Method and apparatus for calibrating a flow cytometer
First Claim
1. A method of continuously verifying proper sort calibration in a droplet sorting flow cytometer that selectively applies or does not apply one of a set of one or more electrical charges to a plurality of droplets as they form from a continuous fluid stream at a droplet break-off location and electrostatically sorts the droplets into two or more separate droplet streams, the selection of the electrical charge for each droplet being dependent on expected particles in the droplet based on a drop delay setting representing an estimate of the time elapsing between the moment a particle contained in the continuous fluid stream is detected at an interrogation location of the flow cytometer and the arrival of that particle at the droplet break-off location, the method comprising:
- selecting a fraction of droplets in one of said separate droplet streams estimated to have substantially zero probability of containing a particle;
applying one charge of said set of charges to the fraction of selected droplets in order to form a calibration test stream out of the selected droplets;
illuminating the droplets in the calibration test stream; and
detecting any light emitted or scattered by any particles in the selected droplets.
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Accused Products
Abstract
A method of continuously verifying proper sort calibration in a droplet sorting flow cytometer by selecting a fraction of droplets estimated to have substantially zero probability of containing a particle; applying one charge of a set of charges to the selected droplets in order to form a test stream out of the selected droplets; illuminating the droplets in the test stream; and detecting any light emitted or scattered by any particles in the selected droplets.
603 Citations
21 Claims
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1. A method of continuously verifying proper sort calibration in a droplet sorting flow cytometer that selectively applies or does not apply one of a set of one or more electrical charges to a plurality of droplets as they form from a continuous fluid stream at a droplet break-off location and electrostatically sorts the droplets into two or more separate droplet streams, the selection of the electrical charge for each droplet being dependent on expected particles in the droplet based on a drop delay setting representing an estimate of the time elapsing between the moment a particle contained in the continuous fluid stream is detected at an interrogation location of the flow cytometer and the arrival of that particle at the droplet break-off location, the method comprising:
- selecting a fraction of droplets in one of said separate droplet streams estimated to have substantially zero probability of containing a particle;
applying one charge of said set of charges to the fraction of selected droplets in order to form a calibration test stream out of the selected droplets;
illuminating the droplets in the calibration test stream; and
detecting any light emitted or scattered by any particles in the selected droplets. - View Dependent Claims (2, 3, 4, 5)
- selecting a fraction of droplets in one of said separate droplet streams estimated to have substantially zero probability of containing a particle;
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6. A method of continuously verifying proper sort calibration in a droplet sorting flow cytometer that selectively applies or does not apply one of a set of one or more electrical charges to a plurality of droplets as they form from a continuous fluid stream at a droplet break-off location and electrostatically sorts the droplets into two or more separate droplet streams, the selection of the electrical charge for each droplet being dependent on expected particles in the droplet based on a drop delay setting representing an estimate of the time elapsing between the moment a particle contained in the continuous fluid stream is detected at an interrogation location of the flow cytometer and the arrival of that particle at the droplet break-off location, the method comprising:
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(a) selecting a fraction of droplets in one of said separate droplet streams estimated to have substantially zero probability of containing a particle and applying one charge of said set of charges to the fraction of selected droplets in order to form a calibration test stream out of the selected droplets; (b) illuminating the selected droplets in said calibration test stream by directing an illumination beam along a beam axis in a forward direction through a lens system to cause emission of fluorescent light by any particles contained in the droplets; (c) using said lens system to collect some of said fluorescent light and direct it in a rearward direction along the beam axis; (d) detecting at least some of the collected fluorescent light and generating an output signal representative of the detected light; and (e) analyzing the output signal and, based on the analysis, automatically adjusting at least one of;
(1) the drop delay setting; and
(2) the amplitude of a charge in said set of charges. - View Dependent Claims (7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. An apparatus for continuously and automatically calibrating a droplet sorting flow cytometer that selectively applies or does not apply one of a set of one or more electrical charges to a plurality of droplets as they break off of a continuous fluid stream at a droplet break-off location and electrostatically sorts the droplets into two or more separate droplet streams, the selection of the electrical charge for each droplet being dependent on expected particles in the droplet based on a drop delay setting representing an estimate of the time elapsing between the moment a particle contained in the continuous fluid stream is detected at an interrogation location of the flow cytometer and the arrival of that particle at the droplet break-off location, the apparatus comprising:
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(a) a sort calibration system for selecting a fraction of droplets in one of said separate droplet streams estimated to have substantially zero probability of containing a particle and for applying one charge of said set of charges to the fraction of selected droplets in order to form a calibration test stream out of the selected droplets; (b) an epi-illumination sensor downstream of the interrogation location for illuminating said calibration test stream, detecting any fluorescence emissions emitted by particles contained in the droplets of the calibration test stream, and generating an output signal representative of the detected fluorescence emissions; and (c) a control operable to analyze the output signal and to automatically adjust at least one of;
(1) the drop delay setting; and
(2) the amplitude of a charge in said set of charges. - View Dependent Claims (17, 18, 19, 20, 21)
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Specification