US5504337A
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Method and apparatus for performing phase fluorescence lifetime measurements in flow cytometry
Assignee: LAKOWICZ JOSEPH R PHD
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Filing: 1991-10-10
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Granted: 1996-04-02
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Expires: 2011-10-10
Abstract
Abstract PCT No. PCT/US91/07259 Sec. 371 Date Apr. 12, 1993 Sec. 102(e) Date Apr. 12, 1993 PCT Filed Oct. 10, 1991 PCT Pub. No. WO92/07245 PCT Pub. Date Apr. 30, 1992.A method and apparatus for identifying individual particles or cells which have been labeled with different fluorochromes, on the basis of the lifetime of their fluorescence, or based on different decay times for a fluorochrome in different cells.
Inventors
- 👤 Joseph R. Lakowicz
- 👤 Klaus W. Berndt
- 👤 Robert A. Hoffman
- 👤 Bertram G. Pinsky
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 07:22
CPC Classifications
G; G01; G01N; G01N15/00; G01N15/10; G01N15/14; G01N15/1434; G01N21/00; G01N21/62; G01N21/63; G01N21/64; G01N21/6408; G01N15/1456; G01N15/1459; G01N2015/1454
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
184 words
A method of determining, in a flow cytometric environment, a fluorescence lifetime of at least one fluorophore associated with a cell or particle, comprising the steps of: directing a stream of a plurality of said cells or particles past an observation point; modulating a light source at a frequency of an input signal to produce modulated exciting light having a wavelength capable of exciting said at least one fluorophore; irradiating at the observation point said cell or particle with said modulated light to produce emitted light from said at least one fluorophore; detecting said emitted light and producing a corresponding modulated emission signal; and comparing said input signal and said modulated emission signal to produce an output signal corresponding to a difference between the phase and/or modulation of said modulated emission signal and said input signal, wherein said output signal is (a) indicative of a value of the fluorescence lifetime of said at least one fluorophore and (b) is independent of the intensity of the emitted light, and wherein said detecting step further comprises adjusting said output signal to reduce the effect of autofluorescence.
Claim 2
Independent Claim
184 words
A method of determining, in a flow cytometric environment, a fluorescence lifetime of at least one fluorophore associated with a cell or particle, comprising the steps of: directing a stream of a plurality of said cells or particles past an observation point; modulating a light source at a frequency of an input signal to produce modulated exciting light having a wavelength capable of exciting said at least one fluorophore; irradiating at the observation point said cell or particle with said modulated light to produce emitted light from said at least one fluorophore; detecting said emitted light and producing a corresponding modulated emission signal; and comparing said input signal and said modulated emission signal to produce an output signal corresponding to a difference between the phase and/or modulation of said modulated emission signal and said input signal, wherein said output signal is (a) indicative of a value of the fluorescence lifetime of said at least one fluorophore and (b) is independent of the intensity of the emitted light, and wherein said detecting step further comprises adjusting said output signal to reduce the effect of autofluorescence.
Claim 3
Independent Claim
269 words
A method of determining, in a flow cytometric environment, a fluorescence lifetime of at least one fluorophore associated with a cell or particle, comprising the steps of: directing a stream of a plurality of said cells or particles past an observation point; modulating a light source at a frequency of an input signal to produce modulated exciting light having a wavelength capable of exciting said at least one fluorophore; irradiating at the observation point said cell or particle with said modulated light to produce emitted light from said at least one fluorophore; detecting said emitted light and producing a corresponding modulated emission signal; and comparing said input signal and said modulated emission signal to produce an output signal corresponding to a difference between the phase and/or modulation of said modulated emission signal and said input signal, wherein said output Signal is (a) indicative of a value of the fluorescence lifetime of said at least one fluorophore and (b) is independent of the intensity of the emitted light, and wherein the comparing step comprises dividing said input signal into first and second signals; dividing said emission signal into third and fourth signals; placing said first signal into phase quadrature with respect to said second signal; mixing said first and third signals and said second and fourth signals, respectively, to generate fifth and sixth signals, respectively, that are in phase quadrature; and determining the ratio of said fifth and sixth signals in order to generate the value which is indicative of the fluorescence lifetime of said at least one fluorophore but which value is independent of intensity of said emitted light.
Claim 4
Independent Claim
269 words
A method of determining, in a flow cytometric environment, a fluorescence lifetime of at least one fluorophore associated with a cell or particle, comprising the steps of: directing a stream of a plurality of said cells or particles past an observation point; modulating a light source at a frequency of an input signal to produce modulated exciting light having a wavelength capable of exciting said at least one fluorophore; irradiating at the observation point said cell or particle with said modulated light to produce emitted light from said at least one fluorophore; detecting said emitted light and producing a corresponding modulated emission signal; and comparing said input signal and said modulated emission signal to produce an output signal corresponding to a difference between the phase and/or modulation of said modulated emission signal and said input signal, wherein said output Signal is (a) indicative of a value of the fluorescence lifetime of said at least one fluorophore and (b) is independent of the intensity of the emitted light, and wherein the comparing step comprises dividing said input signal into first and second signals; dividing said emission signal into third and fourth signals; placing said first signal into phase quadrature with respect to said second signal; mixing said first and third signals and said second and fourth signals, respectively, to generate fifth and sixth signals, respectively, that are in phase quadrature; and determining the ratio of said fifth and sixth signals in order to generate the value which is indicative of the fluorescence lifetime of said at least one fluorophore but which value is independent of intensity of said emitted light.
Claim 5
Dependent Claim
32 words
The method of claim 2, further comprising, prior to said mixing step, amplifying and limiting each of said first, second, third and fourth signals, respectively, by a corresponding amplifier and limiting circuit.
Claim 6
Dependent Claim
27 words
The method of claim 3, further comprising, prior to said determining step, filtering each of said fifth and sixth signals, respectively, by a corresponding low pass filter.
Claim 7
Dependent Claim
20 words
The method of claim 4, wherein said determining step comprises peak detecting said fifth and sixth signal after said filtering.
Claim 8
Independent Claim
327 words
A method of determining, in a flow cytometric environment, a fluorescence lifetime of at least one fluorophore associated with a cell or particle, comprising the steps of: directing a stream of a plurality of said cells or particles past an observation point; modulating a light source at a frequency of an input signal to produce modulated exciting light having a wavelength capable of exciting said at least one fluorophore; irradiating at the observation point said cell or particle with said modulated light to produce emitted light from said at least one fluorophore; detecting said emitted light and producing a corresponding modulated emission signal; and comparing said input signal and said modulated emission signal to produce an output signal corresponding to a difference between the phase and/or modulation of said modulated emission signal and said input signal, wherein said output signal is (a) indicative of a value of the fluorescence lifetime of said at least one fluorophore and (b) is independent of the intensity of the emitted light, and wherein the comparing step comprises dividing said emission signal into first and second signals; filtering (a) said first signal by a high pass filter, followed by amplifying and splitting said first signal into a third signal and a fourth signal and (b) said second signal with a low pass filter; amplifying and limiting said input signal and limiting said third signal, respectively, by passing said input signal through a signal amplifier set at a preset level and a limiting circuit, and by passing said third signal through a limiting circuit; then mixing said third signal and said input signal, respectively, to generate a fifth signal; and determining the ratio of said second and fourth signals as a ratio signal; and comparing said ratio signal and said fifth signal in order to generate a value which is indicative of the fluorescence lifetime of said at least one fluorophore but which value is independent of the intensity of the emitted light.
Claim 9
Independent Claim
327 words
A method of determining, in a flow cytometric environment, a fluorescence lifetime of at least one fluorophore associated with a cell or particle, comprising the steps of: directing a stream of a plurality of said cells or particles past an observation point; modulating a light source at a frequency of an input signal to produce modulated exciting light having a wavelength capable of exciting said at least one fluorophore; irradiating at the observation point said cell or particle with said modulated light to produce emitted light from said at least one fluorophore; detecting said emitted light and producing a corresponding modulated emission signal; and comparing said input signal and said modulated emission signal to produce an output signal corresponding to a difference between the phase and/or modulation of said modulated emission signal and said input signal, wherein said output signal is (a) indicative of a value of the fluorescence lifetime of said at least one fluorophore and (b) is independent of the intensity of the emitted light, and wherein the comparing step comprises dividing said emission signal into first and second signals; filtering (a) said first signal by a high pass filter, followed by amplifying and splitting said first signal into a third signal and a fourth signal and (b) said second signal with a low pass filter; amplifying and limiting said input signal and limiting said third signal, respectively, by passing said input signal through a signal amplifier set at a preset level and a limiting circuit, and by passing said third signal through a limiting circuit; then mixing said third signal and said input signal, respectively, to generate a fifth signal; and determining the ratio of said second and fourth signals as a ratio signal; and comparing said ratio signal and said fifth signal in order to generate a value which is indicative of the fluorescence lifetime of said at least one fluorophore but which value is independent of the intensity of the emitted light.
Claim 10
Dependent Claim
38 words
The method of claim 6, wherein said input signal is provided by one selected from (a) receiving said modulated light by a photomultiplier tube and producing said input signal; and (b) a frequency generator set at said frequency.
Claim 11
Dependent Claim
292 words
A flow cytometer operative to measure a fluorescence lifetime of at least one fluorophore associated with a cell or a particle, comprising: a flow chamber for directing a plurality of cells or particles past an observation point; a light source modulated by a frequency of an input signal for irradiating, at said observation point, said cell or particle with modulated light having a wavelength capable of exciting said at least one fluorophore to produce emitted light; a photodetector for detecting said emitted light and producing a corresponding emission signal; and a phase/modulation detector for generating an output signal corresponding to a difference between the phase and/or modulation of said emission signal and said input signal, wherein said output signal is (a) indicative of a value of the fluorescence lifetime of said at least one fluorophore and (b) is independent of the intensity of the emitted light, and wherein said apparatus further comprises: a first power splitter for dividing said input signal into first and second signals having the same amplitude and frequency; a second power splitter for dividing said emission signal into third and fourth signals, said signals having an equal amplitude and identical frequency; a first phase shifter for providing a predetermined phase shift to said first signal; first and second mixers, said first mixer receiving said first signal of said first power splitter and said third signal from said second power splitter and generating a fifth signal, said second mixer receiving said second signal and said fourth signal and generating a sixth signal; a ratio unit for receiving said fifth signal and said sixth signal from said first mixer and said second mixer, respectively, and generating the ratio of said fifth signal and said sixth signal as said output signal.
Claim 12
Dependent Claim
292 words
A flow cytometer operative to measure a fluorescence lifetime of at least one fluorophore associated with a cell or a particle, comprising: a flow chamber for directing a plurality of cells or particles past an observation point; a light source modulated by a frequency of an input signal for irradiating, at said observation point, said cell or particle with modulated light having a wavelength capable of exciting said at least one fluorophore to produce emitted light; a photodetector for detecting said emitted light and producing a corresponding emission signal; and a phase/modulation detector for generating an output signal corresponding to a difference between the phase and/or modulation of said emission signal and said input signal, wherein said output signal is (a) indicative of a value of the fluorescence lifetime of said at least one fluorophore and (b) is independent of the intensity of the emitted light, and wherein said apparatus further comprises: a first power splitter for dividing said input signal into first and second signals having the same amplitude and frequency; a second power splitter for dividing said emission signal into third and fourth signals, said signals having an equal amplitude and identical frequency; a first phase shifter for providing a predetermined phase shift to said first signal; first and second mixers, said first mixer receiving said first signal of said first power splitter and said third signal from said second power splitter and generating a fifth signal, said second mixer receiving said second signal and said fourth signal and generating a sixth signal; a ratio unit for receiving said fifth signal and said sixth signal from said first mixer and said second mixer, respectively, and generating the ratio of said fifth signal and said sixth signal as said output signal.
Claim 13
Dependent Claim
40 words
The apparatus of claim 8 further comprising: first, second, third and fourth amplifiers and limiters, respectively, for amplifying and limiting each of said first, second, third and fourth signals, respectively, prior to being fed into said first and second mixers.
Claim 14
Dependent Claim
30 words
The method of claim 9 further comprising: first and second low pass filters, respectively, for filtering each of said fifth and sixth signals prior to said generating of said ratio.
Claim 15
Dependent Claim
24 words
The method of claim 8, further comprising, first and second peak detectors for peak detecting said fifth and sixth signals, respectively, after said filtering.
Claim 16
Dependent Claim
306 words
A flow cytometer operative to measure a fluorescence lifetime of at least one fluorophore associated with a cell or a particle, comprising: a flow chamber for directing a plurality of cells or particles past an observation point; a light source modulated by a frequency of an input signal for irradiating, at said observation point, said cell or particle with modulated light having a wavelength capable of exciting said at least one fluorophore to produce emitted light; a photodetector for detecting said emitted light and producing a corresponding emission signal; and a phase/modulation detector for generating an output signal corresponding to a difference between the phase and/or modulation of said emission signal and said input signal, wherein said output signal is (a) indicative of a value of the fluorescence lifetime of said at least one fluorophore and (b) is independent of the intensity of the emitted light, and wherein said apparatus further comprises: a first power splitter for dividing said emission signal into first and second signals, said signals having an equal amplitude and identical frequency; a high pass filter for filtering said first signal; a first amplifier and a second power splitter, respectively, for amplifying and splitting said first signal into a third signal and a fourth signal; a first low pass filter for filtering said second signal; a second amplifier and a first limiting circuit, respectively, for amplifying and limiting said input signal; a second limiting circuit for limiting said third signal; a mixer for mixing said third signal and said input signal, respectively, to generate a fifth signal; a second low pass filter for filtering ,said fifth signal; and a ratio unit for determining the ratio of said second and fourth signals as a ratio signal; wherein said phase/modulation detector is fed said ratio signal and said fifth signal to determine said output signal.
Claim 17
Dependent Claim
306 words
A flow cytometer operative to measure a fluorescence lifetime of at least one fluorophore associated with a cell or a particle, comprising: a flow chamber for directing a plurality of cells or particles past an observation point; a light source modulated by a frequency of an input signal for irradiating, at said observation point, said cell or particle with modulated light having a wavelength capable of exciting said at least one fluorophore to produce emitted light; a photodetector for detecting said emitted light and producing a corresponding emission signal; and a phase/modulation detector for generating an output signal corresponding to a difference between the phase and/or modulation of said emission signal and said input signal, wherein said output signal is (a) indicative of a value of the fluorescence lifetime of said at least one fluorophore and (b) is independent of the intensity of the emitted light, and wherein said apparatus further comprises: a first power splitter for dividing said emission signal into first and second signals, said signals having an equal amplitude and identical frequency; a high pass filter for filtering said first signal; a first amplifier and a second power splitter, respectively, for amplifying and splitting said first signal into a third signal and a fourth signal; a first low pass filter for filtering said second signal; a second amplifier and a first limiting circuit, respectively, for amplifying and limiting said input signal; a second limiting circuit for limiting said third signal; a mixer for mixing said third signal and said input signal, respectively, to generate a fifth signal; a second low pass filter for filtering ,said fifth signal; and a ratio unit for determining the ratio of said second and fourth signals as a ratio signal; wherein said phase/modulation detector is fed said ratio signal and said fifth signal to determine said output signal.
Claim 18
Dependent Claim
20 words
The method of claim 12 further comprising: a photomultiplier tube for receiving said modulated light and producing said input signal.
Claim 19
Dependent Claim
18 words
The method of claim 12 further comprising: a frequency generator for producing said input signal at said frequency.
Claim 20
Dependent Claim
184 words
A flow cytometer operative to measure a fluorescence lifetime of at least one fluorophore associated with a cell or a particle, comprising: a flow chamber for directing a plurality of cells or particles past an observation point; a light source modulated by a frequency of an input signal for irradiating, at said observation point, said cell or particle with modulated light having a wavelength capable of exciting said at least one fluorophore to produce emitted light; a photodetector for detecting said emitted light and producing a corresponding emission signal; and a phase/modulation detector for generating an output signal corresponding to a difference between the phase and/or modulation of said emission signal and said input signal, wherein said output signal is (a) indicative of a value of the fluorescence lifetime of said at least one fluorophore and (b) is independent of the intensity of the emitted light, and wherein said apparatus further comprises: an input optics system operative to shape and direct said modulated light to said observation point; and an output optics system operative to receive and direct said emitted light onto said photodetector.
Claim 21
Dependent Claim
184 words
A flow cytometer operative to measure a fluorescence lifetime of at least one fluorophore associated with a cell or a particle, comprising: a flow chamber for directing a plurality of cells or particles past an observation point; a light source modulated by a frequency of an input signal for irradiating, at said observation point, said cell or particle with modulated light having a wavelength capable of exciting said at least one fluorophore to produce emitted light; a photodetector for detecting said emitted light and producing a corresponding emission signal; and a phase/modulation detector for generating an output signal corresponding to a difference between the phase and/or modulation of said emission signal and said input signal, wherein said output signal is (a) indicative of a value of the fluorescence lifetime of said at least one fluorophore and (b) is independent of the intensity of the emitted light, and wherein said apparatus further comprises: an input optics system operative to shape and direct said modulated light to said observation point; and an output optics system operative to receive and direct said emitted light onto said photodetector.
Claim 22
Dependent Claim
165 words
A flow cytometer operative to measure a fluorescence lifetime of at least one fluorophore associated with a cell or a particle, comprising: a flow chamber for directing a plurality of cells or particles past an observation point; a light source modulated by a frequency of an input signal for irradiating, at said observation point, said cell or particle with modulated light having a wavelength capable of exciting said at least one fluorophore to produce emitted light; a photodetector for detecting said emitted light and producing a corresponding emission signal; and a phase/modulation detector for generating an output signal corresponding to a difference between the phase and/or modulation of said emission signal and said input signal, wherein said output signal is (a) indicative of a value of the fluorescence lifetime of said at least one fluorophore and (b) is independent of the intensity of the emitted light, and wherein said apparatus further comprises: a variable phase shifter operative to shift the phase of said input signal.
Claim 23
Dependent Claim
165 words
A flow cytometer operative to measure a fluorescence lifetime of at least one fluorophore associated with a cell or a particle, comprising: a flow chamber for directing a plurality of cells or particles past an observation point; a light source modulated by a frequency of an input signal for irradiating, at said observation point, said cell or particle with modulated light having a wavelength capable of exciting said at least one fluorophore to produce emitted light; a photodetector for detecting said emitted light and producing a corresponding emission signal; and a phase/modulation detector for generating an output signal corresponding to a difference between the phase and/or modulation of said emission signal and said input signal, wherein said output signal is (a) indicative of a value of the fluorescence lifetime of said at least one fluorophore and (b) is independent of the intensity of the emitted light, and wherein said apparatus further comprises: a variable phase shifter operative to shift the phase of said input signal.
Claim 24
Dependent Claim
18 words
The apparatus of claim 16 wherein said variable phase shifter is operative to reduce the effect of autofluorescence.
Claim 25
Dependent Claim
16 words
The apparatus of claim 17 wherein signals contributed by said autofluorescence are reduced substantially to zero.
References Cited (Backward Citations)
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