High speed flow cytometer droplet formation system and method

• US 6,133,044 A
• Filed: 04/16/1996
• Issued: 10/17/2000
• Est. Priority Date: 10/14/1994
• Status: Expired due to Term

• Current Assignee: University of Washington
• Original Assignee: University of Washington
• Inventors: Van den Engh, Ger
• Primary Examiner(s): LUDLOW, JAN M
• Application Number:
  US08/627,963
• Time in Patent Office: 1,645 Days
• Field of Search: 436/63, 436/52, 436/53, 436/149, 436/164, 436/177, 436/180, 324/76.4, 356/336, 209/3.1-3.3, 209/155, 422/68.1, 422/82.05, 422/82.08, 422/82.09, 422/99, 422/100, 422/101, 73/53.01, 73/61.41, 73/64.56, 73/863.21, 73/DIG. 1, 73/DIG. 2, 264/9, 264/10, 366/114, 366/116, 366/108, 425/6
• US Class Current: 436/177
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1409 Handling samples, e.g. injecting samples
• CPC Class Codes: G01N 2015/1406 Control of droplet point
• CPC Class Codes: Y10T 436/117497 with a continuously flowing sample or carrier stream
• CPC Class Codes: Y10T 436/118339 with formation of a segmented stream
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/2575 Volumetric liquid transfer

Abstract

A droplet forming flow cytometer system allows high speed processing without the need for high oscillator drive powers through the inclusion of an oscillator or piezoelectric crystal such as within the nozzle volume or otherwise unidirectionally coupled to the sheath fluid. The nozzle container continuously converges so as to amplify unidirectional oscillations which are transmitted as pressure waves through the nozzle volume to the nozzle exit so as to form droplets from the fluid jet. The oscillator is directionally isolated so as to avoid moving the entire nozzle container so as to create only pressure waves within the sheath fluid. A variation in substance concentration is achieved through a movable substance introduction port which is positioned within a convergence zone to vary the relative concentration of substance to sheath fluid while still maintaining optimal laminar flow conditions. This variation may be automatically controlled through a sensor and controller configuration. A replaceable tip design is also provided whereby the ceramic nozzle tip is positioned within an edge insert in the nozzle body so as to smoothly transition from nozzle body to nozzle tip. The nozzle tip is sealed against its outer surface to the nozzle body so it may be removable for cleaning or replacement.

Claims

1. A method of creating a droplet from a jet of a flow cytometer comprising the steps of:

a. establishing a nozzle volume defined by a nozzle body;

b. introducing a flow of sheath fluid into said nozzle volume;

c. introducing a flow of a substance within said sheath fluid in said nozzle volume;

d. establishing a substantially isolated unidirectional coupling with said nozzle volume which couples an oscillator to said nozzle volume through use of a directional isolator situated between said nozzle body and said oscillator;

e. creating a substantially isolated unidirectional oscillation within said nozzle volume using an alternating voltage with an amplitude of less than one hundred millivolts for said oscillator;

f. allowing said sheath fluid to exit from said nozzle volume; and

g. forming at least one substance entraining droplet from said sheath fluid after allowing said sheath fluid to exit from said nozzle volume.

2. A method of creating a droplet from a jet of a flow cytometer as described in claim 1 wherein the amplitude of said alternating voltage is about ten millivolts.

3. A method of creating a droplet from a jet of a flow cytometer as described in claim 1 wherein said oscillator coupled to said nozzle volume through the use of a directional isolator situated between said nozzle body and said oscillator is established at least partially within said nozzle volume.

4. A method of creating a droplet from a jet of a flow cytometer as described in claim 1 wherein said nozzle volume has a perpendicular cross sectional area and wherein said oscillator is established substantially throughout said perpendicular cross sectional area.

5. A method of creating a droplet from a jet of a flow cytometer as described in claim 1 wherein said oscillator has an isolated unidirectional coupling to said sheath fluid.

6. A method of creating a droplet from a jet of a flow cytometer as described in claim 1, 4, or 5 and further comprising the step of continuously converging said sheath fluid within said nozzle volume.

7. A system for creating a droplet from a jet of a flow cytometer comprising:

a. a nozzle container establishing a nozzle volume defined by a nozzle body and having a nozzle exit;

b. a sheath fluid port located within said nozzle volume wherein said sheath fluid port introduces a sheath fluid;

c. a substance introduction port located within said nozzle volume;

d. an oscillator to which said sheath fluid is responsive;

e. a substantially isolated unidirectional coupling which couples said oscillator to said nozzle volume through use of a directional isolator situated between said nozzle body and said oscillator wherein said coupling permits said oscillation to create oscillation in substantially one direction;

f. an alternating voltage source having an alternating voltage amplitude of less than one hundred millivolts connected to said oscillator; and

g. a free fall area below said nozzle exit and within which a substance entraining droplet forms.

8. A system for creating a droplet from a jet of a flow cytometer as described in claim 7 wherein said alternating voltage amplitude is about ten millivolts.

9. A system for creating a droplet from a jet of a flow cytometer as described in claim 7 wherein said oscillator coupled to said nozzle volume through the use of a directional isolator situated between said nozzle body and said oscillator is established at least partially within said nozzle container.

10. A system for creating a droplet from a jet of a flow cytometer as described in claim 9 wherein said nozzle container has a cap section and wherein said oscillator comprises a piezoelectric crystal.

11. A system for creating a droplet from a jet of a flow cytometer as described in claim 10 wherein said sheath fluid port introduces a sheath fluid, wherein said oscillator has an oscillator surface which faces said sheath fluid, and further comprising an interface material between said oscillator surface and said sheath fluid.

12. A system for creating a droplet from a jet of a flow cytometer as described in claim 9 wherein said nozzle container has a largest perpendicular cross sectional area and wherein said oscillator is located at said largest perpendicular cross sectional area and is substantially as large as said largest perpendicular cross sectional area.

13. A system for creating a droplet from a jet of a flow cytometer as described in claim 10 wherein said directional isolator comprises a spacer.

14. A system for creating a droplet from a jet of a flow cytometer as described in claim 7 or 12 wherein said nozzle container continuously converges.

15. A system for creating a droplet from a jet of a flow cytometer as described in claim 14 wherein said converging nozzle container continuously converges from said sheath fluid port to said nozzle exit.

16. A system for creating a droplet from a jet of a flow cytometer as described in claim 14 wherein said converging nozzle container comprises:

a. a nozzle body having an inner surface;

b. a nozzle tip having an inner surface; and

c. a seal located off of said inner surface of said nozzle tip and to which both said nozzle body and said nozzle tip are responsive.

17. A method of creating a droplet from a jet of a flow cytometer comprising the steps of:

a. establishing a nozzle volume defined by a nozzle body;

b. introducing a flow of sheath fluid into said nozzle volume;

c. introducing a flow of a substance within said sheath fluid in said nozzle volume;

d. initiating a substantially isolated unidirectional oscillation through use of a directional isolator situated between said nozzle body and an oscillator wherein said substantially unidirectional oscillation occurs within said nozzle volume;

e. allowing said sheath fluid to exit from said nozzle volume; and

f. forming at least one droplet from said sheath fluid after allowing said sheath fluid to exit from said nozzle volume.

18. A method of creating a droplet from a jet of a flow cytometer as described in claim 17 wherein said step of initiating substantially isolated unidirectional oscillation through the use of a directional isolator situated between said nozzle body and an oscillator wherein said substantially unidirectional oscillation occurs within said nozzle volume comprises the step of establishing an isolated unidirectional coupling to said sheath fluid.

19. A method of creating a droplet from a jet of a flow cytometer as described in claim 17 wherein said substantially isolated unidirectional oscillation passes through a material interface and further comprising the step of minimizing the number of material interfaces which said substantially isolated unidirectional oscillation must pass through.

20. A method of creating a droplet from a jet of a flow cytometer as described in claim 19 wherein said substantially isolated unidirectional oscillation is created by an oscillator and wherein said substantially isolated unidirectional oscillation passes through only a protective coating on said oscillator before imparting on said sheath fluid.

21. A method of creating a droplet from a jet of a flow cytometer as described in claim 19 wherein the flow of said sheath fluid has a primary flow direction and wherein said step of minimizing the number of material interfaces which said substantially isolated unidirectional oscillation must pass through comprises the step of coupling said substantially isolated unidirectional oscillation in substantially only said primary flow direction.

22. A method of creating a droplet from a jet of a flow cytometer as described in claim 17 and further comprising the step of directly transferring said substantially isolated unidirectional oscillation to said sheath fluid.

23. A method of creating a droplet from a jet of a flow cytometer as described in claim 17, 18, or 22 and further comprising the step of applying said substantially isolated unidirectional oscillation to said sheath fluid.

24. A method of creating a droplet from a jet of a flow cytometer as described in claim 23 and further comprising the step of directionally isolating said substantially isolated unidirectional oscillation with a spacer.

25. A method of creating a droplet from a jet of a flow cytometer as described in claim 17 and further comprising the step of directionally isolating said substantially isolated unidirectional oscillation with a spacer.

26. A method of creating a droplet from a jet of a flow cytometer as described in claim 24 wherein said step of isolating said substantially isolated unidirectional oscillation comprises the step of coupling said substantially isolated unidirectional oscillation to said sheath fluid along only one plane.

27. A system for creating a droplet from a jet of a flow cytometer comprising:

a. a nozzle container establishing a nozzle volume defined by a nozzle body and having a nozzle exit;

b. a sheath fluid port located within said nozzle volume wherein said sheath fluid port introduces a sheath fluid;

c. a substance introduction port located within said nozzle volume;

d. a substantially isolated unidirectional coupling which couples an oscillator to said nozzle volume through use of a directional isolator situated between said nozzle body and said oscillator wherein said coupling permits said oscillator to create oscillation in substantially one direction;

e. an oscillator to which said substantially isolated unidirectional coupler and said nozzle volume are responsive; and

f. a free fall area below said nozzle exit and within which a substance entraining droplet forms.

28. A system for creating a droplet from a jet of a flow cytometer as described in claim 27 wherein said sheath fluid port introduces a sheath fluid and wherein said oscillator comprises a piezoelectric crystal.

29. A system for creating a droplet from a jet of a flow cytometer as described in claim 27 wherein said sheath fluid port introduces a sheath fluid, wherein said oscillator has an oscillator surface which faces said sheath fluid, and further comprising an interface material between said oscillator surface and said sheath fluid.

30. A system for creating a droplet from a jet of a flow cytometer as described in claim 27 wherein said nozzle container comprises:

a. a cap section;

b. a nozzle body sealed to said cap section; and

c. a nozzle tip having said nozzle exit situated thereon, wherein said nozzle tip is sealed to said nozzle body, and wherein said sheath fluid flows through said nozzle tip.

31. A system for creating a droplet from a jet of a flow cytometer as described in claim 27 wherein said oscillator has an oscillator surface which faces said sheath fluid and wherein said oscillator surface is planar.

32. A system for creating a droplet from a jet of a flow cytometer as described in claim 27 or 28 wherein said sheath fluid port introduces a sheath fluid and further comprising a coupling which is only planar and which couples said oscillator to said sheath fluid.

33. A system for creating a droplet from a jet of a flow cytometer as described in claim 32 wherein said directional isolator between said oscillator and said nozzle body comprises a spacer.

34. A system for creating a droplet from a jet of a flow cytometer as described in claim 27 wherein said oscillator emits a predominant frequency and wherein said directional isolator is effective at said predominant frequency.

35. A system for creating a droplet from a jet of a flow cytometer as described in claim 33 wherein said oscillator has an oscillator side and wherein said spacer maintains said oscillator side detached from said nozzle container.