Methods and systems for determining a drop delay of a flow stream in a flow cytometer

1. A method of determining a drop delay of a flow stream in a flow cytometer, the method comprising:

a) flowing a liquid sample containing particles through a detection region of the flow cytometer, wherein the detection region comprises a flow cell having: an input, into which the liquid sample is introduced, an interrogation point, and a nozzle from which a flow stream of the liquid sample exits the flow cell;

wherein the flow stream exits the nozzle as a continuous flow stream that transitions to a stream of liquid droplets at a break off point, wherein the break off point is a point at which, upstream from the break off point, the flow stream is a continuous and connected fluidic stream and, downstream from the break off point, the flow stream is composed of a plurality of droplets having no physical contact with each other;

b) detecting a particle in the flow stream by illuminating the particle in the flow stream at the interrogation point;

c) capturing images of the flow stream to obtain an imaged flow stream, wherein the images of the flow stream are captured in one or more detection fields by one or more image sensors;

d) identifying a disturbance comprising a change in a break off point in the imaged flow stream, wherein such disturbance further comprises a change that is fanning of the flow stream or a change that is deflection of the flow stream;

e) applying an electrical charge to a droplet forming at the break off point of the flow stream;

f) calculating the drop delay of the flow stream based on the identified disturbance, wherein the drop delay is the amount of time between the time the particle is detected by the flow cytometer at the interrogation point and the time the electrical charge is applied to the droplet forming at the break off point of the flow stream.

2. The method according to claim 1 , wherein the disturbance comprises a change that is fanning of the flow stream.

3. The method according to claim 1 , wherein the disturbance comprises a change that is deflection of the flow stream.

4. The method according to claim 1 , wherein calculating the drop delay of the flow stream comprises:

varying the drop delay of the flow cytometer by drop equivalent increments across a plurality of different drop delays, wherein one or more images of the flow stream is captured corresponding to each drop delay;

calculating the drop delay corresponding to a maximal magnitude of the disturbance; and

adding a predetermined drop equivalent to the drop delay at the maximal magnitude of the disturbance wherein the drop delay at the maximal magnitude of disturbance plus a predetermined drop equivalent provides for an optimized drop delay.

5. The method according to claim 4 , wherein the predetermined drop equivalent is a one-half drop equivalent.

6. The method according to claim 4 , wherein calculating the drop delay at the maximal magnitude of the disturbance comprises applying a phase mask.

7. The method according to claim 6 , wherein the phase mask comprises 1 / 32 drop equivalent increments.

8. The method according to claim 7 , wherein determining the drop delay at the maximal magnitude of the disturbance comprises applying an 8 / 32 drop equivalent phase mask.

9. The method according to claim 4 , wherein calculating the drop delay of the flow stream comprises:

calculating the drop delay corresponding to a maximal magnitude of the disturbance while applying a one-half drop equivalent offset phase mask; and

adding a predetermined drop equivalent to the drop delay at the maximal magnitude of the disturbance to determine the drop delay of the flow stream.

10. The method according to claim 9 , wherein the method comprises adding no drop equivalent to the drop delay at the maximal magnitude of disturbance while the one-half drop equivalent offset phase mask is applied.

11. The method according to claim 1 , wherein a detection field of the one or more detection fields comprises the flow stream upstream from the flow stream break-off point and downstream from the interrogation point.

12. The method according to claim 1 , wherein the flow stream is illuminated with a light source.

13. The method according to claim 12 , wherein the light source is a laser.

14. The method according to claim 1 , further comprising adjusting one or more parameters of the flow cytometer in response to the calculated drop delay.

15. The method according to claim 14 , wherein applying the electrical charge to the flow stream is adjusted in response to the calculated drop delay.

16. The method according to claim 15 , wherein a timing of applying the electrical charge to the flow stream is adjusted in response to the calculated drop delay.

17. The method according to claim 15 , wherein a charging pulse duration of applying the electrical charge to the flow stream is adjusted in response to the calculated drop delay.

18. The method according to claim 1 , wherein capturing images of the flow stream to obtain an imaged flow stream comprises continuously capturing images of the flow stream.

19. The method according to claim 1 , wherein identifying a disturbance comprising a change in a break off point in the imaged flow stream comprises assessing the likeness of one or more images of a feature of a flow stream with one or more images of a disturbance comprising a change in a break off point.

20. The method according to claim 1 , wherein a detection field of the one or more detection fields includes the flow stream downstream from the flow stream break off point.