System for cell-based screening

• US 5,989,835 A
• Filed: 02/27/1997
• Issued: 11/23/1999
• 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): Chin, Christopher L.
• Assistant Examiner(s): NGUYEN, BAO THUY L
• Application Number:
  US08/810,983
• Time in Patent Office: 999 Days
• Field of Search: 435/7.2, 435/7.21, 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: 506/10
• 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 cells comprising;

a) providing an array of locations which contain multiple cells, wherein the cells contain one or more fluorescent reporter molecules;

b) scanning multiple cells in each of the locations containing cells to obtain fluorescent signals from the fluorescent reporter molecules in subcellular compartments of the cells being analyzed wherein the subcellular compartments comprise the cell nucleus and the cell cytoplasm;

c) converting the fluorescent signals into digital data;

d) utilizing the digital data to automatically make measurements, wherein the measurements are used to automatically calculate changes in the distribution, environment or activity of the fluorescent reporter molecules within or between the cell nucleus and the cell cytoplasm of the cells being analyzed, wherein the measurements comprise determining;

i) the fluorescence intensity of reporter molecules within the cell nucleus;

ii) the fluorescence intensity of reporter molecules within the cell cytoplasm;

iii) the area of the cell nucleus; and

iv) the area of the cytoplasm.

2. The method of claim 1 wherein the calculated changes comprise:

a) changes in the fluorescence intensity of reporter molecules within the cell nucleus of the cells being analyzed;

b) changes in the fluorescence intensity of reporter molecules within the cell cytoplasm of the cells being analyzed;

c) changes in the ratio of the fluorescence intensity within the cell cytoplasm to the fluorescence intensity within the cell nucleus of the cells being analyzed; and

d) changes in the difference of the fluorescence intensity within the cytoplasm and the fluorescence intensity within the nucleus of the cells being analyzed.

3. The method of claim 2 wherein the calculated changes provide a measurement of translocation of fluorescent reporter molecules from the cytoplasm to the nucleus of the cells being analyzed.

4. The method of claim 3 wherein the fluorescent reporter molecules comprise a transcription factor.

5. The method of claim 4 wherein the transcription factor comprises NF-kB.

6. An automated method for analyzing cells comprising:

a) providing an array of locations which contain multiple cells, wherein the cells contain one or more fluorescent reporter molecules;

b) scanning multiple cells in each of the locations containing cells to obtain fluorescent signals from the fluorescent reporter molecules in subcellular compartments of the cells, wherein the subcellular compartments comprise the cell nucleus and a subcellular compartment selected from the group consisting of the cell membrane and the cell cytoplasm;

c) converting the fluorescent signals into digital data;

d) utilizing the digital data to automatically make measurements, wherein the measurements are used to automatically calculate changes in the distribution, environment or activity of the fluorescent reporter molecules within or between the cell nucleus and the subcellular compartment selected from the group consisting of the cell membrane and the cell cytoplasm, wherein the digital data are captured as pixels, and wherein;

i) the fluorescence intensity of reporter molecules within the cell nucleus defines a nuclear region, and wherein the total number of pixels within the nuclear region comprises a measure of nuclear area; and

ii) the fluorescence intensity of reporter molecules in the subcellular compartment selected from the group consisting of the cell membrane and the cell cytoplasm defines a cytoplasmic region, and wherein the total number of pixels in the cytoplasmic region comprises a measure of the cytoplasmic area.

7. The method of claim 6 wherein the calculated changes comprise:

(a) changes in the measure of nuclear area;

(b) changes in the measure of cytoplasmic area; and

(c) changes in the ratio of cytoplasmic area to nuclear area.

8. The method of claim 7 wherein the calculated changes provide a measurement of cellular hypertrophy.