Signal generation and mixing electronics for frequency-domain lifetime and spectral fluorometry

Abstract

The present invention additionally comprises a method and apparatus for generating and mixing signals for frequency-domain lifetime and spectral fluorometry. The present invention comprises a plurality of signal generators that generate a plurality of signals where the signal generators modulate the amplitude and/or the frequency of the signals. The present invention uses one of these signals to drive an excitation signal that the present invention then directs and transmits at a target mixture, which absorbs the energy from the excitation signal. The property of fluorescence causes the target mixture to emit an emitted signal that the present invention detects with a signal detector. The present invention uses a plurality of mixers to produce a processor reference signal and a data signal. The present invention then uses a processor to compare the processor reference signal with the data signal by analyzing the differences in the phase and the differences in the amplitude between the two signals. The processor then extracts the fluorescence lifetime and fluorescence spectrum of the emitted signal from the phase and amplitude information using a chemometric analysis.

1. An apparatus for fluorescence lifetime and spectral measurements, comprising:
 a driving/reference signal generator that generates a driving/reference signal, said driving/reference signal is amplitude and/or frequency modulated over time;  
 a mixing signal generator that generates a mixing signal, said mixing signal is amplitude and/or frequency modulated over time;  
 an excitation signal generator that generates an excitation signal, the driving/reference signal drives said excitation signal generator;  
 a signal detector that detects the emitted signal;  
 a mixer that mixes the emitted  mixing signal with the driving/reference signal and produces the processor reference signal;  
 a mixer that mixes the emitted signals with the mixing signal and produces the data signal; and  
 a processor that extracts the fluorescence lifetime and fluorescence spectrum of the emitted signal from the comparison of the processor reference signal with the data signal using a chemometric analysis.  
 
     
     
       2. The apparatus of  claim 1  wherein the driving/reference signal and the mixing signal vary by an adjustable offset frequency. 
     
     
       3. The apparatus of  claim 1  wherein said chemometric analysis extracts the fluorescence lifetime of the emitted signal from the phase difference between the processor reference signal and the data signal. 
     
     
       4. The apparatus of  claim 1  wherein said chemometric analysis extracts the fluorescence spectrum of the emitted signal from the amplitude difference between the processor reference signal and the data signal. 
     
     
       5. The apparatus of  claim 1  wherein said chemometric analysis further comprises a converging iterative solution. 
     
     
       6. A system for fluorescence lifetime and spectral measurements, comprising:
 means for generating a driving/reference signal, said driving/reference signal means modulates the amplitude and/or the frequency of the driving/reference signal over time;  
 means for generating a mixing signal, said mixing signal means modulates the amplitude and/or the frequency of the mixing signal over time,  
 means for generating an excitation signal, the driving/reference signal drives said excitation signal means;  
 means for detecting the emitted signal;  
 means for mixing the emitted  mixing signal with the driving/reference signal to produce the processor reference signal;  
 means for mixing the emitted signal with the mixing signal to produce the data signal; and  
 a processor that extracts the fluorescence lifetime and fluorescence spectrum of the emitted signal from the comparison of the processor reference signal with the data signal using a chemometric analysis.  
 
     
     
       7. The system of  claim 6  wherein the driving/reference signal and the mixing signal vary by an adjustable offset frequency. 
     
     
       8. The system of  claim 6  wherein said chemometric analysis extracts the fluorescence lifetime of the emitted signal from the phase difference between the processor reference signal and the data signal. 
     
     
       9. The system of  claim 6  wherein said chemometric analysis extracts the fluorescence spectrum of the emitted signal from the amplitude difference between the processor reference signal and the data signal. 
     
     
       10. The system of  claim 6  wherein said chemometric analysis further comprises a converging iterative solution. 
     
     
       11. A method for forming the fluorescence lifetime and the fluorescence spectrum, comprising the following steps:
 generating a driving/reference signal and modulating the amplitude and/or the frequency of the driving/reference signal over time;  
 generating a mixing signal and modulating the amplitude and/or the frequency of the mixing signal over time;  
 generating an excitation signal form the driving/reference signal;  
 detecting the emitted signal,  
 mixing the emitted  mixing signal with the driving/reference signal and producing the processor reference signal;  
 mixing the emitted signal with the mixing signal producing the data signal; and  
 extracting the fluorescence lifetime and fluorescence spectrum of the emitted signal from the comparison of the processor reference signal with the data signal to measure using a chemometric analysis.  
 
     
     
       12. The method of  claim 11  wherein the driving/reference signal and the mixing signal vary by an adjustable offset frequency. 
     
     
       13. The method of  claim 11  wherein said chemometric analysis extracts the fluorescence lifetime of the emitted signal from the phase difference between the processor reference signal and the data signal. 
     
     
       14. The method of  claim 11  wherein said chemometric analysis extracts the fluorescence spectrum of the emitted signal from the amplitude difference between the processor reference signal and the data signal. 
     
     
       15. The method of  claim 11  wherein said chemometric analysis further comprises a converging iterative solution. 
     
     
       16. A method of producing an apparatus for fluorescence lifetime and spectral measurements, comprising:
 providing a driving/reference signal generator that generates a driving/reference signal, said driving/reference signal is amplitude and/or frequency modulated over time;  
 providing a mixing signal generator that generates a mixing signal, said mixing signal is amplitude and/or frequency modulated over time;  
 coupling an excitation signal generator that generates an excitation signal and a reference signal to said driving/reference generator;  
 providing a signal detector that detects the emitted signal;  
 coupling a first mixer to said excitation signal generator, said mixer mixes the emitted  mixing signal with the driving/reference signal to produce the processor reference signal,  
 coupling a second mixer to said mixing signal generator, said mixer mixes the emitted signal with the mixing signal to produce the data signal; and  
 coupling a processor to said first mixer and said second mixer, said processor extracts the fluorescence lifetime and fluorescence spectrum of the emitted signal from the comparison of the processor reference signal with the data signal using a chemometric analysis.  
 
     
     
       17. The method of  claim 16  wherein the driving/reference signal and the mixing signal vary by an adjustable offset frequency. 
     
     
       18. The method of  claim 16  wherein said chemometric analysis extracts the fluorescence lifetime of the emitted signal from the phase difference between the processor reference signal and the data signal. 
     
     
       19. The method of  claim 16  wherein said chemometric analysis extracts the fluorescence spectrum of the emitted signal from the amplitude difference between the processor reference signal and the data signal. 
     
     
       20. The method of  claim 16  wherein said chemometric analysis further comprises a converging iterative solution. 
     
     
       21. A program storage device readable by a computer, tangibly embodying a program of instructions executable by the computer to perform method steps for a method for measuring the fluorescence lifetime and the fluorescence spectrum, comprising the following method steps:
 generating a driving/reference signal and modulating the amplitude and/or the frequency of the driving/reference signal over time;  
 generating a mixing signal and modulating the amplitude and/or the frequency of the mixing signal over time;  
 generating an excitation signal from the driving/reference signal;  
 detecting the emitted signal;  
 mixing the emitted  mixing signal with the driving/reference signal and producing the processor reference signal;  
 mixing the emitted signal with the mixing signal producing the data signal; and  
 extracting the fluorescence lifetime and fluorescence spectrum of the emitted signal from the comparison of the processor reference signal with the data signal to measure using a chemometric analysis.  
 
     
     
       22. The program storage device of  claim 21  wherein the driving/reference signal and the mixing signal vary by an adjustable offset frequency. 
     
     
       23. The program storage device of  claim 21  wherein said chemometric analysis extracts the fluorescence lifetime of the emitted signal from the phase difference between the processor reference signal and the data signal. 
     
     
       24. The program storage device of  claim 21  wherein said chemometric analysis extracts the fluorescence spectrum of the emitted signal from the amplitude difference between the processor reference signal and the data signal. 
     
     
       25. The program storage device of  claim 21  wherein said chemometric analysis further comprises a converging iterative solution.