System for cell-based screening

• US 6,727,071 B1
• Filed: 12/08/1999
• Issued: 04/27/2004
• Est. Priority Date: 02/27/1997
• Status: Expired due to Term

• Current Assignee: Cellomics Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Cellomics Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Dunlay, R. Terry, Giuliano, Kenneth A., Taylor, D. Lansing, Gough, Albert H.
• Primary Examiner(s): Chin, Christopher L.
• Assistant Examiner(s): GABEL, GAILENE
• Application Number:
  US09/380,259
• Time in Patent Office: 1,602 Days
• Field of Search: 435/7.2, 435/7.21, 435/29, 435/40.5, 435/40.51, 435/288.3, 435/288.4, 435/4, 435/7.5, 435/6, 435/7.1, 435/183, 435/375, 435/975, 435/377, 436/546, 436/172, 436/800, 436/809, 436/10, 436/17, 436/164, 436/166, 436/174, 436/63, 436/517, 436/56, 356/300, 356/326, 356/328, 382/255, 382/141, 530/350
• US Class Current: 435/7.21
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00743 Cells
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 2015/1497 Particle shape
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]
• CPC Class Codes: Y10T 436/107497 Preparation composition [e.g., lysing or precipitation, etc.]
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

The present invention provides systems, methods, and screens for an optical system analysis of cells to rapidly determine the distribution, environment, or activity of fluorescently labeled reporter molecules in cells for the purpose of screening large numbers of compounds for those that specifically affect particular biological functions. The invention involves providing cells containing fluorescent reporter molecules in an array of locations and scanning numerous cells in each location with a high magnification fluorescence optical system, converting the optical information into digital data, and utilizing the digital data to determine the distribution, environment or activity of the fluorescently labeled reporter molecules in the cells. The array of locations may be an industry standard 96 well or 384 well microtiter plate or a microplate which is microplate having cells in a micropaterned array of locations. The invention includes apparatus and computerized method for processing, displaying and storing the data.

Claims

1. An automated method for analyzing cells comprising:

a) providing an array of locations which contain multiple cells, wherein the cells contain on or within the cells at least a first fluorescent or luminescent reporter molecule that binds to deoxyribonucleic acid and at least a second fluorescent or luminescent reporter molecule that reports on mitochondrial potential by accumulating in and/or binding to mitochondria or actin polymerization by binding to and/or accumulating in actin filaments;

b) automatically obtaining fluorescent or luminescent signals from the at least first fluorescent or luminescent reporter molecule and the at least second fluorescent or luminescent reporter molecule on or in the cells;

c) automatically calculating from the fluorescent or luminescent signals from the at least first fluorescent or luminescent reporter molecule an average nuclear area, and one or both of an average nuclear perimeter; and

an average nuclear intensity; and

d) automatically calculating an average intensity of the fluorescent or luminescent signals from the at least second fluorescent or luminescent reporter molecule,

wherein the calculations made in steps (c) and (d) provide a measure of apoptotic activity in the cells.

2. The method of claim 1 wherein the at least second fluorescent or luminescent reporter molecule reports on mitochondrial potential, and wherein the automatically calculating an average intensity of the fluorescent or luminescent signals from the at least second fluorescent or luminescent reporter molecule provides a measure of mitochondrial potential.

3. The method of claim 2 wherein the cells further contain on or within the cells at least a third fluorescent or luminescent reporter molecule that reports on actin polymerization by binding to and/or accumulating in actin filaments;

and wherein the method further comprises automatically obtaining fluorescent or luminescent signals from the at least third fluorescent or luminescent reporter molecule on or in the cells, and automatically calculating an average intensity of the fluorescent or luminescent signals from the at least third fluorescent or luminescent reporter molecule, wherein the automatically calculating an average intensity of the fluorescent or luminescent signals from the at least third fluorescent or luminescent reporter molecule provides a measure of actin polymerization, and wherein actin polymerization provides an indication of apoptotic activity in the cells.

4. The method of claim 3 wherein the calculations are automatically performed at multiple time points.

5. The method of claim 3 further comprising contacting the cells with a test compound, wherein the calculated changes indicate a test-compound induced change in apoptotic activity.

6. The method of claim 2 wherein the calculations are automatically performed at multiple time points.

7. The method of claim 2 further comprising contacting the cells with a test compound, wherein the calculated changes indicate a test-compound induced change in apoptotic activity.

8. The method of claim 1 wherein the at least second fluorescent or luminescent reporter molecule reports on actin polymerization, and wherein the automatically calculating an average intensity of the fluorescent or luminescent signals from the at least second fluorescent or luminescent reporter molecule provides an indication of actin polymerization.

9. The method of claim 8 wherein the cells further contain on or within the cells at least a third fluorescent or luminescent reporter molecule that reports on mitochondrial potential by accumulating in and/or binding to mitochondria;

and wherein the method further comprises automatically obtaining fluorescent or luminescent signals from the at least third fluorescent or luminescent reporter molecule on or in the cells, and automatically calculating an average intensity of the fluorescent or luminescent signals from the at least third fluorescent or luminescent reporter molecule, wherein the automatically calculating an average intensity of the fluorescent or luminescent signals from the at least third fluorescent or luminescent reporter molecule provides a measure of mitochondrial potential, and wherein mitochondrial potential provides an indication of apoptotic activity in the cells.

10. The method of claim 9 wherein the calculations are automatically performed at multiple time points.

11. The method of claim 9 further comprising contacting the cells with a test compound, wherein the calculated changes indicate a test-compound induced change in apoptotic activity.

12. The method of claim 8 wherein the calculations are automatically performed at multiple time points.

13. The method of claim 8 further comprising contacting the cells with a test compound, wherein the calculated changes indicate a test-compound induced change in apoptotic activity.

14. The method of claim 1 wherein the calculations are automatically performed at multiple time points.

15. The method of claim 1 further comprising contacting the cells with a test compound, wherein the calculated changes indicate a test-compound induced change in apoptotic activity.

16. The method of any one of claims 1, 2, 4, 6-8, and 12-15 wherein the at least first fluorescent or luminescent reporter molecule and the at least second fluorescent or luminescent reporter molecule are fluorescent reporter molecules.

17. The method of any one of claims 3-5 and 9-11 wherein the at least first fluorescent or luminescent reporter molecule, the at least second fluorescent or luminescent reporter molecule, and the at least third fluorescent or luminescent reporter molecule are all fluorescent reporter molecules.