System and method for detecting multiple-excitation-induced light in a flow channel

Patent metadata

Field	Value
Publication number	US-11287379-B2
Application number	US-202016833235-A
Country	US
Kind code	B2
Filing date	Mar 27, 2020
Priority date	Oct 21, 2011
Publication date	Mar 29, 2022
Grant date	Mar 29, 2022

Abstract

A system for detecting signal components of light induced by multiple excitation sources including: a flow channel including at least two spatially separated optical interrogation zones; a non-modulating excitation source that directs a light beam of a first wavelength at a near constant intensity onto a first of the optical interrogation zones; a modulating excitation source that directs a light beam of a second wavelength with an intensity modulated over time at a modulating frequency onto a second of the optical interrogation zones; a detector subsystem comprising a set of detectors configured to detect light emitted from particles flowing through the at least two optical interrogation zones and to convert the detected light into a total electrical signal; and a processor that determines signal components from the light detected from each of the optical interrogation zones.

First claim

What is claimed is:

1. A system for detecting signal components of light induced by multiple excitation sources, comprising:
a flow channel configured for the flow of particles, the flow channel comprising at least two spatially separated optical interrogation zones;
a non-modulating excitation source that directs a light beam of a first wavelength at a near constant intensity onto a first of the optical interrogation zones;
a modulating excitation source that directs a light beam of a second wavelength with an intensity modulated over time at a modulating frequency onto a second of the optical interrogation zones, wherein the second wavelength is different from the first wavelength;
a detector subsystem comprising a set of detectors configured to detect light emitted from particles flowing through the at least two optical interrogation zones and to convert the detected light into a total electrical signal; and
a processor configured to receive the total electrical signal from the detector subsystem, to de-modulate electrical signal that is modulated, and to determine signal components from the light detected from each of the optical interrogation zones.

2. The system according to claim 1 , wherein the optical interrogation zones are spaced from about 30 to about 80 microns apart.

3. The system according to claim 1 , wherein each excitation source is a laser or a light emitting diode (LED).

4. The system according to claim 1 , wherein the first wavelength and the second wavelength are each different wavelengths, each of which is selected from the group consisting of about 325, nm, 355 nm, 365 nm, 375 nm, 405 nm, 407 nm, 488 nm, 532 nm, 561 nm, 595 nm, 633 nm, 635 nm, 640 nm, and 647 nm.

5. The system according to claim 1 , wherein the modulating frequency is between 1 MHz and 100 MHz.

6. The system according to claim 1 , wherein the modulating excitation source is modulated according to a waveform selected from the group consisting of a sine waveform, a square waveform, a triangular waveform and a seesaw waveform.

7. The system according to claim 1 , wherein the set of detectors selectively detect wavelengths of about 421±30 nm, 450±30 nm, 455±40 nm, 519±30 nm, 530±15 nm, 578±15 nm, 585±40 nm, 603±30 nm, 615±30 nm, 620±30 nm, >650 nm, 660±10 nm, 667±30 nm, 668±30 nm, 678±30 nm, 695±25 nm, >750 nm, 780±30 nm and >785 nm.

8. The system according to claim 1 , further comprising a third optical interrogation zone spatially separated from the first and second optical interrogation zones; and another excitation source that directs a light beam onto the third optical interrogation zone.

9. The system according to claim 8 , wherein the excitation source that directs the light beam onto the third optical interrogation zone is a second non-modulating excitation source that directs the light beam of a third wavelength at a near constant intensity onto the third optical interrogation zone, wherein the third wavelength is different from both the first wavelength and the second wavelength.

10. The system according to claim 8 , wherein the excitation source that directs the light beam onto the third optical interrogation zone is a second modulating excitation source that directs the light beam of a third wavelength with an intensity modulated over time at a modulating frequency.

11. The system according to claim 1 , wherein the system further comprises light collecting optics between the flow channel and detector subsystem.

12. The system according to claim 11 , wherein the system further comprises light splitting optics between the flow channel and detector subsystem; and the light collecting and light splitting optics are shared to collect light emitted from each of the optical interrogation zones and to split light into multiple light detection channels.

13. The system according to claim 1 , further comprising a sorting mechanism configured to sort a flowing particle population into one or more chambers in response to commands from the processor.

14. A method of detecting signal components from light induced by multiple excitation sources, the method comprising:
providing a flow channel comprising at least two spatially separated optical interrogation zones;
flowing a population of particles labeled with at least two different fluorescent molecules through each of the optical interrogation zones;
directing a light beam of a first wavelength at a near constant intensity at a first of the optical interrogation zones to induce emission of light from the fluorescence-molecule containing particles;
directing a light beam of a second wavelength with an intensity modulated over time according to a modulating frequency onto a second of the optical interrogation zones to induce emission of light from the fluorescence-molecule containing particles, wherein the second wavelength is different from the first wavelength;
detecting the light emitted from the particles from each of the optical interrogation zone and converting detected light into a total electrical signal;
de-modulating electrical signal from the total electrical signal; and
determining signal components of the light detected from each of the optical interrogation zones.

15. The method according to claim 14 , further comprising:
flowing the population of particles through a third optical interrogation zone spatially separated from the first and second optical interrogation zones;
directing a light beam of a third wavelength at a near constant intensity at the third optical interrogation zone to induce emission of light from the fluorescence-molecule containing particles, wherein the third wavelength is different from the first and second wavelengths; and
detecting the light emitted from the particles flowing through the third optical interrogation zone and converting the detected light into the total electrical signal.

16. The method according to claim 14 , further comprising:
flowing the population of particles through a third optical interrogation zone spatially separated from the first and second optical interrogation zones;
directing a light beam of a third wavelength with an intensity being modulated over time at the modulating frequency onto the third optical interrogation zone to induce emission of light from the fluorescence-molecule containing particles, wherein the third wavelength is different the first and second wavelengths; and
detecting the light emitted from the particles flowing through the third optical interrogation zone and converting the detected light into the total electrical signal.

17. The method according to claim 14 , further comprising sorting the cell population into one or more chambers according to the presence or absence of one or more detected components.

Assignees

• Agilent Technologies Inc

Inventors

• Li Nan
• Wu Jian
• Chen Ye
• Wang Tianxing
• Wang Xiaobo

Classifications

• G01N15/1429

  Signal processing · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N15/1434

  Optical arrangements · CPC title

• G01N2021/6421

  Measuring at two or more wavelengths · CPC title

• G01N2015/1438

  Using two lasers in succession · CPC title