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