System for cell-based screening

• US 6,620,591 B1
• Filed: 04/16/1999
• Issued: 09/16/2003
• 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): GABEL, GAILENE
• Application Number:
  US09/293,210
• Time in Patent Office: 1,614 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, 356/300, 356/326, 356/328, 382/260, 382/133, 382/141, 250/201.3, 348/345
• US Class Current: 435/7.2
• 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 detecting translocation of a cellular component of interest between a first cellular compartment and a second cellular compartment on and/or within individual cells comprising:

a) providing an array of locations which contain multiple cells, wherein the cells contain at least a first fluorescent reporter molecule to identify individual cells, and at least a second fluorescent reporter molecule to report on a cellular component of interest, wherein fluorescent signals from the at least first and the at least second fluorescent reporter molecules are optically distinguishable;

b) automatically scanning multiple cells in each of the locations containing cells to obtain fluorescent signals from the at least first fluorescent reporter molecule, wherein the scanning comprises;

i) acquiring fluorescent signals in a field from the at least first fluorescent reporter molecule at a first wavelength;

ii) automatically converting the fluorescent signals from the at least first fluorescent reporter molecule into digital data; and

iii) automatically utilizing the digital data from the at least first fluorescent reporter molecule to identify individual cells in the field;

c) automatically acquiring fluorescent signals from the at least second fluorescent reporter molecule at a second wavelength in a field containing individual cells;

d) automatically converting the fluorescent signals from the at least second fluorescent reporter molecule into digital data;

e) automatically defining a first cellular compartment mask and a second cellular compartment mask from the fluorescent signals from the at least first reporter molecule and the at least second fluorescent reporter molecule;

f) automatically determining an intensity of the fluorescent signals from the at least second fluorescent reporter molecule in the first cellular compartment mask and the second cellular compartment mask; and

g) automatically calculating one or both of the following;

i) a ratio of the intensity of the fluorescent signals from the at least second fluorescent reporter molecule in the first cellular compartment mask and the second cellular compartment mask; and

ii) a difference of the intensity of the fluorescent signals from the at least second fluorescent reporter molecule in the first cellular compartment mask and the second cellular compartment mask;

wherein the ratio of the intensity of the fluorescent signals from the at least second fluorescent reporter molecule in the first cellular compartment mask and the second cellular compartment mask and/or the difference of the intensity of the fluorescent signals from the at least second fluorescent reporter molecule in the first cellular compartment mask and the second cellular compartment mask provides a measure of the translocation of the cellular component of interest between the first cellular compartment and the second cellular compartment on and/or within the individual cells.

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

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

4. The method of claim 1 wherein the digital data are stored in a database.

5. The method of claim 4 wherein the digital data stored in the database are reviewed for individual cells.

6. The method of claim 4 wherein the digital data stored in the database are reviewed for individual locations containing cells.

7. The method of claim 1, wherein the first cellular compartment and the second cellular compartment consist of a cell nucleus and a cell cytoplasm, and wherein the translocation consists of a translocation between the cell cytoplasm and the cell nucleus.

8. The method of claim 7 wherein the cellular component of interest is a protein.

9. The method of claim 8 wherein the protein is a transcription factor.

10. The method of claim 1 wherein the first cellular compartment and the second cellular compartment consist of a cell cytoplasm and a cell membrane, and wherein the translocation consists of a translocation between the cell cytoplasm and the cell membrane.

11. The method of claim 10 wherein the cellular component of interest is selected from the group consisting of protein kinases and GTP, (guanisinc triphosphate)-binding porteins.

12. The method of claim 11 wherein the cellular component of interest is a protein kinase.

13. The method of claim 11 wherein the cellular component of interest is a GTP-binding protein.

14. The method of claim 13 wherein the GTP-binding protein is a Rho family protein.

15. The method of claim 1, wherein the method further comprises contacting the cells with a test compound, and wherein the ratio of the intensity of the fluorescent signals from the at least second fluorescent reporter molecule in the first cellular compartment mask and the second cellular compartment mask and/or the difference of the intensity of the fluorescent signals from the at least second fluorescent reporter molecule in the first cellular compartment mask and the second cellular compartment mask provides a measure of test compound-induced translocation of the cellular component of interest between the different cellular compartments on and/or within the individual cells.

16. The method of claim 15 wherein the cellular component of interest is a protein.

17. The method of claim 15, wherein the at least first fluorescent reporter molecule identifies nuclei, and wherein the identifying of the individual cells comprises identifying the nucleus of the individual cells.

18. The method of claim 17 wherein the cellular component of interest is a protein.

19. The method of claim 17, wherein the defining a first cellular compartment mask and a second cellular compartment mask comprises creating a nuclear mask from the fluorescent signals from the at least first fluorescent reporter molecule.

20. The method of claim 19 wherein the defining a first cellular compartment mask and a second cellular compartment mask comprises creating a cytoplasmic mask from the fluorescent signals from the at least second fluorescent reporter molecule.

21. The method of claim 20, wherein translocation of the cellular component of interest between the nucleus and the,cytoplasm in the individual cells is detected.

22. The method of claim 19 wherein the cellular component of interest is a protein.

23. The method of claim 20 wherein the cellular component of interest is a protein.

24. The method of claim 21 wherein the cellular component of interest is a protein.

25. The method of claim 19, wherein the defining a first cellular compartment mask and a second cellular compartment mask comprises dilating the nuclear mask to create a cytoplasmic mask.

26. The method of claim 25 wherein the cellular component of interest is a protein.

27. The method of claim 25, wherein translocation of the cellular component of interest between the nucleus and the,cytoplasm in the individual cells is detected.

28. The method of claim 27 wherein the cellular component of interest is a protein.