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 - 90 . (canceled)
91 . 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; and
instructions for generating a sort decision for sorting one or more particles of the sample based on the temporal data signal.
92 . The analysis module according to claim 91 , wherein the temporal data signal is generated from light detected from an irradiated sample in a flow stream.
93 . The analysis module according to claim 91 , wherein light detected from the irradiated sample is fluorescence.
94 . The analysis module according to claim 91 , wherein light detected from the irradiated sample is scattered light.
95 . The analysis module according to claim 91 , wherein the memory comprises instructions for generating the sort decision without forming an image of the sample.
96 . The analysis module according to claim 91 , 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.
97 . The analysis module according to claim 91 , 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.
98 . The analysis module according to claim 97 , wherein the particles are cells in a biological sample.
99 . The analysis module according to claim 98 , wherein the characteristic is associated with an internal organelle.
100 . The analysis module according to claim 99 , 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.
101 . The analysis module according to claim 100 , wherein the cells in the biological sample are stained with two or more fluorescent markers.
102 . The analysis module according to claim 101 , wherein the memory comprises instructions for generating temporal fluorescence waveforms for each of the fluorescent markers.
103 . The analysis module according to claim 102 , 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.
104 . The analysis module according to claim 102 , 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.
105 . The analysis module according to claim 102 , wherein the memory comprises instructions for estimating the cell size based on a temporal duration of a pulse of fluorescence from the cell.
106 . The analysis module according to claim 102 , 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.