Frequency-domain light detection device

• US 6,317,207 B2
• Filed: 01/22/2001
• Issued: 11/13/2001
• Est. Priority Date: 02/23/1999
• Status: Expired due to Fees

• Current Assignee: Molecular Devices
• Original Assignee: LJL Biosystems, Inc. (Molecular Devices)
• Inventors: Stumbo, David P., Modlin, Douglas N., French, Todd E.
• Primary Examiner(s): Font, Frank G.
• Assistant Examiner(s): LAUCHMAN, LAYLA G
• Application Number:
  US09/767,579
• Publication Number:
  US 20010019409A1
• Time in Patent Office: 295 Days
• Field of Search: 356/317, 356/418, 356/417, 356/447, 356/246, 356/303, 356/323, 356/326, 250/458.1, 250/459.1, 250/205, 600/317, 436/172
• US Class Current: 356/317
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence

Abstract

Apparatus and methods for determining temporal properties of photoluminescent samples using frequency-domain photoluminescence measurements, where the measurements may include photon counting and/or the separation of measured luminescence into potentially overlapping time bins.

Claims

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

illuminating the sample with intensity-modulated incident light, where the modulation is characterized by a characteristic time;

detecting luminescence emitted from the sample in response to the illumination with incident light;

counting the number of photons in the detected luminescence during a preselected portion of the characteristic time;

computing a frequency-domain quantity based on the number of counted photons; and

determining the temporal property based on the frequency-domain quantity.

2. The method of claim 1, where the temporal property is a luminescence lifetime or a reorientational correlation time.

3. The method of claim 1, where the intensity of the incident light is modulated periodically with time, and where the characteristic time is the period of the modulation.

4. The method of claim 3, where the incident light is modulated sinusoidally.

5. The method of claim 3, where the period is less than about 10 milliseconds.

6. The method of claim 1, where the detected luminescence is detected substantially exclusively from a sensed volume of the sample.

7. The method of claim 1, where the detected luminescence is detected throughout the characteristic time.

8. The method of claim 1, where the steps of illuminating and detecting are performed simultaneously.

9. The method of claim 1, where the preselected portion is at least one-eighth of the characteristic time.

10. The method of claim 1, the preselected portion being a first preselected portion, further comprising:

counting the number of photons in the detected luminescence during a second preselected portion of the characteristic time, where the first and second portions correspond to at least partially different portions of the characteristic time.

11. The method of claim 10, where the first and second portions overlap.

12. The method of claim 10, where the first and second portions do not overlap.

13. The method of claim 1, further comprising:

counting the number of photons in the detected luminescence during additional preselected portions of the characteristic time, where the total number of portions is an integer power of two.

14. The method of claim 1, where the step of counting the number of photons includes the steps of converting the detected luminescence to a signal, and discriminating photons from noise based on their relative contributions to the signal.

15. The method of claim 1, where the frequency-domain quantity is a phase shift and/or a demodulation of the detected luminescence relative to the incident light.

16. The method of claim 1, where the step of determining the temporal property includes the step of correcting for intensity variations in the light source.

17. The method of claim 1, where the step of determining the temporal property includes the step of correcting for instrumental factors.

18. The method of claim 1, further comprising:

repeating the steps of illuminating, detecting, and counting with the same sample before determining the temporal property, where the number of counted photons used to compute the frequency-domain quantity is the sum of the number of photons counted in each repetition of illuminating and detecting.

19. The method of claim 1, further comprising:

automatically repeating the steps of illuminating, detecting, counting, and determining the temporal property with a series of samples.

20. An apparatus for measuring a temporal property of a luminescent sample, the apparatus comprising:

a light source for producing intensity-modulated excitation light;

an excitation optical relay structure that directs the intensity-modulated excitation light toward the sample, so that the sample may be induced to emit intensity-modulated emission light;

a detector for detecting light;

an emission optical relay structure that directs light from the sample toward the detector, so that intensity-modulated emission light from the sample may be detected; and

a discrete analyzer operatively connected to the detector, where the analyzer includes a counter that determines the number of photons in the detected emission light, and where the analyzer determines the temporal property based on a frequency-domain quantity computed from the number of photons.

21. The apparatus of claim 20, where the temporal property is a luminescence lifetime or a reorientational correlation time.

22. The apparatus of claim 20, where the excitation light is modulated sinusoidally.

23. The apparatus of claim 20, where the frequency-domain quantity is a phase shift and/or a demodulation of the detected luminescence relative to the incident light.

24. The apparatus of claim 20, where the discrete analyzer is configured to correct for at least one of the following:

intensity variations in the light source, and instrumental factors.

25. The apparatus of claim 20, where the emission optical relay structure is capable of transmitting light substantially exclusively from a sensed volume of the sample.

26. A method for measuring a temporal property of a luminescent sample, the method comprising:

illuminating the sample with intensity-modulated incident light capable of exciting luminescence in the sample, where the modulation of the intensity-modulated light is characterized by a characteristic time;

measuring luminescence emitted from the sample during first and second preselected portions of the characteristic time, where the first and second portions overlap; and

determining the temporal property based on the measured luminescence during the first and second portions.

27. The method of claim 26, where the temporal property is a luminescence lifetime or a reorientational correlation time.

28. The method of claim 26, where the step of measuring luminescence includes the step of counting the number of photons in the detected luminescence.

29. The method of claim 26, where the step of measuring luminescence includes the step of performing an analog integration of a signal proportional to the number of photons in the detected luminescence.

30. The method of claim 26, where the step of determining the temporal property includes the step of computing a frequency-domain quantity.

31. The method of claim 30, where the frequency-domain quantity is a phase shift and/or a demodulation of the detected luminescence relative to the incident light.

32. The method of claim 26, further comprising:

measuring luminescence emitted from the sample during additional preselected portions of the characteristic time, where the total number of portions is an integer power of two.

33. An apparatus for measuring a temporal property of a luminescent sample, the apparatus comprising:

a light source for producing intensity-modulated excitation light;

an excitation optical relay structure that directs the intensity-modulated excitation light toward the sample, so that the sample may be induced to emit intensity-modulated emission light;

a detector for detecting light;

an emission optical relay structure that directs light from the sample toward the detector, so that intensity-modulated emission light from the sample may be detected; and

a discrete analyzer operatively connected to the detector, where the analyzer is configured to measure light emitted from the sample during overlapping intervals and to determine the temporal property based on the measured light.

34. The apparatus of claim 33, where the temporal property is a luminescence lifetime or a reorientational correlation time.

35. The apparatus of claim 33, where the discrete analyzer is configured to determine the temporal property based on a frequency-domain quantity computed using the measured light.

36. The apparatus of claim 33, where the frequency-domain quantity is a phase shift and/or a demodulation of the detected luminescence relative to the incident light.