Compositions and methods for disease diagnosis using single cell analysis
Certain embodiments of the invention are directed to evaluating and identifying cells by recording and interpreting a time-dependent signal produced by unique cell respiration and permeability attributes of isolated viable cells.
1. A method for detecting and characterizing microbes in a sample, the method comprising:
(a) isolating microbes from a sample, to form an isolated sample, wherein isolating the microbes comprises separating non-microbe cells, viruses, proteins, and nucleic acids from microbe cells;
(b) dividing the sample into at least two sample portions and suspending a first sample portion in a first growth medium and suspending a second sample portion in a second growth medium, and each sample portion including a reporter, forming a first and a second sample mixture;
(c) partitioning the first and second sample mixture forming first sample partitions and second sample partitions, wherein some of the first and second sample partitions have at most one microbial cell or microbial cell aggregate per partition;
(d) incubating the partitions over time at a specific temperature or temperatures;
(e) monitoring optical characteristics of the partitions during the incubation time, wherein the optical characteristics include the amount of optical signal produced by interaction of the reporter with the microbial cell or microbial cell aggregate in the partition;
(f) constructing an optical signal waveform for each partition resulting in a partition waveform; and
(g) comparing partition waveforms between the first and second sample partition waveforms and assessing the microbes based on partition waveform comparison, wherein the microbes are detected and characterized based on the comparison of the first and second waveforms from each sample portion.
2. The method of claim 1 , further comprising classifying the microbial cell or microbial cell aggregate within each partition using the partition waveform shape.
3. The method of claim 2 , wherein the classifying comprises comparing the waveforms of each sample to a library of stored waveforms.
4. The method of claim 1 , wherein the partitions are droplets in an immiscible fluid.
5. The method of claim 4 , wherein the immiscible fluid is a fluorocarbon comprising a fluorosurfactant.
6. The method of claim 4 , further comprising arranging the droplets in a two-dimensional array monolayer.
7. The method of claim 6 , wherein the two-dimensional array is a static two-dimensional array.
8. The method of claim 1 , wherein partitioning the sample mixture is by using Laplace pressure gradients.
9. The method of claim 1 , wherein partitioning the sample mixture is by using shear stress.
10. The method of claim 1 , wherein monitoring optical characteristics of the partitions is performed using a camera.
11. The method of claim 1 , wherein the reporter is a fluorescent dye.
12. The method of claim 1 , wherein the reporter is resazurin, a resazurin-based dye, or a dye that is structurally related to resazurin.
13. The method of claim 1 , where the reporter is tetrazolium, a tetrazolium-based dye, or a dye structurally related to tetrazolium.
14. The method of claim 1 , wherein the monitoring optical characteristics of the partitions further comprises exciting the reporter using a light source.
15. The method of claim 14 , wherein the light source is a light emitting diode (LED).
16. The method of claim 1 , wherein the incubating of the partitions is at a constant temperature.
17. The method of claim 16 , wherein the constant temperature is 37° C.
18. The method of claim 1 , wherein the target cell within each partition is classified using the partition waveform shape.
19. The method of claim 1 , wherein the second growth media comprising a nutrient or compound not present in the first growth media.