Frequency domain fluorometry using coherent sampling

Claim 2

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 F e and fundamental repetition period T 3 and also including harmonic components varying at harmonic frequencies which are integral multiplies of F e and applying said excitation light to said composition whereby said composition will emit light having repetitive variations in amplitude at modulation frequencies corresponding to F e and to said harmonic frequencies; (b) sampling means for directly sampling the amplitude of light emitted by said composition repetitively at a sampling frequency F s and sampling period T s such that F s is less than F e and T s is greater than T e , and T s is not an integral multiple of T e 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.

Claim 3

Apparatus as claimed in claim 1 wherein said sampling means is operative to sample said response signal so that: T.sub.s T.sub.e N.sub.w +T.sub.e /N.sub.p where N w is a positive integer and N p is a rational number having an absolute value greater than one

Claim 4

Apparatus as claimed in claim 2, wherein said sampling means is operative at a sampling rate F s such that N p is an integer.

Claim 5

Apparatus as claimed in claim 2 wherein said sampling means includes detector means for providing a continuous electrical signal directly related to the amplitude of said emitted light, means for providing a repetitive triggering signal at said sampling frequency F s and analog to digital conversion means for digitizing the instantaneous value of said continuous electrical signal upon each repetition of said triggering signal.

Claim 6

Apparatus as claimed in claim 4 further comprising anti aliasing means for excluding from said sampled response signal components having a frequency greater than F h wherein F h =(N w N p +1)F s /2, said anti aliasing means including means for low pass filtering said continuous electrical signal.

Claim 7

Apparatus as claimed in claim 4 further comprising anti aliasing means for excluding from said sampled response signal components having a frequency higher than F h wherein F h =(NWNp+1)Fs/2

Claim 8

Apparatus as claimed in claim 2 wherein said sampling means includes means for averaging said sampled response signal over a plurality of cycles of the fundamental component therein.

Claim 9

Apparatus as claimed in claim 2 wherein said excitation means includes means for providing excitation light including a plurality of fundamental components varying at a plurality of fundamental frequencies F e and fundamental repetition periods T e , and further including harmonics of each such fundamental frequency, whereby said modulation frequencies will include each of said fundamental frequencies and harmonicas thereof, and wherein said sampling means includes means for providing a plurality of separate series of sample values by sampling the amplitude of light emitted by said composition separately at each of a plurality of sampling frequencies F s and sampling periods T s , each said sampling frequency F s and sampling period T s being associated with one of said fundamental frequencies and fundamental repetition periods, the relationship between each said sampling period T s and the associated fundamental repetition period being in accordance with said relationship T.sub.s =T.sub.e N.sub.w +T.sub.e /Np where N w and N p for the various fundamental frequencies F e and sampling frequencies F s may be the same or different whereby each said separate series of sample values will represent a separate sampled excitation signal corresponding to variations in said emitted light within a separate range of said modulation frequencies incorporating one said fundamental frequency and a preselected number of harmonics of that fundamental frequency.

Claim 10

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.

Claim 11

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.

Claim 12

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.

Claim 13

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..

Claim 14

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.

Claim 15

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 F s 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.

Claim 16

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.

Claim 17

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.

Claim 18

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.

Claim 19

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.

Claim 20

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.

Claim 21

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.

Claim 22

Apparatus for determining a response of a chemical composition to light comprising: (a) excitation for producing excitation light having repetitive variations in amplitude, including a fundamental component varying at a fundamental frequency F e and fundamental repetition period T e and also including harmonic components varying at frequencies which are integral multiples of F e 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 F e 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.

Claim 23

Apparatus for determining a response of a chemical composition to light comprising: (a) excitation for producing excitation light having repetitive variations in amplitude, including a fundamental component varying at a fundamental frequency F e and fundamental repetition period T e and also including harmonic components varying at frequencies which are integral multiples of F e 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 F e 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.

Claim 24

Apparatus as claimed in claim 21, wherein said means for transforming said sampled response signal includes means for providing a value of modulation and a value of phase for each said response component.

Claim 25

Apparatus as claimed in claim 21, further comprising means for sampling said excitation light at said sampling frequency F s to thereby provide an excitation series of sample values constituting a sampled excitation signal corresponding to the variations in amplitude of said excitation light at said modulation frequencies and means or transforming said sampled excitation signal into a frequency domain representation thereof including at least one characteristic of each of a plurality of excitation components at a plurality of said modulation frequencies, said means for determining at least one characteristic of each said response component including means for comparing each characteristic of each said response component with the corresponding characteristic of a corresponding one of said excitation components at the same modulation frequency.

Claim 28

A method as claimed in claim 24 wherein said sampling step is conducted so that: T.sub.s =T.sub.e N.sub.w +T.sub.e /N.sub.p where N w is a positive integer and N p is a rational number having absolute value greater than 1.

Claim 29

A method as claimed in claim 25, wherein said sampling step is conducted at a sampling frequency F s such that N p is an integer.

Claim 30

A method as claimed in claim 25 wherein said sampling step includes the steps of detecting said emitted light and providing a continuous electrical signal directly related to the amplitude of said emitted light, providing a repetitive triggering signal at said sampling frequency F s and digitizing the instantaneous value of said continuous electrical signal upon each repetition of said triggering signal.

Claim 31

A method as claimed in claim 27 wherein said sampling step further includes the step of excluding from said sampled response signal components having a frequency greater than F h wherein F h =(N w N p +1)F s /2 by low filtering said continuous electrical signal.

Claim 32

A method as claimed in claim 25, wherein said exciting step includes the step of providing excitation light including a plurality of fundamental components varying at a plurality of fundamental components varying at a plurality of fundamental frequencies F e and fundamental repetition periods T e , and further including harmonics of each such fundamental frequency, whereby said modulation frequencies will include each of said fundamental frequencies and harmonics thereof, and wherein said sampling step includes the step of providing a plurality of separate series of sample values by sampling the amplitude of light emitted by said composition separately at each of a plurality of sampling frequencies f s and sampling period T s , each said sampling frequency F s and sampling period T s being associated with one of said fundamental frequencies and fundamental repetition period, the relationship between each said sampling period T s and the associated fundamental repetition period being in accordance with said relationship T.sub.s =T.sub.e N.sub.w +T.sub.e /N.sub.p whereby each said separate series of sample values will represent a separate sampled excitation signal corresponding to variations in said emitted light within a separate range of said modulation frequencies incorporating one of said fundamental frequencies and a preselected set of harmonics of that fundamental frequency.

Claim 33

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.

Claim 34

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 F s 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.

Claim 35

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.

Claim 36

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.

Claim 37

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.

Claim 40

A method as claimed in claim 35, wherein said step of transforming said sampled response signal is performed by applying a digital transform to said sampled response values.