Apparatus and methods for time-resolved spectroscopic measurements

• US 6,483,582 B2
• Filed: 01/19/2001
• Issued: 11/19/2002
• Est. Priority Date: 07/27/1998
• Status: Expired due to Fees

• Current Assignee: Molecular Devices
• Original Assignee: LJL Biosystems, Inc. (Danaher Corporation)
• Inventors: Owicki, John C., Modlin, Douglas N., French, Todd E.
• Primary Examiner(s): Font, Frank G.
• Assistant Examiner(s): LAUCHMAN, LAYLA G
• Application Number:
  US09/765,874
• Publication Number:
  US 20010003044A1
• Time in Patent Office: 669 Days
• Field of Search: 356/317, 356/323, 356/418, 356/246, 356/303, 356/318, 250/458.1, 250/459.1, 600/473, 600/317, 435/6
• US Class Current: 356/317
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: G01N 2021/6471 Special filters, filter wheel
• CPC Class Codes: G01N 21/51 inside a container, e.g. in an ampoule G01N21/53 takes precedence
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 21/6445 Measuring fluorescence polarisation
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates

Abstract

Apparatus and methods for measuring light transmitted from a sample. The apparatus may include a stage, a light source, and a detector. The stage may be configured to hold a microplate having a plurality of sample wells. The apparatus may be configured to take frequency-domain time-resolved measurements of one or more of luminescence lifetimes and reorientational correlation times of a luminescent analyte in the sample.

Claims

1. A method for measuring the temporal response properties of a luminescent sample, comprising:

outputting a light beam from a light source, the light beam having a relatively constant intensity;

modulating the light beam to create a modulated incident light, the modulated incident light having a maximum intensity that is substantially equal to the intensity of the light beam from the light source and a minimum intensity that is less than one-quarter of the maximum intensity;

illuminating the sample with the modulated incident light, where the modulated incident light generates a modulated luminescence in the sample;

measuring at least one of an amplitude and a phase of the modulated luminescence relative to the modulated incident light; and

computing a temporal response characteristic of the sample based on the measured amplitude and/or phase.

2. The method of claim 1, wherein the step of measuring includes measuring both of an amplitude and a phase of the modulated luminescence relative to the modulated incident light.

3. The method of claim 1, wherein the step of computing includes calculating a temporal response characteristic of the sample based on the measured amplitude and phase.

4. The method of claim 1, wherein the temporal response characteristic is a luminescence lifetime or a rotational correlation time.

5. The method of claim 1, wherein the step of modulating is carried out at a fundamental frequency.

6. The method of claim 5, wherein the modulating generates a square wave.

7. The method of claim 6, wherein the modulated incident light has approximately zero minimum intensity.

8. The method of claim 5, wherein the step of measuring is carried out at the fundamental frequency and a harmonic thereof.

9. The method of claim 5, wherein the fundamental frequency is less than twenty kilohertz.

10. The method of claim 5, wherein the step of modulating is divided into a first part carried out at one fundamental frequency and a second part carried out at a second fundamental frequency.

11. The method of claim 1 further comprising the step of focusing the light beam into a focal plane during the step of modulating.

12. The method of claim 11, wherein the modulation is carried out with an optical modulator positioned proximal to the focal plane.

13. The method of claim 1, wherein the light source is a continuous arc lamp.

14. The method of claim 13, wherein the continuous arc lamp is a continuous high color temperature xenon arc lamp.

15. The method of claim 1, wherein the light source has a broad spectrum output.

16. The method of claim 1 further including the step of filtering the light beam to generate substantially monochromatic light.

17. An automated apparatus for conducting time-resolved spectroscopy, comprising:

a light source;

a system for directing light from the light source to a measurement region, the system including a light modulator configured to periodically modulate the intensity of light delivered to the measurement region;

a stage configured to hold a plate containing a plurality of sample wells adapted to hold samples, the stage further being configured to place a selected one of the samples in the sample wells into the measurement region;

a detector configured to receive luminescence light from a sample in the measurement region and generate a signal based on the amount of light received; and

a signal processing system configured to track at least one of the phase and modulation of the signal relative to the phase and modulation of the modulated light delivered to the measurement region.

18. The apparatus of claim 17, wherein the signal processing system is configured to track both of the phase and modulation of the signal relative to the phase and modulation of the modulated light delivered to the measurement region.

19. The apparatus of claim 17, wherein the signal processing system includes a phase-locked loop coupled to the signal of the detector.

20. The apparatus of claim 19, wherein the light modulator has a fundamental frequency, and wherein the phase-locked loop is matched to the fundamental frequency.

21. The apparatus of claim 19, wherein the light modulator creates a square wave modulation of a fundamental frequency, and wherein the phase-locked loop is configured to track a harmonic of the fundamental frequency.

22. The apparatus of claim 17, wherein the light modulator is a chopper.

23. The apparatus of claim 22, wherein the system for directing light includes a mechanism focus light from the light source into a focal plane proximal to the chopper.

24. The apparatus of claim 23, wherein the focal plane is aligned with the chopper.

25. The apparatus of claim 22, wherein the chopper is selected from the group consisting of chopper wheels and tuning fork choppers.

26. The apparatus of claim 22, wherein the light modulator includes two choppers with different modulation frequencies.

27. The apparatus of claim 17, wherein the signal processing system includes a filter configured to receive the detector signal and extract a selected frequency component.

28. The apparatus of claim 27, wherein the signal processing system includes a second filter configured to receive the detector signal and extract a second selected frequency component.

29. The apparatus of claim 27, wherein the filter is a Bessel filter.

30. A method for performing a time-resolved spectroscopic assay, comprising:

providing a light source with a broad spectrum output;

extracting a substantially monochromatic component from the output of the light source;

passing the monochromatic component through a chopper to create a periodically modulated incident light with a fundamental frequency;

choosing a chopper duty cycle that produces a harmonic comparable to a selected time constant of the sample;

generating luminescence in a sample by illuminating the sample with the modulated incident light; and

detecting at least one of the phase and modulation of the luminescence relative to the modulated incident light.

31. The method of claim 30, wherein the step of detecting includes detecting both of the phase and modulation of the luminescence relative to the phase and modulation of the modulated incident light.

32. The method of claim 30 further comprising choosing a chopper modulation frequency that is comparable to a selected time constant of the sample.

33. The method of claim 32, wherein the selected time constant is greater than fifty microseconds.

34. The method of claim 30, wherein the light source is a continuous arc lamp.

35. The method of claim 34, wherein the continuous arc lamp is a continuous high color temperature xenon arc lamp.

36. The method of claim 30 further comprising the step of monitoring output intensity variations of the light source.

37. The method of claim 36 further comprising the step of correcting the measured amplitude of the modulated luminescence to compensate for intensity variations in the light source.

38. The method of claim 30, wherein the modulation of the incident light has the general form of a square wave.

39. The method of claim 30, wherein the step of detecting includes detecting the phase and/or modulation of the luminescence relative to the modulated incident light at a harmonic of the fundamental frequency.

40. The method of claim 30 further comprising the step of focusing the monochromatic component into a focal plane adjacent the chopper.

41. The assay of claim 30, wherein the light source has a substantially continuous output.

42. The method of claim 30, wherein the chopper is selected from the group consisting of chopper wheels and tuning fork choppers.

43. The method of claim 30 further comprising passing the monochromatic component through a second chopper to create a periodically modulated incident light with second fundamental frequency.

44. A method for performing a time-resolved spectroscopic assay, comprising:

providing a light source with a broad spectrum output;

extracting a substantially monochromatic component from the output of the light source;

passing the monochromatic component through a chopper to create a periodically modulated incident light with a fundamental frequency;

generating luminescence in a sample by illuminating the sample with the modulated incident light;

monitoring output intensity variations of the light source;

correcting the measured amplitude of the modulated luminescence to compensate for intensity variations in the light source; and

detecting at least one of the phase and modulation of the luminescence relative to the modulated incident light.

45. The method of claim 44, wherein the step of detecting includes detecting both of the phase and modulation of the luminescence relative to the phase and modulation of the modulated incident light.

46. The method of claim 44 further comprising choosing a chopper modulation frequency that is comparable to a selected time constant of the sample.

47. The method of claim 46, wherein the selected time constant is greater than fifty microseconds.

48. The method of claim 44, wherein the light source is a continuous arc lamp.

49. The method of claim 48, wherein the continuous arc lamp is a continuous high color temperature xenon arc lamp.

50. The method of claim 44, wherein the modulation of the incident light has the general form of a square wave.

51. The method of claim 44, wherein the step of detecting includes detecting the phase and/or modulation of the luminescence relative to the modulated incident light at a harmonic of the fundamental frequency.

52. The method of claim 44 further comprising the step of focusing the monochromatic component into a focal plane adjacent the chopper.

53. The assay of claim 44, wherein the light source has a substantially continuous output.

54. The method of claim 44, wherein the chopper is selected from the group consisting of chopper wheels and tuning fork choppers.

55. The method of claim 44 further comprising passing the monochromatic component through a second chopper to create a periodically modulated incident light with second fundamental frequency.

56. A method for performing a time-resolved spectroscopic assay, comprising:

providing a light source with a broad spectrum output;

extracting a substantially monochromatic component from the output of the light source;

passing the monochromatic component through a chopper to create a periodically modulated incident light with a fundamental frequency, wherein the modulation of the incident light has the general form of the square wave;

generating luminescence in a sample by illuminating the sample with the modulated incident light; and

detecting at least one of the phase and modulation of the luminescence relative to the modulated incident light.

57. The method of claim 56, wherein the step of detecting includes detecting both of the phase and modulation of the luminescence relative to the phase and modulation of the modulated incident light.

58. The method of claim 56 further comprising choosing a chopper modulation frequency that is comparable to a selected time constant of the sample.

59. The method of claim 58, wherein the selected time constant is greater than fifty microseconds.

60. The method of claim 56, wherein the light source is a continuous arc lamp.

61. The method of claim 60, wherein the continuous arc lamp is a continuous high color temperature xenon arc lamp.

62. The method of claim 56, wherein the step of detecting includes detecting the phase and/or modulation of the luminescence relative to the modulated incident light at a harmonic of the fundamental frequency.

63. The method of claim 56 further comprising the step of focusing the monochromatic component into a focal plane adjacent the chopper.

64. The assay of claim 56, wherein the light source has a substantially continuous output.

65. The method of claim 56, wherein the chopper is selected from the group consisting of chopper wheels and tuning fork choppers.

66. The method of claim 56 further comprising passing the monochromatic component through a second chopper to create a periodically modulated incident light with second fundamental frequency.

67. A method for performing a time-resolved spectroscopic assay, comprising:

providing a light source with a broad spectrum output;

extracting a substantially monochromatic component from the output of the light source;

passing the monochromatic component through a chopper to create a periodically modulated incident light with a fundamental frequency;

generating luminescence in a sample by illuminating the sample with the modulated incident light; and

detecting at least one of the phase and modulation of the luminescence relative to the modulated incident light, wherein the step of detecting includes detecting the phase and/or modulation of the luminescence relative to the modulated incident light at a harmonic of the fundamental frequency.

68. The method of claim 67, wherein the step of detecting includes detecting both of the phase and modulation of the luminescence relative to the phase and modulation of the modulated incident light.

69. The method of claim 67 further comprising choosing a chopper modulation frequency that is comparable to a selected time constant of the sample.

70. The method of claim 69, wherein the selected time constant is greater than fifty microseconds.

71. The method of claim 67, wherein the light source is a continuous arc lamp.

72. The method of claim 71, wherein the continuous arc lamp is a continuous high color temperature xenon arc lamp.

73. The method of claim 67 further comprising the step of focusing the monochromatic component into a focal plane adjacent the chopper.

74. The assay of claim 67, wherein the light source has a substantially continuous output.

75. The method of claim 67, wherein the chopper is selected from the group consisting of chopper wheels and tuning fork choppers.

76. The method of claim 67 further comprising passing the monochromatic component through a second chopper to create a periodically modulated incident light with second fundamental frequency.

77. A method for performing a time-resolved spectroscopic assay, comprising:

providing a light source with a broad spectrum output;

extracting a substantially monochromatic component from the output of the light source;

focusing the monochromatic component into a focal plane adjacent a chopper;

passing the monochromatic component through a chopper to create a periodically modulated incident light with a fundamental frequency;

generating luminescence in a sample by illuminating the sample with the modulated incident light; and

detecting at least one of the phase and modulation of the luminescence relative to the modulated incident light.

78. The method of claim 77, wherein the step of detecting includes detecting both of the phase and modulation of the luminescence relative to the phase and modulation of the modulated incident light.

79. The method of claim 77 further comprising choosing a chopper modulation frequency that is comparable to a selected time constant of the sample.

80. The method of claim 79, wherein the selected time constant is greater than fifty microseconds.

81. The method of claim 77, wherein the light source is a continuous arc lamp.

82. The method of claim 81, wherein the continuous arc lamp is a continuous high color temperature xenon arc lamp.

83. The assay of claim 77, wherein the light source has a substantially continuous output.

84. The method of claim 77, wherein the chopper is selected from the group consisting of chopper wheels and tuning fork choppers.

85. The method of claim 77 further comprising passing the monochromatic component through a second chopper to create a periodically modulated incident light with second fundamental frequency.

86. A method for performing a time-resolved spectroscopic assay, comprising:

providing a light source with a broad spectrum output;

extracting a substantially monochromatic component from the output of the light source;

passing the monochromatic component through a chopper to create a periodically modulated incident light with a fundamental frequency;

generating luminescence in a sample by illuminating the sample with the modulated incident light; and

detecting at least one of the phase and modulation of the luminescence relative to the modulated incident light.

87. The method of claim 86, wherein the step of detecting includes detecting both of the phase and modulation of the luminescence relative to the phase and modulation of the modulated incident light.

88. The method of claim 86 further comprising choosing a chopper modulation frequency that is comparable to a selected time constant of the sample.

89. The method of claim 86, wherein the selected time constant is greater than fifty microseconds.

90. The method of claim 86, wherein the light source is a continuous arc lamp.

91. The method of claim 90, wherein the continuous arc lamp is a continuous high color temperature xenon arc lamp.

92. The method of claim 86, wherein the chopper is selected from the group consisting of chopper wheels and tuning fork choppers.

93. The method of claim 86 further comprising passing the monochromatic component through a second chopper to create a periodically modulated incident light with second fundamental frequency.

94. A method for performing a time-resolved spectroscopic assay, comprising:

providing a light source with a broad spectrum output;

extracting a substantially monochromatic component from the output of the light source;

passing the monochromatic component through a chopper to create a periodically modulated incident light with a fundamental frequency, wherein the chopper is selected from the group consisting of chopper wheels and tuning fork choppers;

generating luminescence in a sample by illuminating the sample with the modulated incident light; and

detecting at least one of the phase and modulation of the luminescence relative to the modulated incident light.

95. The method of claim 94, wherein the step of detecting includes detecting both of the phase and modulation of the luminescence relative to the phase and modulation of the modulated incident light.

96. The method of claim 94 further comprising choosing a chopper modulation frequency that is comparable to a selected time constant of the sample.

97. The method of claim 96, wherein the selected time constant is greater than fifty microseconds.

98. The method of claim 94, wherein the light source is a continuous arc lamp.

99. The method of claim 98, wherein the continuous arc lamp is a continuous high color temperature xenon arc lamp.

100. The method of claim 94 further comprising passing the monochromatic component through a second chopper to create a periodically modulated incident light with second fundamental frequency.

106. The method of claim 98, wherein the continuous arc lamp is a continuous high color temperature xenon arc lamp.

101. A method for performing a time-resolved spectroscopic assay, comprising:

providing a light source with a broad spectrum output;

extracting a substantially monochromatic component from the output of the light source;

passing the monochromatic component through a chopper to create a periodically modulated incident light with a fundamental frequency;

generating luminescence in a sample by illuminating the sample with the modulated incident light; and

detecting at least one of the phase and modulation of the luminescence relative to the modulated incident light.

102. The method of claim 101, wherein the step of detecting includes detecting both of the phase and modulation of the luminescence relative to the phase and modulation of the modulated incident light.

103. The method of claim 101 further comprising choosing a chopper modulation frequency that is comparable to a selected time constant of the sample.

104. The method of claim 103, wherein the selected time constant is greater than fifty microseconds.

105. The method of claim 101, wherein the light source is a continuous arc lamp.