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US5817462A
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Method for simultaneous detection of multiple fluorophores for in situ hybridization and multicolor chromosome painting and banding
Assignee: Applied Spectral Imaging Ltd
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Filing: 1996-04-22
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Granted: 1998-10-06
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Expires: 2016-04-22
Abstract
Abstract A spectral imaging method for simultaneous detection of multiple fluorophores aimed at detecting and analyzing fluorescent in situ hybridizations employing numerous chromosome paints and/or loci specific probes each labeled with a different fluorophore or a combination of fluorophores for color karyotyping, and at multicolor chromosome banding, wherein each chromosome acquires a specifying banding pattern, which pattern is established using groups of chromosome fragments labeled with various fluorophore or combinations of fluorophores.
Inventors
- 👤 Yuval Garini
- 👤 Dario Cabib
- 👤 Robert A. Buckwald
- 👤 Thomas Ried
- 👤 Dirk G. Soenksen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 22:08
CPC Classifications
G; G01; G01N; G01N21/00; G01N21/62; G01N21/63; G01N21/64; G01N21/645; G01N21/6452; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6841; C12Q1/6876; C12Q1/6883; G01J; G01J3/00; G01J3/12; G01J3/1256; G01J3/26; G01J3/28; G01J3/2823; G01J3/44; G01J3/4406; G01J3/45; G01J3/453; G01N21/6428; G01N21/6456; G01N21/6458; 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; C12Q2600/00; C12Q2600/156; G01J2003/2866; G01J3/02; G01N2021/6417; G01N2021/6439; G01N2021/6441
Claims Analysis
Total Claims: 85
Claim 1
Independent Claim
370 words
A fluorescent in situ hybridization method comprising the steps of: (a) providing a cell nucleus having chromosomes, said chromosomes being hybridized with at least one nucleic acid probe being labeled with at least one fluorophore; (b) viewing said cell nucleus 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, wherein said obtainment of said spectrum of each pixel of said cell nucleus is by: (i) collecting incident light simultaneously from all pixels of said cell nucleus 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 nucleus for the entire duration of the measurement, so that the real image of the cell nucleus 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) scanning 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 nucleus; 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; wherein said method is for detecting chromosomal aberrations.
Claim 2
Dependent Claim
370 words
A fluorescent in situ hybridization method comprising the steps of: (a) providing a cell nucleus having chromosomes, said chromosomes being hybridized with at least one nucleic acid probe being labeled with at least one fluorophore; (b) viewing said cell nucleus 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, wherein said obtainment of said spectrum of each pixel of said cell nucleus is by: (i) collecting incident light simultaneously from all pixels of said cell nucleus 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 nucleus for the entire duration of the measurement, so that the real image of the cell nucleus 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) scanning 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 nucleus; 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; wherein said method is for detecting chromosomal aberrations.
Claim 3
Dependent Claim
32 words
The method as in claim 1, wherein said fluorescence microscope is supplemented with a filter selected from the group consisting of a double band dichroic filter and a triple band dichroic filter.
Claim 4
Dependent Claim
84 words
The method as in claim 1, wherein said at least one nucleic acid probe 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 5
Dependent Claim
29 words
The method as in claim 1, wherein said labeling of said nucleic acid probes is by a strategy selected from the group consisting of combinatorial labeling and combinatorial hybridization.
Claim 6
Dependent Claim
33 words
The method as in claim 1, wherein said cell nucleus is selected from the group consisting of a cell nucleus during interphase, a cell nucleus during mitosis and a cell nucleus during meiosis.
Claim 7
Dependent Claim
69 words
The method as in claim 1, 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 fluorophores.
Claim 8
Dependent Claim
25 words
The method as in claim 1, wherein said chromosomes are selected from the group consisting of interphase chromosomes, chromosomes during mitosis and chromosomes during meiosis.
Claim 9
Dependent Claim
27 words
The method as in claim 1, wherein said mathematical algorithm is a point operation analysis of said spectrum of each of said pixels in said cell nucleus.
Claim 10
Dependent Claim
32 words
The method as in claim 8, wherein said point operation analysis includes mapping said spectrum of each of said pixels in said cell nucleus into a scalar according to a transformation function.
Claim 11
Dependent Claim
32 words
The method as in claim 8, wherein said point operation analysis includes mapping said spectrum of each of said pixels of said cell nucleus into another spectrum according to a transformation function.
Claim 12
Dependent Claim
31 words
The method as in claim 1, wherein said mathematical algorithm is a morphological analysis, said morphological analysis determines the relative size of s a i d chromosomes in said cell nucleus.
Claim 13
Dependent Claim
33 words
The 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 at least one reference spectrum.
Claim 14
Dependent Claim
42 words
The method as in claim 12, 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 15
Dependent Claim
44 words
The method as in claim 13, 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 16
Dependent Claim
15 words
The method as in claim 1, wherein said mathematical algorithm is a principal component analysis.
Claim 17
Dependent Claim
71 words
The method as in claim 15, 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 ba se elements; and (c) finding which of said base elements or a combination thereof tag certain features in said cell nucleus.
Claim 18
Dependent Claim
15 words
The method as in claim 1, wherein said mathematical algorithm is a linear combination analysis.
Claim 19
Dependent Claim
15 words
The method as in claim 17, wherein said linear combination analysis is for spectral normalization.
Claim 20
Dependent Claim
47 words
The method as in claim 17, 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 21
Dependent Claim
43 words
The method as in claim 17, wherein said linear combination analysis is for background subtraction in which a spectrum of a pixel located in a background region of said cell nucleus is subtracted from said. spectra of said pixels of said cell nucleus.
Claim 22
Dependent Claim
41 words
The method as in claim 17, wherein said linear combination analysis is for a calibration procedure in which a spectrum measured prior to said viewing said c ell nucleus is for dividing said spectra of said pixels of said cell nucleus.
Claim 23
Dependent Claim
19 words
The method as in claim 1, wherein said mathematical algorithmn computes a Red-Green-Blue color image using predefined wavelength ranges.
Claim 24
Dependent Claim
18 words
The method as in claim 22, wherein said Red-Green-Blue color image is modified by a contrast stretching algorithm.
Claim 25
Dependent Claim
27 words
The 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 26
Dependent Claim
45 words
The 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 27
Dependent Claim
51 words
The method as in claim 1, wherein said method is for an application selected from the group consisting of providing a color karyotype of embryonic cells, providing a color karyotype of white blood cells, providing a color karyotype of malignant cells and providing a color karyotype of cells examined for malignancy.
Claim 28
Dependent Claim
30 words
The method as in claim 26, wherein said embryonic cells are selected from the group consisting of chorionic villi cells and embryonic cells isolated from a pregnant woman peripheral blood.
Claim 29
Dependent Claim
57 words
The method as in claim 27, wherein said method is for detecting a trisomy of a genetic material selected from the group consisting of human chromosome 21, human chromosomal band 21q22, a fragment of human chromosomal band 21q22, human chromosome 18, a fragment of human chromosome 18, human chromosome 13 and a fragment of human chromosome 13.
Claim 30
Dependent Claim
25 words
The method as in claim 26, wherein said providing said color karyotype of said cells examined for malignancy is for obtaining a color translocation map.
Claim 31
Dependent Claim
23 words
The method as in claim 26, wherein said providing said color karyotype of said malignant cells is for obtaining a color translocation map.
Claim 32
Dependent Claim
21 words
The method as in claim 1, wherein said chromosomal aberrations are selected from the group consisting of deletions, translocations and amplifications.
Claim 33
Dependent Claim
27 words
The method as in claim 1, wherein said method is for detecting chromosomal aberrations selected from the group consisting of recurrent chromosomal aberrations and secondary chromosomal aberrations.
Claim 34
Dependent Claim
17 words
The method as in claim 1, wherein said method is for detecting the stage of a malignancy.
Claim 35
Dependent Claim
23 words
The method as in claim 33, wherein said detection of the stage of said malignancy is for selecting a treatment for said malignancy.
Claim 36
Dependent Claim
18 words
The method as in claim 1, wherein said method is for following the efficiency of an anti-cancer treatment.
Claim 37
Dependent Claim
19 words
The method as in claim 1, wherein said method is for detecting chromosomal aberrations following exposure to mitogenic agents.
Claim 38
Dependent Claim
30 words
The method as in claim 1, wherein said chromosomes are stained with a conventional chromosome banding dye, the method further comprising obtaining a gray level banding image of said chromosomes.
Claim 39
Dependent Claim
14 words
The method as in claim 37, wherein said conventional chromosome banding dye is DAPI.
Claim 40
Dependent Claim
14 words
The method as in claim 1, wherein the method is for comparative genome hybridization.
Claim 41
Independent Claim
402 words
A method for hybridization based multicolor chromosome banding comprising the steps of: (a) providing a cell nucleus of a first species having a genome and chromosomes, said chromosomes being hybridized with at least one group of chromosome fragments covering at least a fraction of said genome of said first species, each of said at least one group of chromosome fragments being labeled with at least one fluorophore; (b) viewing said cell nucleus 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 nucleus, wherein said obtainment of said spectrum of each pixel of said cell nucleus is by: (i) collecting incident light simultaneously from all pixels of said cell nucleus 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 nucleus for the entire duration of the measurement, so that the real image of the cell nucleus 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) scanning 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 nucleus; 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; wherein said method is for detecting chromosomal aberrations.
Claim 42
Independent Claim
402 words
A method for hybridization based multicolor chromosome banding comprising the steps of: (a) providing a cell nucleus of a first species having a genome and chromosomes, said chromosomes being hybridized with at least one group of chromosome fragments covering at least a fraction of said genome of said first species, each of said at least one group of chromosome fragments being labeled with at least one fluorophore; (b) viewing said cell nucleus 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 nucleus, wherein said obtainment of said spectrum of each pixel of said cell nucleus is by: (i) collecting incident light simultaneously from all pixels of said cell nucleus 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 nucleus for the entire duration of the measurement, so that the real image of the cell nucleus 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) scanning 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 nucleus; 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; wherein said method is for detecting chromosomal aberrations.
Claim 43
Dependent Claim
32 words
The method as in claim 40, wherein said fluorescence microscope is supplemented with a filter selected from the group consisting of a double band dichroic filter and a triple band dichroic filter.
Claim 44
Dependent Claim
41 words
The method as in claim 40, wherein said chromosome fragments are from a source selected from the group consisting of radiation hybrid cell lines, YAC-clones, BAC-clones, size separated endonuclease digestion products of said genome of said species and microdissected chromosome fragments.
Claim 45
Dependent Claim
28 words
The method as in claim 40, wherein said labeling of said chromosome fragments is by a strategy selected from the group consisting of combinatorial labeling and combinatorial hybridization.
Claim 46
Dependent Claim
17 words
The method as in claim 40, wherein said labeling of said fragments is by interspersed repetitive sequence--PCR.
Claim 47
Dependent Claim
13 words
The method as in claim 44, wherein said interspersed repetitive sequence is Alu.
Claim 48
Dependent Claim
18 words
The method as in claim 40, wherein said fluorophores are conjugated to a nucleotide or a nucleotide analog.
Claim 49
Dependent Claim
26 words
The method as in claim 40, wherein said fraction is selected from the group consisting of 10-20%, 21-30%, 31-40%, 41-50%, 51-60%, 61-70%, 71-80%, 81-90% and 91-100%.
Claim 50
Dependent Claim
20 words
The method as in claim 40, wherein said first species is selected from the group consisting of human and mouse.
Claim 51
Dependent Claim
15 words
The method as in claim 40, wherein said chromosome fragments are of a second species.
Claim 52
Dependent Claim
28 words
The method as in claim 40, wherein said chromosome fragments are grouped into groups, each of said groups is labeled with a different fluorophore or combination of fluorophores.
Claim 53
Dependent Claim
34 words
The method as in claim 40, wherein said cell nucleus is selected from the group consisting of a cell nucleus during interphase, a cell nucleus during mitosis and a cell nucleus during is meiosis.
Claim 54
Dependent Claim
25 words
The method as in claim 40, wherein said chromosomes are selected from the group consisting of interphase chromosomes, chromosomes during mitosis and chromosomes during meiosis.
Claim 55
Dependent Claim
27 words
The method as in claim 40, wherein said mathematical algorithm is a point operation analysis of said spectrum of each of said pixels in said cell nucleus.
Claim 56
Dependent Claim
32 words
The method as in claim 53, wherein said point operation analysis includes mapping said spectrum of each of said pixels in said cell nucleus into a scalar according to a transformation function.
Claim 57
Dependent Claim
32 words
The method as in claim 53, wherein said point operation analysis includes mapping said spectrum of each of said pixels of said cell nucleus into another spectrum according to a transformation function.
Claim 58
Dependent Claim
28 words
The method as in claim 40, wherein said mathematical algorithm is a morphological analysis, said morphological analysis determines the relative size of said chromosomes in said cell nucleus.
Claim 59
Dependent Claim
33 words
The method as in claim 40, 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 60
Dependent Claim
42 words
The method as in claim 57, 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 61
Dependent Claim
44 words
The method as in claim 58, 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 62
Dependent Claim
15 words
The method as in claim 59, wherein said mathematical algorithm is a principal component analysis.
Claim 63
Dependent Claim
69 words
The method as in claim 60, 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 nucleus.
Claim 64
Dependent Claim
15 words
The method as in claim 40, wherein said mathematical algorithm is a linear combination analysis.
Claim 65
Dependent Claim
15 words
The method as in claim 62, wherein said linear combination analysis is for spectral normalization.
Claim 66
Dependent Claim
47 words
The method as in claim 62, 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 67
Dependent Claim
43 words
The method as in claim 62, wherein said linear combination analysis is for background subtraction in which a spectrum of a pixel located in a background region of said cell nucleus is subtracted from said spectra of said pixels of said cell nucleus.
Claim 68
Dependent Claim
40 words
The method as in claim 62, wherein said linear combination analysis is for a calibration procedure in which a spectrum measured prior to said viewing said cell nucleus is for dividing said spectra of said pixels of said cell nucleus.
Claim 69
Dependent Claim
19 words
The method as in claim 40, wherein said mathematical algorithm computes a Red-Green-Blue color image using predefined wavelength ranges.
Claim 70
Dependent Claim
18 words
The method as in claim 67, wherein said Red-Green-Blue color image is modified by a contrast stretching algorithm.
Claim 71
Dependent Claim
27 words
The method as in claim 40, wherein said mathematical algorithm computes a ratio between intensities at two different wavelengths for each of said spectra of said pixels.
Claim 72
Dependent Claim
45 words
The method as in claim 40, 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 73
Dependent Claim
55 words
The method as in claim 40, wherein said method is for an application selected from the group consisting of providing a color banding karyotype of embryonic cells, providing a color banding karyotype of white blood cells, providing a color banding karyotype of malignant cells and providing a color banding karyotype of cells examined for malignancy.
Claim 74
Dependent Claim
30 words
The method as in claim 71, wherein said embryonic cells are selected from the group consisting of chorionic villi cells and embryonic cells isolated from a pregnant woman peripheral blood.
Claim 75
Dependent Claim
57 words
The method as in claim 72, wherein said method is for detecting a trisomy of a genetic material selected from the group consisting of human chromosome 21, human chromosomal band 21q22, a fragment of human chromosomal band 21q22, human chromosome 18, a fragment of human chromosome 18, human chromosome 13 and a fragment of human chromosome 13.
Claim 76
Dependent Claim
26 words
The method as in claim 72, wherein said providing said color banding karyotype of said cells examined for malignancy, is for obtaining a color translocation map.
Claim 77
Dependent Claim
24 words
The method as in claim 72, wherein said providing said color banding karyotype of said malignant cells is for obtaining a color translocation map.
Claim 78
Dependent Claim
22 words
The method as in claim 40, wherein said chromosomal aberrations are selected from the group consisting of deletions, inversions, translocations and amplifications.
Claim 79
Dependent Claim
27 words
The method as in claim 40, wherein said method is for detecting chromosomal aberrations selected from the group consisting of recurrent chromosomal aberrations and secondary chromosomal aberrations.
Claim 80
Dependent Claim
17 words
The method as in claim 40, wherein said method is for detecting the stage of a malignancy.
Claim 81
Dependent Claim
23 words
The method as in claim 78, wherein said detection of the stage of said malignancy is for selecting a treatment for said malignancy.
Claim 82
Dependent Claim
18 words
The method as in claim 40, wherein said method is for following the efficiency of an anti-cancer treatment.
Claim 83
Dependent Claim
19 words
The method as in claim 40, wherein said method is for detecting chromosomal aberrations following exposure to mitogenic agents.
Claim 84
Dependent Claim
30 words
The method as in claim 40, wherein said chromosomes are stained with a conventional chromosome banding dye, the method further comprising obtaining a gray level banding image of said chromosomes.
Claim 85
Dependent Claim
14 words
The method as in claim 82, wherein said conventional chromosome banding dye is DAPI.
References Cited (Backward Citations)
No citation records indexed.
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