METHOD OF FLUORESCENT MEASUREMENT OF SAMPLES, AND DEVICES THEREFROM

• US 20120104280A1
• Filed: 02/24/2011
• Published: 05/03/2012
• Est. Priority Date: 11/03/2010
• Status: Active Grant

• Current Assignee: ReaMetrix, Inc.
• Original Assignee: ReaMetrix, Inc.
• Inventors: Manian, Bala S.
• Granted Patent: US 9,523,640 B2
• Time in Patent Office: Days
• US Class Current: 250/459.1
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence

Abstract

The invention provides a method and device for fluorescent measurement of a sample. The method includes impinging an incident beam having an excitation wavelength and a focus diameter on the sample to yield a laser spot that defines sample volume. The laser spot is displaced relative to sample volume in three dimensional space defined by sample volume. The laser spot is translated across the sample volume in depth direction to detect one or more emitted fluorescence signals from the sample volume. A depth profile and a thickness of the sample carrier obtained from the one or more emitted fluorescence signals, are used for measuring normalized bulk fluorescence. Further, at least one microvolume of interest is obtained from the depth profile, and the incident beam is focused on the microvolume of interest to obtain at least one concentrated emitted fluorescence signal.

Claims

1. A method for measuring fluorescence comprising:

providing a sample contained in a sample carrier, wherein the sample contains at least one fluorophore;

providing a light source to generate an incident beam having an excitation wavelength and a focus diameter to impinge on the sample to yield a laser spot that defines a sample volume for measuring fluorescence; and

displacing the laser spot relative to the sample volume in a three dimensional space defined by the sample volume, wherein the sample volume comprises at least one individual volume of interest.

2. The method of claim 1 further comprising:

translating the laser spot across the sample volume in a depth direction;

detecting one or more emitted fluorescence signals from the sample volume; and

obtaining a depth profile and a thickness of the sample carrier from the one or more emitted fluorescence signals, wherein the depth profile comprises the at least one individual volume of interest.

3. The method of claim 2, wherein the depth profile and the thickness are used for measuring normalized bulk fluorescence.

4. The method of claim 2 further comprising:

determining at least one microvolume of interest from the depth profile; and

focusing the incident beam on the microvolume of interest to obtain at least one concentrated emitted fluorescence signal.

5. The method of claim 4, wherein the at least one concentrated emitted fluorescence signal is used to measure one or more event fluorescences for the sample.

6. The method of claim 1, wherein the at least one individual volume of interest and the focus diameter of the incident beam have a defined relationship.

7. The method of claim 1 further comprising moving at least one of the sample carrier and the incident beam relative to each other.

8. The method of claim 7, wherein the moving is on at least one of a linear trajectory, an arcuate trajectory, or combinations thereof.

9. The method of claim 4, wherein the one or more emitted fluorescence signals and the at least one concentrated emitted fluorescence signal are split into two or more spectral bands.

10. The method of claim 4, wherein the one or more emitted fluorescence signals and the at least one concentrated emitted fluorescence signal are split into a first spectral band, a second spectral band and a third spectral bands.

11. The method of claim 10, wherein the first spectral band ranges from about 650 nm to about 690 nm, the second spectral band ranges from about 690 nm to about 740 nm, and the third spectral band ranges from about 740 nm to about 800 nm.

12. The method of claim 1, wherein the excitation wavelength ranges from about 300 nanometers to 1200 nanometers.

13. The method of claim 12, wherein the excitation wavelength ranges from about 600 nanometers to 650 nanometers.

14. The method of claim 13, wherein the excitation wavelength is about 640 nanometers.

24. An assay that uses the method of claim 1.

25. An enzymatic assay that uses the method of claim 1.

26. A diagnostic measurement that uses the method of claim 1.

27. An immunoassay that uses the method of claim 1.

28. A sandwich immunoassay that uses the method of claim 1.

29. A device that uses the method of claim 1.

15. A method for measuring fluorescence comprising:

providing a sample contained in a sample carrier, wherein the sample contains at least one fluorophore;

providing a light source to generate an incident beam having an excitation wavelength and a focus diameter to impinge on the sample to yield a laser spot that defines a sample volume for measuring fluorescence;

displacing the laser spot relative to the sample volume in a three dimensional space defined by the sample volume, wherein the sample volume comprises at least one individual volume of interest;

translating the laser spot across the sample volume in a depth direction;

detecting one or more emitted fluorescence signals from the sample volume in the depth direction;

obtaining a depth profile and a thickness of the sample carrier from the one or more emitted fluorescence signals, wherein the depth profile comprises the at least one individual volume of interest;

determining at least one microvolume of interest from the depth profile; and

focusing the incident beam on the microvolume of interest to obtain at least one concentrated emitted fluorescence signal.

16. The method of claim 15 further comprising:

measuring a normalized bulk fluorescence using the depth profile and the thickness; and

measuring one or more event fluorescences for the sample using the at least one concentrated emitted fluorescence signal.

17. The method of claim 15, wherein the at least one individual volume of interest and the focus diameter of the incident beam have a defined relationship.

18. The method of claim 15 further comprising moving at least one of the sample carrier and the incident beam relative to each other.

19. The method of claim 18, wherein the moving is on at least one of a linear trajectory, an arcuate trajectory, or combinations thereof.

20. The method of claim 15, wherein the one or more emitted fluorescence signals and the at least one concentrated emitted fluorescence signal are split into two or more spectral bands.

21. The method of claim 15, wherein the excitation wavelength ranges from about 300 nanometers to 1200 nanometers.

22. The method of claim 21, wherein the excitation wavelength ranges from about 600 nanometers to 850 nanometers.

23. The method of claim 22, wherein the excitation wavelength is about 640 nanometers.

30. A fluorescence measuring device comprising:

a sample assembly for receiving a sample carrier that comprises a sample, wherein the sample comprises at least one fluorophore;

a light source for generating an incident beam having an excitation wavelength and a focus diameter to impinge on the sample to yield a laser spot that defines a sample volume for measuring fluorescence; and

a means for displacing the laser spot relative to the sample volume in a three dimensional space defined by the sample volume, wherein the sample volume comprises at least one individual volume of interest.

31. The device of claim 30, wherein the means further comprises translating means to translate the laser spot across the sample volume in a depth direction and to obtain one or more emitted fluorescence signals from the sample volume.

32. The device of claim 30 further comprising a processor module for obtaining a depth profile and a thickness of the sample carrier from the one or more emitted fluorescence signals, wherein the depth profile comprises the at least one individual volume of interest.

33. The device of claim 32, wherein the processor module uses the depth profile and the thickness for measuring normalized bulk fluorescence.

34. The device of claim 32, wherein the processor module is configured to determine at least one microvolume of interest from the depth profile.

35. The device of claim 34 further comprising a controller module to trigger the light source to focus the incident beam on the microvolume of interest to obtain at least one concentrated emitted fluorescence signal.

36. The device of claim 35, wherein the processor module uses the at least one concentrated emitted fluorescence signal to measure one or more event fluorescences for the sample.

37. The device of claim 30, wherein at least one of the sample assembly or the light source is movable in a linear trajectory, an arcuate trajectory, or combinations thereof relative to each other.

38. The device of claim 30, wherein the at least one individual volume of interest and the focus diameter of the incident beam have a defined relationship.

39. The device of claim 35, further comprising a detector assembly to detect the one or more emitted fluorescence signals and the at least one concentrated emitted fluorescence signal.

40. The device of claim 39, wherein the detector assembly further comprises two or more beam splitters for splitting the one or more emitted fluorescence signals and the at least one concentrated emitted fluorescence signal into two or more spectral bands.

41. The device of claim 40, wherein the detector assembly further comprises three beam splitters for splitting the one or more emitted fluorescence signals and the at least one concentrated emitted fluorescence signal into a first spectral band, a second spectral band and a third spectral bands respectively.

42. The device of claim 41, wherein the first spectral band ranges from about 650 nm to about 690 nm, the second spectral band ranges from about 690 nm to about 740 nm, and the third spectral band ranges from about 740 nm to about 800 nm.

43. The device of claim 40 wherein the detector assembly comprises two or more detectors, each detector configured to receive a respective portion of the one or more emitted fluorescence signals and the at least one concentrated emitted fluorescence signal from the two or more beam splitters.

44. The device of claim 43 wherein the processor module is configured for receiving data representative of each respective portion of the one or more emitted fluorescence signals and the at least one concentrated emitted fluorescence signal from the two or more detectors, and for measuring one or more fluorescence events for the individual volume of interest and normalized bulk fluorescence for the sample, based on the data.

45. The device of claim 30 further comprising a graphical user interface to display a plurality of menu options, to receive inputs from an operator and to display results for the sample.

46. The device of claim 39, wherein the controller module is coupled to the sample assembly, the light source and the detector assembly, wherein the controller module is configured to issue instructions for operation of the sample assembly, the light source and the detector assembly based on a selected menu option.

47. The device of claim 30, wherein the excitation wavelength ranges from about 300 nanometers to 1200 nanometers.

48. The device of claim 47, wherein the excitation wavelength ranges from about 600 nanometers to 800 nanometers.

49. The device of claim 48, wherein the excitation wavelength is about 640 nanometers.

50. A system that comprises the device of claim 30.

51. A method for analyzing a sample comprising:

preparing a sample for analysis, wherein the sample comprises at least one fluorophore;

acquiring simultaneous measurement data from the sample, wherein the simultaneous measurement data is representative of one or more fluorescence events for at least one individual volume of interest and a normalized bulk fluorescence for the sample; and

using the simultaneous measurement data to determine a sample condition.

52. The method of claim 51 wherein the sample condition is an analyte measure for the sample.

53. The method of claim 51 wherein the sample condition is a disease condition.

54. The method of claim 53 wherein the disease condition comprises at least one of an onset, a progression, a regression, stable, an advanced condition.

55. A system that uses the method of claim 51.