System for sensing droplet formation time delay in a flow cytometer

• US 5,643,796 A
• Filed: 10/14/1994
• Issued: 07/01/1997
• 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, Esposito, Richard J.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): FREED, RACHEL
• Application Number:
  US08/323,358
• Time in Patent Office: 991 Days
• Field of Search: 436/164, 436/43, 436/50, 436/63, 209/3.1-3.2, 209/4, 209/571, 209/576-577, 209/127.4, 356/72, 356/73, 250/461.2, 422/68.1, 422/82.05, 422/105, 435/3, 435/1, 435/289, 435/291, 361/226
• US Class Current: 436/50
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: Y10T 436/11 Automated chemical analysis
• CPC Class Codes: Y10T 436/115831 Condition or time responsive

Abstract

A droplet flow cytometer system which includes a system to optimize the droplet formation time delay based on conditions actually experienced includes an automatic droplet sampler which rapidly moves a plurality of containers stepwise through the droplet stream while simultaneously adjusting the droplet time delay. Through the system sampling of an actual substance to be processed can be used to minimize the effect of the substances variations or the determination of which time delay is optimal. Analysis such as cell counting and the like may be conducted manually or automatically and input to a time delay adjustment which may then act with analysis equipment to revise the time delay estimate actually applied during processing. The automatic sampler can be controlled through a microprocessor and appropriate programming to bracket an initial droplet formation time delay estimate. When maximization counts through volume, weight, or other types of analysis exists in the containers, the increment may then be reduced for a more accurate ultimate setting. This may be accomplished while actually processing the sample without interruption.

Claims

1. A method of droplet flow cytometry which senses droplet formation time delay comprising the steps of:

a. establishing a nozzle volume;

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 an oscillation to which said sheath fluid is responsive;

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

f. analyzing a property of said substance entrained within said sheath fluid;

g. forming a plurality of droplets from said sheath fluid after allowing said sheath fluid to exit from said nozzle volume;

h. creating a droplet formation time delay estimate;

i. differentially charging some individual droplets based upon the step of analyzing a property of said substance and said droplet formation time delay estimate;

j. intermittently sampling said droplets while accomplishing the step of analyzing a property of said substance; and

k. analyzing said intermittent sampling;

l. adjusting said droplet formation time delay estimate based upon the step of analyzing said intermittent sampling.

2. A method of droplet flow cytometry which senses droplet formation time delay as described in claim 1 wherein said droplets sampled contain said substance and further comprising the steps of:

a. ascertaining a time delay amount which yields a maximum count of said substance; and

b. adjusting said droplet formation time delay estimate to said time delay amount which yields a maximum count of said substance.

3. A method of droplet flow cytometry which senses droplet formation time delay as described in claim 2 wherein the step of intermittently sampling said droplets while accomplishing the step of analyzing a property of said substance comprises the step of repetitively testing droplets which have been subjected to said step of differentially charging.

4. A method of droplet flow cytometry which senses droplet formation time delay as described in claim 3 wherein the step of repetitively testing droplets which have been subjected to said step of differentially charging comprises the steps of:

a. sampling said droplets a first time;

b. altering said droplet formation time delay estimate; and

then

c. sampling said droplets a second time.

5. A method of droplet flow cytometry which senses droplet formation time delay as described in claim 4 wherein the steps of sampling said droplets, altering said droplet formation time delay estimate, and again sampling said droplets are each accomplished automatically.

6. A method of droplet flow cytometry which senses droplet formation time delay as described in claim 5 wherein the step of altering said droplet formation time delay estimate comprises the step of establishing a incremental amount by which to change said droplet formation time delay estimate.

7. A method of droplet flow cytometry which senses droplet formation time delay as described in claim 6 wherein the step of altering said droplet formation time delay estimate further comprises the steps of:

a. reducing said droplet formation time delay estimate by a multiple of said incremental amount to establish a lower limit droplet formation time delay estimate; and

then

b. repetitively adding increased multiples of said incremental amount to said lower limit droplet formation time delay estimate.

8. A method of droplet flow cytometry which senses droplet formation time delay as described in claim 7 wherein the step of altering said droplet formation time delay estimate further comprises the steps of:

a. creating a reduced incremental amount by which to change said droplet formation time delay estimate;

b. reducing said droplet formation time delay estimate by a multiple of said reduced incremental amount to establish a revised lower limit droplet formation time delay estimate; and

then

c. repetitively adding increased multiples of said reduced incremental amount to said revised limit droplet formation time delay estimate.

9. A droplet flow cytometer system which senses droplet formation time delay comprising:

a. a nozzle container establishing a nozzle volume and having a nozzle exit;

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

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

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

e. a free fall area below said nozzle exit and within which said droplet forms with some droplet formation time delay;

f. a substance property sensor responsive to said free fall area;

g. an electrode to which said sheath fluid is responsive and which is responsive to said substance property sensor; and

h. an automatic substance maximization means to which said electrode is responsive which conducts intermittent analysis and which adjusts said droplet formation time delay based upon said intermittent analysis.

10. A droplet flow cytometer system which senses droplet formation time delay as described in claim 9 wherein said substance maximization means comprises:

a. an automatic droplet sampler which samples said droplets; and

b. a cell counter which is responsive to said automatic droplet sampler.

11. A droplet flow cytometer system which senses droplet formation time delay as described in claim 10 wherein said substance maximization means further comprises a time delay adjustment which is coordinated with said automatic droplet sampler.

13. A droplet flow cytometer system which senses droplet formation time delay as described in claim 12, or 11 wherein said automatic droplet sampler comprises:

a. a movable platter positioned so at to move into said free fall area; and

b. a plurality of containers on said movable platter.

14. A droplet flow cytometer system which senses droplet formation time delay as described in claim 13 wherein said automatic droplet sampler further comprises a stepper wherein said movable platter is responsive to said stepper.

15. A droplet flow cytometer system which senses droplet formation time delay as described in claim 13 wherein said droplet formation time delay adjustment is coordinated with said movable platter.

16. A droplet flow cytometer system which senses droplet formation time delay as described in claim 15 wherein said automatic droplet sampler further comprises an increment control to which said droplet formation time delay adjustment and said movable platter are responsive.

17. A droplet flow cytometer system which senses droplet formation time delay as described in claim 16 wherein said increment control is adjustable.

12. A droplet flow cytometer system which senses droplet formation time delay comprising:

a. a nozzle container establishing a nozzle volume and having a nozzle exit;

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

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

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

e. a free fall area below said nozzle exit and within which said droplet forms;

f. a substance property sensor responsive to said free fall area;

g. an electrode to which said sheath fluid is responsive and which is responsive to said substance property sensor; and

h. an automatic droplet sampler which intermittently samples said droplets;

i. an analysis system which is responsive to said automatic droplet sampler; and

j. a droplet formation time delay adjustment which is coordinated with said analysis system.