Multi-modal fluorescence imaging flow cytometry system

Patent metadata

Field	Value
Publication number	US-12038372-B2
Application number	US-202318198478-A
Country	US
Kind code	B2
Filing date	May 17, 2023
Priority date	Oct 13, 2015
Publication date	Jul 16, 2024
Grant date	Jul 16, 2024

Abstract

Aspects of the disclosure include a system for performing cytometry that can be operated in three operational modes. In one operational mode, a fluorescence image of a sample is obtained by exciting one or more fluorophore(s) with a top-hat-shaped excitation beam with a plurality of beams that are radiofrequency shifted relative to one another. In another operational mode, a sample can be illuminated successively over a time interval by a laser beam at a plurality of excitation frequencies in a scanning fashion to generate an image of the sample from the detected fluorescence. In another operational mode, the system can be operated to illuminate a plurality of locations of a sample concurrently by a single excitation frequency (e.g., a horizontal extent), which is generated, e.g., by shifting the central frequency of a laser beam by a radiofrequency and detected fluorescence is used to analyze the sample.

First claim

The invention claimed is:

1. A method comprising:
generating at least two angularly deflected beams of light with a light beam generator; and
combining the angularly deflected beams of light with an optical combiner onto a sample in a flow stream; and
detecting light from the irradiated sample in the flow stream with a photodetector.

2. The method according to claim 1 , wherein the angularly deflected beams of light are generated by irradiating an acousto-optic deflector with a laser.

3. The method according to claim 2 , wherein at least one of the angularly deflected beams of light is a local oscillator (LO) beam.

4. The method according to claim 3 , wherein the local oscillator beam has a Gaussian intensity profile along a vertical axis.

5. The method according to claim 1 , wherein the angularly deflected beams of light are overlapped at the flow stream by the optical combiner.

6. The method according to claim 5 , wherein overlap of the angularly deflected beams of light is sufficient to generate a beat frequency that comprises a frequency difference between overlapping angularly deflected beams of light.

7. The method according to claim 1 , wherein the method further comprises modulating the intensity of each of the angularly deflected beams of light.

8. The method according to claim 7 , wherein the intensity of each angularly deflected beam of light is modulated with a direct digital synthesizer.

9. The method according to claim 7 , wherein the method comprises modulating the intensity of each angularly deflected beam of light to be equal in intensity.

10. The method according to claim 1 , wherein the method comprises generating a data signal in response to light detected from the sample in the flow stream.

11. The method according to claim 9 , wherein the data signal comprises a time-domain fluorescence emission signal.

12. The method according to claim 11 , wherein the method further comprises generating an image of the sample based on a plurality of time-domain fluorescence emission signals.

13. The method according to claim 12 , wherein the method comprises frequency de-multiplexing each fluorescence emission signal to determine the beat frequencies generated by overlap of the angularly deflected beams of light on the sample.

14. The method according to claim 13 , wherein frequency de-multiplexing comprises obtaining a Fourier Transform of the fluorescence signal to determine the beat frequencies.

15. The method according to claim 14 , wherein the method comprises generating the image by computing the amplitude of each beat frequency to provide a pixel value corresponding to a location of the image.

16. The method according to claim 15 , wherein the method comprises generating the image by generating a darkfield image of the sample and generating a brightfield image of the sample.

17. The method according to claim 1 , wherein particles in the sample are propagated through the flow stream at a flow rate of 1 m/s or more.

18. The method according to claim 1 , wherein the sample comprises one or more fluorophores.

Assignees

• Becton Dickinson Co

Inventors

• Diebold Eric D
• Owsley Keegan
• Lin Jonathan

Classifications

• G02B21/06

  Means for illuminating specimens · CPC title

• G01N2021/6482

  Sample cells, cuvettes · CPC title

• G01N21/645

  Specially adapted constructive features of fluorimeters · CPC title

• G01N15/1429

  Signal processing · CPC title

• G01N15/1433

  using image recognition · CPC title