Systems, methods, and apparatus for in vitro single-cell identification and recovery

Patent metadata

Field	Value
Publication number	US-9953209-B2
Application number	US-201615085175-A
Country	US
Kind code	B2
Filing date	Mar 30, 2016
Priority date	Jan 15, 2015
Publication date	Apr 24, 2018
Grant date	Apr 24, 2018

Abstract

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.

First claim

What is claimed is:

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.

Assignees

• Massachusetts Inst Technology

Inventors

• Adalsteinsson Viktor A
• Loginov Denis
• Love J Christopher
• Stockdale Alan
• Gierahn Todd

Classifications

• B01L3/5085 Primary

  for multiple samples, e.g. microtitration plates · CPC title

• G06V20/698

  Matching; Classification · CPC title

• H04N23/56

  provided with illuminating means · CPC title

• G01N15/1425

  using an analyser being characterised by its control arrangement · CPC title

• G06K9/00147 Primary

  Physics · mapped topic