Phase fluorometry using a modulated electroluminescent lamp as a light source

• US 5,281,825 A
• Filed: 09/05/1991
• Issued: 01/25/1994
• Est. Priority Date: 09/05/1991
• Status: Expired due to Term

• Current Assignee: Joseph R. Lakowicz, Texas Instruments, Inc.
• Original Assignee: University of Maryland School of Medicine (University System of Maryland)
• Inventors: Lakowicz, Joseph R., Berndt, Klaus W.
• Primary Examiner(s): Dzierzynski, Paul M.
• Assistant Examiner(s): HANIG, RICHARD E
• Application Number:
  US07/755,232
• Time in Patent Office: 873 Days
• Field of Search: 250/458.1, 250/459.1, 356/318, 356/417, 315/169.3
• US Class Current: 250/458.1
• CPC Class Codes: G01N 2021/6432 Quenching
• CPC Class Codes: G01N 21/255 Details, e.g. use of specially adapted sources, lighting or optical systems
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 2201/0691 Modulated (not pulsed supply)

Abstract

A method and apparatus for using a square wave- or sinewave-driven electroluminescent lamp (ELL) as a source of periodically pulsed excitation light for a luminescent molecule, wherein changes in decay time, phase angle and/or modulation of the emitted luminescence are detected as parameters that correspond to changes in luminescence lifetimes.

Claims

1. An apparatus for detecting a change in decay time, phase angle and/or modulation, corresponding to a change in a lifetime, of a luminescence emission of at least one luminescent molecule comprising:

an electroluminescent lamp which outputs modulated light when driven by a first electrical signal;

a first electrical signal generator, coupled to said electroluminescent lamp, for generating said first electrical signal to cause said electroluminescent lamp to output said modulated light having a wavelength which excites said at least one luminescent molecule to produce said luminescence emission;

a photosensor for detecting said luminescence emission and generating a second electrical signal corresponding to said luminescence emission;

a meter coupled to said photosensor and said first electrical signal, wherein said meter compares said first electrical signal to said second electrical signal to provide said change in decay time, phase angle and/or modulation of said luminescence emission of said at least one luminescent molecule, wherein said change corresponds to said change in a luminescence lifetime of said luminescent molecule.

2. The apparatus of claim 1, wherein said electroluminescent lamp outputs said modulated light with a plurality of frequency components that are harmonics of said first electrical signal.

3. The apparatus of claim 1, wherein said wavelength of said modulated light is in the range of 250-1000 nm.

4. The apparatus of claim 1, wherein said modulated light is intrinsically or extrinsically modulated at a frequency range of 200 Hz to 10 MHz.

5. The apparatus of claim 4, wherein said modulated light is intrinsically or extrinsically modulated at a frequency range of 1 kHz to 10 MHz.

6. The apparatus according to claim 1, wherein said detecting is done in one selected from frequency-domain, time-domain or phase/modulation.

7. The apparatus of claim 1, wherein said photosensor is optically coupled to said luminescence emission via at least one selected from a lens system and proximity focused geometry.

8. The apparatus of claim 1, wherein said luminescent molecule has a lifetime of at least 25 nanoseconds.

9. The apparatus of claim 1, wherein said first electrical signal is a square wave signal.

10. The apparatus of claim 9, wherein said first electrical signal is modulated at a fundamental frequency of at least 500 Hz.

11. The apparatus of claim 10, wherein a third electrical signal is generated by a second electrical signal generator synchronized to said first electrical signal, and wherein said meter compares said second electrical signal and said third electrical signal to provide said change in decay time, phase angle and/or modulation of said luminescence emission of said at least one luminescent molecule, wherein said change corresponds to said change in a luminescence lifetime of said luminescent molecule.

12. The apparatus of claim 11, wherein said second electrical signal generator is a frequency multiplier.

13. The apparatus of claim 1, wherein said first electrical signal is a sine wave signal.

14. The apparatus of claim 13, wherein said first electrical signal is modulated at a fundamental frequency of at least 10 kHz.

15. The apparatus of claim 13, wherein said third electrical signal is synchronized with said first electrical signal.

16. The apparatus of claim 9 wherein said photosensor comprises a first photodiode.

17. The apparatus of claim 1 wherein said photosensor comprises at least one selected from a photomultiplier tube, a photodiode, or a charged coupled device.

18. The apparatus of claim 16 wherein said first photodiode comprises a PIN photodiode.

50. The apparatus of claim 1, wherein said first electrical signal generator generates said first electrical signal as a square-wave having a frequency at least as high as 500 Hz to produce the modulated light as periodic light pulses which have a pulse repetition frequency equal to twice the frequency of the square-wave signal, and each of which has a full width at half maximum (FWHM) of at least as short as 1 μ

sec.

19. A method of using an electroluminescent lamp as a high-frequency modulated light source, comprising detecting a change in decay time, phase angle and/or modulation, corresponding to a change in a lifetime of a luminescence emission of at least one luminescent molecule.

20. A method according to claim 19, wherein said change in decay time, phase angle and/or modulation corresponds to the presence of an analyte, such that said method provides for sensing said analyte.

21. A method for detecting a change in decay time, phase angle and/or modulation, corresponding to a change in a lifetime, of a luminescence emission of at least one luminescent molecule comprising:

driving an electroluminescent lamp with a first electrical signal to cause said electroluminescent lamp to output modulated light having a wavelength which excites said at least one luminescent -molecule in a sample to produce said luminescence emission;

exciting said at least one luminescent molecule with said modulated light to produce a luminescence emission;

detecting said luminescence emission with a photosensor to produce a second electrical signal corresponding to said luminescence emission;

comparing said first electrical signal to said second electrical signal to provide said change in decay time, phase angle and/or modulation of said luminescence emission of said at least one luminescent molecule, wherein said change corresponds to said change in a luminescence lifetime of said luminescent molecule.

22. The method of claim 21, wherein said electroluminescent lamp outputs said modulated light with a plurality of frequency components that are harmonics of said first electrical signal.

23. The method of claim 21, wherein said wavelength of said modulated light is in the range of 250-1000 nm.

24. The method of claim 21, wherein said output of said modulated light is intrinsically or extrinsically modulated at a frequency range of 200 Hz to 10 MHz.

25. The method of claim 24, wherein said output of said modulated light is intrinsically or extrinsically modulated at a frequency range of 1 kHz to 10 MHz.

26. The method according to claim 21, wherein said method is one selected from frequency-domain, time domain or phase/modulation.

27. The method of claim 21, wherein said photosensor is optically coupled to said luminescence emission via at least one selected from a lens system or proximity focused geometry.

28. The method of claim 21, wherein said luminescent molecule has a lifetime of at least 25 nanoseconds.

29. The method of claim 21, wherein said first electrical signal is a square wave signal.

30. The method of claim 29, wherein said first electrical signal is modulated at a fundamental frequency of at least 100 Hz.

31. The method of claim 30, wherein a third electrical signal, synchronized to said first electrical signal, is generated by a second electrical signal generator.

32. The method of claim 29, wherein said second electrical signal generator is a frequency multiplier.

33. The method of claim 21, wherein said first electrical signal is a sine wave signal.

34. The method of claim 33, wherein said first electrical signal is modulated at a fundamental frequency of at least 10 kHz.

35. The method of claim 33, wherein a third electrical signal is generated by a second electrical signal generator and is synchronized with said first electrical signal.

36. The method of claim 29 wherein said photosensor comprises a first photodiode.

37. The method of claim 21 wherein said photosensor comprises at least one selected from a photomultiplier tube, a photodiode, or a charged coupled device.

38. The method of claim 36 wherein said first photodiode comprises a PIN photodiode.

39. The method of claim 21, wherein said change corresponds to the presence or concentration of an analyte in a sample, wherein said sample is a compound, a mixture, a surface, a solution, an emulsion, a suspension, a mixture, a cell culture, a fermentation culture, a cell, a tissue, a secretion or a derivative or extract thereof.

40. A method according to claim 21, wherein said analyte comprises a salt, a derivative, a polymer, a dissociation product, or aggregate of said analyte.

41. A method according claim 39, wherein said analyte is selected from H⁺, OH^-, CO₂, H₂ HCO₃^3-, Ca⁺⁺, Na⁺, Mg⁺⁺, or K⁺.

42. A method according to claim 39, wherein said method comprises a frequency-domain luminescence lifetime measurement, a time-domain luminescence lifetime measurement, a phase angle luminescence lifetime measurement, a phase-sensitive luminescence lifetime measurement, or a modulation luminescence lifetime measurement.

51. The method claim 21, wherein said first electrical signal is a squarewave signal having a frequency at least as high as 500 Hz to produce the modulated light as periodic light pulses which have a pulse repetition frequency equal to twice the frequency of the square-wave signal, and each of which has a full width at half maximum (FWHM) of at least as short as 1 μ

sec.

43. An apparatus for measuring luminescence comprising:

an electroluminescent lamp (ELL) which produces light when driven by an electrical input signal;

an electrical input signal generator, coupled to said ELL, for generating an electrical input signal which drives said ELL;

at least one luminescent molecule for determining a condition and emitting a characteristic luminescence upon incidence of light output from said ELL; and

light-detecting means for detecting the lifetime of said luminescence as a measurement of said condition.

52. The apparatus of claim 43, wherein said electric input signal generator generates said electrical input signal as a square-wave signal having a frequency at least as high as 500 Hz to produce the modulated light as periodic light pulses which have a pulse repetition frequency equal to twice the frequency of the square-wave signal, and each of which has a full width at half maximum (FWHM) of at least as short as 1 μ

sec.

44. A method of continuously generating light pulses from an electroluminescent lamp (ELL) having a luminescence material disposed between electrodes, said method comprising:

continuously applying a square wave input voltage signal to said electrodes of said ELL; and

rapidly and continuously alternating the polarity of the voltage of said square wave input signal so that said luminescence material continuously generates a corresponding pulsed light output waveform.

53. The method of claim 44, wherein said input voltage signal is a square-wave signal having a frequency at least as high as 500 Hz to produce the modulated light as periodic light pulses which have a pulse repetition frequency equal to twice the frequency of the square-wave signal, and each of which has a full width at half maximum (FWHM) of at least as short as 1 μ

sec.

45. An apparatus for measuring luminescence comprising:

an electroluminescent lamp (ELL) which outputs pulses of light when driven by an amplitude-modulated electrical input signal;

an electrical input signal generator, electrically coupled to said ELL, for generating a square-wave electrical input signal whose transition time from a positive voltage to a negative voltage is less than or equal to 1 μ

s, so that the light pulses have a repetition frequency which is a harmonic of twice the modulation frequency of said electrical input signal;

a chemical sensor for determining a condition and emitting luminescence upon incidence of the light output from said ELL; and

light-detecting means for detecting said luminescence as a measurement of said condition.

46. A method of measuring luminescence, comprising the steps of:

driving an electroluminescent lamp (ELL) with an alternating input voltage signal whose transitions from a positive voltage to a negative voltage cause the ELL to output pulses of light;

exciting a luminescent molecule with the pulses of light from the ELL; and

detecting resulting emitted luminescence radiation from the luminescent molecule.

47. The method of claim 46, further comprising the step of driving the ELL with a sine-wave input voltage signal.

48. The method of claim 46, further comprising the step of driving the ELL with a square wave input voltage.

49. The method of claim 46, further comprising the step of measuring the lifetime of the detected luminescence by measuring a phase difference between said emitted luminescence and the pulses of light from the ELL.

54. The method of claim 48, wherein said square-wave input voltage has a frequency at least as high as 500 Hz to generate the modulated light in the form of periodic light pulses which have a pulse repetition frequency equal to twice the frequency of the square-wave signal, and each of which has a full width at half maximum (FWHM) of at least as short as 1 μ

sec.

55. An apparatus for generating light pulses, comprising:

an electroluminescent lamp having a luminescence material disposed between two electrodes; and

means for applying across said electrodes a square wave voltage signal that continuously alternates in polarity at a frequency of at least 500 Hz to cause said lamp to generate a correspondingly continuous train of periodic light pulses each of which has a full width at half maximum (FWHM) of at least as short as 1 μ

sec.