Silicon microchannel optical flow cytometer

• US 5,726,751 A
• Filed: 09/27/1995
• Issued: 03/10/1998
• Est. Priority Date: 09/27/1995
• Status: Expired due to Term

• Current Assignee: University of Washington
• Original Assignee: University of Washington
• Inventors: Altendorf, Eric H., Yager, Paul
• Primary Examiner(s): Pham, Hoa Q.
• Application Number:
  US08/534,515
• Time in Patent Office: 895 Days
• Field of Search: 356/244, 356/246, 356/335-343, 356/440, 422/82.05, 422/82.09, 422/68.1
• US Class Current: 356/246
• CPC Class Codes: B01L 2200/0636 Focussing flows, e.g. to laminate flows
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0822 Slides
• CPC Class Codes: B01L 2300/0851 Bottom walls
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: G01N 15/1023 Microstructural devices for non-optical measurement
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 2015/1027 Determining speed or velocity of a particle
• CPC Class Codes: G01N 2021/058 Flat flow cell

Abstract

The present invention provides a flow cytometer made of two components: a flow cytometer optical head and a disposable flow module. The flow module utilizes a V-groove flow channel micromachined in a silicon wafer. The optical head comprises a laser to provide an illuminating beam and small and large angle photodetectors. Anisotropically etched facets of the V-groove reflect the illuminating beam. Small angle scattered light is also reflected by the V-groove wall and is collected by the small angle photodetector. Large angle scattered light and fluorescent light can exit the channel without reflection and are collected by the large angle photodetector. In addition, fluorescent light can be back-reflected by the V-groove, which enhances the collection efficiency.

Claims

1. A disposable flow module for use with a flow cytometer optical head, comprising:

a silicon wafer having a surface, said wafer having formed therein;

(1) a V-groove flow channel having a top with width w in the plane of said surface (thereof) and having first and second walls formed in said wafer;

(2) a fluid inlet port coupled to a first end of said V-groove;

(3) a fluid outlet port coupled to the second end of said V-groove; and

(4) a module alignment means for detachably coupling said flow module with said optical head with reproducible optical alignment of said V-groove with said optical head; and

a transparent cover plate sealed to said surface of said wafer and covering said V-groove, but not covering said alignment means.

2. The flow module of claim 1 adapted for use with a particle-containing fluid,, said fluid having particles with largest diameter d, wherein said particles are compliant and wherein said width w is one and one half to two times said diameter d.

3. The flow module of claim 2 wherein said fluid is blood and wherein said width w is between 15 and 20 μ

m.

4. The flow module of claim 1 adapted for use with a particle-containing fluid, said fluid having particles with largest diameter d, wherein said particles are rigid and wherein said width w is two to four times said diameter d.

5. The flow module of claim 1 adapted for use with a sheath flow wherein said fluid inlet port comprises an inlet for a sheath flow and an inlet for injecting a particle-containing fluid core within said sheath flow, said fluid having particles with largest diameter d.

6. The flow module of claim 5 wherein said width w is at least three times said diameter d.

7. The flow module of claim 6 wherein said fluid is blood and wherein said width w is greater than or equal to 50 μ

m.

8. The flow module of claim 1 wherein said module alignment means comprises a groove in said surface of said wafer.

9. The flow module of claim 8 wherein said module alignment means comprises a plurality of grooves in said surface.

10. The flow module of claim 8 wherein said module alignment means further comprises a pit in said surface.

11. The flow module of claim 10 wherein said groove and said pit are configured to couple with a kinematic mount on said optical head.

12. The flow module of claim 11 wherein said groove is a V-groove and wherein said pit is pyramidal.

13. The flow module of claim 1 wherein said module alignment means and said V-groove are simultaneously etched onto said surface of said wafer.

14. The flow module of claim 1 wherein said surface of said wafer is a <

100>

surface and wherein said walls of said V-groove are <

111>

surfaces.

15. The flow module of claim 1 wherein said walls of said V-groove are coated with a reflective coating.

16. The flow module of claim 15 wherein said reflective coating is a metal thin film.

17. The flow module of claim 1 wherein said transparent cover plate is a glass plate.

18. The flow module of claim 17 wherein said glass plate is a pyrex plate.

19. The flow module of claim 18 wherein said pyrex plate is anodically bonded to said surface of said wafer.

20. A flow cytometer for analyzing a particle-containing fluid comprising the flow module of claim 1 and further comprising an optical head, said optical head comprising:

a light source oriented to a direct a light beam onto said first wall of the V-groove, the angle of incidence of said light beam on said wall such that, in the absence of scattering by a particle, the non-scattered light beam is reflected at least once by said walls of said V-groove and is reflected out of said V-groove;

a small angle photodetector oriented to receive light deviating by a small angle from the propagation direction of said non-scattered light beam;

a large angle photodetector oriented to receive light deviating by a large angle from the propagation direction of said non-scattered light beam; and

a mating alignment means for detachably coupling with said module alignment means.

21. The flow cytometer of claim 20 wherein said mating alignment means comprises a plurality of protruding pins.

22. The flow cytometer of claim 21 wherein said module alignment means comprises a groove and a pit in said surface of said wafer.

23. A flow cytometer optical head for analyzing a particle-containing fluid, said optical head adapted for use with the flow module of claim 1, comprising:

a light source oriented to direct a light beam onto a said first wall of the V-groove, the angle of incidence of said light beam on said wall such that, in the absence of scattering by a particle, the non-scattered light beam is reflected at least once by said walls of said V-groove and is reflected out of said V-groove;

a small angle photodetector oriented to receive light deviating by a small angle from the propagation direction of said non-scattered light beam;

a large angle photodetector oriented to receive light deviating by a large angle from the propagation direction of said non-scattered light beam; and

a mating alignment means for detachably coupling with said module alignment means.

24. The optical head of claim 23 wherein the incident beam is in a plane approximately normal to said wall and containing the channel axis of said V-groove.

25. The optical head of claim 24 wherein said incident beam is oriented at about 45°

with respect to the plane normal to said channel axis.

26. The optical head of claim 25 wherein said small angle is less than about 10°

and said large angle is about 90°

.

27. The optical head of claim 23 wherein said small angle is less than about 10°

and said large angle is between about 15° and

150°

.

28. The optical head of claim 27 wherein said small angle is between about 0.5°

and 5°

.

29. The optical head of claim 27 wherein said large angle is between about 60°

and 90°

.

30. The optical head of claim 27 wherein said large angle is about 90°

.

31. The optical head of claim 23 wherein said light source is a diode laser.

32. The optical head of claim 23 wherein said small angle photodetector is an annular detector positioned to receive said non-scattered light beam in its center.

33. The optical head of claim 32 wherein said small angle photodetector is a photodiode.

34. The optical head of claim 23 wherein said large angle photodetector includes a means for separating the wavelengths of received light.

35. The optical head of claim 34 wherein the separating means comprises a dichroic beamsplitter.

36. The optical head of claim 34 wherein said large angle photodetector comprises a plurality of photodetectore for the separated wavelengths of received light.

37. The optical head of claim 23 wherein said large angle photodetector is positioned to receive fluorescence from particles in said channel both directly and on reflection from the second wall of said V-groove.

38. The optical head of claim 23 further comprising a rigid housing on which said laser, said small angle photodetector and said large angle photodetector are mounted.

39. The optical head of claim 23 wherein said mating alignment means comprises a plurality of protruding pins.

40. The optical head of claim 39 wherein said pins form a kinematic mount.

41. A method of using the flow module of claim 1 to analyze a particle-containing fluid sample, comprising the steps of:

providing the flow module of claim 1;

utilizing said module alignment means to align said flow module with a light source;

flowing a sample through said fluid inlet port, said V-groove and said fluid outlet port;

impinging a light beam on said sample in said V-groove; and

detecting light scattered at a small angle by said sample.

42. The method of claim 41 further comprising the step of detecting light scattered at a large angle by said sample.

43. The method of claim 41 further comprising the step of

detecting fluorescence from said sample.

50. The optical head of claim 18 wherein said large angle is between 60°

and 90°

.

52. The optical head of claim 41 wherein said light source is a diode laser.

44. A flow cytometer optical head adapted for use with a V-groove flow channel, said V-groove having reflective walls, said V-groove for carrying a flow of particle-containing fluid, said optical head comprising:

a light source oriented to direct a light beam onto a first wall of the V-groove, the angle of incidence of said light beam on said wall such that, in the absence of scattering by a particle, the non-scattered light beam is reflected at least once by the walls of said V-groove and is reflected out of said V-groove;

a small angle photodetector oriented to receive light deviating by a small angle from the propagation direction of said non-scattered light beam; and

a large angle photodetector oriented to receive light deviating by a large angle from the propagation direction of said non-scattered light beam.

45. The optical head of claim 44 wherein the incident beam is in a plane normal to said wall and containing the channel axis of said V-groove.

46. The optical head of claim 45 wherein said incident beam is oriented at about 45°

with respect to the plane normal to said channel axis.

47. The optical head of claim 46 wherein said small angle is less than 10°

and said large angle is about 90°

.

48. The optical head of claim 44 wherein said small angle is less than 10°

and said large angle is between 15° and

150°

.

49. The optical head of claim 48 wherein said small angle is between 0.5°

and 5°

.

51. The optical head of claim 48 wherein said large angle is about 90°

.

53. The optical head of claim 44 wherein said small angle photodetector is an annular detector positioned to receive said non-scattered light beam in its center.

54. The optical head of claim 53 wherein said small angle photodetector is a photodiode.

55. The optical head of claim 44 wherein said large angle photodetector includes a means for separating the wavelengths of received light.

56. The optical head of claim 55 wherein the separating means comprises a dichroic beamsplitter.

57. The optical head of claim 55 wherein said large angle photodetector comprises a plurality of photodetectors for the separated wavelengths of received light.

58. The optical head of claim 44 wherein said large angle photodetector is positioned to receive fluorescence from particles in said channel both directly and on reflection from the second wall of said V-groove.

59. The optical head of claim 44 further comprising a rigid housing on which said laser, said small angle photodetector and said large angle photodetector are mounted.

60. The optical head of claim 59 wherein said housing further comprises an alignment means for detachably coupling, with a reproducible alignment, said optical head with said V-groove flow channel.

61. The optical head of claim 60 wherein said alignment means comprises a plurality of protruding pins.

62. The optical head of claim 61 wherein said pins form a kinematic mount.