System for cell-based screening

• US 7,060,445 B1
• Filed: 11/22/2000
• Issued: 06/13/2006
• 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, Taylor, D. Lansing
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): CHEU, CHANGHWA J
• Application Number:
  US09/721,168
• Time in Patent Office: 2,029 Days
• Field of Search: 435/7.1, 435/7.21, 435/7.2, 435/29, 435/40.5, 435/40.51, 435/288.3, 435/288.4, 436/546, 436/172, 436/800, 436/809
• US Class Current: 435/7.1
• 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: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6482 Sample cells, cuvettes
• 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 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5035 on sub-cellular localization
• CPC Class Codes: G01N 33/582 : with fluorescent label
• CPC Class Codes: G02B 21/16 : adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/365 : Control or image processing arrangements for digital or video microscopes G02B21/361, G02B21/362 take precedence
• CPC Class Codes: Y10S 436/80 : Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/807 : Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays
• CPC Class Codes: Y10S 977/795 : Composed of biological material
• CPC Class Codes: Y10S 977/881 : Microscopy or spectroscopy, e.g. sem, tem
• CPC Class Codes: Y10S 977/92 : Detection of biochemical
• CPC Class Codes: Y10S 977/941 : including dna logic element
• CPC Class Codes: Y10S 977/942 : including protein logic element

Abstract

The invention relates to an optical system for determining the distribution, environment, or activity of fluorescently labeled reporter molecules in cells for the purpose of screening large numbers of compounds for specific biological activity. The invention involves providing cells containing fluorescent reporter molecules in an array of locations and scanning numerous cells in each location with a fluorescent microscope, 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 a microplate having a 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 distribution of a protein of interest between cell membrane and cell cytoplasm comprising:

a) providing an array of locations which contain multiple cells, wherein the cells contain a plurality of fluorescent reporter molecules, wherein the plurality of fluorescent reporter molecules comprise fluorescent reporter molecules that report on cell cytoplasm, fluorescent reporter molecules that report on cell membrane, and fluorescent reporter molecules that report on the protein of interest;

b) automatically imaging multiple cells in each of the locations containing cells to obtain fluorescent signals from the plurality of fluorescent reporter molecules on or in individual cells, wherein the fluorescent signals from the fluorescent reporter molecules that report on cell cytoplasm are used to create cell cytoplasmic masks of individual cells and the fluorescent signals from the fluorescent reporter molecules that report on cell membrane are used to create cell membrane masks of individual cells;

c) automatically measuring an intensity of the fluorescent signals from the fluorescent reporter molecules that report on the protein of interest in the cell cytoplasmic mask and in the cell membrane mask; and

d) automatically calculating one or both of the following;

i) a ratio of the intensity of the fluorescent signals from the fluorescent reporter molecules that report on the protein of interest in the cell cytoplasmic mask and the cell membrane mask; and

ii) a difference of the intensity of the fluorescent signals from the fluorescent reporter molecules that report on the protein of interest in the cell cytoplasmic mask and the cell membrane mask;

wherein the ratio and/or difference provides a measure of the distribution of the protein of interest between the cytoplasm and the cell membrane in the individual cells.

2. The method of claim 1 further comprising contacting the cells with a test compound, and wherein the ratio and/or difference provides a measure of test compound-induced changes in the distribution of the protein of interest between the cytoplasm and the cell membrane in the individual cells.

3. The method of claim 1 wherein the plurality of fluorescent reporter molecules comprises fluorescently labeled proteins, fluorescently labeled antibodies, and chimeric proteins comprising green fluorescent protein coupled to a protein of interest.

4. The method of claim 1 wherein the cellular protein of interest comprises a protein selected from the group consisting of a GTP binding protein and a protein tyrosine kinase.

5. The method of claim 4 wherein the cellular protein of interest is a GTP binding protein.

6. The method of claim 5 wherein the GTP binding protein is a Rho protein.

7. The method of claim 4 wherein the cellular protein of interest comprises a protein tyrosine kinase.

8. The method of claim 7 wherein the protein tyrosine kinase is a src protein.

9. The method of claim 1 further comprising automatically storing an image of each individual cell.

10. The method of claim 1 wherein calculations made in step (d) are stored in a database.

11. The method of claim 10 wherein the calculations made in step (d) stored in a database can be reviewed for individual cells.

12. The method of claim 10 wherein the calculations made in step (d) stored in a database can be reviewed for individual locations containing cells.