Method for imaging and differential analysis of cells

• US 8,824,770 B2
• Filed: 03/13/2013
• Issued: 09/02/2014
• Est. Priority Date: 03/16/2004
• Status: Active Grant

• Current Assignee: Cytek Biosciences Inc
• Original Assignee: Amnis Corporation (DiaSorin S.p.A.)
• Inventors: George, Thaddeus C., Basiji, David A., Hall, Brian E., Ortyn, William E., Seo, Michael J., Morrissey, Philip J., Zimmerman, Cathleen A.
• Primary Examiner(s): Bella, Matthew
• Assistant Examiner(s): HEIDEMANN, JASON E
• Application Number:
  US13/802,328
• Publication Number:
  US 20130251234A1
• Time in Patent Office: 538 Days
• Field of Search: None
• US Class Current: 382/133
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 2021/6423 Spectral mapping, video display
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6482 Sample cells, cuvettes
• CPC Class Codes: G01N 21/47 Scattering, i.e. diffuse reflection G01N21/25, G01N21/41 take precedence G01N21/55 takes precedence
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G06V 20/69 Microscopic objects, e.g. biological cells or cellular parts
• CPC Class Codes: G06V 20/698 Matching; Classification

Abstract

Provided are methods for determining and analyzing photometric and morphometric features of small objects, such as cells to, for example, identify different cell states. In particularly, methods are provided for identifying apoptotic cells, and for distinguishing between cells undergoing apoptosis versus necrosis.

Claims

1. A method for identifying a specific cell, to enable a determination to be made as to whether the specific cell corresponds to a known cell type, wherein the known cell type relates to the viability of the specific cell, comprising:

directing incident light at the specific cell and using a detector to image the specific cell;

calculating a darkfield spatial frequency for an image of the specific cell;

determining a cytoplasmic size for the specific cell from imagery of the cell; and

based upon the darkfield spatial frequency and the cytoplasmic size of the specific cell, determining if the specific cell corresponds to the known cell type.

2. The method of claim 1, further comprising determining a granularity of the specific cell from the darkfield spatial frequency, to determine a known cell type to which the specific cell corresponds.

3. The method of claim 1, wherein there is relative motion between the specific cell and the detector.

4. The method of claim 1, wherein the specific cell is contained within a heterogeneous cell population, further comprising producing images for the heterogeneous cell population.

5. The method of claim 1, further comprising identifying the specific cell as an apoptotic cell.

6. The method of claim 5, further comprising determining whether the apoptotic cell is an early stage apoptotic cell or a late stage apoptotic cell.

7. The method of claim 1, further comprising identifying the specific cell as a necrotic cell.

8. The method of claim 1, wherein determining if the specific cell corresponds to the known cell type comprises determining whether the specific cell is an apoptotic cell or a necrotic cell.

9. The method of claim 1, wherein the darkfield spatial frequency is for a cell nucleus.

10. The method of claim 1, wherein the cytoplasmic size of the specific cell is determined by:

(a) staining the nucleus of the specific cell before the specific cell is imaged;

(b) determining an area of the stained nucleus of the specific cell from imagery of the specific cell; and

(c) determining the cytoplasmic size of the specific cell by subtracting the area of the stained nucleus from a brightfield area of the specific cell included in the imagery of the specific cell, wherein the brightfield area corresponds to a size of the specific cell.

11. The method of claim 1, wherein calculating a darkfield spatial frequency for an image of the specific cell comprises computing a standard deviation of individual pixel intensities within a segmented dark field image mask of the specific cell.

12. A method for determining whether a specific cell in a population of cells corresponds to a known cell type, wherein the known cell type relates to the viability of the specific cell, comprising:

(a) illuminating the specific cell;

(b) using light received from the specific cell for forming an image of the specific cell on a detector;

(c) based on the image of the specific cell formed on the detector, determining;

(i) a cytoplasmic size of the specific cell; and

(ii) a darkfield spatial frequency of the specific cell; and

(d) identifying the specific cell as being in one of four different types of cells, at least in part, as a function of the cytoplasmic size of the specific cell and its darkfield spatial frequency.

13. The method of claim 12, wherein identifying the specific cell as being in one of four different types of cells comprises determining whether the specific cell is:

(a) a live cell;

(b) an early apoptotic cell;

(c) a late apoptotic cell;

or

(d) a necrotic cell.

14. The method of claim 12, wherein imaging the specific cell includes forming a brightfield image of the specific cell on the detector, a brightfield area of the specific cell being evident in the brightfield image.

15. The method of claim 14, further comprising applying a stain to the specific cell before forming the image of the specific cell on the detector, so that an area of a nucleus of the specific cell is evident in the image of the specific cell.

16. The method of claim 15, wherein the cytoplasmic size is determined by subtracting the area of the nucleus from the brightfield area of the specific cell.

17. The method of claim 14, wherein a specific cell in which the nucleus excludes the stain that is applied, as evident by a small area of the nucleus in the image of the specific cell formed on the detector, is identified to be either a live cell or an early apoptotic cell, the specific cell being then identified as a live cell or an early apoptotic cell based upon the total brightfield area and a darkfield peak intensity of the specific cell, the total brightfield area of an early apoptotic cell being smaller compared to the total brightfield area of a live cell, and the darkfield peak intensity an early apoptotic cell being greater than the spatial scatter peak intensity of a live cell.

18. The method of claim 14, wherein a specific cell in which the nucleus readily absorbs the stain that is applied, as evident by a large area of the nucleus in an image of the specific cell formed on the detector, is thereby identified to be either a necrotic cell or a late apoptotic cell, the specific cell being then identified as a necrotic cell or a late apoptotic cell based upon a darkfield peak intensity of the specific cell, the darkfield peak intensity of a necrotic cell having a lower complexity compared to that of a late apoptotic cell.

19. The method of claim 12, wherein the darkfield spatial frequency is determined based upon a standard deviation of individual pixel intensities of the image of the specific cell within a segmented dark field image mask.

20. The method of claim 12, wherein the darkfield spatial frequency is used to determine at least one of an internal cell complexity or cell granularity of the specific cell, for use in identifying the specific cell as being in one of the four different types of cells.