MULTI-SPECTRAL FILTER PROFILING AND QUALITY CONTROL FOR FLOW CYTOMETRY

• US 20170045436A1
• Filed: 08/11/2016
• Published: 02/16/2017
• Est. Priority Date: 08/12/2015
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Fox, Daniel Nelson, Hunter, Susan
• Granted Patent: US 10,180,385 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: G01J 2003/104 Monochromatic plural sources
• CPC Class Codes: G01J 3/0218 using optical fibers
• CPC Class Codes: G01J 3/027 Control of working procedures of a spectrometer; Failure detection; Bandwidth calculation
• CPC Class Codes: G01J 3/10 Arrangements of light sources specially adapted for spectrometry or colorimetry
• CPC Class Codes: G01J 3/36 Investigating two or more bands of a spectrum by separate detectors
• CPC Class Codes: G01N 15/01 specially adapted for biological cells, e.g. blood cells investigating sedimentation of particle suspensions in blood G01N15/05
• CPC Class Codes: G01N 15/1012 Calibrating particle analysers; References therefor
• CPC Class Codes: G01N 15/1434 Optical arrangements
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/1006 for cytology

Abstract

Disclosed is a system and method for characterizing optical filters in a flow cytometer and optionally checking the operation of detectors in the flow cytometer. In some embodiments, the system may utilize an LED board having an opening through which the fluorescence and side scatter beams, rays, or images pass and light emitting diodes around the opening that emit light having different spectral profiles. The different spectral profiles allow the system to identify the optical filters that are placed in the flow cytometer, to verify detector operation, to assist in instrumentation troubleshooting, and to provide a quantitative reference for detector comparison.

Claims

1. A flow cytometry system comprising:

at least one sample illumination source, wherein each sample illumination source is configured to deliver light to a corresponding sample location, thereby causing sample light to be emitted by or scattered off of particles in the corresponding sample location;

focusing optics that are configured to direct the sample light from each sample location along one or more optical paths, wherein each optical path passes through a corresponding one or more optical filter elements and terminates at a corresponding detector that is configured to produce output data indicative of the measured intensity of light reaching that detector;

a calibration light source that is configured to independently emit different spectral profiles of calibration light, each spectral profile of calibration light having one or more peaks at different wavelengths, such that the emitted calibration light is directed along at least a portion of each of the optical paths;

one or more processors; and

a memory that stores computer-executable instructions that, when executed by the one or more processors, cause the one or more processors to;

receive the output data from each of the detectors responsive to that detector receiving calibration light from the calibration light source, and

determine filtering characteristics for the optical filter elements along each optical path by analyzing the output data produced during the emission of at least two different spectral profiles of calibration light by the calibration light source.

2. The flow cytometry system of claim 1, wherein the computer-executable instructions further include instructions that, when executed by the one or more processors, cause the one or more processors to:

cause the calibration light source to emit, at a first time, first calibration light having a first spectral profile;

obtain first output data from each of the detectors responsive to the detectors receiving the first calibration light from the calibration light source at the first time;

cause the calibration light source to emit, at a second time after the first time, second calibration light having the first spectral profile;

obtain second output data from each of the detectors responsive to the detectors receiving the second calibration light from the calibration light source at the second time; and

compare the second output data to the first output data to determine if there has been a change in optical performance of the flow cytometry system.

3. The flow cytometry system of claim 1, wherein:

the one or more detectors includes a first detector, and

the computer-executable instructions further include instructions that, when executed by the one or more processors, cause the one or more processors to;

obtain first output data from the first detector produced in response to detection by the first detector of first calibration light having a first spectral profile;

obtain second output data from the first detector produced in response to detection by the first detector of second calibration light having a second spectral profile that is different from the first spectral profile;

compare the first output data and the second output data against a database of spectral fingerprints, each spectral fingerprint associated with a particular filtering characteristic and having a first component associated with the first spectral profile and a second component associated with the second spectral profile; and

determine the filtering characteristics for the optical filter elements along the optical path corresponding to the first detector by identifying the particular filtering characteristic by, at least in part, correlating the first output data and the second output data with the first component and the second component, respectively.

4. The flow cytometry system of claim 1, wherein:

the one or more detectors includes a first detector, and the computer-executable instructions further include instructions that, when executed by the one or more processors, cause the one or more processors to;

obtain first output data from the first detector produced in response to detection by the first detector of first calibration light having a first spectral profile;

compare the first output data against a database of spectral fingerprints, each spectral fingerprint associated with a particular filtering characteristic and having a first component associated with the first spectral profile; and

determine the filtering characteristics for the optical filter elements along the optical path corresponding to the first detector by identifying the particular filtering characteristic by, at least in part, correlating the first output data with the first component, respectively.

5. The flow cytometry system of claim 1, wherein:

the one or more detectors includes a first detector, and

the computer-executable instructions further include instructions that, when executed by the one or more processors, cause the one or more processors to;

obtain first output data from the first detector produced in response to detection by the first detector of first calibration light having a first spectral profile;

obtain second output data from the first detector produced in response to detection by the first detector of second calibration light having a second spectral profile that is different from the first spectral profile and that also overlaps with the first spectral profile;

compare the first output data and the second output data by determining a ratio of the second output data to the first output data; and

determine the filtering characteristics for the optical filter elements along the optical path corresponding to the first detector by, at least in part, comparing the ratio against intensity ratios of the first spectral profile and the second spectral profile corresponding with a plurality of wavelengths.

6. The flow cytometry system of claim 5, wherein the computer-executable instructions further include instructions that, when executed by the one or more processors, cause the one or more processors to:

obtain additional output data from the first detector produced in response to detection by the first detector of one or more additional emissions of calibration light having spectral profiles other than the first spectral profile and the second spectral profile; and

determine that none of the additional output data indicates any detection of the additional emissions of calibration light by the first detector.

7. The flow cytometry system of claim 5, wherein the computer-executable instructions further include instructions that, when executed by the one or more processors, cause the one or more processors to:

compare the filtering characteristics for the optical filter elements along the optical path with predefined filtering characteristics associated with that optical path; and

provide an indication via a user interface as to whether the filtering characteristics for the optical filter elements along the optical path are within a threshold amount of the predefined filtering characteristics associated with that optical path.

8. The flow cytometry system of claim 1, wherein a different subset of the one or more optical paths passes through each optical filter element.

9. The flow cytometry system of claim 1, wherein each of the one or more optical paths is defined, at least in part, by an optical fiber.

10. The flow cytometry system of claim 9, wherein the focusing optics include objective optics configured to focus the sample light and the calibration light onto the ends of the one or more optical fibers.

11. The flow cytometry system of claim 1, wherein each of the one or more optical paths pass through a pinhole aperture.

12. The flow cytometry system of claim 1, wherein:

the calibration light source includes a printed circuit board having an opening through it and a plurality of light emitting diodes (LEDs) placed around the periphery of the opening,

the plurality of LEDs are configured to emit the calibration light, and

the one or more optical paths pass through the opening.

13. The flow cytometry system of claim 1, wherein the calibration light source is configured to emit calibration light that is directed through the sample location and into the focusing optics.

14. A method for determining filtering characteristics for a plurality of optical filter elements in a flow cytometry system, wherein a different subset of a plurality of optical paths passes through each of the optical filter elements, and wherein each optical path directs emitted or scattered sample light from a sample location to a corresponding detector, the method comprising:

emitting different spectral profiles of calibration light from a calibration light source, wherein each spectral profile of calibration light has one or more intensity peaks at different wavelengths;

directing some of the calibration light along at least a portion of each of the optical paths;

measuring, for each different spectral profile of calibration light, the light intensity at each of the detectors, wherein each of the detectors produces output data that is indicative of the measured light intensity of the calibration light that reaches the detector; and

analyzing the output data from one of the detectors produced during the emission of at least two different spectral profiles of calibration light by the calibration light source to determine the filtering characteristics of the optical filter elements along the optical path corresponding to that detector.

15. The method of claim 14, further comprising:

causing the calibration light source to emit, at a first time, first calibration light having a first spectral profile;

obtaining first output data from each of the detectors responsive to the detectors receiving the first calibration light from the calibration light source at the first time;

causing the calibration light source to emit, at a second time after the first time, second calibration light having the first spectral profile;

obtaining second output data from each of the detectors responsive to the detectors receiving the second calibration light from the calibration light source at the second time; and

comparing the second output data to the first output data to determine if there has been a change in optical performance of the flow cytometry system

16. The method of claim 14, further comprising:

obtaining first output data from a first detector of the one or more detectors produced in response to detection by the first detector of first calibration light having a first spectral profile;

obtaining second output data from the first detector produced in response to detection by the first detector of second calibration light having a second spectral profile that is different from the first spectral profile;

comparing the first output data and the second output data against a database of spectral fingerprints, each spectral fingerprint associated with a particular filtering characteristic and having a first component associated with the first spectral profile and a second component associated with the second spectral profile; and

determining the filtering characteristics for the optical filter elements along the optical path corresponding to the first detector by identifying the particular filtering characteristic by, at least in part, correlating the first output data and the second output data with the first component and the second component, respectively.

17. The method of claim 14, further comprising:

obtaining first output data from a first detector of the one or more detectors produced in response to detection by the first detector of first calibration light having a first spectral profile;

comparing the first output data against a database of spectral fingerprints, each spectral fingerprint associated with a particular filtering characteristic and having a first component associated with the first spectral profile; and

determining the filtering characteristics for the optical filter elements along the optical path corresponding to the first detector by identifying the particular filtering characteristic by, at least in part, correlating the first output data with the first component, respectively.

18. The method of claim 14, further comprising:

obtaining first output data from a first detector of the detectors responsive to detection by the first detector of first calibration light having a first spectral profile;

obtaining second output data from the first detector responsive to detection by the first detector of second calibration light having a second spectral profile that is different from the first spectral profile and that also overlaps with the first spectral profile;

comparing the first output data and the second output data by determining a ratio of the second output data to the first output data; and

determining the filtering characteristics for the optical filter elements along the optical path corresponding to the first detector by comparing the ratio against intensity ratios of the first spectral profile and the second spectral profile corresponding with a plurality of wavelengths.

19. The method of claim 18, further comprising:

obtaining additional output data from the first detector responsive to detection by the first detector of one or more additional emissions of calibration light having spectral profiles other than the first spectral profile and the second spectral profile; and

determining that none of the additional output data indicates any detection of the additional emissions of calibration light by the first detector.