CELL SORTING USING A HIGH THROUGHPUT FLUORESCENCE FLOW CYTOMETER

• US 20170268981A1
• Filed: 03/17/2017
• Published: 09/21/2017
• Est. Priority Date: 03/17/2016
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: BD Biosciences
• Inventors: Diebold, Eric, Owsley, Keegan, Lin, Jonathan
• Granted Patent: US 10,324,019 B2
• Time in Patent Office: Days
• CPC Class Codes: G01N 15/1429 Signal processing
• CPC Class Codes: G01N 15/1434 Optical arrangements
• CPC Class Codes: G01N 15/147 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1447 Spatial selection
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6402 Atomic fluorescence; Laser induced fluorescence
• CPC Class Codes: G01N 21/6428 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

Abstract

In one aspect, a method of sorting cells in a flow cytometry system is disclosed, which includes illuminating a cell with radiation having at least two optical frequencies shifted from one another by a radiofrequency to elicit fluorescent radiation from the cell, detecting the fluorescent radiation to generate temporal fluorescence data, and processing the temporal fluorescence data to arrive at a sorting decision regarding the cell without generating an image (i.e., a pixel-by-pixel image) of the cell based on the fluorescence data. In other words, while the fluorescence data can contain image data that would allow generating a pixel-by-pixel fluorescence intensity map, the method arrives at the sorting decision without generating such a map. In some cases, the sorting decision can be made with a latency less than about 100 microseconds. In some embodiments, the above method of sorting cells can have a sub-cellular resolution, e.g., the sorting decision can be based on characteristics of a component of the cell. In some embodiments in which more than two frequency-shifted optical frequencies are employed, a single radiofrequency shift is employed to separate the optical frequencies while in other such embodiments a plurality of different radiofrequency shifts are employed.

Claims

1. A method of determining one or more characteristics of a particle, comprising:

illuminating a particle as it flows through a flow cytometry system with radiation having at least two optical frequencies shifted from one another by a radiofrequency to elicit fluorescent radiation from the particle,

detecting the fluorescent radiation from the particle to generate temporal fluorescence data, and

processing said temporal fluorescence data to obtain an estimate of at least one characteristic of said particle.

2. The method of claim 1, wherein said processing step comprises analyzing one or more beat frequencies modulating said temporal fluorescence data to obtain said estimate of the at least one characteristic of the particle.

3. The method of claim 1, wherein said processing step is performed without generating a fluorescence image based on said temporal fluorescence data

5. The method of claim 1, wherein said at least one characteristic comprises at least one of a dimensional size of the particle, a ratio of sizes of the particle along two different dimensions, co-localization of fluorescent radiation emitted by two or more markers associated with the particle, a degree of punctateness of the fluorescent radiation, a measure of the spatial distribution of the fluorescent radiation, a measure of location or orientation of the particle, a measure of the eccentricity of the particle, a measure of the particle'"'"'s similarity to a reference particle, a combination of one or more spatial Fourier components of the particle, a measure of the degree to which the particle lies in a focal point of the illuminating radiation.

6. The method of claim 1, wherein said processing step is sufficiently fast such that a latency associated with obtaining said estimate of at least one characteristic of the particle is less than about 100 microseconds.

15. The method of claim 1, wherein the step of processing comprises obtaining an estimate of a lateral size of the particle along a direction substantially perpendicular to direction of particle flow in the flow cytometry system.

4. (canceled)

7. (canceled)

8-14. -14. (canceled)

16-30. -30. (canceled)

31. A method of determining a characteristic of a particle, comprising:

illuminating a particle as it flows through a flow cytometry system with a radiofrequency-modulated optical beam so as to elicit at least one radiative response from the particle,

detecting said radiative response emanating from the particle to generate temporal waveform data associated with said radiative response,

processing said waveform to obtain an estimate of at least one characteristic of said particle without generating an image of the particle based on said waveform data.

32. The method of claim 31, wherein said radiative response comprises any of fluorescent radiation and scattered radiation emanated from the particle in response to said illumination.

33. The method of claim 31, wherein said radiofrequency-modulated optical beam comprises at least two optical frequencies separated from one another by at least one radiofrequency.

34. The method of claim 33, wherein said processing step comprises analyzing at least one beat frequency associated with said at least one radiofrequency detected in said radiative response to determine said estimate of the at least one characteristic of the particle.

35. The method of claim 31, wherein said at least one characteristic comprises at least one of a dimensional size of the particle, a ratio of sizes of the particle along two different dimensions, co-localization of fluorescent radiation emitted by two or more markers associated with the particle, a degree of punctateness of the fluorescent radiation, a measure of the spatial distribution of the fluorescent radiation, a measure of location or orientation of the particle, a measure of the eccentricity of the particle, a measure of the particle'"'"'s similarity to a reference particle, a combination of one or more spatial Fourier components of the particle, a measure of the degree to which the particle lies in a focal point of the illuminating radiation.

36-63. -63. (canceled)

64. A method for sorting cells in a flow cytometry system, comprising:

illuminating a cell with radiation having two or more optical frequencies shifted from one another by one or more radiofrequencies to elicit fluorescent radiation from the cell,

detecting the fluorescent radiation to generate temporal fluorescence data, and

processing said temporal fluorescence data to arrive at a sorting decision regarding said cell with a latency equal to or less than about 100 microseconds.

65-79. -79. (canceled)

80. A system for determining a characteristic of a particle, comprising:

an illumination system for illuminating a particle with radiofrequency-modulated optical radiation,

a detection system for detecting any of fluorescent and scattered radiation emanating from the particle in response to said illumination to generate fluorescence or scattering data,

an analysis module in communication with said detection system for receiving said fluorescence and scattering data and processing said data to calculate an estimate of at least one characteristic.

81. The system of claim 80, wherein said analysis module calculates an estimate of at least one characteristic of the particle without forming an image of the particle based on said fluorescence or scattering data.

82. The system of claim 80, wherein said at least one characteristic of the particle comprises at least one of a dimensional size of the particle, a ratio of sizes of the particle along two different dimensions, co-localization of fluorescence radiation emitted by two or more markers associated with the particle, a degree of punctateness of fluorescent radiation emitted from the particle, a measure of the spatial distribution of the fluorescent radiation, a measure of location or orientation of the particle, a measure of the eccentricity of the particle, a measure of the particle'"'"'s similarity to a reference particle, a combination of one or more spatial Fourier components of the particle, a measure of the degree to which the particle lies in a focal point of the illuminating radiation

83. The system of claim 80, wherein said particle comprises any of a cell, a micro-vesicle, a cellular fragment, a liposome, a bead, and a small organism.

84. The system of claim 80, wherein said particle comprises a cell and said at least one characteristic is a ratio of sizes of the cell'"'"'s cytoplasm and nucleus.

85. The system of claim 80, wherein said illumination system generates an optical beam comprising a plurality of angularly-separated beamlets having optical frequencies separated from one another by at least one radiofrequency.

86. The system of claim 80, wherein said illumination system comprises:

a source for generating a laser beam,

a single acousto-optic deflector (AOD) receiving said laser beam, and

a radiofrequency (RF) comb generator for applying a plurality of RF drive signals

to said AOD diffract said received laser beam into said plurality of angularly-separated beamlets.

87-90. -90. (canceled)