System for cell-based screening

• US 6,902,883 B2
• Filed: 05/06/2003
• Issued: 06/07/2005
• 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
• Assistant Examiner(s): Padmanabhan, Kartic
• Application Number:
  US10/430,534
• Publication Number:
  US 20030204316A1
• Time in Patent Office: 763 Days
• Field of Search: 435/4, 435/7.2, 435/7.21, 435/29, 435/40.5, 435/40.51, 435/288.3, 435/501, 435/800, 435/807, 435/809, 436/63, 436/172, 436/501, 436/800, 436/807, 436/809, 250/201.3, 348/80, 348/345, 356/300, 356/319, 356/326, 356/328, 382/128, 382/129, 382/133, 382/173, 382/254, 382/255, 382/286, 382/291
• US Class Current: 435/4
• 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 translocation of a cellular component of interest between nucleus and cytoplasm in cells comprising:

a) providing an array of locations which contain multiple cells, wherein the cells contain at least a first fluorescent reporter molecule that reports on a cellular component of interest;

b) contacting the cells with at least one test compound of interest;

c) scanning multiple cells in each of the locations containing cells to obtain fluorescent signals from the at least first fluorescent reporter molecule within the nucleus and the cytoplasm in the cells;

d) measuring a fluorescence intensity of the fluorescent signals from the at least first fluorescent reporter molecule within the nucleus and within the cytoplasm of the cells; and

e) automatically calculating test compound-induced changes in one or more of the following;

i) a ratio of fluorescent signal intensity from the at least first fluorescent reporter molecule in the cytoplasm to fluorescent signal intensity from the at least first fluorescent reporter molecule in the nucleus; and

ii) a difference between fluorescent signal intensity from the at least first fluorescent reporter molecule in the cytoplasm and fluorescent signal intensity from the at least first fluorescent reporter molecule in the nucleus;

wherein the test compound-induced changes indicate an effect of the test compound on translocation of the cellular component of interest between the nucleus and the cytoplasm in the cells.

2. The method of claim 1 wherein the cellular component of interest is a protein.

3. The method of claim 2 wherein the protein is a transcription factor.

4. The method of claim 1 wherein the cells contain at least a second reporter molecule that identifies cells, wherein the method further comprises obtaining fluorescent signals from the at least second reporter molecule and using the fluorescent signals from the at least second reporter molecule to define individual cells, and wherein the translocation is analyzed in the individual cells.

5. The method of claim 1 wherein measuring a fluorescence intensity of the fluorescent signals from the at least first fluorescent reporter molecule within the nucleus comprises measuring the intensity of the fluorescent signal from the at least first fluorescent reporter molecule within a nuclear mask.

6. The method of claim 1 wherein measuring a fluorescence intensity of the fluorescent signals from the at least first fluorescent reporter molecule within the cytoplasm comprises measuring the intensity of the fluorescent signal from the at least first fluorescent reporter molecule within a cytoplasmic mask.

7. The method of claim 2 wherein the at least first reporter molecule comprises an antibody that reports on the protein of interest.