Machine-readable storage medium for analyzing distribution of macromolecules between the cell membrane and the cell cytoplasm

• US 7,117,098 B1
• Filed: 11/27/2000
• Issued: 10/03/2006
• 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: Gough, Albert H., Dunlay, R. Terry, Giuliano, Kenneth A., Taylor, D. Lansing
• Primary Examiner(s): Moran, Marjorie A.
• Assistant Examiner(s): Smith, Carolyn L.
• Application Number:
  US09/723,256
• Time in Patent Office: 2,136 Days
• Field of Search: 702/21, 702/19, 382/133, 382/165, 382/212, 422/68.1, 435/4, 436/172
• US Class Current: 702/21
• CPC Class Codes: G01N 2021/6417 Spectrofluorimetric devices
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• 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 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5035 on sub-cellular localization

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 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. A machine readable storage medium comprising a program containing a set of instructions for causing a cell screening system to execute procedures for detecting the distribution of one or more cellular macromolecule of interest between a cell cytoplasm and a plasma membrane in individual cells comprising:

a) scanning multiple cells in an array of locations which contain multiple cells to obtain fluorescent signals from fluorescent reporter molecules in the cells, wherein the cells possess a plurality of fluorescent reporter molecules, wherein the plurality of fluorescent reporter molecules comprises one or more fluorescent reporter molecules to report on

(i) one or more cellular macromolecule of interest;

(ii) the cell cytoplasm and

(iii) the plasma membrane;

b) identifying individual cells from the fluorescent signals from the plurality of fluorescent reporter molecules;

c) creating a plasma membrane mask and a cell cytoplasm mask in the individual cells from the fluorescent signals from the plurality of fluorescent reporter molecules;

d) determining an intensity of the fluorescent signals from the fluorescent reporter molecules that report on the one or more cellular macromolecule of interest within the plasma membrane mask and the cell cytoplasm mask in the individual cells in response to contacting the cells with a test stimulus;

e) determining a translocation quotient between the cell cytoplasm and the plasma membrane for the cellular macromolecule of interest by calculating a ratio of the intensity of the fluorescent signals from the fluorescent reporter molecules that report on the one or more cellular macromolecule of interest within the plasma membrane mask and the intensity of the fluorescent signals from the fluorescent reporter molecules that report on the one or more cellular macromolecule of interest within the cell cytoplasm mask in the individual cells in response to contacting the cells at a first time point with a test stimulus;

(f) comparing the translocation quotient determined in step (e) to;

i) one or more translocation quotients for the cellular macromolecule of interest between the cell cytoplasm and the plasma membrane, which are determined by calculating a ratio of an intensity of fluorescent signals from the fluorescent reporter molecules that report on the one or more cellular macromolecule of interest within the plasma membrane mask and an intensity of fluorescent signals from the fluorescent reporter molecules that report on the one or more cellular macromolecule of interest within the cell cytoplasm mask (A) in the individual cells in response to contacting the cells with the test stimulus from at least a second time point; and

(B) in individual cells that have not been contacted with the test stimulus;

or

ii) one or more translocation quotients for the cellular macromolecule of interest between the cell cytoplasm and the plasma membrane, which are determined by calculating a ratio of an intensity of fluorescent signals from the fluorescent reporter molecules that report on the one or more cellular macromolecule of interest within the plasma membrane mask and an intensity of fluorescent signals from the fluorescent reporter molecules that report on the one or more cellular macromolecule of interest within the cell cytoplasm mask in the individual cells that have not been contacted with the test stimulus; and

g) determining the effect of the test stimulus on the distribution of the one or more cellular macromolecule of interest between the plasma membrane and the cell cytoplasm in the individual cells as a function of the translocation quotient determined in step (e), and the one or more translocation quotients determined in step (f)(i)(A) and (B), or step (f)(ii).

2. The machine readable storage medium of claim 1, wherein the identifying of the individual cells comprises identifying the nucleus of the individual cells.

3. The machine readable storage medium of claim 1, wherein the cellular macromolecule of interest is a protein.

4. The machine readable storage medium of claim 1, wherein the fluorescent reporter molecules that report on the one or more cellular macromolecule of interest comprises a fluorescently labeled antibody.

5. The machine readable storage medium of claim 1, wherein the multiple cells are fixed cells.

6. The machine readable storage medium of claim 1, wherein the translocation quotient determined in step (e) is compared to the one or more translocation quotients determined in step (f)(i).

7. The machine readable storage medium of claim 6, wherein the multiple cells are live cells.

8. The machine readable storage medium of claim 1 wherein the translocation quotient determined in step (e) is compared to the one or more translocation quotients determined in step (f)(ii).