Multi-photon counting for high sensitivity flow cytometer systems and methods for using the same

• US 10,578,542 B2
• Filed: 09/19/2018
• Issued: 03/03/2020
• Est. Priority Date: 10/16/2017
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Cao, Jianying, Chen, Wei
• Primary Examiner(s): Nguyen, Sang H
• Application Number:
  US16/135,819
• Publication Number:
  US 20190113435A1
• Time in Patent Office: 531 Days
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1427 with the synchronisation of components, a time gate for operation of components, or suppression of particle coincidences
• CPC Class Codes: G01N 15/1429 Signal processing
• CPC Class Codes: G01N 15/1436 the optical arrangement forming an integrated apparatus with the sample container, e.g. a flow cell
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 33/4915 using flow cells flow cytometry G01N15/14

Abstract

Aspects of the present disclosure include methods and systems for detecting light from a sample in a flow stream by multi-photon counting. Methods according to certain embodiments include irradiating a sample in a flow stream with a light source and detecting light from the sample in the flow stream and counting photons of the detected light by integrating photo-electron charge over a time interval. Methods also include irradiating a sample in a flow stream with a light source, detecting light from the sample in the flow stream and outputting a digital output signal and an analog output signal produced by the detected light. Systems for detecting light from a sample in a flow stream with a detector and counting photons by integrating photo-electron charge over a time interval are also described. Kits having a detector, a photon counter and a flow cell configured to propagate a sample in flow stream are also provided.

Claims

1. A method comprising:

irradiating a sample in a flow stream with a light source;

detecting light from the sample in the flow stream with a multi-photon counting detector;

generating a plurality of data signals from the detected light;

integrating photo-electron charge of the plurality of data signals over a time interval; and

counting photons of the detected light with one or more photon counters based on the integrated photo-electron charge over the time interval,

wherein counting photons comprises;

integrating photo-electron charge with a first photon counter over a plurality of time intervals, each time interval separated by a reset period therebetween; and

integrating photo-electron charge with a second photon counter during each reset period of the first photon counter.

2. The method according to claim 1, wherein counting photons comprises integrating photo-electron charge over a plurality of time intervals.

3. The method according to claim 2, wherein counting photons comprises a reset period between each of the time intervals.

4. The method according to claim 2, wherein two or more of the time intervals overlap.

5. The method according to claim 2, wherein each time interval is the same.

6. The method according to claim 2, wherein each time interval is different.

7. The method according to claim 2, wherein two or more of the time intervals are the same and two or more of the time intervals are different.

8. The method according to claim 2, wherein counting photons comprises:

integrating photo-electron charge with a first photon counter over a first time interval; and

integrating photo-electron charge with a second photon counter over a second time interval.

9. The method according to claim 8, wherein the first time interval and the second time interval at least partially overlap.

10. The method according to claim 1, wherein counting photons comprises integrating photo-electron charge when the detected light exceeds a predetermined threshold.

11. The method according to claim 10, wherein counting photons comprises stopping integration of photo-electron charge when the number of photons detected light falls below the predetermined threshold.

12. The method according to claim 1 wherein counting photons comprises counting a plurality of pulses, wherein each pulse is produced by a single photon.

13. The method according to claim 12, wherein light intensity from the sample in the flow stream is determined by the number of counted pulses.

14. The method according to claim 1, further comprising measuring the detected light at one or more wavelengths.

15. The method according to claim 1, wherein the detected light is forward scattered light, side scattered light, transmitted light, emitted light or a combination thereof.

16. The method according to claim 15, wherein the detected light is side scattered light.

17. The method according to claim 1, wherein the light is detected by a detector array comprising a plurality of detectors.

18. The method according to claim 17, wherein the detector array comprises a plurality of solid-state detectors.

19. The method according to claim 18, wherein the solid-state detectors are avalanche photodiodes.