Instrument for selecting and depositing multicellular organisms and other large objects

• US 6,400,453 B1
• Filed: 08/20/1999
• Issued: 06/04/2002
• Est. Priority Date: 08/21/1998
• Status: Expired due to Term

• Current Assignee: Union Biometrica, Inc. (Harvard Bioscience, Inc.)
• Original Assignee: Union Biometrica, Inc. (Harvard Bioscience, Inc.)
• Inventors: Hansen, W. Peter
• Primary Examiner(s): Rosenberger, Richard A.
• Application Number:
  US09/378,634
• Time in Patent Office: 1,019 Days
• Field of Search: 356/237.1, 356/72, 356/73, 209/639
• US Class Current: 356/237.1
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1456 without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/0057 of filaments in liquids
• CPC Class Codes: G01N 2015/1413 Hydrodynamic focussing

Abstract

An instrument for analyzing and dispensing objects larger than about 70 μm in diameter is based on a flow cytometer with a novel fluidic switch arrangement for diverting a portion of a sample stream in response to detector signals in a flow cell. The instrument is particularly adapted for dispensing multicellular test organisms like nematodes or large microspheres for use in screening large libraries of potential pharmaceutical agents. Hydrodynamic focussing is used to center and align the objects in the flow cell. The objects pass through a sensing zone where optical or other characteristics of the objects are detected. The detector signals are processed and used to operate a fluidic switch that is located downstream from the sensing zone. The fluid stream containing the detected objects emerges from the flow cell into air where a fluid stream controlled by the fluidic switch diverts portions of the stream containing no sample objects or sample objects not meeting predetermined characteristics. The undiverted sample stream deposits selected sample objects into a plurality of containers.

Claims

1. An instrument for analyzing and selectively dispensing sample objects greater than about 50 μ

m in diameter encapsulated in a continuous liquid sample stream in air, the instrument comprising;

a sample object source containing sample objects greater than about 50 μ

m in diameter in a fluid suspension, said source having an output opening;

an alignment chamber having an input opening in fluidic communication with the output opening of the sample object source and having an output opening;

a sample object sensing zone having an input opening in fluidic communication with the output opening of the alignment chamber, said object sensing zone having an output opening which produces a continuous liquid stream in air;

a sample detector responsive to presence of sample objects in the sensing zone; and

a fluid switch downstream of the output of the sample object sensing zone, said fluid switch controlling a fluid stream intersecting the continuous liquid stream in air for disrupting said stream such disrupting physically isolated from the sample object sensing zone and the alignment chamber to prevent introduction of fluidic instabilities therein, said switch responsive to the sample detector to turn off intermittently said intersecting fluid stream thereby allowing lengths of the continuous liquid sample stream in air containing detected objects to pass undisrupted to a sample container.

2. The instrument of claim 1, wherein the fluid switch comprises a source of compressed gas and an electrically operated valve to interrupt a gas stream from the source of compressed gas.

3. The instrument of claim 2, wherein the fluid switch is operative to interact with the sample stream with sufficient force to convert said stream into a spray of droplets.

4. The instrument of claim 1, wherein the fluid switch is responsive to a delayed detection signal from the sample detector.

5. The instrument of claim 1, wherein the fluid switch is operative to include only predetermined amounts of fluid with detected objects passing to the sample container.

6. The instrument of claim 1 further comprising a controller connected between the sample detector and the fluid switch and operative to cause said switch to select one sample object at a time.

7. The instrument of claim 1, wherein the alignment chamber has an input opening for a sheath fluid.

8. The instrument of claim 7, wherein a relationship between the alignment chamber and the output of the sample source is defined to maintain a Reynolds number of about one hundred or less between said output opening and a volume of the sensing zone.

9. The instrument of claim 1 further comprising an illumination source directed toward the sensing zone, and wherein the sample detector is an optical detector.

10. The instrument of claim 9 further comprising a processor operative to determine the length of at least one of the sample objects by measuring the time that the at least one of the sample objects takes to pass between the detector and the illumination source.

11. The instrument of claim 9 further comprising an off-axis detector, located across the sensing zone along an illumination axis of the illumination source.

12. The instrument of claim 11, wherein the off- axis detector is generally perpendicular t o an illumination axis of the illumination source.

13. The instrument of claim 9, wherein the illumination source is a focused low-power laser.

14. The instrument of claim 9, wherein the sensing zone has a height of about 10-40 μ

m.

15. The instrument of claim 9, wherein the sensing zone is defined by a set of walls having a square cross-section.

16. The instrument of claim 1, wherein the output of the sample object source is separated from the sensing zone by a total conduit volume of less than 500 microliters.

17. An instrument for analyzing and selectively dispensing sample objects greater than about 50 μ

m in diameter encapsulated in a continuous liquid sample stream in air, the instrument comprising;

means for aligning the sample objects in a fluid stream, in a direction parallel to a flow direction of the fluid stream;

means for detecting the presence of the sample objects in the fluid stream, the means for detecting being located downstream from the means for aligning with respect to the fluid stream;

means for forming a continuous liquid sample stream in air; and

means for selectively disrupting the continuous liquid sample stream in air, the means for selectively disrupting located downstream from the means for detecting with respect to the fluid stream, such disrupting being physically isolated from said means for detecting so that fluidic instabilities cannot be transmitted from the means for selectively disrupting to the means for detecting, and being responsive to the means for detecting so as intermittently to cease disrupting the continuous fluid stream in air thereby allowing undisrupted lengths of said stream in air containing sample objects to pass undisturbed into a container.

18. The instrument of claim 17, wherein the means for selectively disrupting includes only a predetermined amount of fluid with each sample object.

19. An instrument for analyzing and selectively dispensing sample objects encapsulated in a continuous liquid sample stream in air, the instrument comprising:

a source of objects having an output;

a sensing zone responsive to sample objects from the output;

a detector responsive to sample objects in the sensing zone;

means for forming a continuous liquid sample stream in air; and

a switched source of compressed gas having a gas output directed toward the continuous liquid sample stream in air downstream from the detector so as to disrupt said stream, such disruption physically isolated from the detector to prevent transmissions of fluidic instabilities to the detector, and having a control input responsive to the detector to cease disrupting said stream thereby allowing lengths of the liquid sample stream containing detected objects to pass undisrupted to a sample container.

20. The instrument of claim 19, wherein the switched source of compressed gas includes a source of compressed gas and an electrically operated valve to interrupt a gas stream from the source of compressed gas.

21. The instrument of claim 19, wherein the switched source of compressed gas is operative to interact with a fluid stream from the detector with sufficient force to convert the fluid stream into a spray of droplets.

22. The instrument of claim 19, wherein the switched source of compressed gas is responsive to a delayed detection signal from the detector.

23. The instrument of claim 19, wherein the switched source is operative to repeatedly leave predetermined amounts of fluid undisrupted.

24. The instrument of claim 19, further comprising a second switched source of compressed gas positioned to disrupt fluid left undisrupted by the switched source of compressed gas.

25. The instrument of claim 19, wherein the source of objects is a source of elongate, multicellular animals.

26. An instrument for selectively dispensing sample objects encapsulated in a continuous liquid sample stream in air, the instrument comprising:

means for providing a fluid stream carrying sample objects;

means for detecting the presence of the sample objects in the fluid stream, the means for detecting being located downstream from the means for providing with respect to the fluid stream;

means for forming a continuous liquid sample stream in air; and

means for selectively directing a gas stream toward the fluid stream to disrupt portions of the continuous sample stream in air, the means for selectively directing being located downstream from the means for detecting with respect to the fluid stream, physically isolating sample stream in air disruption from the means for detecting so as not to introduce fluidic instabilities to the means for detecting, and being responsive to the means for detecting intermittently to cease sample stream in air disruption thereby allowing lengths of the continuous liquid sample stream in air containing detected objects to pass undisrupted to a sample container.

27. The instrument of claim 26, wherein the means for selectively directing operates so as to include only a predetermined amount of the continuous sample stream in air with each of the detected objects.

28. The instrument of claim 26, wherein the means for providing provides live elongate, multicellular animals as the objects, and wherein the means for selectively directing is operative to select said animals while leaving viability of the animals unimpaired.

29. A method of dispensing sample objects encapsulated in a continuous liquid sample stream in air comprising the steps of:

centering and orienting the sample objects in a flowing fluid stream;

passing the fluid stream through a sensing zone with a surrounding sheath fluid;

detecting the presence of the sample objects in the sensing zone;

forming the fluid stream into a continuous liquid sample stream in air;

diverting at least some portion of the sample stream in air with a switched fluid stream , wherein said diverting is physically isolated from the sensing zone so as to not induce fluidic instabilities therein; and

collecting ones of the sample objects by intermittently ceasing to divert at least some portion of the sample stream in air based on the step of detecting so that remaining portions of the sample stream are not diverted by the step of diverting are collected.

30. The method of claim 29, wherein the step of diverting includes a step of converting the sample stream in air into a spray of droplets.

31. The method of claim 29, wherein the step of collecting takes place for a predetermined period of time for each of the sample objects detected in the sample zone.

32. The method of claim 29 further comprising a step of illuminating the sensing zone, and wherein the step of detecting detects light from the step of illuminating.

33. The method of claim 29, wherein the step of detecting employs an on-axis detector and an off-axis detector and combines signals from these detectors.

34. The method of claim 29, wherein the step of centering includes a step of conveying a sheath fluid past a nozzle.

35. The method of claim 34, wherein the step of conveying is performed with a maximum Reynolds number of around one hundred.

36. The method of claim 29 further comprising a step of sorting the sample objects into a plurality of categories wherein the step of collecting collects the categories in a plurality of different containers.

37. The method of claim 29, wherein the sample objects are multicellular organisms and further comprising the step of exposing the multicellular organisms collected in the step of collecting to a pharmaceutical agent.

38. The method of claim 29, wherein the step of collecting the sample objects further includes dispensing predetermined numbers of sample objects into each of a plurality of containers.

39. The method of claim 29, wherein the step of centering includes reference particles with the sample objects.

40. The method of claim 29, wherein the step of collecting includes dispensing only sample objects having a predetermined characteristic into a container.

41. An instrument for analyzing and selectively dispensing sample objects encapsulated in a continuous liquid sample stream in air, the instrument comprising:

a sample source containing sample objects in a fluid suspension for dispensing a fluid suspension of sample objects;

an alignment chamber having an input in fluidic communication with an output of the sample source for aligning a longitudinal axis of the sample sources with a direction of flow of the fluid suspension;

a sample object sensing zone having an input in fluidic communication with an output of the alignment chamber;

a sample detector for detecting sample objects in the sensing zone;

an orifice downstream from the sensing zone for producing a continuous sample stream in air from the fluid suspension; and

a fluid switch downstream of the orifice, said fluid switch controlling a fluid stream intersecting the continuous liquid stream in air for disrupting said stream, such disrupting physically isolated from the sample object sensing zone and the alignment chamber to prevent introduction of fluidic instabilities therein, said switch responsive to the sample detector to turn off intermittently said intersecting fluid stream thereby allowing lengths of the continuous liquid sample stream in air containing detected objects to pass undisrupted to a sample container.

42. The instrument of claim 41, wherein the fluid switch comprises a source of compressed gas and an electrically operated valve to interrupt a gas stream from the source of compressed gas.

43. The instrument of claim 42, wherein the fluid switch is operative to interact with the sample stream with sufficient force to convert said stream into a spray of droplets.

44. The instrument of claim 41, wherein the fluid switch is responsive to a delayed detection signal from the sample detector.

45. The instrument of claim 41, wherein the fluid switch is operative to include only predetermined amounts of fluid with detected objects passing to the sample container.

46. The instrument of claim 41 further comprising a controller connected between the sample detector and the fluid switch operative to cause said switch to pass one sample object at a time.

47. The instrument of claim 41, wherein the alignment chamber has an input for a sheath fluid.

48. The instrument of claim 47, wherein a relationship between the sample object sensing zone and the output of the sample source is defined to maintain a Reynolds number of about one hundred or less between said output and a volume of the sensing zone.

49. The instrument of claim 41 further comprising an illumination source directed toward the sensing zone, and wherein the sample detector is an optical detector.

50. The instrument of claim 49 further comprising a processor operative to determine the length of at least one of the sample objects by measuring the time that the at least one of the sample objects takes to pass between the detector and the illumination source.

51. The instrument of claim 49 further comprising an off-axis detector, located across the sensing zone along an illumination axis of the illumination source.

52. The instrument of claim 51, wherein the off- axis detector is generally perpendicular to an illumination axis of the illumination source.

53. The instrument of claim 49, wherein the illumination source is a focused low-power laser.

54. The instrument of claim 49, wherein the sensing zone has a height of about 10-40 μ

m.

55. The instrument of claim 49, wherein the sensing zone is defined by a set of walls having a square cross-section.

56. The instrument of claim 41, wherein the output of the sample object source is separated from the sensing zone by a total conduit volume of less than 500 microliters.