Apparatus for analyzing and sorting biological particles

• US 20020171827A1
• Filed: 05/17/2001
• Published: 11/21/2002
• Est. Priority Date: 05/17/2001
• Status: Active Grant

• Current Assignee: Cytopeia, Inc. (Becton, Dickinson & Co)
• Original Assignee: Cytopeia, Inc. (Becton, Dickinson & Co)
• Inventors: van den Engh, Ger
• Granted Patent: US 7,345,758 B2
• Time in Patent Office: Days
• US Class Current: 356/317
• CPC Class Codes: G01N 15/1434 Optical arrangements
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties

Abstract

The invention provides an apparatus including (a) a frame having a boundary plane; (b) a flow chamber supported by the frame, the flow chamber placed a distance from the boundary plane; (c) a radiation source, the radiation source directed away from the flow chamber and away from the exterior side of the boundary plane, and (d) a first reflective surface placed to direct a radiation beam in a path crossing the boundary plane to the flow chamber; (e) one or more reflective surfaces placed to direct a radiation beam from the radiation source to the first reflective surface, the path from the radiation source to the flow chamber being at least 1.5 times the distance from the flow chamber to the boundary plane.

Claims

1. A flow cytometer, comprising:

(a) a frame having a boundary plane;

(b) a flow chamber supported by said frame, said flow chamber placed a distance from said boundary plane;

(c) a radiation source, said radiation source directed away from said flow chamber and away from the exterior side of said boundary plane,

(d) a first reflective surface placed to direct a radiation beam in a path crossing said boundary plane to said flow chamber;

(e) one or more reflective surfaces placed to direct a radiation beam from said radiation source to said first reflective surface, the path from said radiation source to said flow chamber being at least 1.5 times the distance from said flow chamber to said boundary plane.

2. The flow cytometer of claim 1, further comprising two or more openings, said openings capable of passing said radiation beam.

3. The flow cytometer of claim 2, wherein said two or more openings are aligned vertically.

4. The flow cytometer of claim 2, comprising 3 openings in said frame.

5. The flow cytometer of claim 2, comprising 4 openings in said frame.

6. The flow cytometer of claim 1, wherein said radiation source is a laser.

7. The flow cytometer of claim 1, further comprising a sample tube having a discharge opening directed to said flow chamber.

8. The flow cytometer of claim 7, wherein said discharge opening has a diameter of 300 micrometers or less.

9. The flow cytometer of claim 7, further comprising a pressure source providing pressure to said sample tube.

10. The flow cytometer of claim 9, wherein said pressure source provides at least 10 pounds per square inch to said sample tube.

11. The flow cytometer of claim 7, further comprising an oscillator removably connected to said sample tube, said oscillator capable of vibrating at a frequency that produces droplets from a stream emerging from said sample tube.

12. The flow cytometer of claim 1, further comprising a second radiation source, said second radiation source directed away from said flow chamber and away from the exterior side of said boundary plane.

13. The flow cytometer of claim 12, further comprising 2 separate lenses placed to separately focus excitation radiation beams from said first and second radiation sources on said flow chamber.

14. The flow cytometer of claim 12, further comprising a comprising a third radiation source, said third radiation source directed away from said flow chamber and away from the exterior side of said boundary plane.

15. The flow cytometer of claim 14, further comprising 3 separate lenses placed to separately focus excitation radiation beams from said first, second and third radiation sources on said flow chamber.

16. The flow cytometer of claim 14, wherein said radiation sources are directed parallel to each other.

17. The flow cytometer of claim 1, wherein said beam path from said flow chamber to said boundary plane is at least 2 times the distance from said flow chamber to said boundary plane.

18. The flow cytometer of claim 1, further comprising a camera placed to observe a radiation beam contacting said flow chamber.

19. The flow cytometer of claim 18, wherein said camera is placed to observe said flow chamber at a vantage opposite an entry point of said radiation beam.

20. A flow cytometer, comprising:

(a) a flow chamber;

(b) a first reflective surface placed to direct an excitation radiation beam to said flow chamber;

(c) one or more devices for directing an excitation radiation beam to said first reflective surface, wherein said excitation radiation beam passes orthogonally through a vertical plane in a forward direction, and

(d) a device for directing an emission radiation beam from said flow chamber, wherein said emission radiation beam passes through said vertical plane in a reverse direction compared to said forward direction.

21. The flow cytometer of claim 20, further comprising

(e) a second reflective surface placed to direct an excitation radiation beam to said first reflective surface, wherein said second reflective surface is placed at a different elevation compared to said first reflective surface.

22. The flow cytometer of claim 20, further comprising

(e) a radiation source placed to direct a radiation beam to said first reflective surface.

23. The flow cytometer of claim 22, wherein said radiation source is a laser.

24. The flow cytometer of claim 20, further comprising a sample tube having a discharge opening directed to said flow chamber.

25. The flow cytometer of claim 24, wherein said discharge opening has a diameter of 300 micrometers or less.

26. The flow cytometer of claim 24, further comprising a pressure source providing pressure to said sample tube.

27. The flow cytometer of claim 26, wherein said pressure source provides at least 10 pounds per square inch to said sample tube.

28. The flow cytometer of claim 24, further comprising an oscillator removably connected to said sample tube, said oscillator capable of vibrating at a frequency that produces droplets from a stream emerging from said sample tube.

29. The flow cytometer of claim 20, further comprising a second reflective surface placed to direct a second excitation radiation beam to said flow chamber.

30. The flow cytometer of claim 29, further comprising a second device for directing said second excitation radiation beam to said second reflective surface, wherein said second excitation radiation beam passes through said vertical plane in a forward direction.

31. The flow cytometer of claim 29, further comprising separate lenses placed to separately focus said first and second excitation radiation beams on said flow chamber.

32. The flow cytometer of claim 29, further comprising a third reflective surface placed to direct a third excitation radiation beam to said flow chamber.

33. The flow cytometer of claim 32, further comprising a third device for directing said third excitation radiation beam to said third reflective surface, wherein said third excitation radiation beam passes through said vertical plane in a forward direction.

34. The flow cytometer of claim 32, further comprising separate lenses placed to separately focus said first second and third excitation radiation beams on said flow chamber.

35. The flow cytometer of claim 20, further comprising an alignment pin hole placed to pass an excitation radiation beam from said first reflective surface to said flow chamber.

36. The flow cytometer of claim 20, wherein said flow chamber, said first reflective surface, said device for directing an excitation radiation beam, and said device for directing an emission radiation beam occur within an area having dimensions of 15 inches by 9 inches or smaller.

37. The flow cytometer of claim 20, further comprising a camera placed to observe a radiation beam contacting said flow chamber.

38. The flow cytometer of claim 37, wherein said camera is placed to observe said flow chamber at a vantage opposite an entry point of said radiation beam.

40. The flow cytometer of claim 39, further comprising a separate filter cassette, wherein said separate filter cassette attaches to an opening in said detector module.

41. The flow cytometer of claim 40, further comprising a separate filter, wherein said separate filter attaches to said filter cassette at an internal surface.

42. The flow cytometer of claim 41, further comprising a separate attachment module, said separate attachment module attaching to an outer surface of said filter cassette, wherein a radiation beam passing through said opening can pass through said filter cassette and said attachment module.

43. The flow cytometer of claim 42, further comprising a separate detector attached to said attachment module.

44. The flow cytometer of claim 39, further comprising a sample tube having a discharge opening directed to said flow chamber.

45. The flow cytometer of claim 44, wherein said discharge opening has a diameter of 300 micrometers or less.

46. The flow cytometer of claim 44, further comprising a pressure source providing pressure to said sample tube.

47. The flow cytometer of claim 46, wherein said pressure source provides at least 10 pounds per square inch to said sample tube.

48. The flow cytometer of claim 44, further comprising an oscillator removably connected to said nozzle, said oscillator capable of vibrating at a frequency that produces droplets from a stream emerging from said sample tube.

49. The flow cytometer of claim 39, further comprising a screen having a mirrored surface interrupted by one or more pin holes, said screen placed to pass said separate radiation beams from said lens to said detector module.

50. The flow cytometer of claim 49, wherein said screen having a mirrored surface is interrupted by 3 pin holes, wherein said separate radiation beams pass through two of said three pin holes.

51. The flow cytometer of claim 50, further comprising a third detector placed to detect forward scattered radiation passing through the third of said three pin holes.

52. The flow cytometer of claim 49, further comprising a camera placed to observe said mirrored surface.

53. The flow cytometer of claim 39, further comprising a camera placed to observe a radiation beam contacting said flow chamber.

54. The flow cytometer of claim 53, wherein said camera is placed to observe said flow chamber at a vantage opposite an entry point of said radiation beam.

39. A flow cytometer, comprising

(a) a flow chamber capable of being contacted by a radiation beam;

(b) a reflective surface, wherein said reflective surface is responsive to radiation in the UV, VIS and IR regions of the spectrum;

(c) a lens placed to direct a radiation beam from said flow chamber to said reflective surface;

(d) a detector module;

(e) a second reflective surface placed to reflect said radiation beam from said reflective surface to said detector module, wherein said reflective surface is responsive to radiation in the UV, VIS and IR regions of the spectrum, and

(f) a detector removably connected to said detector module, wherein said detector is placed to detect said radiation beam.

55. A flow cytometer, comprising

(a) a flow chamber having two separate points capable of being contacted by separate radiation beams;

(b) a detector module;

(c) a lens placed to direct separate radiation beams from said two separate points to said detector module;

(d) a first detector removably connected to said detector module, wherein said first detector is placed to detect said first radiation beam, and

(e) a second detector removably connected to said detector module, said second detector being placed to detect said second radiation beam, wherein said first and second detectors are placed on the same side of said first and second radiation beams directed from said lens to said first pair of reflective surfaces.

56. The flow cytometer of claim 55, further comprising a pair of reflective surfaces placed to reflect each of said separate radiation beams from said lens to said detector module.

57. The flow cytometer of claim 55, further comprising a separate filter cassette, wherein said separate filter cassette attaches to an opening in said detector module.

58. The flow cytometer of claim 57, further comprising a separate filter, wherein said separate filter attaches to said filter cassette at an internal surface.

59. The flow cytometer of claim 58, further comprising a separate attachment module, said separate attachment module attaching to an outer surface of said filter cassette, wherein a radiation beam passing through said opening can pass through said filter cassette and said attachment module.

60. The flow cytometer of claim 59, further comprising a separate detector attached to said attachment module.

61. The flow cytometer of claim 55, further comprising a sample tube having a discharge opening directed to said flow chamber.

62. The flow cytometer of claim 61, wherein said discharge opening has a diameter of 300 micrometers or less.

63. The flow cytometer of claim 61, further comprising a pressure source providing pressure to said sample tube.

64. The flow cytometer of claim 63, wherein said pressure source provides at least 10 pounds per square inch to said sample tube.

65. The flow cytometer of claim 61, further comprising an oscillator removably connected to said nozzle, said oscillator capable of vibrating at a frequency that produces droplets from a stream emerging from said sample tube.

66. The flow cytometer of claim 55, further comprising a screen having a mirrored surface interrupted by one or more pin holes, said screen placed to pass said separate radiation beams from said lens to said detector module.

67. The flow cytometer of claim 66, wherein said screen having a mirrored surface is interrupted by 3 pin holes, wherein said separate radiation beams pass through two of said three pin holes.

68. The flow cytometer of claim 67, further comprising a third detector placed to detect forward scattered radiation passing through the third of said three pin holes.

69. The flow cytometer of claim 66, further comprising a camera placed to observe said mirrored surface.

70. The flow cytometer of claim 66, further comprising a means for detecting radiation reflected by said mirrored surface, wherein said detecting means determines a position of a radiation beam relative to said pin hole.

71. The flow cytometer of claim 55, further comprising a camera placed to observe a radiation beam contacting said flow chamber.

72. The flow cytometer of claim 71, wherein said camera is placed to observe said flow chamber at a vantage opposite an entry point of said radiation beam.

73. A flow cytometer capable of attaching a nozzle having a discharge opening, comprising

(a) a means for directing a radiation beam in a horizontal path;

(b) an adjustable bracket, said adjustable bracket having a means for attaching a nozzle having a discharge opening, wherein said adjustable bracket is placed to direct said discharge opening of said nozzle above said horizontal path, and

(c) a means for adjusting said adjustable bracket, said adjusting means pivoting said discharge opening of said attached nozzle in an arc parallel to said horizontal path of said radiation beam.

74. The flow cytometer of claim 73, further comprising a nozzle attached to said adjustable bracket.

75. The flow cytometer of claim 73, further comprising

(e) a second means for adjusting said adjustable bracket, wherein said second adjusting means moves said discharge opening in a vertical path orthogonal to said horizontal path of said radiation beam.

76. The flow cytometer of claim 73, further comprising

(e) a second means for adjusting said adjustable bracket, wherein said second adjusting means moves said discharge opening in a horizontal path orthogonal to said horizontal path of said radiation beam.

77. The flow cytometer of claim 73, further comprising a sample tube within said nozzle, said sample tube having a discharge opening in said nozzle.

78. The flow cytometer of claim 77, wherein said discharge opening of said sample tube has a diameter of 300 micrometers or less.

79. The flow cytometer of claim 77, further comprising a pressure source providing pressure at said discharge opening.

80. The flow cytometer of claim 79, wherein said pressure source provides at least 10 pounds per square inch to said sample tube.

81. The flow cytometer of claim 77, further comprising an oscillator, said oscillator capable of vibrating at a frequency that produces droplets from a stream emerging from said sample tube.

82. The flow cytometer of claim 73, further comprising a oscillator removably connected to said nozzle, said oscillator capable of vibrating at a frequency that produces droplets from a stream emerging from said sample tube.

83. A flow system, comprising

(a) a vacuum manifold having an internal cavity with 3 openings;

(b) a sample container seal;

(c) a first tube removably connected to a first of said 3 openings in said vacuum manifold, said first tube removably connected to said sample container seal, wherein said first tube passes through said sample container seal;

(d) a second tube removably connected to a second of said 3 openings in said vacuum manifold;

(e) a nozzle removably connected to said second tube, wherein a discharge opening of said second tube occurs in said nozzle, and

(f) a third tube removably connected to a third of said 3 openings in said vacuum manifold, wherein a vacuum applied to said third tube can evacuate said first and second tubes.

84. The flow cytometer of claim 83, further comprising zero volume pinch valves attached to one or more of said tubes.

85. The flow cytometer of claim 83, further comprising a sample container having an internal surface attached to said sample container seal.

86. The flow cytometer of claim 85, further comprising a pressure source providing pressure to said sample container.

87. The flow cytometer of claim 86, wherein said pressure source provides at least 10 pounds per square inch to said sample container.

88. The flow cytometer of claim 83, wherein said discharge opening of said second tube has a diameter of 300 micrometers or less.

89. The flow cytometer of claim 83, further comprising an oscillator removably connected to said nozzle, said oscillator capable of vibrating at a frequency that produces droplets from a stream emerging from said sample tube.

90. A flow cytometer comprising,

(a) a flow chamber, having two separate points capable of being contacted by separate radiation beams;

(b) a first reflective surface placed to direct a first excitation radiation beam to a first of said two separate points of said flow chamber;

(c) a second reflective surface placed to direct a second excitation radiation beam to a second of said two separate points of said flow chamber;

(d) two or more devices for separately directing said first and second excitation radiation beams to said two separate points of said flow chamber, wherein said first excitation radiation beam passes orthogonally through a vertical plane in a forward direction, wherein said second excitation radiation beam passes through said vertical plane in a forward direction;

(e) a device for directing first and second emission radiation beams from said two separate points of said flow chamber, wherein said emission radiation beams pass through said vertical plane in a reverse direction compared to said forward direction;

(f) a detector module, said detector module placed to contact said first and second radiation beams;

(g) a first detector connected to said detector module, wherein said first detector is placed to detect said first radiation beam;

(h) a second detector connected to said detector module, said second detector being placed to detect said second radiation beam, wherein said first and second detectors are placed on the same side of said vertical plane;

(i) an adjustable bracket, said adjustable bracket having a means for attaching a nozzle, wherein said adjustable bracket is placed to direct an attached nozzle toward said two separate points of said flow chamber, and

(j) a means for adjusting said adjustable bracket, said adjusting means pivoting said nozzle in an arc parallel to said horizontal path of said radiation beam.