Instrument for selecting and depositing multicellular organisms and other large objects

• US 6,657,713 B2
• Filed: 11/21/2001
• Issued: 12/02/2003
• 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:
  US09/991,394
• Publication Number:
  US 20020033939A1
• Time in Patent Office: 741 Days
• Field of Search: 356/72, 356/73, 356/625, 356/635, 356/237.1, 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 comprising:

a detector;

a structure adapted to provide a first control stream; and

a first fluidic valve located downstream of the detector, wherein the first fluidic valve is in operative relationship with the detector and the structure so that, when a continuous fluid stream carrying sample objects is introduced into the instrument, the continuous fluid stream;

(i) flows past the detector so that sample objects in the continuous fluid stream are detected and a detection signal is generated, and then

(ii) flows past the first fluidic valve, which responds to the detection signal by interrupting the first control stream that operationally intersects the continuous fluid stream and deflects the continuous fluid stream, thereby allowing lengths of the continuous fluid stream containing detected sample objects to flow past the first fluidic valve undeflected by the first control stream.

2. The instrument of claim 1 further comprising:

a source located upstream of the detector, wherein the source is adapted to contain sample objects in a fluid suspension and has an output opening that produces a continuous fluid stream carrying sample objects when the source contains sample objects in a fluid suspension.

3. The instrument of claim 2 further comprising:

a flow chamber having an input opening in fluidic communication with the output opening of the source and having an output opening, wherein the flow chamber centers and aligns sample objects in a continuous fluid stream when a continuous fluid stream carrying sample objects is flown there through.

4. The instrument of claim 3 further comprising:

a sensing chamber having an input opening in fluidic communication with the output opening of the flow chamber and having an output opening, wherein the detector is adapted to detect sample objects in the sensing chamber when a continuous fluid stream carrying sample objects is flown there through.

5. The instrument of claim 4, wherein a relationship between the flow chamber and the output opening of the source is defined to maintain a Reynolds number of about one hundred or less between the output opening of the source and a volume of the sensing chamber.

6. The instrument of claim 4 further comprising:

an illumination source directed toward the sensing chamber, wherein the detector is an optical detector.

7. The instrument of claim 6 further comprising:

a processor in operative relationship with the detector that determines the length of at least one of the sample objects by measuring the time that at least one of the sample objects takes to pass between the detector and the illumination source when a continuous fluid stream carrying sample objects is introduced into the instrument.

8. The instrument of claim 6, wherein the detector is an on-axis detector that is located across the sensing chamber from the illumination source along an illumination axis of the illumination source.

9. The instrument of claim 8 further comprising:

an off-axis detector, located generally perpendicular to an illumination axis of the illumination source.

10. The instrument of claim 6, wherein the illumination source is a focused low-power laser.

11. The instrument of claim 6, wherein the sensing chamber has a width of about 10 to 40 microns.

12. The instrument of claim 6, wherein the sensing chamber is defined by a set of walls having a square cross-section.

13. The instrument of claim 6, wherein the output opening of the source is separated from the input opening of the sensing chamber by a total conduit volume of less than about 500 microliters.

14. The instrument of claim 3, wherein the flow chamber further includes an input opening for a sheath fluid.

15. The instrument of claim 1 wherein the structure includes a source of compressed gas and the first control stream originates from the source of compressed gas.

16. The instrument of claim 1 further comprising:

at least one container located downstream of the first fluidic valve, wherein the at least one container is adapted to receive lengths of the continuous fluid stream containing sample objects that pass undeflected by the first control stream when a continuous fluid stream carrying sample objects is introduced into the instrument.

17. The instrument of claim 16 further comprising:

a computer system in operative relationship with the detector and the first fluidic valve, wherein the computer system is adapted to test the detection signal for a detection condition so that, when target objects that meet the detection condition are identified by the computer system, the computer system triggers the first fluidic valve to interrupt the first control stream, thereby allowing lengths of the continuous fluid stream containing target objects to flow past the first fluidic valve undeflected by the first control stream.

18. The instrument of claim 17, further comprising:

a second fluidic valve located downstream of the first fluidic valve, wherein the second fluidic valve is adapted to control a second control stream that is directed at the lengths of continuous fluid stream containing target objects that pass undeflected by the first control stream, wherein the computer system is adapted to test the detection signal for a set of different detection conditions, and wherein the second fluidic valve is in operative relationship with the computer system so that, different lengths of the continuous fluid stream containing different target objects can be sorted into different containers by the second control stream based on the different detection conditions identified by the computer system.

19. The instrument of claim 1, wherein the first control stream operationally intersects the continuous fluid stream with sufficient force to convert the continuous fluid stream into a spray of droplets.

20. An instrument comprising:

means for providing a continuous fluid stream carrying sample objects;

means for detecting the sample objects in the continuous fluid stream, the means for detecting being located downstream from the means for providing;

means for providing a first gas stream, wherein the first gas stream operationally impinges on the continuous fluid stream and diverts the continuous fluid stream; and

first means for interrupting the first gas stream, wherein the first means for interrupting is located downstream from the means for detecting, and is in operative relationship with the means for detecting so that, when the means for detecting detects sample objects in the continuous fluid stream, the first means for interrupting responds by selectively interrupting the first gas stream, thereby allowing lengths of the continuous fluid stream containing detected sample objects to pass undiverted by the first gas stream.

21. The instrument of claim 20 further comprising:

means for aligning sample objects in a continuous fluid stream.

22. The instrument of claim 20 further comprising:

means for collecting the lengths of continuous fluid stream containing sample objects that pass undiverted by the first gas stream.

23. The instrument of claim 22 further comprising:

means for testing sample object detection signals from the means for detecting for a detection condition, the means for testing being in operative relationship with the means for detecting and the first means for interrupting so that, when target objects that meet the detection condition are identified by the means for testing, the means for testing triggers the first means for interrupting to selectively interrupt the first gas stream, thereby allowing lengths of the continuous fluid stream containing target objects to flow past the first means for interrupting undeflected by the first gas stream.

24. The instrument of claim 23 further comprising:

second means for interrupting a second gas stream, the second means for interrupting being located downstream from the first means for interrupting, the second means for interrupting being adapted to control a second gas stream that is directed at the lengths of continuous fluid stream containing target objects that pass undeflected by the first gas stream, wherein the means for testing is adapted to test sample object detection signals from the means for detecting for a set of different detection conditions, and wherein the second means for interrupting is in operative relationship with the means for testing so that, different lengths of the continuous fluid stream containing different target objects can be sorted into different means for collecting by the second gas stream based on the different detection conditions identified by the means for testing.

25. The instrument of claim 23, wherein the first means for interrupting includes only a predetermined amount of fluid with each target object that is identified by the means for testing.

26. The instrument of claim 20, wherein the means for providing provides live elongate, multicellular animals as the sample objects, and wherein the gas stream is operative to divert the multicellular animals while leaving the viability of the multicellular animals unimpaired.

27. The instrument of claim 20, wherein the means for providing provides micro spheres having a dimension of between about 70 and 500 microns as the sample objects.

28. A method comprising steps of:

providing large objects in a continuous carrier fluid;

passing the continuous carrier fluid and large objects through a sensing chamber;

detecting the presence of large objects in the sensing chamber;

diverting the continuous carrier fluid with a switched control stream that is located downstream of the sensing chamber; and

collecting at least one large object by temporarily ceasing to divert the continuous carrier fluid based on the step of detecting.

29. The method of claim 28 further comprising a step of centering and orienting the large objects in the continuous carrier fluid before the step of passing the continuous carrier fluid and large objects through a sensing chamber.

30. The method of claim 29, wherein the step of centering and orienting the large objects in the continuous carrier fluid includes a step of conveying a sheath fluid past a nozzle.

31. The method of claim 30, wherein the step of conveying is performed wit a Reynolds number of about one hundred or less.

32. The method of claim 28, wherein the step of diverting includes a step of converting the continuous carrier fluid into a spray of droplets.

33. The method of claim 28, wherein the step of diverting is temporarily ceased for a predetermined period of time for each of the large objects that is collected.

34. The method of claim 28 further comprising a step of illuminating the sensing chamber, wherein the step of detecting detects light from the step of illuminating.

35. The method of claim 28, wherein the step of detecting employs an on-axis detector and an off-axis detector and combines signals from these detectors.

36. The method of claim 28 further comprising a step of sorting the large objects into a plurality of categories following the step of diverting.

37. The method of claim 36, wherein the step of collecting collects the categories in a plurality of different containers.

38. The method of claim 28, wherein the large objects are multicellular organisms, the method further comprising a step of exposing the multicellular organisms collected in the step of collecting to a pharmaceutical agent.

39. The method of claim 28, wherein the step of collecting the large objects further includes dispensing a predetermined number of large objects into each of a plurality of containers.

40. The method of claim 39, wherein the large objects are multicellular organisms, the method further comprising steps of:

contacting the multicellular organisms dispensed in the step of dispensing to a pharmaceutical agent; and

detecting a change in the multicellular organisms after the step of contacting.

41. The method of claim 40, wherein each of the plurality of containers includes a different pharmaceutical agent.

42. The method of claim 40, wherein each of the plurality of containers includes the same pharmaceutical agent.

43. The method of claim 40, wherein the multicellular organisms are nematode worms.

44. The method of claim 40, wherein the multicellular organisms are fruit fly larvae.

45. The method of claim 40, wherein the multicellular organisms are zebrafish embryos.

46. The method of claim 40, wherein the step of dispensing includes dispensing multicellular organisms having a predetermined characteristic into each of the plurality of containers.

47. The method of claim 46, wherein the step of dispensing includes dispensing only multicellular organisms of a predetermined developmental stage into each of the plurality of containers.

48. The method of claim 46, wherein the step of dispensing includes dispensing only multicellular organisms having a predetermined genetic mutation into each of the plurality of containers.

49. The method of claim 46, wherein the step of dispensing includes dispensing multicellular organisms having a predetermined phenotypic defect into each of the plurality of containers.

50. The method of claim 28, wherein the step of providing provides reference particles with the large objects.

51. The method of claim 28, wherein the step of collecting includes collecting only large objects having a predetermined characteristic into a container.