Method and apparatus for selectively targeting specific cells within a mixed cell population

• US 6,534,308 B1
• Filed: 11/30/1999
• Issued: 03/18/2003
• Est. Priority Date: 03/27/1997
• Status: Expired due to Term

• Current Assignee: Intrexon Corporation
• Original Assignee: Oncosis, LLC
• Inventors: Eisfeld, Timothy M., Palsson, Bernhard O., Koller, Manfred R.
• Primary Examiner(s): Beisner, William H.
• Application Number:
  US09/451,659
• Time in Patent Office: 1,204 Days
• Field of Search: 435/4, 435/29, 435/7.2, 435/173.1, 435/34, 435/285.1, 435/288.7, 435/287.2, 435/173.4, 435/173.5, 435/173.7, 435/460, 382/129, 382/133, 382/134
• US Class Current: 435/288.7
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 5/0087 Purging against subsets of blood cells, e.g. purging alloreactive T cells
• CPC Class Codes: C12N 5/0093 Purging against cancer cells
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G02B 21/0088 Inverse microscopes
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/365 Control or image processing arrangements for digital or video microscopes G02B21/361, G02B21/362 take precedence

Abstract

This invention provides a method and apparatus for selectively identifying, and targeting with an energy beam, specific cells within a mixed cell population, for the purpose of inducing a response in the targeted cells. Using the present invention, every detectable cell in a population can be identified and affected, without substantially affecting non-targeted cells within the mixture.

Claims

1. An apparatus for selectively inducing a response in one or more targeted cells within a biological specimen, comprising:

a lens that defines a field of view that comprises a plurality of frames of cells within said biological specimen;

an illumination source for illuminating a frame of cells within said field of view;

an image capture system that captures an image of said frame of cells;

first commands for determining the locations of said one or more targeted cells within said image;

an energy source that emits an energy beam sufficient to induce a response in at least one of said one or more targeted cells within said frame of cells; and

second commands for steering said energy beam to said locations of said one or more targeted cells.

2. The apparatus of claim 1, wherein said one or more targeted cells are identified by the presence of, or absence of, a label.

3. The apparatus of claim 2, wherein said label is directed at the cell surface.

4. The apparatus of claim 3, wherein said label comprises a monoclonal antibody.

5. The apparatus of claim 2, wherein said label comprises a membrane permeable reagent.

6. The apparatus of claim 1, wherein said illumination source comprises a diffused laser light.

7. The apparatus of claim 1, wherein said illumination source comprises an arc lamp.

8. The apparatus of claim 1, wherein said illumination source comprises a light-emitting diode.

9. The apparatus of claim 1, wherein said illumination source comprises an incandescent lamp.

10. The apparatus of claim 1, wherein said illumination source comprises a fluorescent lamp.

11. The apparatus of claim 1, wherein said illumination source only illuminates a sub-population of cells in said biological specimen.

12. The apparatus of claim 1, wherein said one or more targeted cells comprises tumor cells.

13. The apparatus of claim 1, wherein said one or more targeted cells comprises fibroblasts.

14. The apparatus of claim 1, wherein said one or more targeted cells comprises T-cells.

15. The apparatus of claim 1, wherein said one or more targeted cells comprises teratoma-forming cells.

16. The apparatus of claim 1, wherein said one or more targeted cells comprises non-tumor cells.

17. The apparatus of claim 1, wherein said image capture system comprises a camera.

18. The apparatus of claim 17, wherein said camera comprises a charge-coupled device.

19. The apparatus of claim 1, wherein said energy source comprises a laser.

20. The method of claim 19, wherein the wavelength of energy emitted from said laser is approximately between 100 nanometers and 30 micrometers.

21. The method of claim 19, wherein the wavelength of energy emitted from said laser is about 349 nanometers.

22. The method of claim 19, wherein the wavelength of energy emitted from said laser is about 355 nanometers.

23. The method of claim 19, wherein the wavelength of energy emitted from said laser is about 523 nanometers.

24. The method of claim 19, wherein the wavelength of energy emitted from said laser is about 532 nanometers.

25. The method of claim 19, wherein the wavelength of energy emitted from said laser is about 1046.

26. The method of claim 19, wherein the wavelength of energy emitted from said laser is about 1064 nanometers.

27. The apparatus of claim 1, wherein said first commands comprise commands for determining the two-dimensional locations of said one or more targeted cells within said biological specimen.

28. The apparatus of claim 1, wherein said first commands comprise commands for determining the three-dimensional locations of said one or more targeted cells within said biological specimen.

29. The apparatus of claim 1, further comprising one or more energy beam steering devices that are controlled by said second commands.

30. The apparatus of claim 29, wherein said energy beam steering devices comprise one or more elements that reflect the energy beam.

31. The apparatus of claim 29, wherein said energy beam steering devices comprise one or more elements that refract the energy beam.

32. The apparatus of claim 29, wherein said energy beam steering devices comprise one or more elements that diffract the energy beam.

33. The apparatus of claim 1, wherein said first commands are executed under the control of a computer.

34. The apparatus of claim 1, wherein said second commands are executed under the control of a computer.

35. The apparatus of claim 1, wherein said apparatus processes at least 1 square centimeter of biological specimen area per minute.

36. The apparatus of claim 1, wherein said apparatus images at least 0.25 million cells of said biological specimen per minute.

37. The apparatus of claim 1, wherein said apparatus can induce a response in at least 50 targeted cells per second.

38. The apparatus of claim 1, wherein said response induced in said one or more targeted cells is cell death.

39. The apparatus of claim 1, wherein said response induced in said one or more targeted cells comprises activation of a photosensitive agent.

40. The apparatus of claim 1, wherein said response induced in said one or more targeted cells comprises optoporation to allow entry of a substance.

41. The apparatus of claim 40, wherein said substance is genetic material.

42. The apparatus of claim 1, wherein said response induced in said one or more targeted cells comprises inactivation of a cell component.

43. The apparatus of claim 1, wherein said response induced in said one or more targeted cells comprises controlled movement of said one or more targeted cells.

44. The apparatus of claim 1, wherein said response is excitation of fluorescent dye within said one or more targeted cells.

45. The apparatus of claim 1, further comprising an energy absorbing dye in contact with said one or more targeted cells.

46. The apparatus of claim 1, further comprising third commands that control an autofocus mechanism linked to said image capture system.

47. The apparatus of claim 46, wherein said third commands are executed under the control of a computer.

48. The apparatus of claim 1, wherein said apparatus processes at least 7 square centimeters of biological specimen area per minute.

49. The apparatus of claim 1, wherein said apparatus images at least 4 million cells of said biological specimen per minute.

50. The apparatus of claim 1, wherein said apparatus can induce a response in at least 400 targeted cells per second.

51. The apparatus of claim 1, wherein said apparatus processes at least 4 square centimeters of biological specimen area per minute.

52. The apparatus of claim 1, wherein said apparatus images at least 2 million cells of said biological specimen per minute.

53. An apparatus for selectively inducing a response in one or more targeted cells within a biological specimen, comprising:

a scanning lens which focuses on a field-of-view within said biological specimen, said field-of-view comprising a plurality of frames of cells;

an illumination source that illuminates one or more frames of cells;

an image capture system that captures an image of a first frame of cells in said field-of-view;

first commands for determining the locations of said one or more targeted cells in said first frame of cells;

an energy source that emits an energy beam sufficient to induce a response in at least one of said one or more targeted cells; and

second commands for steering said energy beam to said locations of said one or more targeted cells within said first frame of cells.

54. The apparatus of claim 53, further comprising:

means for directing the output of said illumination source to a second frame of cells in said field-of-view;

means for capturing an image of said second frame of cells;

third commands for determining the locations of said one or more targeted cells in said second frame of cells; and

fourth commands for steering said energy beam to said locations of said one or more targeted cells within said second frame of cells.

55. The apparatus of claim 53, further comprising means for moving the position of said biological specimen with respect to said scanning lens.

56. The apparatus of claim 53, wherein said scanning lens is of F-theta design with a diameter of at least 8 mm.

57. The apparatus of claim 53, wherein said scanning lens is of F-theta design with a diameter of at least 18 mm.

58. The apparatus of claim 53, wherein said scanning lens is of F-theta design with a diameter of at least 10 mm.

59. The apparatus of claim 53, wherein said scanning lens is of F-theta design with a diameter of at least 12 mm.

60. The apparatus of claim 53, wherein said scanning lens is of F-theta design with a diameter of at least 14 mm.

61. The apparatus of claim 53, wherein said scanning lens is of F-theta design with a diameter of at least 16 mm.

62. An apparatus for identifying a first population of cells having a label within a mixture comprising said first population and a second population of cells lacking said label, comprising:

a lens that defines a field of view that comprises a plurality of frames of cells within said mixture;

an illumination source for illuminating a frame of cells in said field of view;

an image capture system that captures an image of said frame of cells; and

first commands for determining the locations of at least one cell in said first population of cells having said label by reference to said image.

63. The apparatus of claim 62, further comprising a memory for storing said locations of said at least one cell in said first population of cells having said label.

64. The apparatus of claim 62, further comprising a memory for storing the quantity of cells in said first population of cells having said label.

65. The apparatus of claim 62, further comprising an energy source that emits an energy beam sufficient to induce a response in at least one cell in said first population of cells having said label.

66. An apparatus for determining a morphological or physiological characteristic of individual cells in a biological specimen, comprising:

a lens that defines a field of view that comprises a plurality of frames of cells within said biological specimen;

an illumination source for illuminating a frame of cells within said field of view;

an image capture system that captures an image of said frame of cells;

first commands for determining the location of a first targeted cell in said biological specimen by reference to said image;

second commands for intercepting said first targeted cell with an energy beam sufficient to interrogate said first targeted cell for the presence of a morphological or physiological characteristic; and

a detector for measuring the response of said first targeted cell to said interrogation.

67. The apparatus of claim 66, wherein said first targeted cell is labeled with a fluorescently-tagged antibody.

68. The apparatus of claim 66, wherein said first targeted cell is labeled with two labels, wherein the first label is activated by said illumination source and the second label is activated by said energy beam.

69. The apparatus of claim 66, wherein said morphological or physiological characteristic is calcium flux.

70. The apparatus of claim 66, wherein said morphological or physiological characteristic is cell cycle status.

71. The apparatus of claim 66, wherein said morphological or physiological characteristic is cell mitotic history.

72. The apparatus of claim 66, wherein said morphological or physiological characteristic is viability.

73. The apparatus of claim 66, wherein said morphological or physiological characteristic is membrane integrity.

74. The apparatus of claim 66, wherein said morphological or physiological characteristic is intracellular pH.

75. The apparatus of claim 66, wherein said morphological or physiological characteristic is transmembrane potential.

76. The apparatus of claim 66, wherein said morphological or physiological characteristic is glutathione reductive stage.

77. The apparatus of claim 66, wherein said morphological or physiological characteristic is thiol activity.

78. The apparatus of claim 66, wherein said morphological or physiological characteristic is expression of a gene.

79. An apparatus for selectively inducing a response in one or more targeted cells within a biological specimen, comprising:

an illumination source for illuminating a frame of cells in said biological specimen;

an image capture system that captures an image of said frame of cells;

first commands for determining the locations of said one or more targeted cells within said image;

an energy source that emits an energy beam sufficient to induce a response in at least one of said one or more targeted cells within said frame of cells; and

a sensor that detects the amount of energy emitted by said energy source.

80. A method for selectively inducing a response in one or more targeted cells within a biological specimen, comprising:

defining a field of view of view that comprises a plurality of frames of cells in said biological specimen;

illuminating a frame of cells within said field of view;

capturing an image of said frame of cells;

determining the locations of said one or more targeted cells within said image; and

steering an energy beam to the locations of said one or more targeted cells, wherein said energy beam is sufficient to induce a response in at least one of said one or more targeted cells.

81. The method of claim 80, wherein determining the locations of said one or more targeted cells comprises determining the presence of, or absence of, a label attached to said one or more targeted cells.

82. The method of claim 80, wherein capturing an image of said frame of cells comprises capturing an image of labeled cells.

83. The method of claim 80, wherein illuminating said frame of cells comprises illuminating said frame of cells with an excitation laser light source.

84. The method of claim 80, wherein said response is selected from the group consisting of cell death, optoporation, activation of a photosensitive agent, inactivation of a cell component, controlled movement, and excitation of a fluorescent reagent.

85. A method for rapidly inducing a response in cells within a cell population, said cell population being held in a container, comprising:

defining a first field of cells that comprises a plurality of frames of cells in said biological specimen;

illuminating said first field of cells in said cell population;

capturing an image of a first fame of cells within said first field of cells;

determining the locations of first targeted cells within said first frame of cells by reference to said image; and

steering an energy beam to the locations of said first targeted cells, wherein said energy beam is sufficient to induce a response in at least one of said first targeted cells.

86. The method of claim 85, further comprising:

capturing an image of a second frame of cells within said first field of cells;

determining the locations of second targeted cells within said second frame of cells by reference to said image; and

steering an energy beam to the locations of said second targeted cells within said second frame of cells, wherein said energy beam is sufficient to induce a response in at least one of said second targeted cells.

87. The method of claim 86, further comprising:

illuminating a second field of cells in said cell population;

capturing an image of a first frame of cells within said second field of cells;

determining the locations of first targeted cells within said first frame of cells by reference to said image; and

steering an energy beam to the locations of said first targeted cells, wherein said energy beam is sufficient to induce a response in at least one of said first targeted cells.

88. The method of claim 87, wherein illuminating said second field of cells comprises moving said container.

89. A method for selectively inducing a response in one or more targeted cells within a biological specimen, comprising:

focusing a scanning lens on a field-of-view within said biological specimen, said field-of-view comprising a plurality of frames of cells;

illuminating one or more frames of cells within said field-of-view;

capturing an image of a first frame of cells in said field-of-view;

determining the locations of said one or more targeted cells in said first frame of cells;

emitting an energy beam sufficient to induce a response in at least one of said one or more targeted cells; and

steering said energy beam to the locations of said one or more targeted cells within said first frame of cells.

90. The method of claim 89, wherein said response is selected from the group consisting of cell death, optoporation, activation of a photosensitive agent, inactivation of a cell component, controlled movement, and excitation of a fluorescent reagent.

91. The method of claim 89, wherein capturing an image of said first frame of cells comprises capturing an image of labeled cells.

92. The method of claim 89, wherein said illuminating one or more frames of cells comprises illuminating said one or more frames of cells with an excitation laser light source.

93. A method for inducing a response in one or more targeted cells within a cell population, comprising:

illuminating cells in said cell population;

capturing an image of the illuminated cells;

determining the locations of said one or more targeted cells within said image;

emitting an energy beam sufficient to induce a response in at least one of said one or more targeted cells; and

measuring the amount of energy contained in said energy beam; and

steering said energy beam to the locations of said one or more targeted cells.

94. The method of claim 93, comprising measuring the amount of energy contained in said energy beam prior to steering said energy beam.