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. An analysis module comprising a processor comprising memory operably coupled to the processor wherein the memory comprises:
instructions for receiving a temporal data signal generated from light detected from a sample comprising particles irradiated with a first beam of radiofrequency-modulated optical radiation and a second beam of radiofrequency-modulated optical radiation; and
instructions for generating a sort decision for sorting one or more particles of the sample based on the temporal data signal.
2. The analysis module according to claim 1 , wherein the first beam of radiofrequency-modulated optical radiation comprises an optical frequency which differs from the second beam of radiofrequency-modulated optical radiation by a radiofrequency.
3. The analysis module according to claim 1 , wherein the temporal data signal is generated from light detected from an irradiated sample in a flow stream.
4. The analysis module according to claim 1 , wherein light detected from the irradiated sample is fluorescence.
5. The analysis module according to claim 1 , wherein light detected from the irradiated sample is scattered light.
6. The analysis module according to claim 1 , wherein the memory comprises instructions for generating the sort decision without forming an image of the sample.
7. The analysis module according to claim 1 , wherein the memory comprises instructions for generating the sort decision based on the temporal signal with a latency equal to or less than about 100 microseconds.
8. The analysis module according to claim 1 , wherein the memory further comprises instructions for determining a characteristic of the one or more particles in the sample based on the temporal data signal.
9. The analysis module according to claim 8 , wherein the particles are cells in a biological sample.
10. The analysis module according to claim 9 , wherein the characteristic is associated with an internal organelle.
11. The analysis module according to claim 10 , wherein the 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.
12. The analysis module according to claim 11 , wherein the cells in the biological sample are stained with two or more fluorescent markers.
13. The analysis module according to claim 12 , wherein the memory comprises instructions for generating temporal fluorescence waveforms for each of the fluorescent markers.
14. The analysis module according to claim 13 , wherein the memory comprises:
instructions for measuring co-localization of fluorescence signals corresponding to the fluorescent markers; and
instructions for generating the sort decision based on the measure of co-localization.
15. The analysis module according to claim 13 , wherein the memory comprises:
instructions for applying a high-pass or a band-pass filter to at least one of the waveforms to generate at least one filtered waveform;
instructions for point-wise multiplication of the waveforms to generate a resultant multiplicative waveform;
instructions for integrating the filtered waveform to obtain an integrated value; and
instructions for comparing the integrated value with a predefined threshold to obtain the measure of co-localization.
16. The analysis module according to claim 13 , wherein the memory comprises:
instructions for applying a high-pass or a band-pass filter to at least one of the waveforms to generate at least one filtered waveform;
instructions for point-wise multiplication of the waveforms to generate a resultant multiplicative waveform;
instructions for integrating said multiplicative waveform to obtain an integrated value;
instructions for subtracting a background value from the integrated value and scaling the resultant value by intensity to generate a finalized value; and
instructions for comparing the finalized value with a predefined threshold to obtain said measure of co-localization.
17. The analysis module according to claim 13 , wherein the memory comprises:
instructions for obtaining an estimate of a size of cells in the biological sample; and
instructions for generating the sort decision based on the estimated cell size.
18. The analysis module according to claim 13 , wherein the memory comprises instructions for estimating the cell size based on a temporal duration of a pulse of fluorescence from the cell.
19. The analysis module according to claim 13 , wherein the memory comprises:
instructions for squaring the detected temporal fluorescence data;
instructions for applying a bandpass filter to the squared fluorescence data;
instructions for integrating the filtered data; and
instructions for comparing the filtered data with a predefined threshold.