Method and apparatus for identifying fluorophores
First Claim
1. A method of identifying a plurality of fluorophores having a different excitation spectra in a fluorophore containing substance comprising:
- a. stimulating at least one of the plurality of fluorophores with an incident optical signal fixed in relationship to a fluorophore substance holder, the incident optical signal having an optical spectrum comprising in spectral components wherein m is an integer greater than 1;
b. modulating each of said spectral components in intensity according to a respective one of a set of linearly independent time-domain functions;
c. exciting at least one of the plurality of fluorophores with at least one of said spectral components modulated in intensity according to said respective one of a set of linearly independent time-domain functions to produce fluorescence emission over a range of wavelengths;
d. providing excitation efficiencies and fluorescence quantum yields for each of the plurality of fluorophores for each of the m spectral components;
e. detecting substantially all of the entire fluorescence emission excited by the incident optical signal to produce a response signal which corresponds to the intensity of substantially all of said fluorescence emission over said range of wavelengths in the time-domain of the linearly independent time-domain functions; and
f. analyzing the response signal utilizing the excitation efficiencies and quantum yields to identify at least one fluorophore of the plurality of fluorophores.
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Abstract
The present invention is a unique method for identifying the presence, and preferably the identity, of a fluorophore by optically stimulating one or more fluorophores with an optical signal which has been modulated in intensity in the time domain. The stimulated fluorophore produces a resulting fluorescence which is demodulated to produce an electrical signal corresponding to the intensity modulation of the fluorescence. Finally, the electrical signal is compared to the modulation of the optical signal to determine whether or not the fluorophore is present. The present method can be used alone or in conjunction with known methods of optically analyzing fluorescence of fluorophores to determine the presence of fluorophores.
131 Citations
24 Claims
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1. A method of identifying a plurality of fluorophores having a different excitation spectra in a fluorophore containing substance comprising:
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a. stimulating at least one of the plurality of fluorophores with an incident optical signal fixed in relationship to a fluorophore substance holder, the incident optical signal having an optical spectrum comprising in spectral components wherein m is an integer greater than 1; b. modulating each of said spectral components in intensity according to a respective one of a set of linearly independent time-domain functions; c. exciting at least one of the plurality of fluorophores with at least one of said spectral components modulated in intensity according to said respective one of a set of linearly independent time-domain functions to produce fluorescence emission over a range of wavelengths; d. providing excitation efficiencies and fluorescence quantum yields for each of the plurality of fluorophores for each of the m spectral components; e. detecting substantially all of the entire fluorescence emission excited by the incident optical signal to produce a response signal which corresponds to the intensity of substantially all of said fluorescence emission over said range of wavelengths in the time-domain of the linearly independent time-domain functions; and f. analyzing the response signal utilizing the excitation efficiencies and quantum yields to identify at least one fluorophore of the plurality of fluorophores. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19)
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16. Apparatus for detecting a plurality of flurophores in a fluorophore containing substance comprising:
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a. a source of incident optical signal having an optical spectrum comprising m spectral components where m is an integer greater than 1, at least one of said spectral components capable of exciting fluorophores; b. modulator for modulating the intensity of each of said spectral components according to a respective one of a set of linearly independent time-domain functions; c. a fluorophore substance holder for securing a fluorophore-containing substance fixed in relationship to said incident optical signal and making said fluorophore accessible to said incident optical signal from said source and for detection of fluorescence resulting from excitation of said fluorophores; d. a detector optically coupled to said fluorophore substance holder for detection of substantially all of the fluorescence emitted by excited fluorophores and conversion of substantially all of said emitted fluorescence to an electrical signal; and e. means for analyzing the electrical signal utilizing provided quantities derived from excitation efficiencies and quantum yields for each of the fluorophores for each of the spectral components. - View Dependent Claims (17, 20, 21, 22, 23, 24)
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Specification