Method for imaging and differential analysis of cells

• US 8,953,866 B2
• Filed: 04/30/2012
• Issued: 02/10/2015
• 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 C
• Assistant Examiner(s): Heidemann, Jason
• Application Number:
  US13/460,682
• Publication Number:
  US 20120328177A1
• Time in Patent Office: 1,016 Days
• Field of Search: None
• US Class Current: 382/133
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• 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 21/53 within a flowing fluid, e.g. smoke
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging

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 as being of a known cell type, wherein the known cell type relates to the viability of the cell, comprising:

staining a nucleus of the specific cell with a fluorescence stain;

directing incident light at the specific cell to obtain a nuclear image and a brightfield image of the specific cell with a light detector; and

determining the known cell type to which the specific cell corresponds, based on morphologic features of the specific cell that are derived from the nuclear image and the brightfield image, wherein the morphologic features include at least one morphologic feature selected from the group consisting of a size of the specific cell, a size of a nucleus in the specific cell, and a brightfield image contrast of the specific cell.

2. The method of claim 1, wherein the specific cell is stained with a nucleic acid-binding dye consisting of one of 7-aminoactinomycin D (7-AAD) or propidium iodide.

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 identified is contained within a heterogeneous cell population, and the nuclear image and the brightfield image are collected for the specific cell from nuclear images and brightfield images of the heterogeneous cell population.

5. The method of claim 1, wherein the specific cell identified is an apoptotic cell.

6. The method of claim 5, wherein the apoptotic cell is an early stage apoptotic cell or a late stage apoptotic cell.

7. The method of claim 1, wherein the specific cell identified is a necrotic cell.

8. The method of claim 1, wherein the specific cell identified is at least one of an apoptotic cell and a necrotic cell.

9. 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 cell, comprising:

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

calculating a contrast metric for a brightfield image of the specific cell;

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

based upon the contrast metric for the brightfield image and on the nuclear size of the specific cell, determining if the specific cell corresponds to the known cell type.

10. The method of claim 9, wherein there is relative motion between the specific cell and the detector.

11. The method of claim 9, wherein the specific cell identified is contained within a heterogeneous cell population, and images are produced for the heterogeneous cell population.

12. The method of claim 9, wherein the specific cell is identified as an apoptotic cell.

13. The method of claim 12, wherein the apoptotic cell is an early stage apoptotic cell or a late stage apoptotic cell.

14. The method of claim 9, wherein the specific cell identified as a necrotic cell.

15. The method of claim 9, wherein the specific cell identified is as at least one of an apoptotic cell and a necrotic cell.