SUB-DIFFRACTION LIMIT IMAGE RESOLUTION IN THREE DIMENSIONS

• US 20110002530A1
• Filed: 12/19/2008
• Published: 01/06/2011
• Est. Priority Date: 12/21/2007
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Bates, Wilfred M., Huang, Bo, Wang, Wenqin, Zhuang, Xiaowei
• Granted Patent: US 8,564,792 B2
• Time in Patent Office: Days
• US Class Current: 382/154
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• 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 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/361 Optical details, e.g. image relay to the camera or image sensor G02B21/364 takes precedence; illumination details G02B21/06 and subgroups
• CPC Class Codes: G02B 21/367 providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
• CPC Class Codes: G02B 27/58 Optics for apodization or superresolution; Optical synthetic aperture systems
• CPC Class Codes: H04N 13/204 using stereoscopic image cameras stereoscopic photography G03B35/00
• CPC Class Codes: H04N 7/18 Closed-circuit television [CCTV] systems, i.e. systems in which the video signal is not broadcast

Abstract

The present invention generally relates to sub-diffraction limit image resolution and other imaging techniques, including imaging in three dimensions. In one aspect, the invention is directed to determining and/or imaging light from two or more entities separated by a distance less than the diffraction limit of the incident light. For example, the entities may be separated by a distance of less than about 1000 nm, or less than about 300 nm for visible light. In some cases, the position of the entities can be determined in all three spatial dimensions (i.e., in the x, y, and z directions), and in certain cases, the positions in all three dimensions can be determined to an accuracy of less than about 1000 nm. In one set of embodiments, the entities may be selectively activatable, i.e., one entity can be activated to produce light, without activating other entities. A first entity may be activated and determined (e.g., by determining light emitted by the entity), then a second entity may be activated and determined. The emitted light may be used to determine the x and y positions of the first and second entities, for example, by determining the positions of the images of these entities, and in some cases, with sub-diffraction limit resolution. In some cases, the z positions may be determined using one of a variety of techniques that uses intensity information or focal information (e.g., a lack of focus) to determine the z position. Non-limiting examples of such techniques include astigmatism imaging, off-focus imaging, or multi-focal-plane imaging.

Claims

1. A method, comprising:

providing a first entity and a second entity separated by a distance of less than about 1000 nm;

determining light emitted by the first entity;

determining light emitted by the second entity; and

determining x, y, and z positions of the first entity and the second entity using the light emitted by the first entity and the light emitted by the second entity.

2. A method, comprising:

providing a first entity and a second entity separated by a distance of separation;

determining light emitted by the first entity;

determining light emitted by the second entity; and

determining x, y, and z positions of the first entity and the second entity using the light emitted by the first entity and the light emitted by the second entity.

40. The method of claim 2, wherein the first entity and a second entity separated by a distance of less than about 1000 nm.

3. A method, comprising:

providing a first entity and a second entity separated by a distance of less than about 1000 nm;

activating the first entity but not the second entity;

determining light emitted by the first entity;

activating the second entity;

determining light emitted by the second entity; and

determining x, y, and z positions of the first entity and the second entity using the light emitted by the first entity and the light emitted by the second entity.

6. The method of claim 3, further comprising constructing an image of the first entity and the second entity using their respective x, y, and z positions.

7. The method of claim 3, further comprising determining the z positions of first entity and/or the second entity from the shapes of their images at a detector.

8. The method of claim 3, further comprising determining the z positions of first entity and/or the second entity from the intensities of their images at a detector.

9. The method of claim 3, comprising passing light emitted by the first entity through a lens that is non-circularly symmetric with respect to the direction light emitted by the first entity passes through the lens.

10. The method of claim 3, further comprising passing light emitted by the first entity through a cylindrical lens.

11. The method of claim 3, further comprising collecting the light emitted by the first entity with a detector, wherein the light emitted by the first entity is not focused at the detector.

13. The method of claim 11, further comprising collecting the light emitted by the second entity by the detector, wherein the light emitted by the second entity by the detector is not focused in a detection region of the detector.

15. The method of claim 11, comprising passing the light emitted by the second entity through a lens that defines a focal region that does not contain the second entity.

16. The method of claim 11, comprising passing the light emitted by the second entity through a lens that is non-circularly symmetric with respect to the direction light emitted by the second entity passes through the lens.

17. The method of claim 11, comprising passing the light emitted by the second entity through a cylindrical lens.

18. The method of claim 9, further comprising collecting the light emitted by the second entity by a detector, wherein the light emitted by the second entity by the detector is not focused in a detection region of the detector.

20. The method of claim 9, comprising passing the light emitted by the second entity through a lens that defines a focal region that does not contain the second entity.

21. The method of claim 9, comprising passing the light emitted by the second entity through a lens that is non-circularly symmetric with respect to the direction light emitted by the second entity passes through the lens.

22. The method of claim 9, comprising passing the light emitted by the second entity through a cylindrical lens.

23. The method of claim 3, further comprising collecting the light emitted by the first entity by a plurality of detectors, wherein the light emitted by the first entity is not in a focal region defined by at least one of the plurality of detectors.

24. The method of claim 3, further comprising collecting the light emitted by the first entity by a plurality of detectors, wherein at one or more of the plurality of detectors, the light emitted by the first entity is not focused.

25. The method of claim 24, further comprising collecting the light emitted by the second entity by the plurality of detectors, wherein the light emitted by the second entity by the detector is not focused in a detection region of at least one of the plurality of detectors.

26. The method of claim 24, further comprising collecting the light emitted by the second entity by the plurality of detectors, wherein the light emitted by the second entity by the detector is not focused in a conjugate plane of at least one of the plurality of detectors.

27. The method of claim 3, wherein the light emitted by the first entity and the light emitted by the second entity have different wavelengths.

28. The method of claim 3, wherein the light emitted by the first entity and the light emitted by the second entity have substantially the same wavelength.

29. The method of claim 3, wherein the first entity and the second entity are activated by different wavelengths.

30. The method of claim 3, wherein the first entity and the second entity are activated by substantially the same wavelength.

31. The method of claim 3, wherein the first entity is chemically different from the second entity.

32. The method of claim 3, wherein the first entity is substantially chemically identical from the second entity.

33. The method of claim 3, wherein the first entity is photoactivatable or photoswitchable probe, or the first entity is photoactivatable or photoswitchable.

34. The method of claim 3, wherein the first entity comprises a first, light-emitting portion and a second, activation portion that activates the first portion upon exposure to an external stimulus.

35. The method of claim 3, wherein the second entity is photoactivatable or photoswitchable probe, or the second entity is photoactivatable or photoswitchable.

36. The method of claim 3, wherein the second entity comprises a first, light-emitting portion and a second, activation portion that activates the first portion upon exposure to an external stimulus.

37. The method of claim 3, wherein the light emitted by the first entity has a wavelength of between about 400 nm and about 1000 nm.

38. The method of claim 3, wherein the first entity and the second entity are immobilized relative to a common entity.

39. The method of claim 3, wherein the positions of the first entity and the second entity are determined to an accuracy of at least about 300 nm.

41. The method of claim 3, or 4, wherein the positions of the first entity and the second entity are determined to an accuracy of at least about 100 nm.

42. The method of claim 3, wherein the positions of the first entity and the second entity are determined to an accuracy of at least about 50 nm.

43. The method of claim 3, wherein the positions of the first entity and the second entity are determined to an accuracy of at least about 20 nm.

44. The method of claim 3, wherein the positions of the first entity and the second entity are determined to an accuracy of at least about 10 nm.

45. The method of claim 3, wherein the positions of the first entity and the second entity are determined to an accuracy that is less than the wavelength of the light emitted by the first entity and/or the second entity.

46. The method of claim 3, comprising determining the positions of the first entity and the second entity at a first point of time, and determining the positions of the first entity and the second entity at a second point of time.

47. The method of claim 3, comprising determining the positions of the first entity and the second entity at more than one points of time and/or as a function of time.

48. The method of claim 3, wherein the acts are performed in the order recited.

78. The method of claim 3, comprising passing light emitted by the first entity and/or the second entity through an optical element that is non-circularly symmetric with respect to the direction light emitted by the first entity.

4. A method, comprising:

providing a first entity and a second entity separated by a distance of separation;

activating the first entity but not the second entity;

determining light emitted by the first entity;

activating the second entity;

determining light emitted by the second entity; and

determining x, y, and z positions of the first entity and the second entity using the light emitted by the first entity and the light emitted by the second entity.

5. The method of claim 4, wherein the light emitted by the first entity has a wavelength greater than the distance of separation.

12. (canceled)

14. (canceled)

19. (canceled)

49. A method, comprising:

providing a plurality of entities able to emit light, at least some of which are separated by a distance of less than about 1000 nm;

activating a fraction of the plurality of entities to emit light;

determining the emitted light;

deactivating the activated fraction of the plurality of entities; and

repeating the acts of activating and deactivating the plurality of entities to determine x, y, and z positions of the plurality of entities.

50. A method, comprising:

providing a plurality of entities able to emit light;

activating a fraction of the plurality of entities to emit light;

determining the emitted light;

deactivating the activated fraction of the plurality of entities; and

repeating the acts of activating and deactivating the plurality of entities to determine x, y, and z positions of the plurality of entities.

51. The method of claim 50, wherein at least some of the plurality of entities are separated by a distance of separation less than the wavelength of the emitted light.

52. The method of claim 50, further comprising constructing an image of the plurality of entities using their respective x, y, and z positions.

53. The method of claim 50, further comprising determining the z positions of the plurality of entities from the shapes of their images at a detector.

54. The method of claim 50, further comprising determining the z positions of the plurality of entities from the intensities of their images at a detector.

55. The method of claim 50, comprising passing light emitted by the fraction of the plurality of entities through a lens that is non-circularly symmetric with respect to the direction light emitted by the some of the plurality of entities passes through the lens.

56. The method of claim 50, further comprising passing light emitted by the fraction of the plurality of entities through a cylindrical lens.

57. The method of claim 50, further comprising collecting the light emitted by the fraction of the plurality of entities with a detector, wherein the light emitted by some of the fraction of the plurality of entities is not focused at the detector.

58. The method of claim 50, further comprising collecting the light emitted by the fraction of the plurality of entities by a plurality of detectors, wherein some of the fraction of the plurality of entities are not in a focal region of at least one of the plurality of detectors.

59. The method of claim 50, comprising passing the light emitted by the fraction of the plurality of entities through a lens that defines a focal region that does not contain at least a portion of the fraction of the plurality of entities.

60. The method of claim 50, further comprising collecting the light emitted by the fraction of the plurality of entities with a detector, wherein the light emitted by at least a portion of the fraction of the plurality of entities is not in a conjugate plane of the detector.

61. The method of claim 50, further comprising collecting the light emitted by the fraction of the plurality of entities by a plurality of detectors, wherein at one or more of the plurality of detectors, the light emitted by at least a portion of the fraction of the plurality of entities is not focused.

62. The method of claim 50, wherein at least some of the plurality of entities emit light at different wavelengths.

63. The method of claim 50, wherein the plurality of entities each emit light at substantially the same wavelength.

64. The method of claim 50, wherein at least some of the plurality of entities are activated by light at different wavelengths.

65. The method of claim 50, wherein the plurality of entities are each activated by light at substantially the same wavelength.

66. The method of claim 50, wherein at least some of the plurality of entities are photoactivatable or photoswitchable probes.

67. The method of claim 50, wherein the light emitted by at least some of the plurality of entities has a wavelength of between about 490 nm and about 1000 nm.

68. The method of claim 50, wherein the positions of the plurality of entities are determined to an accuracy of at least about 1000 nm.

69. The method of claim 50, wherein the positions of the plurality of entities are determined to an accuracy of at least about 300 nm.

70. The method of claim 50, wherein the positions of the plurality of entities are determined to an accuracy of at least about 100 nm.

71. The method of claim 50, wherein the positions of the plurality of entities are determined to an accuracy of at least about 50 nm.

72. The method of claim 50, wherein the positions of the plurality of entities are determined to an accuracy of at least about 20 nm.

73. The method of claim 50, wherein the positions of the plurality of entities are determined to an accuracy of at least about 10 nm.

74. The method of claim 50, wherein the positions of the plurality of entities are determined to an accuracy that is less than the wavelength of the light emitted by the plurality of entities.

75. The method of claim 50, comprising determining the positions of the plurality of entities at a first point of time, and determining the positions of the plurality of entities at a second point of time.

76. The method of claim 50, comprising determining the positions of the plurality of entities at more than one points of time and/or as a function of time.

77. The method of claim 50, wherein the acts are performed in the order recited.

79. The method of claim 50, comprising passing light emitted by the fraction of the plurality of entities through an optical element that is non-circularly symmetric with respect to the direction light emitted by the some of the plurality of entities.