Frequency domain fluorometry using coherent sampling
First Claim
1. Apparatus for determining a response of a chemical composition to light comprising:
- (a) excitation means for producing excitation light having repetitive variations in amplitude, including a fundamental component varying at a fundamental frequency Fe and fundamental repetition period T3 and also including harmonic components varying at harmonic frequencies which are integral multiplies of Fe and applying said excitation light to said composition whereby said composition will emit light having repetitive variations in amplitude at modulation frequencies corresponding to Fe and to said harmonic frequencies;
(b) sampling means for directly sampling the amplitude of light emitted by said composition repetitively at a sampling frequency Fs and sampling period Ts such that Fs is less than Fe and Ts is greater than Te, and Ts is not an integral multiple of Te and providing a response series of sample values representing said amplitude of said emitted light, whereby said response series of samples will represent a sampled response signal corresponding to the variations in amplitude of said emitted light at said modulation frequencies and;
(c) means for determining at least one characteristic of said sampled response signal.
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Accused Products
Abstract
Apparatus and methods for measuring response of a chemical composition to light, as by frequency domain, phase angle, or modulation fluorometry and/or phosphorimetry. Pulsed light having a known fundamental frequency is applied to the composition thereby eliciting response light varying at the fundamental frequency and at harmonics thereof. The response light signal may be sampled according to a wave-skipping sampling scheme, and the sampled response signal may be transformed into a frequency domain representation, as by a digital fast Fourier transform technique. A reference signal derived from the excitation light may be similarly processed and the results used as a basis for comparison to derive properties of the chemical composition such as phase and modulation response of the composition to the fundamental and harmonic frequencies. The invention provides rapid monitoring and determination of luminescence characteristics. Multiple excitation frequencies may be applied simultaneously and the resulting response signals may be separated and separately sampled.
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Citations
36 Claims
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1. Apparatus for determining a response of a chemical composition to light comprising:
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(a) excitation means for producing excitation light having repetitive variations in amplitude, including a fundamental component varying at a fundamental frequency Fe and fundamental repetition period T3 and also including harmonic components varying at harmonic frequencies which are integral multiplies of Fe and applying said excitation light to said composition whereby said composition will emit light having repetitive variations in amplitude at modulation frequencies corresponding to Fe and to said harmonic frequencies; (b) sampling means for directly sampling the amplitude of light emitted by said composition repetitively at a sampling frequency Fs and sampling period Ts such that Fs is less than Fe and Ts is greater than Te, and Ts is not an integral multiple of Te and providing a response series of sample values representing said amplitude of said emitted light, whereby said response series of samples will represent a sampled response signal corresponding to the variations in amplitude of said emitted light at said modulation frequencies and; (c) means for determining at least one characteristic of said sampled response signal. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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9. Apparatus as claimed in claim 8 wherein said excitation means includes means for providing said excitation light including all of said fundamental components and all of said harmonics simultaneously.
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10. Apparatus as claimed in claim 9 wherein said sampling means includes means for detecting the emitted light and generating a composite analog signal representing variations in said emitted light in all of said ranges of frequencies simultaneously, a plurality of sampling devices, and bandpass filter means for separating components of said composite analog signal in different ones of said ranges of modulation frequencies into separate parts of said composite analog signal and directing different parts of said composite analog signal to different ones of said sampling devices.
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11. Apparatus as claimed in claim 1 wherein said excitation means includes means for providing repetitive pulses of light at a pulse repetition frequency equal to said fundamental frequency Fe.
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12. Apparatus as claimed in claim 11 wherein said excitation means includes means for controlling said excitation light pulses so that said excitation light pulses have a duty cycle less than or equal to 1/N.sub..
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13. Apparatus as claimed in claim 1 further comprising means for providing a reference signal having at least one characteristic representing a corresponding characteristics of said excitation light, said means for determining at least one characteristic of said sampled response signal including means for comparing said at least one characteristic of said sampled response signal with a corresponding characteristic of said reference signal.
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14. Apparatus as claimed in claim 13 wherein said means for providing a reference signal includes means for sampling said excitation light at said sampling frequency Fs to thereby provided an excitation series of sample values, whereby said excitation series of sample values will represent a sampled excitation signal corresponding to the variations in amplitude of said excitation light at said modulation frequencies.
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15. Apparatus as claimed in claim 14 wherein said means for comparing includes means for determining the degree of modulation for components at each of a plurality of said modulation frequencies in each of said sampled response signal and said sampled excitation signal and comparing the degree of modulation for each component in said sampled response signal with the degree of modulation for a corresponding component at the same modulation frequency in said sampled excitation signal.
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16. Apparatus as claimed in claim 14 wherein said means for comparing includes means for determining the phase relationship between each component in said sampled response signal and a corresponding component in said sampled excitation signal at the same modulation frequency.
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17. Apparatus as claimed in claim 14, wherein said means for comparing at least one characteristic of said sampled response signal with a corresponding characteristic of said reference signal includes means for transforming said response signal into a frequency domain representation including values of at least one characteristic of each of a plurality of response components at a plurality of said modulation frequencies, means for transforming said sampled excitation signal into a frequency domain representation thereof including values of at least one characteristic for each of a plurality of excitation components at said plurality of modulation frequencies and means for comparing the value of each such characteristic for each such response component with the value of the corresponding characteristic for the corresponding excitation component at the same modulation frequency.
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18. Apparatus as claimed in claim 17, wherein said means for transforming said sampled response signal and said means for transforming said sampled excitation signal includes means for providing modulation and phase values of each said response component and for each said excitation component respectively.
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19. Apparatus as claimed in claim 17, wherein means for transforming said sampled response signal includes means for applying a digital transformation to said response series of values and said means for transforming said sampled excitation signal includes means for applying a digital transformation to said excitation series of values.
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20. Apparatus as claimed in claim 13 wherein said means for providing a reference signal includes means for directing said excitation light through a light path of predetermined characteristics and providing said reference signal representing said excitation light after such passage.
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21. Apparatus for determining a response of a chemical composition to light comprising:
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(a) excitation for producing excitation light having repetitive variations in amplitude, including a fundamental component varying at a fundamental frequency Fe and fundamental repetition period Te and also including harmonic components varying at frequencies which are integral multiples of Fe and applying said excitation light to said composition whereby said composition will emit light having repetitive variations in amplitude of said modulation frequencies corresponding to Fe and to said frequencies of said harmonic components; (b) sampling means for directly sampling the amplitude of light emitted by said composition to thereby provide a response series of samples representing said amplitude of light emitted by said composition, whereby said response series of samples will represent a sampled response signal corresponding to the variations in amplitude of said emitted light at said modulation frequencies; (c) means for transforming said response series of sample values to a frequency domain representation thereof including value for at least one characteristic of each of plural response components at a plurality of said modulation frequencies by applying a digital transformation to said response series of values; and (d) means for determining a luminescence function of said chemical composition from said characteristics of said response components. - View Dependent Claims (22, 23)
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24. A method for determining a response of a chemical composition to light comprising the steps of:
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(a) exciting said chemical composition with light having repetitive variations in amplitude at a fundamental frequency Fe and fundamental repetition period Te and also including harmonic components varying in amplitude at frequencies which are integral multiples of Fe, whereby said composition will emit light having repetitive variations in amplitude at modulation frequencies corresponding to Fe and to said frequencies of said harmonic components; (b) directly sampling the amplitude of light emitted by said composition repetitively at a sampling frequency Fs less than Fe and sampling period Ts greater than Te so as to provide a response series of sample values representing said amplitude, whereby said response series of sample values will constitute a sampled response signal corresponding to the variations in amplitude of said emitted light at said modulation frequencies; and (d) determining at least one characteristic of said sampled response signal. - View Dependent Claims (25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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30. A method as claimed in claim 24 further comprising the steps of providing a reference signal having at least one characteristic representing a corresponding characteristic of said excitation light, said step of determining at least one characteristic of said sampled response signal including the steps of comparing said at least one characteristic of said sampled response signal with a corresponding characteristic of said reference signal.
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31. A method as claimed in claim 30 wherein said step of providing a reference signal includes the step of sampling said excitation light at said sampling frequency Fs to thereby provide an excitation series of sample values, whereby said excitation series of sample values will represent a sampled excitation signal corresponding to the variations in amplitude of said excitation light at said modulation frequencies.
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32. A method as claimed in claim 31 wherein said step of comparing at least one characteristic of said sampled response signal with a corresponding characteristic of said reference signal includes the steps of transforming said response signal into a frequency domain representation including values of at least one characteristic of each of a plurality of response components at a plurality of said modulation frequencies, transforming said sampled excitation signal into a frequency domain representation thereof including values of at least one characteristic for each of a plurality of excitation components at a plurality of said modulation frequencies and comparing the value of each such characteristic for each such response component with the value of the corresponding characteristic for the corresponding excitation component at the same modulation frequency.
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33. A method as claimed in claim 29, wherein said exciting step includes the step of providing said excitation light including all of said fundamental components and all of said harmonics simultaneously.
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34. A method as claimed in claim 33 wherein said sampling step includes the step of detecting the emitted light and generating a composite analog signal representing variations in said emitted light in all of said ranges of modulation frequencies simultaneously, separating components of said composite analog signal in different ones of said ranges of modulation frequencies into separate parts of said composite analog signal and directing said separate parts to different sampling devices.
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35. A method of determining a response of a chemical composition to light comprising steps of:
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(a) applying excitation light having repetitive variations in amplitude at a fundamental frequency Fe and fundamental repetition period Te and also including harmonic components varying at frequencies which are integral multiples of Fe to the composition so that the composition emits light including response components having repetitive variations in amplitude at modulation frequencies corresponding to Fe and to said frequencies of said harmonic components; (b) directly sampling the amplitude of light emitted by the composition to thereby provide a response series of sample values representing the amplitude of the emitted light whereby the response series of sample values will constitute a sampled response signal corresponding to the variations in amplitude of the emitted light at said modulation frequencies; (c) transforming said sampled response signal to a frequency domain representation thereof including values for at least one characteristic of each of said plurality of response components; and (d) determining a luminescence characteristic of said chemical composition from said characteristics of said response components. - View Dependent Claims (36)
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Specification