Timing and/or phase adjustment of the separation and/or charging of drops from a fluid stream in a flow cytometer

Patent metadata

Field	Value
Publication number	US-10451535-B2
Application number	US-201815883229-A
Country	US
Kind code	B2
Filing date	Jan 30, 2018
Priority date	Aug 16, 2013
Publication date	Oct 22, 2019
Grant date	Oct 22, 2019

Abstract

Disclosed is a system for setting the timing or phase of the separation of droplets from a fluid stream in a flow cytometer, or the timing or phase of a charge pulse generator, based upon the collected charge of charged droplets. In one embodiment, a conductive mesh can be used to collect the charged droplets that are either deflected or not deflected by the deflection plates. In another embodiment, the charge can be collected from metal plates in the waste collection device. In addition, a defanning device is disclosed that allows substantially uniform deflection of charged cells.

First claim

What is claimed is:

1. A method of adjusting the timing or phase of a charge pulse signal for charging droplets from a fluid stream of sheath fluid in a flow cytometer, the method comprising:
forming a fluid stream by flowing the sheath fluid through a nozzle, wherein the fluid stream separates into droplets after exiting the nozzle;
applying the charge pulse signal to the nozzle during a calibration phase, wherein each droplet that separates from the stream when the charge pulse signal causes a charge to be applied to the nozzle becomes a charged droplet and each droplet that separates from the stream when the charge pulse signal does not cause a charge to be applied to the nozzle remains an uncharged droplet;
receiving the charged and uncharged droplets in a charge collector during the calibration phase;
collecting charges during the calibration phase from the charged droplets for a plurality of timing or phase settings using an electrometer that is connected with the charge collector;
measuring the charges collected from the charged droplets for each of the timing or phase settings to create a plurality of collected charged droplet signals that are each indicative of an accumulated charge signal produced by the electrometer;
determining a collected charged droplet signal of the plurality of collected charged droplet signals that has the greatest magnitude; and
operating the flow cytometer with a timing or phase setting of the plurality of timing or phase settings that corresponds to the collected charge droplet signal that has the greatest magnitude.

2. The method of claim 1 , wherein the charge collector is a thin conductive mesh.

3. The method of claim 1 , wherein the thin conductive mesh is disposed in a waste stream collector in the flow cytometer.

4. The method of claim 1 , wherein
the charge collector comprises metal surfaces of a droplet collector.

5. The method of claim 4 wherein the process of adjusting the timing or phase of the charge signal comprises adjusting the timing of the charge pulses in the charge pulse signal.

6. The method of claim 4 wherein the process of adjusting the timing or phase of the charge pulse signal comprises adjusting the phase of the charge pulse signal.

7. A method of adjusting the amplitude of a charge signal that is applied to droplets formed from a fluid stream in a flow cytometer to create a substantially uniform charge on the droplets in a fluid stream comprising:
controlling a charge pulse signal to impart a positive charge, a negative charge, or a neutral charge to each droplet generated during a calibration phase to produce a plurality of calibration charge series, each calibration charge series including a sequence of droplets;
subjecting each calibration charge series to an electric field to cause the droplets in that calibration charge series having a positive charge imparted to them by the charge pulse signal to be collected in a first conductive droplet collector, the droplets in that calibration charge series having a negative charge imparted to them by the charge pulse signal to be collected in a second conductive droplet collector, and the droplets in that calibration charge series having a neutral charge imparted to them by the charge pulse signal to be collected in a third conductive droplet collector;
detecting accumulated charges in the first conductive collector, the second conductive collector, and the third conductive collector for one or more instances of each calibration charge series
determining, for each calibration charge series and based on the accumulated charges for the first conductive collector for that calibration charge series, the accumulated charges for the second conductive collector for that calibration charge series, and the accumulated charges for the third conductive collector for that calibration charge series, an average measured charge magnitude for each droplet of that calibration charge series,
adjusting an amplitude of the charge pulse signal used to impart positive, negative, or neutral charges to each droplet of a first charge series based on the average measured charge magnitude for droplets in the calibration charge series that corresponds with the first charge series.

8. The method of claim 7 , further comprising:
controlling the charge pulse signal to impart a droplet in one or more of the charge series with a charge polarity opposite the charge polarity of an adjacent preceding droplet in the one or more of the charge series.

9. The method of claim 8 , wherein the process of detecting the accumulated charges comprises using an electrometer that measures the accumulated charge for each of the first conductive collector, the second conductive collector, and the third conductive collector.

10. The method of claim 7 , further comprising:
determining an amount by which the average measured charge magnitude for a droplet of a charge series differs from an expected average charge magnitude and modifying the charge pulse signal amplitude based on the amount.

11. The method of claim 7 , further comprising:
generating a first calibration charge series of the plurality of calibration charge series that includes charged droplets that are not affected by electric fields of other droplets in the first calibration charge series;
collecting the charged droplets of the first calibration charge series in a conductive collector selected from the group consisting of: the first conductive collector and the second conductive collector;
comparing the average measured charge magnitude of the charged droplets of the first calibration charge series with the average measured charge magnitude of similarly charged droplets in one or more other calibration charge series to obtain a charge variation amount;
using the charge variation amount to adjust the amplitude of the charge signal.

12. A system for adjusting a charge amplitude of a charge pulse signal that is applied to droplets formed from a fluid stream in a flow cytometer to create a substantially uniform charge on said droplets comprising:
a charge pulse generator that generates said charge pulse signal in response to a control signal, that has a charge magnitude that charges droplets just prior to separation from said fluid stream to obtain charged droplets;
at least one conductive collector that collects said charged droplets and accumulates a charge having a charge magnitude;
an electrometer connected to said at least one conductive collector that detects said charge magnitude and generates a charge magnitude signal;
a processor that receives said charge magnitude signal and generates said control signal, said control signal producing a plurality of charge series of said droplets that produces affected droplets that have a charge that has been affected by electric fields of preceding droplets and non-affected droplets that have not been affected by electric fields of preceding droplets, and compares said charge magnitude signal for said affected droplets and said non-affected droplets for said plurality of charge series to alter said charge magnitude of said control signal, which charges said droplets to a uniform magnitude during operation of said flow cytometer.

Assignees

• Bio Rad Laboratories

Inventors

• Marquette Edward

Classifications

• G01N15/1427

  with the synchronisation of components, a time gate for operation of components, or suppression of particle coincidences · CPC title

• G01N15/1404 Primary

  Handling flow, e.g. hydrodynamic focusing · CPC title

• G01N2015/1406

  Control of droplet point · CPC title

• G01N15/1012

  Calibrating particle analysers; References therefor · CPC title

• G01R5/28

  Electrostatic instruments · CPC title