High resolution flow cytometer

• US 7,855,078 B2
• Filed: 08/15/2003
• Issued: 12/21/2010
• Est. Priority Date: 08/15/2002
• Status: Active Grant

• Current Assignee: XY, LLC (Inguran LLC)
• Original Assignee: XY, LLC (Inguran LLC)
• Inventors: Evans, Kenneth M.
• Primary Examiner(s): Menon; Krishnan S
• Assistant Examiner(s): FRITCHMAN, REBECCA M
• Application Number:
  US10/524,793
• Publication Number:
  US 20060141628A1
• Time in Patent Office: 2,685 Days
• Field of Search: 436/63, 436/10, 738/655, 738/658, 324/71.4, 356440-442, 422/73, 435/287.1
• US Class Current: 436/63
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]

Abstract

High resolution particle differentiation process and separation system that provides enhanced resolution of particles based upon selected particle characteristics. In particular, the system may include an enhanced resolution flow cytometer. In an embodiment, the invention can include at least one fluid source conduit (24) that introduces 0 fluid source stream (24) into an enhanced resolution nozzle (25) at an angle that enhances particle resolution by the cell sensing system (13).

Claims

1. A method of flow cytometry, comprising the steps of:

a. obtaining sperm cells of a mammal;

b. injecting said sperm cells at an injection point along the central longitudinal axis of a fluid stream within a nozzle of a flow cytometer;

c. forming a plurality of droplets in said fluid stream;

d. entraining in each of a portion of said plurality of droplets one of said sperm cells injected into said fluid stream;

e. analyzing said sperm cells entrained in said portion of said plurality of droplets;

f. discriminating between said sperm cells to generate two populations based upon at least one sperm cell characteristic; and

g. adjustably varying axial location of said injection point of said sperm cells along said central longitudinal axis in said fluid stream within said nozzle of said flow cytometer to increase resolution of said at least two populations of said sperm cells based upon said at least one sperm cell characteristic.

2. A method of flow cytometry as described in claim 1, wherein said mammal is selected from the group consisting of a bovine mammal, an equine mammal, a ovine mammal, a canine mammal, a feline mammal, a swine mammal, a marine mammal, and a deer mammal.

3. A method of flow cytometry as described in claim 1, wherein said fluid stream comprises a sheath fluid.

4. A method of flow cytometry as described in claim 3, wherein said sheath fluid comprises a sheath fluid having a buffer selected from the group consisting of a citrate buffer, a phosphate buffer, and a HEPES buffer.

5. A method of flow cytometry as described in claim 1, wherein said step of obtaining sperm cells of a mammal comprises the step of obtaining sperm cells of a first species of mammal and obtaining sperm cells of a second species of mammal and wherein said step of adjustably varying axial location of said injection point of said sperm cells along said central longitudinal axis in said fluid stream to increase resolution of said at least two populations of said sperm cells comprises the step of establishing a first injection point for said sperm cells of said first species of mammal at a first location along said central longitudinal axis in said fluid stream to increase resolution of said at least two populations of sperm cells and the step of establishing a second injection point for said sperm cells of said second species of mammal at a second location along said central longitudinal axis in said fluid stream to increase resolution of said at least two populations of sperm cells.

6. A method of flow cytometry as described in claim 1, wherein said step of adjustably varying axial location of said injection point of said sperm cells along said central longitudinal axis in said fluid stream to increase resolution of said at least two populations of said sperm cells comprises the step of adjustably varying axial location at which a particle injector introduces said sperm cells in said fluid stream.

7. A method of flow cytometry as described in claim 6, wherein said step of adjustably varying axial location at which a particle injector introduces said sperm cell in said fluid stream further comprises the step of providing an axially slidly adjustable coupling between said particle injector and a nozzle body.

8. A method of flow cytometry as described in claim 6, wherein said step of adjustably varying axial location at which a particle injector introduces said sperm cell in said fluid stream further comprises the step of operating a mated pair of spiral threads between said particle injector and a nozzle body.

9. A method of flow cytometry as described in claim 6, wherein said step of adjustably varying axial location at which a particle injector introduces said sperm cells in said fluid stream further comprises the step of replacing said particle injector with a second particle injector to alter distance between said injection point of said sperm cells into said fluid stream and a nozzle orifice through which said fluid stream flows.

10. A method of flow cytometry as described in claim 1, wherein said step of adjustably varying location of said injection point of said sperm cells along said central longitudinal axis in said fluid stream to increase resolution of said at least two populations of said sperm cells further comprises the step of adjustably varying axial distance between said injection point of said sperm cells along the central longitudinal axis in said fluid stream and a nozzle orifice through which said fluid stream flows.

11. A method of flow cytometry as described in claim 1, wherein said fluid stream has fluid stream characteristics and wherein said step of adjustably varying axial location of said injection point of said sperm cells in said fluid stream along said central longitudinal axis to increase resolution of said at least two populations of said sperm cells further comprises the step of adjustably varying axial location of said injection point of said sperm cells in response to said fluid stream characteristics.

12. A method of flow cytometry as described in claim 1, wherein said fluid stream has altered fluid stream characteristics and wherein said step of adjustably varying axial location of said injection point of said sperm cells in said fluid stream along said central longitudinal axis to increase resolution of said at least two populations of said sperm cells further comprises the step of adjustably varying axial location of said injection point of said sperm cells in response to said altered fluid stream characteristics.

13. A method of flow cytometry as described in claim 1, further comprising the step of separating said sperm cells into a first population of sperm cells and a second population of sperm cells.

14. A method of flow cytometry as described in claim 13, wherein said step of discriminating between said sperm cells to generate two populations based upon at least one sperm cell characteristic further comprises the step of discriminating between said sperm cells based upon a sex characteristic, and wherein said first population of sperm cells bear an X-chromosome and said second population of sperm cells bear a Y-chromosome.

15. A method of flow cytometry as described in claim 6 wherein said step of adjustably varying axial location at which a particle injector introduces said sperm cell in said fluid stream further comprises the step of adjustably varying axial location of said particle injector with a keyed stop mated with a nozzle body.