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US4745285A
Expired / Lapsed
US
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Multi-color fluorescence analysis with single wavelength excitation
Assignee: Becton Dickinson and Co
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Filing: 1986-08-21
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Granted: 1988-05-17
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Expires: 2006-08-21
Abstract
Abstract A method for determining one or more characteristics of particles using multiple fluorescence analysis comprises directing an incident light beam at the particles under analysis. The particles include at least three fluorescent markers each having different emission spectra. The incident light beam causes the excitation of the markers by light at a single wavelength whereby different wavelengths of fluorescence are emitted from the particles. Different fluorescence emissions associated with the particles under analysis are simultaneously detected. This method further includes associating the detected fluorescence with one or more characteristics of the particles. An apparatus is also part of the present invention for carrying out the aforementioned method.
Inventors
- 👤 Diether J. Recktenwald
- 👤 Chia H. Chen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 23:45
CPC Classifications
G; G01; G01N; G01N21/00; G01N21/62; G01N21/63; G01N21/64; G01N21/6428; G01N15/00; G01N15/10; G01N15/14; G01N15/1456; G01N15/1459; G01N2015/1006; G01N2015/1477; G01N2015/1479; G01N2021/6417; G01N2021/6421; G01N2021/6439; G01N2021/6441
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
160 words
A method for determining one or more characteristics of cells or the like using multiple fluorescence analysis comprising: moving cells, substantially one at a time, in a liquid flow stream, said cells including at least three fluorescent markers each having different emission spectra wherein the markers, when excited, produce emission maxima sufficiently spectrally separated to permit detection thereof without substantial spectral overlap; directing an incident light beam of a single wavelength at the cells in said flow stream to excite all of the fluorescent markers with the single wavelength of light such that different wavelengths of fluorescence are emitted from said cells; spectrally separating with optical elements the different wavelengths of emitted fluorescence to simultaneously refine the emitted fluorescence to enable the separate detection of the different wavelengths; simultaneously detecting the three or more different fluorescence emissions associated with the cells moving in the flow stream; and using said detected fluorescence to determine one or more characteristics of said cells.
Claim 2
Independent Claim
160 words
A method for determining one or more characteristics of cells or the like using multiple fluorescence analysis comprising: moving cells, substantially one at a time, in a liquid flow stream, said cells including at least three fluorescent markers each having different emission spectra wherein the markers, when excited, produce emission maxima sufficiently spectrally separated to permit detection thereof without substantial spectral overlap; directing an incident light beam of a single wavelength at the cells in said flow stream to excite all of the fluorescent markers with the single wavelength of light such that different wavelengths of fluorescence are emitted from said cells; spectrally separating with optical elements the different wavelengths of emitted fluorescence to simultaneously refine the emitted fluorescence to enable the separate detection of the different wavelengths; simultaneously detecting the three or more different fluorescence emissions associated with the cells moving in the flow stream; and using said detected fluorescence to determine one or more characteristics of said cells.
Claim 3
Dependent Claim
20 words
The method of claim 1 wherein the fluorescent markers on said cells are cell surface markers suitable for immunofluorescence analysis.
Claim 4
Dependent Claim
37 words
The method of claim 2 wherein there are at least four fluorescent markers associated with the cells, at least one of said markers being a dye for providing fluorescence analysis of the interior structure of the cells.
Claim 5
Dependent Claim
12 words
The method of claim 3 wherein said dye is a DNA dye.
Claim 6
Dependent Claim
26 words
The method of claim 4 wherein the spectral spacing between the emission maxima of adjacent fluorescent markers along the fluorescence spectrum is at least 15 nm.
Claim 7
Dependent Claim
24 words
The method of claim 1 wherein the light beam is provided by a laser which generates a wavelength of excitation light at 488 nm.
Claim 8
Dependent Claim
35 words
The method of claim 6 wherein the fluorescent markers are selected from the group of dyes consisting of fluorescein, phycoerythrin, Texas red, phycocyanin, allo-phycocyanin, peridinin-chlorophyll protein and dyes in tandem with any of the foregoing.
Claim 9
Dependent Claim
15 words
The method of claim 7 wherein there are four fluorescent markers selected from said group.
Claim 10
Dependent Claim
15 words
The method of claim 7 wherein there are five fluorescent markers selected from said group.
Claim 11
Dependent Claim
15 words
The method of claim 7 wherein there are six fluorescent markers selected from said group.
Claim 12
Independent Claim
128 words
A method for determining one or more characteristics of particles using multiple fluorescence analysis comprising: directing an incident light beam at particles under analysis, the particles including at least three fluorescent markers each having different emission spectra, but each marker being excitable by light at the same wavelength, said incident light beam causing the excitation of said markers such that spectrally different wavelengths of fluorescence are emitted from said particles without substantial spectral overlap; spectrally separating with optical elements the different wavelengths of emitted fluorescence to simultaneously refine the emitted fluorescence to enable the separate detection of the different wavelengths; simultaneously detecting the three or more different fluorescence emissions associated with the particles under analysis; and associating said detected fluorescence with one or more characteristics of said particles.
Claim 13
Independent Claim
128 words
A method for determining one or more characteristics of particles using multiple fluorescence analysis comprising: directing an incident light beam at particles under analysis, the particles including at least three fluorescent markers each having different emission spectra, but each marker being excitable by light at the same wavelength, said incident light beam causing the excitation of said markers such that spectrally different wavelengths of fluorescence are emitted from said particles without substantial spectral overlap; spectrally separating with optical elements the different wavelengths of emitted fluorescence to simultaneously refine the emitted fluorescence to enable the separate detection of the different wavelengths; simultaneously detecting the three or more different fluorescence emissions associated with the particles under analysis; and associating said detected fluorescence with one or more characteristics of said particles.
Claim 14
Independent Claim
157 words
A method for determining one or more immunofluorescent characteristics of cells or the like flowing in a liquid stream using multiple fluorescence analysis comprising: moving cells, substantially one at time, in a liquid flow stream, said cells including at least three cell-surface fluorescent markers thereon suitable for immunofluorescence analysis, each marker having different emission spectra in which the emission maxima are sufficiently spectrally separated so that there is no substantial spectral overlap; directing a light beam of a single wavelength at the cells in said flow stream to excite all of the fluorescent markers such that different wavelengths of fluorescence are emitted from said cells; spectrally separating with optical elements the different wavelengths of emitted fluorescence to enable the separate detection of the different wavelengths; simultaneously detecting the three or more different fluorescence emissions associated with the cells moving in the flow streams; and using said detected fluorescence to determine one or more characteristics of said cells.
Claim 15
Independent Claim
157 words
A method for determining one or more immunofluorescent characteristics of cells or the like flowing in a liquid stream using multiple fluorescence analysis comprising: moving cells, substantially one at time, in a liquid flow stream, said cells including at least three cell-surface fluorescent markers thereon suitable for immunofluorescence analysis, each marker having different emission spectra in which the emission maxima are sufficiently spectrally separated so that there is no substantial spectral overlap; directing a light beam of a single wavelength at the cells in said flow stream to excite all of the fluorescent markers such that different wavelengths of fluorescence are emitted from said cells; spectrally separating with optical elements the different wavelengths of emitted fluorescence to enable the separate detection of the different wavelengths; simultaneously detecting the three or more different fluorescence emissions associated with the cells moving in the flow streams; and using said detected fluorescence to determine one or more characteristics of said cells.
Claim 16
Dependent Claim
26 words
The method of claim 12 wherein the flourescent markers are chosen from a select combination of cell-surface fluorescent markers excitable by a single wavelength of light.
Claim 17
Independent Claim
133 words
An apparatus for determining one or more characteristics of cells or the like flowing in a liquid stream using multiple fluorescence analysis comprising: means for moving cells, substantially one at a time, in a liquid flow stream; means for directing a light beam at the cells in said flow stream in order to excite multiple fluorescence markers on said cells by a single wavelength of light; at least three fluorescence detection means for detecting spectrally separated fluorescence without substantial spectral overlap emitted by the cells moving the flow stream, each detection means including a select combination of optical light filter means which permit the simultaneous collection of spectrally separate fluorescence emission of at least three spectrally wavelengths; and means for using said detected fluorescence to determine one or more characteristics of said cells.
Claim 18
Independent Claim
133 words
An apparatus for determining one or more characteristics of cells or the like flowing in a liquid stream using multiple fluorescence analysis comprising: means for moving cells, substantially one at a time, in a liquid flow stream; means for directing a light beam at the cells in said flow stream in order to excite multiple fluorescence markers on said cells by a single wavelength of light; at least three fluorescence detection means for detecting spectrally separated fluorescence without substantial spectral overlap emitted by the cells moving the flow stream, each detection means including a select combination of optical light filter means which permit the simultaneous collection of spectrally separate fluorescence emission of at least three spectrally wavelengths; and means for using said detected fluorescence to determine one or more characteristics of said cells.
Claim 19
Dependent Claim
14 words
The apparatus of claim 14 wherein said means for directing light includes a laser.
Claim 20
Dependent Claim
37 words
The apparatus of claim 15 wherein the select combination of filter means includes filters selected from the group of filters which permit light to pass therethrough at wavelengths at least 15 nm spectrally spaced from each other.
Claim 21
Dependent Claim
17 words
The apparatus of claim 15 wherein the laser provides a wavelength of excitation light at 480 nm.
Claim 22
Dependent Claim
51 words
The apparatus of claim 17 wherein the select combination of light filter means includes filters selected from the group of filters which permit light at wavelengths of essentially 525 nm, 575 nm, 610 nm, 630 nm, 660 nm and 680 nm to pass therethrough for collection in the respective detection means.
Claim 23
Independent Claim
104 words
An apparatus for determining one or more characteristics of particles using multiple flourescence analysis comprising: means for directing a light beam at particles under analysis in order to excite multiple flourescence markers on said cells by a single wavelength of light; at least three fluorescence detection means for detecting spectrally separated fluorescence without substantial spectral overlap emitted by the particles, each detection means including a select combination of optical light filter means which permit the simultaneous collection of spectrally separate fluorescence emission of at least three spectrally separated wavelengths; and means for associating said detected fluorescence with one or more characteristics of said particles.
Claim 24
Independent Claim
104 words
An apparatus for determining one or more characteristics of particles using multiple flourescence analysis comprising: means for directing a light beam at particles under analysis in order to excite multiple flourescence markers on said cells by a single wavelength of light; at least three fluorescence detection means for detecting spectrally separated fluorescence without substantial spectral overlap emitted by the particles, each detection means including a select combination of optical light filter means which permit the simultaneous collection of spectrally separate fluorescence emission of at least three spectrally separated wavelengths; and means for associating said detected fluorescence with one or more characteristics of said particles.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-05-17 | 1991-11-16 | 1992-05-17 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-05-17 | 1995-11-16 | 1996-05-17 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-05-17 | 1999-11-16 | 2000-05-17 | $7700.00 | Window Closed / Lapsed |