Cell sorting using a high throughput fluorescence flow cytometer
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.
1. A system for sorting particles, the system comprising:
a light source configured to concurrently illuminate a plurality of spatial locations on a particle as the particle passes through two or more discrete positions along a flow cell;
a detector configured to measure a temporal waveform emission of the illuminated particle;
a deflection system configured to direct the illuminated particle to one of two or more destinations; and
a computer-readable storage medium comprising instructions that, when executed by one or more processing devices:
receive the temporal waveform emission;
determine a characteristic of the particle based on the temporal waveform emission; and
control the deflection system to direct the particle to the destination based on the characteristic.
2. The system according to claim 1 , wherein the instructions control the deflection system to direct the particle to the destination without basing the direction on an image of the particle.
3. The system according to claim 1 , wherein the detector is configured to detect fluorescent light.
4. The system according to claim 1 , wherein the detector is configured to detect scattered light.
5. The system according to claim 1 , wherein the instructions control the deflection system to direct the particle to the destination with a latency equal to or less than about 100 microseconds.
6. The system according to claim 1 , wherein the light source is a laser.
7. The system according to claim 1 , wherein the light source is an LED.
8. The system according to claim 1 , wherein the particle is a cell.
9. The system according to claim 8 , wherein the characteristic is morphology of a cell.
10. The system according to claim 8 , wherein the characteristic is associated with an internal organelle of a cell.
11. The system according to claim 8 , wherein the characteristic comprises any of a dimensional size of a cell, a ratio of sizes of a cell in along two different dimensions, co-localization of fluorescence radiation emitted by two or more markers associated with a cell, a ratio of sizes of a cell's cytoplasm and nucleus, a degree of punctateness of fluorescent radiation emitted from a cell, a measure of the spatial distribution of the fluorescent radiation from a cell, a measure of location or orientation of a cell, a measure of the eccentricity of a cell, a measure of a cell's similarity to a reference cell, a combination of one or more spatial Fourier components of a cell, and a measure of the degree to which a cell lies in a focal point of the illuminating radiation.
12. A method for sorting particles in a sample, the method comprising:
concurrently illuminating a plurality of spatial locations on a particle with a light source as the particle passes through two or more discrete positions along a flow cell;
measuring with a detector a temporal waveform emission of the illuminated particle;
determining a characteristic of the particle based on the temporal waveform emission; and
directing the illuminated particle to one of two or more destinations with a deflection system based on the characteristic.
13. The method according to claim 12 , wherein the particle is directed to the destination without basing the direction on an image of the particle.
14. The method according to claim 12 , wherein the detector is configured to detect fluorescent light.
15. The method according to claim 12 , wherein the detector is configured to detect scattered light.
16. The method according to claim 12 , wherein the directing occurs with a latency of 100 microseconds or less.
17. The method according to claim 12 , wherein the light source is a laser.
18. The method according to claim 12 , wherein the light source is an LED.
19. The method according to claim 12 , wherein the particles are cells in a biological sample.
20. The method according to claim 19 , wherein the characteristic is morphology of a cell.
21. The method according to claim 19 , wherein the characteristic is associated with an internal organelle of a cell.
22. The method according to claim 19 , wherein the characteristic comprises any of a dimensional size of a cell, a ratio of sizes of a cell in along two different dimensions, co-localization of fluorescence radiation emitted by two or more markers associated with a cell, a ratio of sizes of a cell's cytoplasm and nucleus, a degree of punctateness of fluorescent radiation emitted from a cell, a measure of the spatial distribution of the fluorescent radiation from a cell, a measure of location or orientation of a cell, a measure of the eccentricity of a cell, a measure of a cell's similarity to a reference cell, a combination of one or more spatial Fourier components of a cell, and a measure of the degree to which a cell lies in a focal point of the illuminating radiation.