Image-based quantitation of molecular translocation

• US 9,528,989 B2
• Filed: 03/30/2012
• Issued: 12/27/2016
• 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., Frost, Keith, Hall, Brian E., Ortyn, William E., Seo, Michael J.
• Primary Examiner(s): Kassa, Yosef
• Application Number:
  US13/436,371
• Publication Number:
  US 20120244550A1
• Time in Patent Office: 1,733 Days
• Field of Search: 382/128, 382/129, 382/133, 382/134, 382/209, 382/278, 128/922, 128/923
• US Class Current: 1/1
• CPC Class Codes: G01N 33/56966 Animal cells

Abstract

The use of an imaging system, cell compartment markers, and molecular markers in methods for correlating the movement of molecules within a cell to a particular compartment are provided, including measuring and correlating molecule movement in adherent and non-adherent cells.

Claims

1. A method for measuring molecular movement in a cell, comprising:

contacting the cell with at least one marker;

imaging the cell with a detector;

creating a mask; and

measuring the molecular movement in the cell based at least in part on a correlation between the mask and the at least one marker.

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

3. The method of claim 1 wherein the at least one marker is a fluorescent labeled antibody.

4. The method of claim 1 wherein the at least one marker is a fluorescent molecule.

5. The method of claim 1 wherein the mask is a compartment mask and the compartment is nucleus, cytoplasm, or membrane.

6. The method of claim 1 wherein the at least one marker is marked NF-κ

B.

7. The method of claim 1 further comprising the step of inducing molecular movement in the cell.

8. The method of claim 7 wherein the induced molecular movement is nuclear translocation.

9. The method of claim 7 wherein the molecular movement is induced with LPS or IL-1β

/TNF-α

.

17. The method according to any one of claims 1-16 wherein the detector is a time delay integration charge-coupled detector.

18. A system configured to perform the method according to claim 1 or claim 10.

10. A method for measuring nuclear translocation in a cell, comprising:

contacting the cell with at least one marker;

imaging the cell with a detector;

creating a nuclear mask; and

measuring the nuclear translocation in the cell based at least in part on a correlation between the nuclear mask and the at least one marker.

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

12. The method of claim 10 further comprising the step of inducing molecular movement in the cell.

13. The method of claim 12 wherein the induced molecular movement is the nuclear translocation.

14. The method of claim 12 wherein the molecular movement is induced with LPS or IL-1β

/TNF-α

.

15. The method of claim 10 wherein the at least one marker is 7-AAD.

16. The method of claim 10 wherein the at least one marker is marked NF-κ

B.

19. An imaging system adapted to measure molecular movement in a cell, comprising:

a light source that produces light incident on the cell, the cell contacted with at least one marker;

a collection lens disposed so that light emitted by the light source and traveling from the cell passes through the collection lens and travels along a collection path;

a spectral decomposition optical system disposed so that light traveling along the collection path is separated into multiple spectral bands;

a detector disposed to receive the multiple spectral bands, producing a first signal corresponding to a first one of the multiple spectral bands and producing a second signal corresponding to a second one of the multiple spectral bands; and

a processor configured to measure the molecular movement in the cell based at least in part on a correlation between the first signal and the second signal.

20. The imaging system of claim 19, wherein the light source is a spectrally-limited light source.

21. The imaging system of claim 19, wherein the light that has passed through the collection lens is dispersed in a plane that is orthogonal to a direction of relative movement between the cell and the imaging system.

22. The imaging system of claim 19, wherein the detector is a time delay integration charge-coupled detector.

23. The imaging system of claim 19, wherein the detector produces the first signal and the second signal simultaneously.

24. The imaging system of claim 19, wherein the first one of the multiple spectral bands corresponds to a spectral band of the cell contacted with the at least one marker.

25. The imaging system of claim 19, wherein the second one of the multiple spectral bands corresponds to a spectral band for a specific cellular compartment of the cell.

26. The imaging system of claim 19, wherein the processor is further configured to create an image mask from the second signal.