Instrument for selecting and depositing multicellular organisms and other large objects

• US 20020033939A1
• Filed: 11/21/2001
• Published: 03/21/2002
• Est. Priority Date: 08/21/1998
• Status: Active Grant

• Current Assignee: Union Biometrica, Inc. (Harvard Bioscience, Inc.)
• Original Assignee: Union Biometrica, Inc. (Harvard Bioscience, Inc.)
• Inventors: Hansen, W. Peter
• Granted Patent: US 6,657,713 B2
• Time in Patent Office: Days
• US Class Current: 356/73
• 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 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 a input opening in fluidic communication with the output opening of the object source and having an output opening;

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

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 and 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 allow detected objects to pass to a sample container.

2. The instrument of claim 1, wherein the fluid switch includes a switched source of fluid having a fluid output directed toward a sample stream in air, said sample stream in fluidic communication with the output of the sample object sensing zone.

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

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

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

6. 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.

7. 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.

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

9. The instrument of claim 8, 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.

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

11. The instrument of claim 10 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.

12. The instrument of claim 10 further comprising an off-axis detector, located across the sensing volume along an illumination axis of the illumination source.

13. The instrument of claim 12, wherein the off-axis detector is generally perpendicular to an illumination axis of the illumination source.

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

15. The instrument of claim 10, wherein the sensing zone has a width of about 10-40 μ

m.

16. The instrument of claim 10, wherein the sensing zone is defined by a set of walls having a square cross-section.

17. 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.

18. The instrument of claim 1 further comprising a second fluid switch downstream of the fluid switch, for dispensing the sample objects into different containers.

20. The instrument of claim 19 further comprising means for redirecting an output of the means for determining relative to a first container to thereby dispense further ones of the sample objects into a second container.

21. The instrument of claim 19, wherein the means for selectively diverting includes only a predetermined amount of fluid with each sample object selected by the means for selectively diverting.

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

m in diameter 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, and

means for selectively diverting portions of the fluid, the means for selectively diverting being located downstream from the means for detecting with respect to the fluid stream, being physically isolated from said means for detecting so that fluidic instabilities cannot be transmitted from the means for selectively diverting to the means for detecting, and being responsive to the means for detecting.

22. An instrument for analyzing and selectively dispensing sample objects 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; and

a switched source of compressed gas having a gas output directed toward a location downstream from the detector, being physically isolated from the detector to prevent transmissions of fluidic instabilities to the detector, and having a control input responsive to the detector.

23. The instrument of claim 22, 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.

24. The instrument of claim 22, 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.

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

26. The instrument of claim 22, wherein the switched source is operative to repeatedly leave predetermined amounts of fluid undiverted.

27. The instrument of claim 22, further comprising a second switched source of compressed gas positioned to divert fluid left undiverted by the switched source of compressed gas.

28. The instrument of claim 22, wherein the source of objects is a source of elongate, multicellular animals.

30. The instrument of claim 29 further comprising a second means for selectively directing an output of the first means for selectively directing, relative to a first container to thereby dispense portions of the fluid stream into a second container.

31. The instrument of claim 29, wherein the means for selectively diverting includes only a predetermined amount of the fluid stream with each of the objects selected by the means for selectively diverting.

32. The instrument of claim 29, wherein the means for providing provides live elongate, multicellular animals as the objects, and wherein the means for selectively diverting is operative to select said animals while leaving viability of the animals unimpaired.

29. An instrument for selectively dispensing sample objects 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; and

first means for selectively directing a gas stream toward the fluid stream to divert portions of the fluid stream, the means for selectively directing being located downstream from the means for detecting with respect to the fluid stream, being physically isolated 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.

33. A method of dispensing sample objects 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;

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

collecting ones of the sample objects remaining in portions of the sample stream that were not diverted by the step of diverting.

34. The method of claim 33, wherein the step of diverting includes a step of converting the sample stream and sheath fluid into a spray of droplets.

35. The method of claim 33, wherein the step of diverting takes place for a predetermined period of time for each of the sample objects.

36. The method of claim 33 further comprising a step of illuminating the sensing zone, and wherein the step of detecting detects light from the step of illuminating.

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

38. The method of claim 33, wherein the step of centering includes a step of conveying a sheath fluid past a nozzle.

39. The method of claim 38, wherein the step of conveying is performed with a maximum Reynolds number of around one hundred.

40. The method of claim 33 further comprising a step of sorting the sample objects into a plurality of categories following the step of diverting and wherein the step of collecting collects the categories in a plurality of different containers.

41. The method of claim 33, 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.

42. The method of claim 33, wherein the step of dispensing the sample objects further includes dispensing predetermined numbers of sample objects into each of a plurality of containers.

43. The method of claim 33, wherein the step of feeding feeds reference particles with the sample objects.

44. The method of claim 33, wherein the step of dispensing includes dispensing only sample objects having a predetermined characteristic into a container.