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 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. In some embodiments, 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, comprising:
an illumination system for illuminating a particle with radiofrequency-modulated optical radiation having two or more optical frequencies which differ from one another by one or more radiofrequencies,
a detection system for detecting fluorescent radiation emanating from the particle in response to said illumination to generate temporal fluorescence data,
an analysis module in communication with said detection system for receiving said temporal fluorescence data and processing said data to arrive at a sorting decision regarding said particle, and
an actuator capable of diverting the particles from their flow path to separate containers based upon said sorting decision.
2. The system of claim 1 , wherein said analysis module arrives at the sorting decision without forming an image of the particle based on said fluorescence data.
3. The system of claim 1 , wherein said illumination system generates an optical beam comprising a plurality of angularly-separated beamlets having optical frequencies which differ from one another by one or more radiofrequencies.
4. The system of claim 3 , 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.
5. The system of claim 1 , wherein the analysis module processes said temporal fluorescence data to arrive at a sorting decision regarding said particles with a latency equal to or less than about 100 microseconds.
6. The system of claim 1 , wherein the analysis module processes said temporal fluorescence data to analyze at least one beat frequency associated with said radiofrequency in said temporal fluorescence data to obtain an estimate of a characteristic of the particles.
7. The system of claim 6 , wherein said particles are cells in a biological sample.
8. The system of claim 7 , wherein said characteristic is associated with an internal organelle.
9. The system of claim 7 , wherein said characteristic of the cells in the biological sample comprises any of a dimensional size of the cell, a ratio of sizes of the cell in along two different dimensions, co-localization of fluorescence radiation emitted by two or more markers associated with the cell, a ratio of sizes of the cell's cytoplasm and nucleus, a degree of punctateness of fluorescent radiation emitted from the cell, a measure of the spatial distribution of the fluorescent radiation, a measure of location or orientation of the cell, a measure of the eccentricity of the cell, a measure of the cell's similarity to a reference cell, a combination of one or more spatial Fourier components of the cell, a measure of the degree to which the cell lies in a focal point of the illuminating radiation.
10. The system of claim 7 , wherein said optical beam is configured such that an optical frequency at which each of a plurality of spatial locations within a cell in the biological sample is illuminated corresponds to a different one of said different optical frequencies.
11. The system of claim 7 , wherein said cells in the biological sample are stained with at least two fluorescence markers and said optical radiation is configured to elicit fluorescent radiation from said markers.
12. The system of claim 11 , wherein said detection system collects and digitizes fluorescent radiation emanated from said markers to generate temporal fluorescence waveforms each corresponding to one of said markers.
13. The system of claim 12 , wherein said analysis module operates on said fluorescence waveforms to obtain a measure of co-localization of said fluorescence signals corresponding to said fluorescence markers and makes said sorting decision based on said co-localization measure.
14. The system of claim 13 , wherein said analysis module operates on said waveforms by applying a high-pass or a band-pass filter to at least one of said waveforms to generate at least one filtered waveform followed by point-wise multiplication of said waveforms to generate a resultant multiplicative waveform, integrating said filtered waveform to obtain an integrated value, and comparing the integrated value with a predefined threshold to obtain said measure of co-localization.
15. The system of claim 13 , wherein said analysis module operates on said waveforms by applying a high-pass or a band-pass filter to at least one of said waveforms to generate at least one filtered waveform followed by point-wise multiplication of said waveforms to generate a resultant multiplicative waveform, integrating said multiplicative waveform to obtain an integrated value, subtracting a background value from the integrated value and scaling the resultant value by intensity to generate a finalized value, and comparing the finalized value with a predefined threshold to obtain said measure of co-localization.
16. The system of claim 7 , wherein said analysis modules operates on said fluorescence data to obtain an estimate of a size of cells in the biological sample and making said sorting decision based on said estimated cell size.
17. The system of claim 16 , wherein said estimate of the cell size can be any of an estimate of the cell size in a direction of flow in a flow stream and a lateral size of the cell.
18. The system of claim 17 , wherein said analysis modules estimates said cell size in the direction of flow based on a temporal duration of a pulse of fluorescent radiation emanated from the cell.
19. The system of claim 17 , wherein said estimate of the lateral size of the cell is obtained by squaring the detected temporal fluorescence data, applying a bandpass filter to the squared fluorescence data, integrating the filtered data, and comparing the filtered data with a predefined threshold.
20. The system of claim 7 , wherein said analysis module operates on said fluorescence data to obtain a ratio of the size of one or more cells in the biological sample along two different dimensions and utilizing said ratio to make the sorting decision.