Systems, methods, and apparatus for in vitro single-cell identification and recovery
Described herein are systems, methods, and apparatus for automatically identifying and recovering individual cells of interest from a sample of biological matter, e.g., a biological fluid. Also described are methods of enriching a cell type of interest. These systems, methods, and apparatus allow for coordinated performance of two or more of the following, e.g., all with the same device, thereby enabling high throughput: cell enrichment, cell identification, and individual cell recovery for further analysis (e.g., sequencing) of individual recovered cells.
1. A system for performing spectral spillover compensation in multicolor slide cytometry, the system comprising at least one memory and a processor of a computing device communicatively coupled to the at least one memory, wherein the processor is operable to perform steps (i) to (xi) as follows:
(i) identify location of one or more beads;
(ii) extract a signal intensity of each pixel in each of a plurality of spectral channels for each bead;
(iii) create one or more 3D probability matrices relating intensity of signal in the spectral channel assigned to a fluorophore to the signal in each of the other channels;
(iv) identify a location of cells in one or more images;
(v) extract a signal in each of the plurality of spectral channels for each cell;
(vi) extract a background signal;
(vii) determine an amount of each fluorophore on each cell using one or more average spillover values extracted from the one or more probability matrices;
(viii) create n-replicas of the compensated fluorophore content of each cell;
(ix) sample at least one of the one or more 3D probability matrices to calculate an expected distribution of raw fluorescent signal in each channel based on concentration of each fluorophore;
(x) compensate reconstructed pseudo-raw fluorescent values to create a distribution of calculated signal on cells identified as having no actual fluorophores present; and
(xi) resample a plurality of times for each cell to generate an expected negative cell distribution for each individual cell.
2. The system of claim 1 , wherein the processor is operable to perform step (iii) by performing (a) to (e), as follows:
(a) determine an average amount of light emitted in channel B by fluorophore A;
(b) normalize B signal to 0;
(c) bin data into overlapping bins based on fluorophore A concentration;
(d) create a 2D probability distribution of B signal for each bin; and
(e) combine the 2D distributions into a 3D spectral probability matrix.
3. The system of claim 1 , further comprising an imaging device.
4. The system of claim 1 , wherein the processor is operable to perform five or more of steps (i) to (xi).
5. The system of claim 1 , wherein the plurality of spectral channels comprises from 10 to 30 spectral channels.
6. The system of claim 1 , wherein the background signal in step (vi) is extracted from one or more areas similar in size to an area from which a cell signal is extracted.
7. The system of claim 1 , wherein step (viii) comprises, for each replica, one fluorophore content being zeroed by replacing the value with a sample taken from the background signal distribution.
8. The system of claim 1 , wherein the plurality of times in step (xi) is at least 5k times.
9. The system of claim 2 , wherein the average amount of light in step (a) is determined through linear regression.
10. The system of claim 2 , wherein the B signal is normalized in step (b) by subtracting a product fluorophore A concentration and slope of the linear regression in step (a).
11. The system of claim 2 , wherein the 2D probability distribution of B signal for each bin is normalized to 1.
12. The system of claim 1 , wherein the processor is operable to perform at least 5 of steps (i) to (xi).
13. The system of claim 1 , wherein the plurality of spectral channels comprises from 10 to 30 spectral channels.
14. The system of claim 1 , wherein the background signal in step (vi) is extracted from one or more areas similar in size to an area from which a cell signal is extracted.
15. The system of claim 1 , wherein step (viii) comprises, for each replica, one fluorophore content is zeroed by replacing the value with a sample taken from the background signal distribution.
16. The system of claim 1 , wherein the plurality of times in step (xi) is at least 5k times.
17. A method for performing spectral spillover compensation in multicolor slide cytometry, the method comprising performing steps (i) to (xi) as follows using a processor of a computing device:
(i) identifying location(s) of one or more beads;
(ii) extracting a signal intensity of each pixel in each of a plurality of spectral channels for each bead;
(iii) creating one or more 3D probability matrices relating intensity of signal in the spectral channel assigned to a fluorophore to the signal in each of the other channels;
(iv) identifying a location of cells in one or more images;
(v) extracting a signal in each of the plurality of spectral channels for each cell;
(vi) extracting a background signal;
(vii) determining an amount of each fluorophore on each cell using one or more average spillover values extracted from the one or more probability matrices;
(viii) creating n-replicas of the compensated fluorophore content of each cell;
(ix) sampling at least one of the one or more 3D probability matrices to calculate an expected distribution of raw fluorescent signal in each channel based on concentration of each fluorophore;
(x) compensating reconstructed pseudo-raw fluorescent values to create a distribution of calculated signal on cells identified as having no actual fluorophores present; and
(xi) resampling a plurality of times for each cell to generate an expected negative cell distribution for each individual cell.