Imaging and analyzing parameters of small moving objects such as cells

• US 6,249,341 B1
• Filed: 01/24/2000
• Issued: 06/19/2001
• Est. Priority Date: 01/25/1999
• Status: Expired due to Term

• Current Assignee: Cytek Biosciences Inc
• Original Assignee: Amnis Corporation (DiaSorin S.p.A.)
• Inventors: Basiji, David A., Ortyn, William E.
• Primary Examiner(s): EVANS, FANNIE L
• Application Number:
  US09/490,478
• Time in Patent Office: 512 Days
• Field of Search: 356/73, 356/319, 356/326, 356/328, 356/338, 356/317, 356/318
• US Class Current: 356/73
• CPC Class Codes: G01J 3/2803 using photoelectric array detector
• CPC Class Codes: G01J 3/2823 Imaging spectrometer
• CPC Class Codes: G01N 15/075 by optical means
• CPC Class Codes: G01N 15/147 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/0294 Particle shape
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1472 with colour
• CPC Class Codes: G01N 2015/1486 Counting the particles
• 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 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/85 Investigating moving fluids or granular solids
• CPC Class Codes: G02B 27/145 having sequential partially reflecting surfaces
• CPC Class Codes: G02B 27/148 including stacked surfaces having at least one double-pass partially reflecting surface
• CPC Class Codes: G02B 7/28 Systems for automatic generation of focusing signals
• CPC Class Codes: G06V 20/69 Microscopic objects, e.g. biological cells or cellular parts

Abstract

Light from an object such as a cell moving through an imaging system is collected and dispersed so that it can be imaged onto a time delay and integration (TDI) detector. The light can be emitted from a luminous object or can be light from a light source that has been scattered by the object or can be a fluorescent emission by one or more FISH probes, frequently used to detect substances within cells. Further, light that is absorbed or reflected by the object can also be used to produce images for determining specific characteristics of the object. The movement of the object matches the rate at which a signal is read from the TDI detector. Multiple objects passing through the imaging system can be imaged, producing both scatter images and spectrally dispersed images at different locations on one or more TDI detectors.

Claims

1. An imaging system adapted to determine one or more characteristics of an object from an image of the object in which at least one component of the object is detected while there is relative movement between the object and the imaging system, comprising:

(a) a collection lens disposed so that light traveling from the object passes through the collection lens and travels along a collection path, said collection lens substantially collimating light from the object;

(b) a light dispersing element disposed in the collection path so as to disperse the light that has passed through the collection lens, producing dispersed light;

(c) an imaging lens disposed to receive the dispersed light, producing an image from the dispersed light; and

(d) a time delay integration (TDI) detector disposed to receive the image produced by the imaging lens, said image clearly separately distinguishing components included in the object, producing an output signal that is indicative of at least one characteristic of the object, said TDI detector producing the output signal by integrating light from at least a portion of the object over time, while the relative movement between the object and the imaging system occurs.

2. The imaging system of claim 1, wherein the light dispersing element spectrally disperses the light that has passed through the collection lens, and wherein the dispersed light is spectrally dispersed across the TDI detector.

3. The imaging system of claim 1, wherein the light that has passed through the collection lens is dispersed in a plane that is orthogonal to a direction of the relative movement between the object and the imaging system.

4. The imaging system of claim 1, wherein the image of the object produced by the imaging lens moves across the TDI detector as the relative movement between the object and the imaging system occurs.

5. The imaging system of claim 1, wherein the light from the object comprises an unstimulated emission from the object.

6. The imaging system of claim 1, further comprising a light source that is disposed to provide an incident light that illuminates the object.

7. The imaging system of claim 6, wherein the object scatters the incident light, said light that is scattered from the object at least in part passing through the collection lens.

8. The imaging system of claim 6, wherein the incident light illuminating the object stimulates the object to emit the light that passes through the collection lens.

9. The imaging system of claim 6, wherein the incident light is at least partially absorbed by the object, so that the light passing through the collection lens does not include a portion of the light absorbed by the object.

10. The imaging system of claim 6, wherein the incident light is reflected from the object toward the collection lens.

11. The imaging system of claim 6, wherein the light source comprises at least one of:

(a) a coherent light source;

(b) a non-coherent light source;

(c) a pulsed light source; and

(d) a continuous light source.

12. The imaging system of claim 1, wherein the object is entrained within a fluid stream that moves the object past the collection lens.

13. The imaging system of claim 1, wherein the object is carried on a support past the collection lens.

14. The imaging system of claim 1, wherein the TDI detector responds to the image of the object by producing a signal that propagates through the TDI detector.

15. The imaging system of claim 14, wherein a propagation rate of the signal through the TDI detector is synchronized with a motion of the image of the object on the TDI detector as a result of the relative movement between the object and the imaging system.

16. The imaging system of claim 14, wherein a propagation rate of the signal through the TDI detector is not synchronized with a motion of the image of the object on the TDI detector as a result of the relative movement between the object and the imaging system.

17. The imaging system of claim 1, wherein the dispersing element comprises a prism.

18. The imaging system of claim 1, further comprising an objective lens disposed between the object and the collection lens, having a focal point at which the object is imaged;

and an optical slit aligned with a direction of the relative movement between the object and the imaging system and disposed between the objective lens and the collection lens at the focal point of the objective lens, said slit substantially preventing extraneous light reaching the collection lens by transmitting to the collection lens the light from the object that is focussed on the slit by the objective lens.

19. An imaging system adapted to determine one or more characteristics of an object from a scatter pattern image of the object while there is relative movement between the object and the imaging system, comprising:

(a) a light source that produces light incident on the object;

(b) a collection lens disposed so that light emitted by the light source and traveling from the object passes through the collection lens and travels along a collection path, said collection lens having a focal plane that is conjugate to the object;

(c) a cylindrical lens disposed in the collection path to receive the light that has passed through the collection lens from the object, said cylindrical lens having a central axis around which the cylindrical lens is curved, said central axis being generally orthogonal to a direction of the relative movement between the object and the imaging system, so that the cylindrical lens produces a scattered pattern image of the object along a direction that is substantially parallel to said central axis of the cylindrical lens; and

(d) a time delay integration (TDI) detector disposed to receive the scattered pattern image produced by the cylindrical lens, producing an output signal that is indicative of at least one characteristic of the object, said TDI detector producing the output signal by integrating light from at least a portion of the object over time.

20. The imaging system of claim 19, wherein the TDI detector includes a plurality of light sensitive regions arranged in an array of rows and columns, said scattered pattern image being distributed along either a row or a column of the light sensitive regions, said array being oriented with either the rows or the columns of the array aligned with the central axis of the cylindrical lens.

21. The imaging system of claim 19, wherein light emitted by the light source illuminates a plurality of objects so that the light from the plurality of objects passes through the collection lens and the cylindrical lens, forming a plurality of separate scattered pattern images on the TDI detector, the signals produced by the TDI detector in response to the separate scattered pattern images indicating at least one characteristic of each of the plurality of objects.

22. The imaging system of claim 19, wherein the object is entrained within a fluid stream that moves the object past the collection lens.

23. The imaging system of claim 19, wherein a signal propagates through the TDI detector, and wherein a propagation rate of said signal through the TDI detector is synchronized with a motion of the scattered pattern image of the object on the TDI detector caused by the relative movement of the object relative to the imaging system.

24. The imaging system of claim 19, wherein a signal propagates through the TDI detector, and wherein a propagation rate of the signal through the TDI detector is not synchronized with a motion of the scattered pattern image of the object on the TDI detector caused by the relative movement of the object relative to the imaging system.

25. The imaging system of claim 19, wherein the object is carried on a support that moves the object past the collection lens.

26. The imaging system of claim 19, wherein the light source comprises at least one of:

(a) a coherent light source;

(b) a non-coherent light source;

(c) a pulsed light source; and

(d) a continuous light source.

27. The imaging system of claim 19, wherein the scattered pattern image is distributed along the TDI detector along a line that is aligned with the central axis of the cylindrical lens, said line moving across the TDI detector in accord with the relative movement between the object and the imaging system.

28. An imaging system adapted to determine different characteristics of an object from images of the object in which components of the object are detected while there is relative movement between the object and the imaging system, comprising:

(a) a first collection lens disposed so that light from the object passes through the first collection lens and travels along a first collection path, said first collection lens substantially collimating the light traveling along the first collection path;

(b) a first light dispersing element disposed in the first collection path so as to disperse the light that has passed through the first collection lens, producing first dispersed light;

(c) a first imaging lens disposed to receive the first dispersed light, producing at least one image from the first dispersed light;

(d) a first time delay integration (TDI) detector disposed to receive said at least one image produced by the first imaging lens, said at least one image clearly separately distinguishing components included in the object, producing a first output signal that is indicative of at least one characteristic of the object, said first TDI detector producing the first output signal by integrating the first dispersed light from at least a portion of the object over time, while the relative movement between the object and the imaging system occurs;

(e) a second collection lens disposed so that light from the object passes through the second collection lens and travels along a second collection path different than the first collection path, said second collection lens substantially collimating the light traveling along the second collection path;

(f) a second light dispersing element disposed in the second collection path so as to disperse the light that has passed through the second collection lens, producing second dispersed light;

(g) a second imaging lens disposed to receive the second dispersed light from the second light dispersing element, producing at least one image from said second dispersed light; and

(h) a second TDI detector disposed to receive said at least one image produced by the second imaging lens, said at least one image clearly separately distinguishing components included in the object, producing a second output signal that is indicative of at least one other characteristic of the moving object, said second TDI detector producing the second output signal by integrating the second dispersed light from at least a portion of the object over time while the relative movement between the object and the imaging system occurs, said first and second output signals being indicative of substantially different characteristics of the moving object.

29. The imaging system of claim 28, further comprising a light source that produces light incident on the object, wherein the light from the object includes an emission from the object stimulated by the light from the light source that is incident on the object.

30. The imaging system of claim 28, wherein the object is entrained within a fluid stream that moves the object past the first and the second collection lens to achieve the relative movement between the object and the imaging system.

31. The imaging system of claim 28, wherein the object is carried on a support.

32. An imaging system adapted to determine characteristics of an object from images of the object, while there is relative movement between the object and the imaging system, comprising:

(a) a light source that emits light incident on the object;

(b) a collection lens disposed so that light from the light source that has illuminated the object and is traveling from the object passes through the collection lens and travels along a collection path;

(c) a beam splitter that is disposed in the collection path so that light from the light source that is scattered from the object is directed along a different path, while light that is emitted by the object continues through the beam splitter along the collection path;

(d) a spectral dispersing element disposed beyond the beam splitter in the collection path, said spectral dispersing element spectrally dispersing the light emitted by the object, producing spectrally dispersed light;

(e) an imaging lens disposed to receive the spectrally dispersed light, producing an image thereof;

(f) a cylindrical lens disposed to receive the light scattered from the object, said cylindrical lens having a central axis around which the cylindrical lens is curved, said central axis being generally orthogonal to a direction of the relative movement between the object and the imaging system, so that the cylindrical lens produces a scattered pattern image of the object along a direction that is substantially parallel to said central axis of the cylindrical lens; and

(g) at least one time delay integration (TDI) detector disposed to receive the image produced by the imaging lens, producing an output signal that is indicative of at least one characteristic of the object, and to receive the scattered pattern image produced by the cylindrical lens, producing a different output signal that is indicative of at least one other characteristic of the object, said at least one TDI detector producing said output signals by integrating light from the object over time while the object is moving.

33. The imaging system of claim 32, wherein the object is entrained within a fluid stream that moves the object past the first and the second collection lens.

34. The imaging system of claim 32, wherein the object is carried on a support.

35. The imaging system of claim 32, further comprising at least one beam conditioning lens disposed so that the light scattered by the object passes therethrough before reaching the cylindrical lens, said at least one beam conditioning lens improving a quality of the scattered pattern image on said at least one TDI detector.

36. The imaging system of claim 32, wherein a portion of said at least one TDI detector receives the scattered pattern image, and a different portion of said at least one TDI detector receives the image produced by the imaging lens from the spectrally dispersed light.

37. The imaging system of claim 36, wherein said at least one TDI detector responds to said images of the object by producing signals that propagate through said at least one TDI detector.

38. The imaging system of claim 37, wherein propagation rates of the signals through said at least one TDI detector are synchronized with a motion of the scattered pattern image and of the image of the object on said at least one TDI detector, while the relative movement between the object and the imaging system occurs.

39. The imaging system of claim 37, wherein propagation rates of the signals through said at least one TDI detector are not synchronized with a motion of the scattered pattern image and of the image of the object on said at least one TDI detector produced from the spectrally dispersed light, while the relative movement between the object and the imaging system occurs.

40. The imaging system of claim 32, wherein said dispersion element comprises a diffraction grating.

41. The imaging system of claim 32, wherein the beam splitter comprises a dichroic element that reflects the light scattered by the object and transmits the light emitted by the object.

42. The imaging system of claim 32, wherein the imaging lens images the spectrally dispersed light on a first TDI detector, and the cylindrical lens images the scattered pattern image on a second TDI detector.

43. A method for determining one or more characteristics of a moving object from an image of the object in which at least one component of the object is detected, while there is relative movement between the object and the imaging system, comprising the steps of:

(a) focusing light from the object along a collection path that is in a different direction than the relative movement between the object and the imaging system. said light traveling along the collection path being substantially collimated;

(b) dispersing the light that is traveling along the collection path, producing dispersed light;

(c) focusing the dispersed light to produce an image, said image clearly separately distinguishing components included in the object;

(d) providing a time delay integration (TDI) detector disposed to receive the image; and

(e) analyzing an output signal from the TDI detector to determine at least one characteristic of the object.

44. The method of claim 43, wherein the step of dispersing the light comprises the step of spectrally dispersing the light.

45. The method of claim 43, wherein the image of the object produced by the step of focusing moves across the TDI detector, while the relative movement between the object and the imaging system occurs.

46. The method of claim 43, wherein the light from the object comprises an unstimulated emission from the object.

47. The method of claim 43, further comprising the steps of:

(a) providing a light source; and

(b) illuminating the object with incident light from the light source while the object is moving.

48. The method of claim 47, wherein the object scatters the incident light, said light that is scattered from the object at least in part passing through the collection lens.

49. The method of claim 47, wherein the incident light illuminating the object stimulates the object to em it the light from the object that is focussed along the collection path.

50. The method claim 47, wherein the incident light is at least partially absorbed by the object, so that the light that is foussed along the collection path does not include light absorbed by the object.

51. The method of claim 47, wherein the light focussed along the collection path is the incident light produced by the light source that has been reflected from the object .

52. The method of claim 47, where in the light source comprises at least one of:

(a ) a coherent light source;

(b) a non-coherent light source;

(c) a pulsed light source; and

(d) a continuous light source.

53. The method of claim 43, further comprising the step of entraining the object within a fluid stream that moves the object.

54. The method of claim 43, further comprising the step of carrying the object on a substrate during the step of focusing the light from the object along the collection path.

55. The method of claim 43, wherein the TDI detector responds to the imag(e of the object by producing a signal that propagates through the TDI detector.

56. The method of claim 55, further comprising the step of synchronizing a motion of the image of the object on the TDI detector with a propagation rate of the signal through the TDI detector, while the image of the object moves over the TDI detector.

57. The met hod of claim 43, further comprising the step of preventing extraneous light from reaching the TDI detector by transmitting substantially only the light from the object along the collection path.

58. A method for determining one or more characteristics of an object from a moving scattered pattern image of the object, comprising the steps of:

(a) providing and positioning a light source to produce light that is incident on the object;

(b) focusing light from the light source that is scattered from the object, along a collection path;

(c) providing a cylindrical lens disposed in the collection path;

(d) producing a scattered pattern image of the object with the cylindrical lens;

(e) providing a time delay integration (TDI) detector disposed to receive the scattered pattern image produced by the cylindrical lens; and

(f) analyzing an output signal from the TDI detector that has been produced by integrating light from at least a portion of the object over time, as the scattered pattern image of the object moves over the TDI detector.

59. The method of claim 58, wherein the TDI detector includes a plurality of light sensitive regions arranged in an array of rows and columns, further comprising the step of distributing said scattered pattern image along either a row or a column of the array.

60. The method of claim 58, further comprising the steps of:

(a) illuminating a plurality of objects with the light emitted by the light source;

(b) forming a plurality of separate scattered pattern images on the TDI detector; and

(c) analyzing signals produced by the TDI detector in response to the separate scattered pattern images of the plurality of objects, each signal indicating at least one characteristic of a different one of the plurality of objects.

61. The method of claim 58, further comprising the step of entraining the object within a fluid stream that moves the object.

62. The method of claim 58, further comprising the step of synchronizing a motion of the scattered pattern image of the object on the TDI detector with a propagation rate of a signal through the TDI detector as the scattered pattern image of the object moves over the TDI detector.

63. The method of claim 58, further comprising the step of carrying the object on a support during the step of illuminating the object with light.

64. The method of claim 58, wherein the light source comprises at least one of:

(a) a coherent light source;

(b) a non-coherent light source;

(c) a pulsed light source; and

(d) a continuous light source.

65. The method of claim 58, further comprising the step of distributing the scattered pattern image over the TDI detector along a line aligned with a central axis of the cylindrical lens.

66. A method for determining characteristics of an object from images of the object, wherein components of the object are detected in the images while the images of the object move, comprising the steps of:

(a) focusing light from the object along a first collection path in which the light is substantially collimated, and along a second collection path in which the light is substantially collimated, said second collection path being in a different direction than the first collection path;

(c) producing first dispersed light from the light traveling along the first collection path;

(d) producing an image from the first dispersed light;

(e) providing a first time delay integration (TDI) detector disposed to receive the image;

(f) analyzing a first output signal from the first TDI detector produced in response to the image of the first dispersed light by integrating the first dispersed light from at least a portion of the object over time while the image of the object is moving over the first TDI detector;

(g) producing second dispersed light from the light traveling along the second collection path;

(h) producing an image from said second dispersed light said image clearly separately distinguishing components included in the object;

(i) providing a second TDI detector disposed to receive the image produced from the second dispersed light; and

(j) analyzing a second output signal from the second TDI detector that was produced in response to the image of the second dispersed light by integrating the second dispersed light from at least a portion of the object over time while the image of the object is moving over the second TDI detector, said steps of analyzing the first and second output signals determining at least one characteristic of the object.

67. The method of claim 66, wherein the light emitted from the object is stimulated by light from a light source that is incident on the object.

68. The method of claim 66, further comprising the step of entraining the object within a fluid stream that moves the object.

69. The method of claim 66, further comprising the step of carrying the object on a support.

70. A method for determining characteristics of an object from images of the object, while the images of the object are moving, comprising the steps of:

(a) providing a light source that emits light incident on the object;

(b) focusing light from the object along a collection path;

(c) splitting light scattered from the object apart from light that is emitted by the object and directing the light scattered from the object along a different path than the collection path;

(d) spectrally dispersing the light emitted by the object to produce spectrally dispersed light;

(e) producing an image of the spectrally dispersed light;

(f) providing a cylindrical lens disposed to receive the light scattered from the object;

(g) using the cylindrical lens to produce a scattered pattern image of the object;

(h) providing a time delay integration (TDI) detector disposed to receive the image produced from the spectrally dispersed light, and to receive the scattered pattern image produced by the cylindrical lens;

(i) analyzing output signals from the TDI detector to determine at least one characteristic of the moving object for each image by integrating light from the object over time while the images of the object move over the TDI detector.

71. The method of claim 70, further comprising the step of entraining the object within a fluid stream.

72. The method of claim 70, further comprising the step of carrying the object on a support.

73. The method of claim 70, wherein a portion of the TDI detector receives the scattered pattern image, and a different portion of the TDI detector receives the image produced from the spectrally dispersed light.

74. The method of claim 70, wherein the TDI detector responds to said images of the object by producing signals that propagate through the TDI detector.

75. The method of claim 70, further comprising the step of synchronizing a motion of the scattered pattern image and of the image of the object on the TDI detector produced by imaging the spectrally dispersed light with propagation rates of the signals through the TDI detector, as the images of the object move over the TDI detector.

76. The method of claim 70, wherein the step of splitting comprises the step of using a dichroic element to reflect the light scattered by the object and to transmit the light emitted by the object.