording signals of each of the detector elements as function of time using a recording device to form a first spectral cube of data; and (c) interpreting the first spectral cube of data using a mathematical algorithm.
Inventors
- 👤 Dario Cabib
- 👤 Robert A. Buckwald
- 👤 David Wine
- 👤 Yuval Garini
- 👤 Nir Katzir
- 👤 Dirk G. Soeknsen
- 👤 Moshe Lavi
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 12:06
CPC Classifications
G; G01; G01N; G01N21/00; G01N21/62; G01N21/63; G01N21/64; G01N21/645; G01N21/6456; G01N21/6458; A; A61; A61B; A61B3/00; A61B3/10; A61B3/12; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6841; C12Q1/6876; C12Q1/6883; G01B; G01B11/00; G01B11/02; G01B11/06; G01B11/0616; G01B11/0675; G01J; G01J3/00; G01J3/12; G01J3/1256; G01J3/26; G01J3/28; G01J3/2823; G01J3/44; G01J3/4406; G01J3/45; G01J3/453; G01J3/4535; G01N21/6428; G01N33/00; G01N33/48; G01N33/50; G01N33/5005; G01N33/53; G01N33/569; G01N33/56966; G01N33/58; G01N33/582; G06; G06V; G06V10/00; G06V10/88; G06V20/00; G06V20/60; G06V20/69; A61B3/1241; C12Q2600/00; C12Q2600/156; G01J2003/2866; G01J3/02; G01N2021/6417; G01N2021/6439; G01N2021/6441
Claims Analysis
Total Claims: 69
Claim 1
Independent Claim
334 words
A spectral bio-imaging method characterized by high spatial and high spectral resolutions, the method comprising the steps of: (a) preparing a sample to be spectrally imaged; (b) viewing said sample through an optical device, said optical device being optically connected to an imaging spectrometer, said optical device and said imaging spectrometer being for obtaining a spectrum of each pixel of said sample by: (i) collecting incident light simultaneously from all pixels of said sample using collimating optics; (ii) passing said incident collimated light through an interferometer system having a number of elements, so that said light is first split into two coherent beams which travel in different directions inside said interferometer and then said two coherent beams recombine to interfere with each other to form an exiting light beam; (iii) passing said exiting light beam through a focusing optical system which focuses said exiting light beam on a detector having a two-dimensional array of detector elements, so that at each instant each of said detector elements is the image of one and always the same pixel of said sample for the entire duration of the measurement, so that the real image of the sample is stationary on the plane of the detector array and at any time during the measurement the image is still visible and recognizable, and so that each of said detector elements produces a signal which is a particular linear combination of light intensity emitted by said pixel at different wavelengths, wherein said linear combination is a function of the instantaneous optical path difference; (iv) translating one or more of said elements of said interferometer system, so that said optical path difference between said two coherent beams generated by said interferometer system is scanned simultaneously for all said pixels of said sample; and (v) recording signals of each of said detector elements as function of time using a recording device to form a first spectral cube of data; and (c) interpreting said first spectral cube of data using a mathematical algorithm.
Claim 2
Dependent Claim
334 words
A spectral bio-imaging method characterized by high spatial and high spectral resolutions, the method comprising the steps of: (a) preparing a sample to be spectrally imaged; (b) viewing said sample through an optical device, said optical device being optically connected to an imaging spectrometer, said optical device and said imaging spectrometer being for obtaining a spectrum of each pixel of said sample by: (i) collecting incident light simultaneously from all pixels of said sample using collimating optics; (ii) passing said incident collimated light through an interferometer system having a number of elements, so that said light is first split into two coherent beams which travel in different directions inside said interferometer and then said two coherent beams recombine to interfere with each other to form an exiting light beam; (iii) passing said exiting light beam through a focusing optical system which focuses said exiting light beam on a detector having a two-dimensional array of detector elements, so that at each instant each of said detector elements is the image of one and always the same pixel of said sample for the entire duration of the measurement, so that the real image of the sample is stationary on the plane of the detector array and at any time during the measurement the image is still visible and recognizable, and so that each of said detector elements produces a signal which is a particular linear combination of light intensity emitted by said pixel at different wavelengths, wherein said linear combination is a function of the instantaneous optical path difference; (iv) translating one or more of said elements of said interferometer system, so that said optical path difference between said two coherent beams generated by said interferometer system is scanned simultaneously for all said pixels of said sample; and (v) recording signals of each of said detector elements as function of time using a recording device to form a first spectral cube of data; and (c) interpreting said first spectral cube of data using a mathematical algorithm.
Claim 3
Dependent Claim
22 words
A method as in claim 1, further comprising the step of: (d) displaying a map of said interpreted spectral cube of data.
Claim 4
Dependent Claim
14 words
A method as in claim 1, wherein said optical device is a fluorescence microscope.
Claim 5
Dependent Claim
18 words
A method as in claim 1, wherein said collimated light is a fluorescence light emitted from said sample.
Claim 6
Dependent Claim
19 words
A method as in claim 4, wherein said collimated light emitted from said sample is an administered probe fluorescence.
Claim 7
Dependent Claim
34 words
A method as in claim 1, wherein said light originates from a source selected from the group consisting of laser, white light, filtered light, ultraviolet light and a light having a small wavelength range.
Claim 8
Dependent Claim
20 words
A method as in claim 1, wherein said light originates from a multiplicity of light sources, said sources operate simultaneously.
Claim 9
Dependent Claim
20 words
A method as in claim 1, wherein said light originates from a multiplicity of light sources, said sources operate successively.
Claim 10
Dependent Claim
36 words
A method as in claim 1, wherein said two-dimensional array is selected from the group consisting of a video rate CCD, a cooled high dynamic range CCD, an intensified CCD and a time gated intensified CCD.
Claim 11
Dependent Claim
29 words
A method as in claim 1, wherein said sample is selected from the group consisting of a cell during interphase, a cell during mitosis and a cell during meiosis.
Claim 12
Dependent Claim
13 words
A method as in claim 10, wherein said cell is from a human.
Claim 13
Dependent Claim
32 words
A method as in claim 10, wherein said cell is selected from the group consisting of a cancerous cell, a blood cell, a fetal cell and a cell suspected of being malignant.
Claim 14
Dependent Claim
41 words
A method as in claim 1, wherein said sample is a cell, said light is induced by a probe, said probe binds to a specific cellular constituent, the method is for detecting the presence or the level of said cellular constituent.
Claim 15
Dependent Claim
26 words
A method as in claim 13, wherein said probe includes a conjugated fluorescent moiety and said induction is a fluorescence light emission of said fluorescent moiety.
Claim 16
Dependent Claim
36 words
A method as in claim 14, wherein said probe further includes a nucleic acid molecule, the method is for detecting the presence or the level of a cellular nucleic acid hybridizing with said nucleic acid molecule.
Claim 17
Dependent Claim
23 words
A method as in claim 15, wherein said cellular nucleic acid is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid.
Claim 18
Dependent Claim
32 words
A method as in claim 14, wherein said fluorescent moiety is selected from the group consisting of Aqua, Texas-Red, FITC, rhodamine, rhodamine derivative, fluorescein, fluorescein derivative, cascade blue and any combination thereof.
Claim 19
Dependent Claim
26 words
A method as in claim 1, wherein said mathematical algorithm is a point operation analysis of said spectrum of each of said pixels in said sample.
Claim 20
Dependent Claim
31 words
A method as in claim 18, wherein said point operation analysis includes mapping said spectrum of each of said pixels in said sample into a scalar according to a transformation function.
Claim 21
Dependent Claim
31 words
A method as in claim 18, wherein said point operation analysis includes mapping said spectrum of each of said pixels of said sample into another spectrum according to a transformation function.
Claim 22
Dependent Claim
14 words
A method as in claim 1, wherein said mathematical algorithm is a morphological analysis.
Claim 23
Dependent Claim
32 words
A method as in claim 1, wherein said mathematical algorithm is a similarity mapping analysis for computing for each of said pixels in said sample a spectral difference from a reference spectrum.
Claim 24
Dependent Claim
40 words
A method as in claim 22, wherein said similarity mapping analysis results in generating a gray level or a pseudocolor image, in which bright pixels correspond to a small spectral difference and dark pixels correspond to a large spectral difference.
Claim 25
Dependent Claim
40 words
A method as in claim 22, wherein said similarity mapping analysis results in generating a gray level or a pseudocolor image, in which bright pixels correspond to a large spectral difference and dark pixels correspond to a small spectral difference.
Claim 26
Dependent Claim
41 words
A method as in claim 22, wherein said spectral difference is a scalar defined as the integral over a predefined wavelength range of the absolute value of the difference between said spectrum of each of said pixels and said reference spectrum.
Claim 27
Dependent Claim
31 words
A method as in claim 1, wherein said mathematical algorithm is a classification mapping analysis computing for said spectrum of each of said pixels a spectral difference from several reference spectra.
Claim 28
Dependent Claim
42 words
A method as in claim 26, wherein said classification mapping analysis results in generating a multicolor image, in which groups of pixels having a predetermined maximal spectral differences from one of said several reference spectra are colored with a predetermined artificial color.
Claim 29
Dependent Claim
44 words
A method as in claim 26, wherein said spectral difference is a scalar defined as the integral over a predefined wavelength range of the absolute value of the difference between said spectrum of each of said pixels and one of said several reference spectra.
Claim 30
Dependent Claim
15 words
A method as in claim 1, wherein said mathematical algorithm is a principal component analysis.
Claim 31
Dependent Claim
68 words
A method as in claim 29, wherein said principal component analysis includes: (a) building a covariant matrix for all of said pixels and said wavelengths of said measurement, including wavelengths of exciting sources when multiple wavelengths are used; (b) diagonalizing said covariant matrix and finding all independent orthogonal spectral base elements; (c) finding which of said base elements or a combination thereof tag certain features in said sample.
Claim 32
Dependent Claim
15 words
A method as in claim 1, wherein said mathematical algorithm is a linear combination analysis.
Claim 33
Dependent Claim
49 words
A method as in claim 31, wherein said linear combination analysis includes applying an arithmetical function between corresponding wavelengths of corresponding pairs of pixels belonging to said first spectral cube of data and to a second spectral cube of data, to obtain a resulting third spectral cube of data.
Claim 34
Dependent Claim
34 words
A method as in claim 31, wherein said linear combination analysis is for a purpose selected from the group consisting of averaging two spectral cubes of data and time changes follow-up and spectral normalization.
Claim 35
Dependent Claim
47 words
A method as in claim 36, wherein said linear combination analysis includes applying a given scalar to every wavelength of said spectra of each of said pixels by an arithmetical function, said function is selected from the group consisting of addition, subtraction, multiplication, division and combinations thereof.
Claim 36
Dependent Claim
41 words
A method as in claim 31, wherein said linear combination analysis is for background subtraction in which a spectrum of a pixel located in a background region of said sample is subtracted from said spectra of said pixels of said sample.
Claim 37
Independent Claim
38 words
A method as in claims 31, wherein said linear combination analysis is for a calibration procedure in which a spectrum measured prior to said viewing said sample is for dividing said spectra of said pixels of said sample.
Claim 38
Dependent Claim
15 words
A method as in claim 1, wherein said mathematical algorithm is an optical density analysis.
Claim 39
Dependent Claim
23 words
A method as in claim 37, wherein said optical density analysis is for obtaining an interpreted image which is an optical density map.
Claim 40
Dependent Claim
19 words
A method as in claim 1, wherein said mathematical algorithm computes a Red-Green-Blue color image using predefined wavelength ranges.
Claim 41
Dependent Claim
18 words
A method as in claim 39, wherein said Red-Green-Blue color image is modified by a contrast stretching algorithm.
Claim 42
Dependent Claim
27 words
A method as in claim 1, wherein said mathematical algorithm computes a ratio between intensities at two different wavelengths for each of said spectra of said pixels.
Claim 43
Dependent Claim
45 words
A method as in claim 1, wherein said mathematical algorithm computes a ratio between intensities at two different wavelengths for each of said spectra of said pixels and paints each of said pixels in a lighter or darker artificial color, according to said computed ratio.
Claim 44
Dependent Claim
20 words
A method as in claim 1, wherein the method is for spectral identification of multiple fluorophores administered to said sample.
Claim 45
Dependent Claim
376 words
A fluorescent in situ hybridization method comprising the steps of: (a) providing a cell nuclei having chromosomes, said chromosomes being hybridized with at least one nucleic acid probe, each of said at least one nucleic acid probe including at least one nucleic acid molecule, each of said at least one nucleic acid molecule being labeled with at least one fluorophore; (b) viewing said cell nuclei through a fluorescence microscope, said fluorescence microscope being optically connected to an imaging spectrometer, said fluorescence microscope and said imaging spectrometer being for obtaining a spectrum of each pixel of said cell nuclei by: (i) collecting incident light simultaneously from all pixels of said cell nuclei using collimating optics; (ii) passing said incident collimated light through an interferometer system having a number of elements, so that said light is first split into two coherent beams which travel in different directions inside said interferometer and then said two coherent beams recombine to interfere with each other to form an exiting light beam; (iii) passing said exiting light beam through a focusing optical system which focuses said exiting light beam on a detector having a two-dimensional array of detector elements, so that at each instant each of said detector elements is the image of one and always the same pixel of said cell nuclei for the entire duration of the measurement, so that the real image of the cell nuclei is stationary on the plane of the detector array and at any time during the measurement the image is still visible and recognizable, and so that each of said detector elements produces a signal which is a particular linear combination of light intensity emitted by said pixel at different wavelengths, wherein said linear combination is a function of the instantaneous optical path difference; (iv) rotating or translating one or more of said elements of said interferometer system, so that said optical path difference between said two coherent beams generated by said interferometer system is scanned simultaneously for all said pixels of said cell nuclei; and (v) recording signals of each of said detector elements as function of time using a recording device to form a first spectral cube of data; and (c) interpreting said first spectral cube of data using a mathematical algorithm.
Claim 46
Dependent Claim
376 words
A fluorescent in situ hybridization method comprising the steps of: (a) providing a cell nuclei having chromosomes, said chromosomes being hybridized with at least one nucleic acid probe, each of said at least one nucleic acid probe including at least one nucleic acid molecule, each of said at least one nucleic acid molecule being labeled with at least one fluorophore; (b) viewing said cell nuclei through a fluorescence microscope, said fluorescence microscope being optically connected to an imaging spectrometer, said fluorescence microscope and said imaging spectrometer being for obtaining a spectrum of each pixel of said cell nuclei by: (i) collecting incident light simultaneously from all pixels of said cell nuclei using collimating optics; (ii) passing said incident collimated light through an interferometer system having a number of elements, so that said light is first split into two coherent beams which travel in different directions inside said interferometer and then said two coherent beams recombine to interfere with each other to form an exiting light beam; (iii) passing said exiting light beam through a focusing optical system which focuses said exiting light beam on a detector having a two-dimensional array of detector elements, so that at each instant each of said detector elements is the image of one and always the same pixel of said cell nuclei for the entire duration of the measurement, so that the real image of the cell nuclei is stationary on the plane of the detector array and at any time during the measurement the image is still visible and recognizable, and so that each of said detector elements produces a signal which is a particular linear combination of light intensity emitted by said pixel at different wavelengths, wherein said linear combination is a function of the instantaneous optical path difference; (iv) rotating or translating one or more of said elements of said interferometer system, so that said optical path difference between said two coherent beams generated by said interferometer system is scanned simultaneously for all said pixels of said cell nuclei; and (v) recording signals of each of said detector elements as function of time using a recording device to form a first spectral cube of data; and (c) interpreting said first spectral cube of data using a mathematical algorithm.
Claim 47
Dependent Claim
84 words
A method as in claim 44, wherein said at least one nucleic acid molecule is selected from the group consisting of at least one locus, at least one fragmented chromosome, at least one yeast artificial chromosome including an insert, at least one plasmid including an insert, at least one cosmid including an insert, at least one phagemid including an insert, at least one viral vector including an insert, a complete genome of a species, a complete genome of a cancerous tissue and combinations thereof.
Claim 48
Dependent Claim
19 words
A method as in claim 44, wherein said at least one fluorophore is at least one fluorescent combinatorial dye.
Claim 49
Dependent Claim
33 words
A method as in claim 44, wherein said cell nuclei is selected from the group consisting of a cell nuclei during interphase, a cell nuclei during mitosis and a cell nuclei during meiosis.
Claim 50
Dependent Claim
70 words
A method as in claim 44, wherein the number of nucleic acid probes is selected from the group of numbers consisting of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty one, twenty two, twenty three, twenty four and higher than twenty four, each of said probes includes a different fluorophore or a different combination of said fluorophores.
Claim 51
Dependent Claim
25 words
A method as in claim 44, wherein said chromosomes are selected from the group consisting of interphase chromosomes, chromosomes during mitosis and chromosomes during meiosis.
Claim 52
Dependent Claim
27 words
A method as in claim 44, wherein said mathematical algorithm is a point operation analysis of said spectrum of each of said pixels in said cell nuclei.
Claim 53
Dependent Claim
32 words
A method as in claim 50, wherein said point operation analysis includes mapping said spectrum of each of said pixels in said cell nuclei into a scalar according to a transformation function.
Claim 54
Dependent Claim
32 words
A method as in claim 51, wherein said point operation analysis includes mapping said spectrum of each of said pixels of said cell nuclei into another spectrum according to a transformation function.
Claim 55
Dependent Claim
28 words
A method as in claim 44, wherein said mathematical algorithm is a morphological analysis, said morphological analysis determines the relative size of said chromosomes in said cell nuclei.
Claim 56
Dependent Claim
33 words
A method as in claim 44, wherein said mathematical algorithm is a classification mapping analysis computing for said spectrum of each of said pixels a spectral difference from at least one reference spectrum.
Claim 57
Dependent Claim
42 words
A method as in claim 54, wherein said classification mapping analysis results in generating a multicolor image, in which groups of pixels having a predetermined maximal spectral differences from one of said several reference spectra are colored with a predetermined artificial color.
Claim 58
Dependent Claim
44 words
A method as in claim 55, wherein said spectral difference is a scalar defined as the integral over a predefined wavelength range of the absolute value of the difference between said spectrum of each of said pixels and one of said several reference spectra.
Claim 59
Dependent Claim
15 words
A method as in claim 44, wherein said mathematical algorithm is a principal component analysis.
Claim 60
Dependent Claim
69 words
A method as in claim 57, wherein said principal component analysis includes: (a) building a covariant matrix for all of said pixels and said wavelengths of said measurement, including wavelengths of exciting sources when multiple wavelengths are used; (b) diagonalizing said covariant matrix and finding all independent orthogonal spectral base elements; (c) finding which of said base elements or a combination thereof tag certain features in said cell nuclei.
Claim 61
Dependent Claim
15 words
A method as in claim 44, wherein said mathematical algorithm is a linear combination analysis.
Claim 62
Dependent Claim
15 words
A method as in claim 59, wherein said linear combination analysis is for spectral normalization.
Claim 63
Dependent Claim
47 words
A method as in claim 59, wherein said linear combination analysis includes applying a given scalar to every wavelength of said spectra of each of said pixels by an arithmetical function, said function is selected from the group consisting of addition, subtraction, multiplication, division and combinations thereof.
Claim 64
Dependent Claim
43 words
A method as in claim 59, wherein said linear combination analysis is for background subtraction in which a spectrum of a pixel located in a background region of said cell nuclei is subtracted from said spectra of said pixels of said cell nuclei.
Claim 65
Dependent Claim
40 words
A method as in claim 59, wherein said linear combination analysis is for a calibration procedure in which a spectrum measured prior to said viewing said cell nuclei is for dividing said spectra of said pixels of said cell nuclei.
Claim 66
Dependent Claim
19 words
A method as in claim 44, wherein said mathematical algorithm computes a Red-Green-Blue color image using predefined wavelength ranges.
Claim 67
Dependent Claim
18 words
A method as in claim 64, wherein said Red-Green-Blue color image is modified by a contrast stretching algorithm.
Claim 68
Dependent Claim
27 words
A method as in claim 44, wherein said mathematical algorithm computes a ratio between intensities at two different wavelengths for each of said spectra of said pixels.
Claim 69
Dependent Claim
45 words
A method as in claim 44, wherein said mathematical algorithm computes a ratio between intensities at two different wavelengths for each of said spectra of said pixels and paints each of said pixels in a lighter or darker artificial color, according to said computed ratio.
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