Method for imaging and differential analysis of cells

• US 8,103,080 B2
• Filed: 03/16/2005
• Issued: 01/24/2012
• 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): Perungavoor, Sath V
• Assistant Examiner(s): HEIDEMANN, JASON E
• Application Number:
  US10/593,016
• Publication Number:
  US 20080240539A1
• Time in Patent Office: 2,505 Days
• Field of Search: 382/128, 382/129, 382/133, 356/326
• 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 morphogenic 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 cell, comprising the steps of:

providing spatial frequency content data from a side scatter image of the known cell type;

directing incident light at the specific cell, using a detector to obtain the side scatter image of the specific cell;

calculating the spatial frequency content data of the side scatter image of the specific cell by computing a standard deviation of individual pixel intensities within the side scatter image of the specific cell; and

comparing the spatial frequency content data of the side scatter image of the specific cell to the spatial frequency content data of the side scatter image of the known cell type to determine if the specific cell corresponds to the known cell type.

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

3. The method of claim 1 wherein the specific cell identified is contained within a heterogeneous cell population, and side scatter image data is collected for the heterogeneous cell population.

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

5. The method of claim 4 wherein the apoptotic cell is an early stage apoptotic cell or a late stage apoptotic cell.

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

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

8. 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 the steps of:

providing spatial frequency content data from a brightfield image of the known cell type;

directing incident light at the specific cell, using a detector to obtain the brightfield image of the specific cell;

calculating spatial frequency content data of the brightfield image of the specific cell by computing a standard deviation of individual pixel intensities within the brightfield image of the specific cell; and

comparing the spatial frequency content data of the brightfield image of the specific cell to the spatial frequency content data of the brightfield image of the known cell type to determine if the specific cell corresponds to the known cell type.

9. The method of claim 8 wherein there is relative motion between the specific cell and the detector.

10. The method of claim 8 wherein the specific cell identified is contained within a heterogeneous cell population, and brightfield image data is collected for the heterogeneous cell population.

11. The method of claim 8 wherein the specific cell identified is an apoptotic cell.

12. The method of claim 11 wherein the apoptotic cell is an early stage apoptotic cell or a late stage apoptotic cell.

13. The method of claim 8 wherein the specific cell identified is a necrotic cell.

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

15. The method of claim 8 wherein the spatial frequency content is of the nucleus.

16. 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 the steps of:

providing an image of the known cell type that has been marked with a single nuclear marker;

providing spatial frequency content data from the image of the known cell type that has been marked with the nuclear marker;

contacting the specific cell with the nuclear marker;

directing incident light at the marked specific cell;

using a detector to obtain an image of the marked specific cell;

calculating spatial frequency content data of the image of the marked specific cell by computing a standard deviation of individual pixel intensities within the image of the marked specific cell; and

comparing the image of the marked specific cell and the spatial frequency content data of the image of the marked specific cell to the marked image of the known cell type and the spatial frequency content data of the marked image of the known cell type to determine if the specific cell corresponds to the known cell type.

17. The method of claim 16 wherein there is relative motion between the specific cell and the detector.

18. The method of claim 16 wherein the specific cell identified is contained within a heterogeneous cell population, and image data is collected for the heterogeneous cell population.

19. The method of claim 16 wherein the specific cell identified is an apoptotic cell.

20. The method of claim 19 wherein the apoptotic cell is an early stage apoptotic cell or a late stage apoptotic cell.

21. The method of claim 16 wherein the specific cell identified is a necrotic cell.

22. The method of claim 16 wherein the specific cell identified is at least one of an apoptotic cell and a necrotic cell.

23. The method of claim 16 wherein a single nuclear marker is used, the single nuclear marker and the spatial frequency content of the image enabling one to classify the specific cell as one of the following cell types:

a viable cell having a cellular membrane that is impermeable to the nuclear marker;

a cell in an early stage of apoptosis and which has a cellular membrane that is impermeable to the nuclear marker;

a cell in a late stage of apoptosis and which has a cellular membrane that is permeable to the nuclear marker; and

a necrotic cell which has a cellular membrane that is permeable to the nuclear marker.

24. The method of claim 23 wherein the single nuclear marker is 7-aminoactinomycin D.

25. A method for classifying a specific cell as one of the following four types of cells, a viable cell, a necrotic cell, an early apoptotic cell in which a cellular membrane of the cell is still intact, and a late apoptotic cell in which the cellular membrane of the cell is not intact, using only a single nuclear marker and image data from the cell, the method comprising the steps of:

exposing the specific cell to only a single nuclear marker that will bind to DNA in a nucleus of the cell in the event that the cellular membrane of the cell is not intact;

collecting image data from the specific cell;

using the image data of the specific cell to determine spatial frequency content data of a side scatter image of the specific cell by computing a standard deviation of individual pixel intensities within the side scatter image of the specific cell; and

analyzing the image data of the specific cell and the spatial frequency content data of the side scatter image of the specific cell to classify the cell as one of a viable cell, a necrotic cell, an early apoptotic cell, and a late apoptotic cell.

26. The method of claim 25 wherein the step of analyzing determines that the specific cell is a viable cell, when:

the image data indicates that the nuclear marker has not crossed the cellular membrane; and

the spatial frequency content of the side scatter image of the specific cell corresponds to a previously determined spatial frequency content of a side scatter image of a viable cell.

27. The method of claim 25 wherein the step of analyzing determines that the specific cell is a viable cell, when:

the image data indicates that the nuclear marker has not crossed the cellular membrane; and

the spatial frequency content data of the side scatter image of the specific cell does not correspond to a previously determined spatial frequency content data of a side scatter image of an early apoptotic cell.

28. The method of claim 25 wherein the step of analyzing determines that the specific cell is a cell in an early apoptotic cell when:

the image data indicates that the nuclear marker has not crossed the cellular membrane; and

the spatial frequency content data of the side scatter image of the specific cell does not correspond to a previously determined spatial frequency content data of a side scatter image of a viable cell.

29. The method of claim 25 wherein the step of analyzing determines that the specific cell is a cell in an early apoptotic cell when:

the image data indicates that the nuclear marker has not crossed the cellular membrane; and

the spatial frequency content data of the side scatter image of the specific cell corresponds to a previously determined spatial frequency content data of a side scatter image of an early apoptotic cell.

30. The method of claim 25 wherein the step of analyzing determines that the specific cell is a late apoptotic cell when:

the image data indicates that the nuclear marker has crossed the cellular membrane; and

the spatial frequency content data of the side scatter image of the specific cell corresponds to a previously determined spatial frequency content data of a side scatter image of a late apoptotic cell.

31. The method of claim 25 wherein the step of analyzing determines that the specific cell is a late apoptotic cell when:

the image data indicates that the nuclear marker has crossed the cellular membrane; and

the spatial frequency content data of the side scatter image of the specific cell does not correspond to a previously determined spatial frequency content data of a side scatter image of a necrotic cell.

32. The method of claim 25 wherein the step of analyzing determines that the specific cell is a necrotic cell when:

the image data indicates that the nuclear marker has crossed the cellular membrane; and

the spatial frequency content data of the side scatter image of the specific cell does not correspond to a previously determined spatial frequency content data of a side scatter image of a late apoptotic cell.

33. The method of claim 25 wherein the step of analyzing determines that the specific cell is a necrotic cell when:

the image data indicates that the nuclear marker has crossed the cellular membrane; and

the spatial frequency content data of the side scatter image of the specific cell corresponds to a previously determined spatial frequency content data of a side scatter image of a necrotic cell.