U.S. patent application number 10/686161 was filed with the patent office on 2004-04-01 for system for cell-based screening.
This patent application is currently assigned to Cellomics, Inc.. Invention is credited to Dunlay, R. Terry, Giuliano, Kenneth A., Gough, Albert H., Taylor, D. Lansing.
Application Number | 20040063162 10/686161 |
Document ID | / |
Family ID | 25205216 |
Filed Date | 2004-04-01 |
United States Patent
Application |
20040063162 |
Kind Code |
A1 |
Dunlay, R. Terry ; et
al. |
April 1, 2004 |
System for cell-based screening
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.
Inventors: |
Dunlay, R. Terry; (New
Kensington, PA) ; Taylor, D. Lansing; (Pittsburght,
PA) ; Gough, Albert H.; (Glenshaw, PA) ;
Giuliano, Kenneth A.; (Pittsburgh, PA) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Assignee: |
Cellomics, Inc.
|
Family ID: |
25205216 |
Appl. No.: |
10/686161 |
Filed: |
October 15, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10686161 |
Oct 15, 2003 |
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09724376 |
Nov 27, 2000 |
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6671624 |
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09724376 |
Nov 27, 2000 |
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09031271 |
Feb 27, 1998 |
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09031271 |
Feb 27, 1998 |
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08810983 |
Feb 27, 1997 |
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5989835 |
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Current U.S.
Class: |
506/39 ; 435/7.2;
506/41; 702/19 |
Current CPC
Class: |
B01J 2219/00317
20130101; C12Q 1/68 20130101; Y10S 977/92 20130101; G01N 2021/6482
20130101; Y10S 436/80 20130101; Y10S 977/881 20130101; G01N 33/582
20130101; B01J 2219/00707 20130101; Y10S 977/941 20130101; B01L
3/5085 20130101; G01N 21/6428 20130101; G01N 15/1475 20130101; Y10S
707/99943 20130101; C12Q 1/6813 20130101; Y10S 977/795 20130101;
B01J 2219/00743 20130101; G02B 21/365 20130101; G01N 2015/1497
20130101; Y10S 977/942 20130101; B82Y 30/00 20130101; Y10S 436/807
20130101; G01N 21/6452 20130101; G01N 21/6458 20130101; Y10S
707/99931 20130101; B01J 2219/00315 20130101; G01N 33/5005
20130101; C40B 60/14 20130101; G02B 21/16 20130101; Y10S 436/809
20130101; G01N 2021/6419 20130101; G01N 33/5035 20130101; G01N
2015/1486 20130101; C12Q 1/68 20130101; C12Q 2525/179 20130101 |
Class at
Publication: |
435/007.2 ;
702/019 |
International
Class: |
G01N 033/53; G01N
033/567; G06F 019/00; G01N 033/48; G01N 033/50 |
Claims
What is claimed is:
1. A 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 molecule in the
cells; (c) converting the fluorescent signals into digital data;
and (d) utilizing the digital data to determine the distribution,
environment or activity of the fluorescent reporter molecules
within the cells.
2. The method of claim 1 wherein the array of locations are wells
in a microtiter plate.
3. The method of claim 1 wherein the array of locations are
microwells on a microplate.
4. The method of claim 1 wherein the fluorescent reporter is added
to the cell.
5. The method of claim 1 wherein the fluorescent reporter is
produced by the cell.
6. The method of claim 1 wherein a computer means converts the
digital data into the difference between the average cytoplasmic
reporter molecule fluorescent intensity and the average nucleus
fluorescent reporter molecule intensity.
7. The method of claim 1 wherein a computer means converts the
digital data into the average cytoplasmic fluorescent reporter
molecule intensity within the nucleus region.
8. The method of claim 1 wherein a computer means converts the
digital data into the average fluorescent reporter molecule
intensity within the cytoplasmic mask.
9. The method of claim 1 wherein multiple different fluorescent
reporter molecules are in the cell.
10. A cell screening system comprising: (a) a high magnification
fluorescence optical system having an objective lens, an XY stage
adapted for holding a plate with an array of locations for holding
cells and having a means for moving the plate to align the
locations with the microscope objective and a means for moving the
plate in the direction to effect focusing; (b) a digital camera;
(c) a light source having optical means for directing excitation
light to cells in the array locations and a means for directing
fluorescent light emitted from the cells to the digital camera; and
(d) a computer means for receiving and processing digital data from
the digital camera wherein the computer means includes: i) a
digital frame grabber for receiving the images from the camera, ii)
a display for user interaction and display of assay results, iii)
digital storage media for data storage and archiving, and iv) means
for control, acquisition, processing and display of results.
11. The cell screening system of claim 10 having a PC screen
operatively associated with the computer means, for displaying
graphs of data and images of cells having fluorescent reporter
molecules.
12. The cell screening system of claim 10 wherein the computer
means stores the data in a bioinformatics data base.
13. The method of claim 1 further comprising scanning multiple
cells in each of the array of locations containing cells in high
throughput mode, and selectively scanning only a subset of the
locations containing cells in a high content mode.
14. The cell screening system of claim 10 further comprising an
attached reader which measures a signal from the array of locations
which contain multiple cells, and a method to transfer both the
array of locations which contain multiple cells and the
measurements to the cell screening system.
15. The cell screening system of claim 10 wherein the optical means
comprise a mechanical-optical means for changing the magnification
of the system.
16. The cell screening system of claim 10 further comprising a
chamber and control system to maintain the temperature, CO.sub.2
concentration and humidity surrounding the array of ocations which
contain multiple cells.
17. The cell screening system of claim 10 wherein the optical means
comprises a confocal scanning illumination and detection
system.
18. A machine readable storage medium comprising a program
containing a set of instructions for causing a cell screening
system to execute the procedures set forth in FIG. 9, wherein the
cell screening system comprises a high magnification fluorescence
optical system with a stage adapted for holding cells and a means
for moving the stage, a digital camera, a light source for
receiving and processing the digital data from the digital camera,
and a computer means for receiving and processing the digital data
from the digital camera.
19. A machine readable storage medium comprising a program
containing a set of instructions for causing a cell screening
system to execute the procedures set forth in FIG. 11, wherein the
cell screening system comprises a high magnification fluorescence
optical system with a stage adapted for holding cells and a means
for moving the stage, a digital camera, a light source for
receiving and processing the digital data from the digital camera,
and a computer means for receiving and processing the digital data
from the digital camera.
20. A machine readable storage medium comprising a program
containing a set of instructions for causing a cell screening
system to execute the procedures set forth in FIG. 12, wherein the
cell screening system comprises a high magnification fluorescence
optical system with a stage adapted for holding cells and a means
for moving the stage, a digital camera, a light source for
receiving and processing the digital data from the digital camera,
and a computer means for receiving and processing the digital data
from the digital camera.
21. A machine readable storage medium comprising a program
containing a set of instructions for causing a cell screening
system to execute the procedures set forth in FIG. 13, wherein the
cell screening system comprises a high magnification fluorescence
optical system with a stage adapted for holding cells and a means
for moving the stage, a digital camera, a light source for
receiving and processing the digital data from the digital camera,
and a computer means for receiving and processing the digital data
from the digital camera.
22. A machine readable storage medium comprising a program
containing a set of instructions for causing a cell screening
system to execute the procedures set forth in FIG. 14, wherein the
cell screening system comprises a high magnification fluorescence
optical system with a stage adapted for holding cells and a means
for moving the stage, a digital camera, a light source for
receiving and processing the digital data from the digital camera,
and a computer means for receiving and processing the digital data
from the digital camera.
23. A machine readable storage medium comprising a program
containing a set of instructions for causing a cell screening
system to execute the procedures set forth in FIG. 15, wherein the
cell screening system comprises a high magnification fluorescence
optical system with a stage adapted for holding cells and a means
for moving the stage, a digital camera, a light source for
receiving and processing the digital data from the digital camera,
and a computer means for receiving and processing the digital data
from the digital camera.
24. 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 and
activity of specific cellular constituents and processes, wherein
the cell screening system comprises a high magnification
fluorescence optical system with a stage adapted for holding cells
and a means for moving the stage, a digital camera, a light source
for receiving and processing the digital data from the digital
camera, and a computer means for receiving and processing the
digital data from the digital camera.
25. The machine readable storage medium of claim 24, wherein the
specific cellular process comprises the nuclear translocation of a
protein.
26. The machine readable storage medium of claim 25 wherein said
protein comprises a membrane protein.
27. The machine readable storage medium of claim 24, wherein the
specific cellular process comprises cellular hypertrophy.
28. The machine readable storage medium of claim 24, wherein the
specific cellular process comprises apoptosis.
29. The machine readable storage medium of claim 24, wherein the
specific cellular process comprises protease-induced translocation
of a protein.
30. A machine readable storage medium comprising a program
containing a set of instructions for causing a cell screening
system to execute procedures for identifying novel receptor
agonists and antagonists, wherein the cell screening system
comprises a high magnification fluorescence optical system with a
stage adapted for holding cells and a means for moving the stage, a
digital camera, a light source for receiving and processing the
digital data from the digital camera, and a computer means for
receiving and processing the digital data from the digital
camera.
31. A machine readable storage medium comprising a program
containing a set of instructions for causing the cell screening
system of claim 10 to execute the procedures set forth in FIG.
9.
32. A machine readable storage medium comprising a program
containing a set of instructions for causing the cell screening
system of claim 10 to execute the procedures set forth in FIG.
11.
33. A machine readable storage medium comprising a program
containing a set of instructions for causing the cell screening
system of claim 10 to execute the procedures set forth in FIG.
12.
34. A machine readable storage medium comprising a program
containing a set of instructions for causing the cell screening
system of claim 10 to execute the procedures set forth in FIG.
13.
35. A machine readable storage medium comprising a program
containing a set of instructions for causing the cell screening
system of claim 10 to execute the procedures set forth in FIG.
14.
36. A machine readable storage medium comprising a program
containing a set of instructions for causing the cell screening
system of claim 10 to execute the procedures set forth in FIG.
15.
37. A machine readable storage medium comprising a program
containing a set of instructions for causing the cell screening
system of claim 10 to execute procedures for detecting the
distribution and activity of specific cellular constituents and
processes.
38. The machine readable storage medium of claim 37, wherein the
specific cellular process comprises the nuclear translocation of a
protein.
39. The machine readable storage medium of claim 37 wherein said
protein comprises a membrane protein.
40. The machine readable storage medium of claim 37, wherein the
specific cellular process comprises cellular hypertrophy.
41. The machine readable storage medium of claim 37, wherein the
specific cellular process comprises apoptosis.
42. The machine readable storage medium of claim 37, wherein the
specific cellular process comprises protease-induced translocation
of a protein.
43. A machine readable storage medium comprising a program
containing a set of instructions for causing the cell screening
system of claim 10 to execute procedures for identifying novel
receptor agonists and antagonists, wherein the cell screening
system comprises a high magnification fluorescence optical system
with a stage adapted for holding cells and a means for moving the
stage, a digital camera, a light source for receiving and
processing the digital data from the digital camera, and a computer
means for receiving and processing the digital data from the
digital camera.
Description
CROSS REFERENCE
[0001] This application is a continuation-in-part of U.S.
Applications for Pat. Ser. No. 08/810983, filed on Feb. 27, 1997,
and Ser. No. 08/865,341 filed on May 29, 1997; PCT Application WO
97/45730 filed May 29, 1997, and a continuation-in-part of U.S.
Provisional Applications filed Dec. 11, 1997 (97,022-A and
97,223-A), serial numbers to be assigned.
FIELD OF THE INVENTION
[0002] This invention is in the field of fluorescence-based cell
and molecular biochemical assays for drug discovery.
BACKGROUND OF THE INVENTION
[0003] Drug discovery, as currently practiced in the art, is a
long, multiple step process involving identification of specific
disease targets, development of an assay based on a specific
target, validation of the assay, optimization and automation of the
assay to produce a screen, high throughput screening of compound
libraries using the assay to identify "hits", hit validation and
hit compound optimization. The output of this process is a lead
compound that goes into pre-clinical and, if validated, eventually
into clinical trials. In this process, the screening phase is
distinct from the assay development phases, and involves testing
compound efficacy in living biological systems.
[0004] Historically, drug discovery is a slow and costly process,
spanning numerous years and consuming hundreds of millions of
dollars per drug created. Developments in the areas of genomics and
high throughput screening have resulted in increased capacity and
efficiency in the areas of target identification and volume of
compounds screened. Significant advances in automated DNA
sequencing, PCR application, positional cloning, hybridization
arrays, and bioinformatics have greatly increased the number of
genes (and gene fragments) encoding potential drug screening
targets. However, the basic scheme for drug screening remians the
same.
[0005] Validation of genomic targets as points for therapeutic
intervention using the existing methods and protocols has become a
bottleneck in the drug discovery process due to the slow, manual
methods employed, such as in vivo functional models, functional
analysis of recombinant proteins, and stable cell line expression
of candidate genes. Primary DNA sequence data acquired through
automated sequencing does not permit identification of gene
function, but can provide information about common "motifs" and
specific gene homology when compared to known sequence databases.
Genomic methods such as subtraction hybridization and RADE (rapid
amplification of differential expression) can be used to identify
genes that are up or down regulated in a disease state model.
However, identification and validation still proceed down the same
pathway. Some proteomic methods use protein identification (global
expression arrays, 2D electrophoresis, combinatorial libraries) in
combination with reverse genetics to identify candidate genes of
interest. Such putative "disease associated sequences" or DAS
isolated as intact cDNA are a great advantage to these methods, but
they are identified by the hundreds without providing any
information regarding type, activity, and distribution of the
encoded protein. Choosing a subset of DAS as drug screening targets
is "random", and thus extremely inefficient, without functional
data to provide a mechanistic link with disease. It is necessary,
therefore, to provide new technologies to rapidly screen DAS to
establish biological function, thereby improving target validation
and candidate optimization in drug discovery.
[0006] There are three major avenues for improving early drug
discovery productivity. First, there is a need for tools that
provide increased information handling capability. Bioinformatics
has blossomed with the rapid development of DNA sequencing systems
and the evolution of the genomics database. Genomics is beginning
to play a critical role in the identification of potential new
targets. Proteomics has become indispensible in relating structure
and function of protein targets in order to predict drug
interactions. However, the next level of biological complexity is
the cell. Therefore, there is a need to acquire, manage and search
multi-dimensional information from cells. Secondly, there is a need
for higher throughput tools. Automation is a key to improving
productivity as has already been demonstrated in DNA sequencing and
high throughput primary screening. The instant invention provides
for automated systems that extract multiple parameter information
from cells that meet the need for higher throughput tools. The
instant invention also provides for miniaturizing the methods,
thereby allowing increased throughput, while decreasing the volumes
of reagents and test compounds required in each assay.
[0007] Radioactivity has been the dominant read-out in early drug
discovery assays. However, the need for more information, higher
throughput and miniaturization has caused a shift towards using
fluorescence detection. Fluorescence-based reagents can yield more
powerful, multiple parameter assays that are higher in throughput
and information content and require lower volumes of reagents and
test compounds. Fluorescence is also safer and less expensive than
radioactivity-based methods.
[0008] Screening of cells treated with dyes and fluorescent
reagents is well known in the art. There is a considerable body of
literature related to genetic engineering of cells to produce
fluorescent proteins, such as modified green fluorescent protein
(GFP), as a reporter molecule. Some properties of wild-type GFP are
disclosed by Morise et al. (Biochemistry 13 (1974), p. 2656-2662),
and Ward et al. (Photochem. Photobiol. 31 (1980), p. 611-615). The
GFP of the jellyfish Aequorea victoria has an excitation maximum at
395 nm and an emission maximum at 510 nm, and does not require an
exogenous factor for fluorescence activity. Uses for GFP disclosed
in the literature are widespread and include the study of gene
expression and protein localization (Chalfie et al., Science 263
(1994), p. 12501-12504)), as a tool for visualizing subcellular
organelles (Rizzuto et al., Curr. Biology 5 (1995), p. 635-642)),
visualization of protein transport along the secretory pathway
(Kaether and Gerdes, FEBS Letters 369 (1995), p. 267-271)),
expression in plant cells (Hu and Cheng, FEBS Letters 369 (1995),
p. 331-334)) and Drosophila embryos (Davis et al., Dev. Biology 170
(1995), p. 726-729)), and as a reporter molecule fused to another
protein of interest (U.S. Pat. No. 5,491,084). Similarly,
W096/23898 relates to methods of detecting biologically active
substances affecting intracellular processes by utilizing a GFP
construct having a protein kinase activation site. This patent, and
all other patents referenced in this application are incorporated
by reference in their entirety
[0009] Numerous references are related to GFP proteins in
biological systems. For example, WO 96/09598 describes a system for
isolating cells of interest utilizing the expression of a GFP like
protein. WO 96/27675 describes the expression of GFP in plants. WO
95/21191 describes modified GFP protein expressed in transformed
organisms to detect mutagenesis. U.S. Pat. Nos. 5,401,629 and
5,436,128 describe assays and compositions for detecting and
evaluating the intracellular transduction of an extracellular
signal using recombinant cells that express cell surface receptors
and contain reporter gene constructs that include transcriptional
regulatory elements that are responsive to the activity of cell
surface receptors.
[0010] Performing a screen on many thousands of compounds requires
parallel handling and processing of many compounds and assay
component reagents. Standard high throughput screens ("HTS") use
mixtures of compounds and biological reagents along with some
indicator compound loaded into arrays of wells in standard
microtiter plates with 96 or 384 wells. The signal measured from
each well, either fluorescence emission, optical density, or
radioactivity, integrates the signal from all the material in the
well giving an overall population average of all the molecules in
the well.
[0011] Science Applications International Corporation (SAIC) 130
Fifth Avenue, Seattle, Wash. 98109) describes an imaging plate
reader. This system uses a CCD camera to image the whole area of a
96 well plate. The image is analyzed to calculate the total
fluorescence per well for all the material in the well.
[0012] Molecular Devices, Inc. (Sunnyvale, Calif.) describes a
system (FLIPR) which uses low angle laser scanning illumination and
a mask to selectively excite fluorescence within approximately 200
microns of the bottoms of the wells in standard 96 well plates in
order to reduce background when imaging cell monolayers. This
system uses a CCD camera to image the whole area of the plate
bottom. Although this system measures signals originating from a
cell monolayer at the bottom of the well, the signal measured is
averaged over the area of the well and is therefore still
considered a measurement of the average response of a population of
cells. The image is analyzed to calculate the total fluorescence
per well for cell-based assays. Fluid delivery devices have also
been incorporated into cell based screening systems, such as the
FLIPR system, in order to initiate a response, which is then
observed as a whole well population average response using a
macro-imaging system.
[0013] In contrast to high throughput screens, various high-content
screens ("HCS") have been developed to address the need for more
detailed information about the temporal-spatial dynamics of cell
constituents and processes. High-content screens automate the
extraction of multicolor fluorescence information derived from
specific fluorescence-based reagents incorporated into cells
(Giuliano and Taylor (1995), Curr. Op. Cell Biol. 7:4; Giuliano et
al. (1995) Ann. Rev. Biophys. Biomol. Struct. 24:405). Cells are
analyzed using an optical system that can measure spatial, as well
as temporal dynamics. (Farkas et al. (1993) Ann. Rev. Physiol.
55:785; Giuliano et al. (1990) In Optical Microscopy for Biology.
B. Herman and K. Jacobson (eds.), pp. 543-557. Wiley-Liss, New
York; Hahn et al (1992) Nature 359:736; Waggoner et al. (1996) Hum.
Pathol. 27:494). The concept is to treat each cell as a "well" that
has spatial and temporal information on the activities of the
labeled constituents.
[0014] The types of biochemical and molecular information now
accessible through fluorescence-based reagents applied to cells
include ion concentrations, membrane potential, specific
translocations, enzyme activities, gene expression, as well as the
presence, amounts and patterns of metabolites, proteins, lipids,
carbohydrates, and nucleic acid sequences (DeBiasio et al., (1996)
Mol. Biol. Cell. 7:1259;Giuliano et al., (1995) Ann. Rev. Biophys.
Biomol. Struct. 24:405; Heim and Tsien, (1996) Curr. Biol.
6:178).
[0015] High-content screens can be performed on either fixed cells,
using fluorescently labeled antibodies, biological ligands, and/or
nucleic acid hybridization probes, or live cells using multicolor
fluorescent indicators and "biosensors." The choice of fixed or
live cell screens depends on the specific cell-based assay
required.
[0016] Fixed cell assays are the simplest, since an array of
initially living cells in a microtiter plate format can be treated
with various compounds and doses being tested, then the cells can
be fixed, labeled with specific reagents, and measured. No
environmental control of the cells is required after fixation.
Spatial information is acquired, but only at one time point. The
availability of thousands of antibodies, ligands and nucleic acid
hybridization probes that can be applied to cells makes this an
attractive approach for many types of cell-based screens. The
fixation and labeling steps can be automated, allowing efficient
processing of assays.
[0017] Live cell assays are more sophisticated and powerful, since
an array of living cells containing the desired reagents can be
screened over time, as well as space. Environmental control of the
cells (temperature, humidity, and carbon dioxide) is required
during measurement, since the physiological health of the cells
must be maintained for multiple fluorescence measurements over
time. There is a growing list of fluorescent physiological
indicators and "biosensors" that can report changes in biochemical
and molecular activities within cells (Giuliano et al., (1995) Ann.
Rev. Biophys. Biomol. Struct. 24:405; Hahn et al., (1993) In
Fluorescent and Luminescent Probes for Biological Activity. W. T.
Mason, (ed.), pp. 349-359, Academic Press, San Diego).
[0018] The availability and use of fluorescence-based reagents has
helped to advance the development of both fixed and live cell
high-content screens. Advances in instrumentation to automatically
extract multicolor, high-content information has recently made it
possible to develop HCS into an automated tool. An article by
Taylor, et al. (American Scientist 80 (1992), p. 322-335) describes
many of these methods and their applications. For example, Proffitt
et. al. (Cytometry 24: 204-213 (1996)) describe a semi-automated
fluorescence digital imaging system for quantifying relative cell
numbers in situ in a variety of tissue culture plate formats,
especially 96-well microtiter plates. The system consists of an
epifluorescence inverted microscope with a motorized stage, video
camera, image intensifier, and a microcomputer with a PC-Vision
digitizer. Turbo Pascal software controls the stage and scans the
plate taking multiple images per well. The software calculates
total fluorescence per well, provides for daily calibration, and
configures easily for a variety of tissue culture plate formats.
Thresholding of digital images and reagents which fluoresce only
when taken up by living cells are used to reduce background
fluorescence without removing excess fluorescent reagent.
[0019] Scanning confocal microscope imaging (Go et al., (1997)
Analytical Biochemistry 247:210-215; Goldman et al., (1995)
Experimental Cell Research 221:311-319) and multiphoton microscope
imaging (Denk et al., (1990) Science 248:73; Gratton et al., (1994)
Proc. of the Microscopical Society of America, pp. 154-155) are
also well established methods for acquiring high resolution images
of microscopic samples. The principle advantage of these optical
systems is the very shallow depth of focus, which allows features
of limited axial extent to be resolved against the background. For
example, it is possible to resolve internal cytoplasmic features of
adherent cells from the features on the cell surface. Because
scanning multiphoton imaging requires very short duration pulsed
laser systems to achieve the high photon flux required,
fluorescence lifetimes can also be measured in these systems
(Lakowicz et al., (1992) Anal. Biochem. 202:316-330; Gerrittsen et
al. (1997), J. of Fluorescence 7:11-15)), providing additional
capability for different detection modes. Small, reliable and
relatively inexpensive laser systems, such as laser diode pumped
lasers, are now available to allow multiphoton confocal microscopy
to be applied in a fairly routine fashion.
[0020] A combination of the biological heterogeneity of cells in
populations (Bright, et al., (1989). J Cell. Physiol. 141:410;
Giuliano, (1996) Cell Motil. Cytoskel. 35:237)) as well as the high
spatial and temporal frequency of chemical and molecular
information present within cells, makes it impossible to extract
high-content information from populations of cells using existing
whole microtiter plate readers. No existing high-content screening
platform has been designed for multicolor, fluorescence-based
screens using cells that are analyzed individually. Similarly, no
method is currently available that combines automated fluid
delivery to arrays of cells for the purpose of systematically
screening compounds for the ability to induce a cellular response
that is identified by HCS analysis, especially from cells grown in
microtiter plates. Furthermore, no method exists in the art
combining high throughput well-by-well measurements to identify
"hits" in one assay followed by a second high content cell-by-cell
measurement on the same plate of only those wells identified as
hits.
[0021] The instant invention provides systems, methods, and screens
that combine high throughput screening (ITS) and high content
screening (HCS) that significantly improve target validation and
candidate optimization by combining many cell screening formats
with fluorescence-based molecular reagents and computer-based
feature extraction, data analysis, and automation, resulting in
increased quantity and speed of data collection, shortened cycle
times, and, ultimately, faster evaluation of promising drug
candidates. The instant invention also provides for miniaturizing
the methods, thereby allowing increased throughput, while
decreasing the volumes of reagents and test compounds required in
each assay.
SUMMARY OF THE INVENTION
[0022] In one aspect, the present invention relates to a method for
analyzing cells comprising
[0023] providing cells containing fluorescent reporter molecules in
an array of locations,
[0024] treating the cells in the array of locations with one or
more reagents,
[0025] imaging numerous cells in each location with fluorescence
optics,
[0026] converting the optical information into digital data,
[0027] utilizing the digital data to determine the distribution,
environment or activity of the fluorescently labeled reporter
molecules in the cells and the distribution of the cells, and
[0028] interpreting that information in terms of a positive,
negative or null effect of the compound being tested on the
biological function
[0029] In this embodiment, the method rapidly determines 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 array of locations may be a microtiter
plate or a microchip which is a microplate having cells in an array
of locations. In a preferred embodiment, the method includes
computerized means for acquiring, processing, displaying and
storing the data received. In a preferred embodiment, the method
further comprises automated fluid delivery to the arrays of cells.
In another preferred embodiment, the information obtained from high
throughput measurements on the same plate are used to selectively
perform high content screening on only a subset of the cell
locations on the plate.
[0030] In another aspect of the present invention, a cell screening
system is provided that comprises:
[0031] a high magnification fluorescence optical system having a
microscope objective,
[0032] an XY stage adapted for holding a plate containing an array
of cells and having a means for moving the plate for proper
alignment and focusing on the cell arrays;
[0033] a digital camera;
[0034] a light source having optical means for directing excitation
light to cell arrays and a means for directing fluorescent light
emitted from the cells to the digital camera; and
[0035] a computer means for receiving and processing digital data
from the digital camera wherein the computer means includes a
digital frame grabber for receiving the images from the camera, a
display for user interaction and display of assay results, digital
storage media for data storage and archiving, and a means for
control, acquisition, processing and display of results.
[0036] In a preferred embodiment, the cell screening system further
comprises a computer screen operatively associated with the
computer for displaying data. In another preferred embodiment, the
computer means for receiving and processing digital data from the
digital camera stores the data in a bioinformatics data base. In a
further preferred embodiment, the cell screening system further
comprises a reader that measures a signal from many or all the
wells in parallel. In another preferred embodiment, the cell
screening system further comprises a mechanical-optical means for
changing the magnification of the system, to allow changing modes
between high throughput and high content screening. In another
preferred embodiment, the cell screening system further comprises a
chamber and control system to maintain the temperature, CO.sub.2
concentration and humidity surrounding the plate at levels required
to keep cells alive. In a further preferred embodiment, the cell
screening system utilizes a confocal scanning illumination and
detection system.
[0037] In another aspect of the present invention, a machine
readable storage medium comprising a program containing a set of
instructions for causing a cell screening system to execute
procedures for defining the distribution and activity of specific
cellular constituents and processes is provided. In a preferred
embodiment, the cell screening system comprises a high
magnification fluorescence optical system with a stage adapted for
holding cells and a means for moving the stage, a digital camera, a
light source for receiving and processing the digital data from the
digital camera, and a computer means for receiving and processing
the digital data from the digital camera. Preferred embodiments of
the machine readable storage medium comprise programs consisting of
a set of instructions for causing a cell screening system to
execute the procedures set forth in FIGS. 9, 11, 12, 13, 14 or 15.
Another preferred embodiment comprises a program consisting of a
set of instructions for causing a cell screening system to execute
procedures for detecting the distribution and activity of specific
cellular constituents and processes. In most preferred embodiments,
the cellular processes include, but are not limited to, nuclear
translocation of a protein, cellular hypertrophy, apoptosis, and
protease-induced translocation of a protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 shows a diagram of the components of the cell-based
scanning system.
[0039] FIG. 2 shows a schematic of the microscope subassembly.
[0040] FIG. 3 shows the camera subassembly.
[0041] FIG. 4 illustrates cell scanning system process.
[0042] FIG. 5 illustrates a user interface showing major functions
to guide the user.
[0043] FIG. 6 is a block diagram of the two platform architecture
of the Dual Mode System for Cell Based Screening in which one
platform uses a telescope lens to read all wells of a microtiter
plate and a second platform that uses a higher magnification lens
to read individual cells in a well.
[0044] FIG. 7 is a detail of an optical system for a single
platform architecture of the Dual Mode System for Cell Based
Screening that uses a moveable `telescope` lens to read all wells
of a microtiter plate and a moveable higher magnification lens to
read individual cells in a well.
[0045] FIG. 8 is an illustration of the fluid delivery system for
acquiring kinetic data on the Cell Based Screening System.
[0046] FIG. 9 is a flow chart of processing step for the cell-based
scanning system.
[0047] FIGS. 10 A-J illustrates the strategy of the Nuclear
Translocation Assay.
[0048] FIG. 11 is a flow chart defining the processing steps in the
Dual Mode System for Cell Based Screening combining high throughput
and high content screening of microtiter plates.
[0049] FIG. 12 is a flow chart defining the processing steps in the
High Throughput mode of the System for Cell Based Screening.
[0050] FIG. 13 is a flow chart defining the processing steps in the
High Content mode of the System for Cell Based Screening.
[0051] FIG. 14 is a flow chart defining the processing steps
required for acquiring kinetic data in the High Content mode of the
System for Cell Based Screening.
[0052] FIG. 15 is a flow chart defining the processing steps
performed within a well during the acquisition of kinetic data.
[0053] FIG. 16 is an example of data from a known inhibitor of
translocation.
[0054] FIG. 17 is an example of data from a known stimulator of
translocation.
[0055] FIG. 18 illustrates data presentation on a graphical
display.
[0056] FIG. 19 is an illustration of the data from the High
Throughput mode of the System for Cell Based Screening, an example
of the data passed to the High Content mode, the data acquired in
the high content mode, and the results of the analysis of that
data.
[0057] FIG. 20 shows the measurement of a drug-induced cytoplasm to
nuclear translocation.
[0058] FIG. 21 illustrates a graphical user interface of the
measurement shown in FIG. 20.
[0059] FIG. 22 illustrates a graphical user interface, with data
presentation, of the measurement shown in FIG. 20.
[0060] FIG. 23 is a graph representing the kinetic data obtained
from the measurements depicted in FIG. 20.
[0061] FIG. 24 details a high-content screen of drug-induced
apoptosis.
DETAILED DESCRIPTION OF THE INVENTION
[0062] All cited patents, patent applications and other references
are hereby incorporated by reference in their entirety.
[0063] As used herein, the following terms have the specified
meaning:
[0064] Markers of Cellular Domains.
[0065] Luminescent probes that have high affinity for specific
cellular constituents including specific organelles or molecules.
These probes can either be small luminescent molecules or
fluorescently tagged macromolecules used as "labeling reagents",
"environmental indicators", or "biosensors."
[0066] Labeling Reagents.
[0067] Labeling reagents include, but are not limited to,
luminescently labeled macromolecules including fluorescent protein
analogs and biosensors, luminescent macromolecular chimeras
including those formed with the green fluorescent protein and
mutants thereof, luminescently labeled primary or secondary
antibodies that react with cellular antigens involved in a
physiological response, luminescent stains, dyes, and other small
molecules.
[0068] Markers of Cellular Translocations.
[0069] Luminescently tagged macromolecules or organelles that move
from one cell domain to another during some cellular process or
physiological response. Translocation markers can either simply
report location relative to the markers of cellular domains or they
can also be "biosensors" that report some biochemical or molecular
activity as well.
[0070] Biosensors.
[0071] Macromolecules consisting of a biological functional domain
and a luminescent probe or probes that report the environmental
changes that occur either internally or on their surface. A class
of luminescently labeled macromolecules designed to sense and
report these changes have been termed "fluorescent-protein
biosensors". The protein component of the biosensor provides a
highly evolved molecular recognition moiety. A fluorescent molecule
attached to the protein component in the proximity of an active
site transduces environmental changes into fluorescence signals
that are detected using a system with an appropriate temporal and
spatial resolution such as the cell scanning system of the present
invention. Because the modulation of native protein activity within
the living cell is reversible, and because fluorescent-protein
biosensors can be designed to sense reversible changes in protein
activity, these biosensors are essentially reusable.
[0072] Disease Associated Sequences ("DAS").
[0073] This term refers to nucleic acid sequences identified by
standard techniques, such as primary DNA sequence data, genomic
methods such as subtraction hybridization and RADE, and proteomic
methods in combination with reverse genetics, as being of drug
candidate compounds. The term does not mean that the sequence is
only associated with a disease state.
[0074] High content screening (HCS) can be used to measure the
effects of drugs on complex molecular events such as signal
transduction pathways, as well as cell functions including, but not
limited to, apoptosis, cell division, cell adhesion, locomotion,
exocytosis, and cell-cell communication. Multicolor fluorescence
permits multiple targets and cell processes to be assayed in a
single screen. Cross-correlation of cellular responses will yield a
wealth of information required for target validation and lead
optimization.
[0075] In one aspect of the present invention, a cell screening
system is provided comprising a high magnification fluorescence
optical system having a microscope objective, an XY stage adapted
for holding a plate with an array of locations for holding cells
and having a means for moving the plate to align the locations with
the microscope objective and a means for moving the plate in the
direction to effect focusing; a digital camera; a light source
having optical means for directing excitation light to cells in the
array of locations and a means for directing fluorescent light
emitted from the cells to the digital camera; and a computer means
for receiving and processing digital data from the digital camera
wherein the computer means includes: a digital frame grabber for
receiving the images from the camera, a display for user
interaction and display of assay results, digital storage media for
data storage and archiving, and means for control, acquisition,
processing and display of results.
[0076] FIG. 1 is a schematic diagram of a preferred embodiment of
the cell scanning system. An inverted fluorescence microscope is
used i, such as a Zeiss Axiovert inverted fluorescence microscope
which uses standard objectives with magnification of 1-100.times.
to the camera, and a white light source (e.g. 100W mercury-arc lamp
or 75W xenon lamp) with power supply 2. There is an XY stage 3 to
move the plate 4 in the XY direction over the microscope objective.
A Z-axis focus drive 5 moves the objective in the Z direction for
focusing. A joystick 6 provides for manual movement of the stage in
the XYZ direction. A high resolution digital camera 7 acquires
images from each well or location on the plate. There is a camera
power supply 8. an automation controller 9 and a central processing
unit 10. The PC 11 provides a display 12 and has associated
software. The printer 13 provides for printing of a hard copy
record.
[0077] FIG. 2 is a schematic of one embodiment of the microscope
assembly 1 of the invention, showing in more detail the XY stage 3,
Z-axis focus drive 5, joystick 6, light source 2, and automation
controller 9. Cables to the computer 15 and microscope 16,
respectively, are provided. In addition, FIG. 2 shows a 96 well
microtiter plate 17 which is moved on the XY stage 3 in the XY
direction. Light from the light source 2 passes through the PC
controlled shutter 18 to a motorized filter wheel 19 with
excitation filters 20. The light passes into filter cube 25 which
has a dichroic mirror 26 and an emission filter 27. Excitation
light reflects off the dichroic mirror to the wells in the
microtiter plate 17 and fluorescent light 28 passes through the
dichroic mirror 26 and the emission filter 27 and to the digital
camera 7.
[0078] FIG. 3 shows a schematic drawing of a preferred camera
assembly. The digital camera 7, which contains an automatic shutter
for exposure control and a power supply 31, receives fluorescent
light 28 from the microscope assembly. A digital cable 30
transports digital signals to the computer.
[0079] The standard optical configurations described above use
microscope optics to directly produce an enlarged image of the
specimen on the camera sensor in order to capture a high resolution
image of the specimen. This optical system is commonly referred to
as `wide field` microscopy. Those skilled in the art of microscopy
will recognize that a high resolution image of the specimen can be
created by a variety of other optical systems, including, but not
limited to, standard scanning confocal detection of a focused point
or line of illumination scanned over the specimen (Go et al. 1997,
supra), and multi-photon scanning confocal microscopy (Denk et al.,
1990, supra), both of which can form images on a CCD detector or by
synchronous digitization of the analog output of a photomultiplier
tube.
[0080] In screening applications, it is often necessary to use a
particular cell line, or primary cell culture, to take advantage of
particular features of those cells. Those skilled in the art of
cell culture will recognize that some cell lines are contact
inhibited, meaning that they will stop growing when they become
surrounded by other cells, while other cell lines will continue to
grow under those conditions and the cells will literally pile up,
forming many layers. An example of such a cell line is the HEK 293
(ATCC CRL-1573) line. An optical system that can acquire images of
single cell layers in multilayer preparations is required for use
with cell lines that tend to form layers. The large depth of field
of wide field microscopes produces an image that is a projection
through the many layers of cells, making analysis of subcellular
spatial distributions extremely difficult in layer-forming cells.
Alternatively, the very shallow depth of field that can be achieved
on a confocal microscope, (about one micron), allows discrimination
of a single cell layer at high resolution, simplifying the
determination of the subcellular spatial distribution. Similarly,
confocal imaging is preferable when detection modes such as
fluorescence lifetime imaging are required.
[0081] The output of a standard confocal imaging attachment for a
microscope is a digital image that can be converted to the same
format as the images produced by the other cell screening system
embodiments described above, and can therefore be processed in
exactly the same way as those images. The overall control,
acquisition and analysis in this embodiment is essentially the
same. The optical configuration of the confocal microscope system,
is essentially the same as that described above, except for the
illuminator and detectors. Illumination and detection systems
required for confocal microscopy have been designed as accessories
to be attached to standard microscope optical systems such as that
of the present invention (Zeiss, Germany). These alternative
optical systems therefore can be easily integrated into the system
as described above.
[0082] FIG. 4 illustrates an alternative embodiment of the
invention in which cell arrays are in microwells 40 on a microplate
4, described ion co-pending U.S. patent application Ser. No.
08/865,341, incorporated by reference herein in its entirety.
Typically the microplate is 20 mm by 30 mm as compared to a
standard 96 well microtiter plate which is 86 mm by 129 mm. The
higher density array of cells on a microplate allows the microplate
to be imaged at a low resolution of a few microns per pixel for
high throughput and particular locations on the microplate to be
imaged at a higher resolution of less than 0.5 microns per pixel.
These two resolution modes help to improve the overall throughput
of the system.
[0083] The microplate chamber 42 serves as a microfluidic delivery
system for the addition of compounds to cells. The microplate 41 in
the microplate chamber 42 is placed in an XY microplate reader 43.
Digital data is processed as described above. The small size of
this microplate system increases throughput, minimizes reagent
volume and allows control of the distribution and placement of
cells for fast and precise cell-based analysis. Processed data can
be displayed on a PC screen 11 and made part of a bioinformatics
data base 44. This data base not only permits storage and retrieval
of data obtained through the methods of this invention, but also
permits acquisition and storage of external data relating to cells.
FIG. 5 is a PC display which illustrates the operation of the
software.
[0084] In an alternative embodiment, a high throughput system (HTS)
is directly coupled with the HCS either on the same platform or on
two separate platforms connected electronically (e.g. via a local
area network). This embodiment of the invention, referred to as a
dual mode optical system, has the advantage of increasing the
throughput of a HCS by coupling it with a HTS and thereby requiring
slower high resolution data acquisition and analysis only on the
small subset of wells that show a response in the coupled HTS.
[0085] High throughput `whole plate` reader systems are well known
in the art and are commonly used as a component of an HTS system
used to screen large numbers of compounds (Beggs (1997), J. of
Biomolec. Screening 2:71-78, Macaffrey et al., (1996) J. Biomolec.
Screening 1:187-190).
[0086] In one embodiment of dual mode cell based screening, a two
platform architecture in which high throughput acquisition occurs
on one platform and high content acquisition occurs on a second
platform is provided (FIG. 6). Processing occurs on each platform
independently, with results passed over a network interface, or a
single controller is used to process the data from both
platforms.
[0087] As illustrated in FIG. 6, an exemplified two platform dual
mode optical system consists of two light optical instruments, a
high throughput platform 60 and a high content platform 65, which
read fluorescent signals emitted from cells cultured in microtiter
plates or microwell arrays on a microplate, and communicate with
each other via an electronic connection 64. The high throughput
platform 60 analyzes all the wells in the whole plate either in
parallel or rapid serial fashion. Those skilled in the art of
screening will recognize that there are a many such commercially
available high throughput reader systems that could be integrated
into a dual mode cell based screening system (Topcount (Packard
Instruments, Meriden, Conn.); Spectramax, Lumiskan (Molecular
Devices, Sunnyvale, Calif.); Fluoroscan (Labsystems, Beverly,
Mass.)). The high content platform 65, as described above, scans
from well to well and acquires and analyzes high resolution image
data collected from individual cells within a well.
[0088] The HTS software, residing on the system's computer 62,
controls the high throughput instrument, and results are displayed
on the monitor 61. The HCS software, residing on it's computer
system 67 controls the high content instrument hardware 65,
optional devices (e.g. plate loader, environmental chamber, fluid
dispenser), analyzes digital image data from the plate, displays
results on the monitor 66 and manages data measured in an
integrated database. The two systems can also share a single
computer, in which case all data would be collected, processed and
displayed on that computer, without the need for a local area
network to transfer the data. Microtiter plates are transferred
from the high throughput system to the high content system 63
either manually or by a robotic plate transfer device, as is well
known in the art (Beggs (1997), supra; Mcaffrey (1996), supra).
[0089] In a preferred embodiment, the dual mode optical system
utilizes a single platform system (FIG. 7). It consists of two
separate optical modules, an HCS module 203 and an HTS module 209
that can be independently or collectively moved so that only one at
a time is used to collect data from the microtiter plate 201. The
microtiter plate 201 is mounted in a motorized X,Y stage so it can
be positioned for imaging in either HTS or HCS mode. After
collecting and analyzing the HTS image data as described below, the
HTS optical module 209 is moved out of the optical path and the HCS
optical module 203 is moved into place.
[0090] The optical module for HTS 209 consists of a projection lens
214, excitation wavelength filter 213 and dichroic mirror 210 which
are used to illuminate the whole bottom of the plate with a
specific wavelength band from a conventional microscope lamp system
(not illustrated). The fluorescence emission is collected through
the dichroic mirror 210 and emission wavelength filter 211 by a
lens 212 which forms an image on the camera 216 with sensor
215.
[0091] The optical module for HCS 203 consists of a projection lens
208 excitation wavelength filter 207 and dichroic mirror 204 which
are used to illuminate the back aperture of the microscope
objective 202, and thereby the field of that objective, from a
standard microscope illumination system (not shown). The
fluorescence emission is collected by the microscope objective 202,
passes through the dichroic mirror 204 and emission wavelength
filter 205 and is focused by a tube lens 206 which forms an image
on the same camera 216 with sensor 215.
[0092] In an alternative embodiment of the present invention, the
cell screening system further comprises a fluid delivery device for
use with the live cell embodiment of the method of cell screening
(see below). FIG. 8 exemplifies a fluid delivery device for use
with the system of the invention. It consists of a bank of 12
syringe pumps 701 driven by a single motor drive. Each syringe 702
is sized according to the volume to be delivered to each well,
typically between 1 and 100 .mu.L. Each syringe is attached via
flexible tubing 703 to a similar bank of connectors which accept
standard pipette tips 705. The bank of pipette tips are attached to
a drive system so they can be lowered and raised relative to the
microtiter plate 706 to deliver fluid to each well. The plate is
mounted on an X,Y stage, allowing movement relative to the optical
system 707 for data collection purposes. This set-up allows one set
of pipette tips, or even a single pipette tip, to deliver reagent
to all the wells on the plate. The bank of syringe pumps can be
used to deliver fluid to 12 wells simultaneously, or to fewer wells
by removing some of the tips.
[0093] In another aspect, the present invention provides a method
for analyzing cells comprising providing an array of locations
which contain multiple cells wherein the cells contain one or more
fluorescent reporter molecules; scanning multiple cells in each of
the locations containing cells to obtain fluorescent signals from
the fluorescent reporter molecule in the cells; converting the
fluorescent signals into digital data; and utilizing the digital
data to determine the distribution, environment or activity of the
fluorescent reporter molecule within the cells.
[0094] Cell Arrays
[0095] Screening large numbers of compounds for activity with
respect to a particular biological function requires preparing
arrays of cells for parallel handling of cells and reagents.
Standard 96 well microtiter plates which are 86 mm by 129 mm, with
6 mm diameter wells on a 9 mm pitch, are used for compatibility
with current automated loading and robotic handling systems. The
microplate is typically 20 mm by 30 mm, with cell locations that
are 100-200 microns in dimension on a pitch of about 500 microns.
Methods for making microplates are described in U.S. patent
application Ser. No. 08/865,341, incorporated by reference herein
in its entirety. Microplates may consist of coplanar layers of
materials to which cells adhere, patterned with materials to which
cells will not adhere, or etched 3-dimensional surfaces of
similarly pattered materials. For the purpose of the following
discussion, the terms `well` and `microwell`0 refer to a location
in an array of any construction to which cells adhere and within
which the cells are imaged. Microplates may also include fluid
delivery channels in the spaces between the wells. The smaller
format of a microplate increases the overall efficiency of the
system by minimizing the quantities of the reagents, storage and
handling during preparation and the overall movement required for
the scanning operation. In addition, the whole area of the
microplate can be imaged more efficiently, allowing a second mode
of operation for the microplate reader as described later in this
document.
[0096] Fluorescence Reporter Molecules
[0097] A major component of the new drug discovery paradigm is a
continually growing family of fluorescent and luminescent reagents
that are used to measure the temporal and spatial distribution,
content, and activity of intracellular ions, metabolites,
macromolecules, and organelles. Classes of these reagents include
labeling reagents that measure the distribution and amount of
molecules in living and fixed cells, environmental indicators to
report signal transduction events in time and space, and
fluorescent protein biosensors to measure target molecular
activities within living cells. A multiparameter approach that
combines several reagents in a single cell is a powerful new tool
for drug discovery.
[0098] The method of the present invention is based on the high
affinity of fluorescent or luminescent molecules for specific
cellular components. The affinity for specific components is
governed by physical forces such as ionic interactions, covalent
bonding (which includes chimeric fusion with protein-based
chromophores, fluorophores, and lumiphores), as well as hydrophobic
interactions, electrical potential, and, in some cases, simple
entrapment within a cellular component. The luminescent probes can
be small molecules, labeled macromolecules, or genetically
engineered proteins, including, but not limited to green
fluorescent protein chimeras.
[0099] Those skilled in this art will recognize a wide variety of
fluorescent reporter molecules that can be used in the present
invention, including, but not limited to, fluorescently labeled
biomolecules such as proteins, phospholipids and DNA hybridizing
probes. Similarly, fluorescent reagents specifically synthesized
with particular chemical properties of binding or association have
been used as fluorescent reporter molecules (Barak et al., (1997),
J. Biol. Chem. 272:27497-27500; Southwick et al., (1990), Cytometry
11:418-430; Tsien (1989) in Methods in Cell Biology, Vol. 29 Taylor
and Wang (eds.), pp. 127-156). Fluorescently labeled antibodies are
particularly useful reporter molecules due to their high degree of
specificity for attaching to a single molecular target in a mixture
of molecules as complex as a cell or tissue.
[0100] The luminescent probes can be synthesized within the living
cell or can be transported into the cell via several non-mechanical
modes including diffusion, facilitated or active transport,
signal-sequence-mediated transport, and endocytotic or pinocytotic
uptake. Mechanical bulk loading methods, which are well known in
the art, can also be used to load luminescent probes into living
cells (Barber et al. (1996), Neuroscience Letters 207:17-20; Bright
et al. (1996), Cytometry 24:226-233; McNeil (1989) in Methods in
Cell Biology, Vol. 29, Taylor and Wang (eds.), pp. 153-173). These
methods include electroporation and other mechanical methods such
as scrape-loading, bead-loading, impact-loading, syringe-loading,
hypertonic and hypotonic loading. Additionally, cells can be
genetically engineered to express reporter molecules, such as GFP,
coupled to a protein of interest as previously described (Chalfie
and Prasher U.S. Pat. No. 5,491,084; Cubitt et al. (1995), Trends
in Biochemical Science 20:448-455).
[0101] Once in the cell, the luminescent probes accumulate at their
target domain as a result of specific and high affinity
interactions with the target domain or other modes of molecular
targeting such as signal-sequence-mediated transport. Fluorescently
labeled reporter molecules are useful for determining the location,
amount and chemical environment of the reporter. For example,
whether the reporter is in a lipophilic membrane environment or in
a more aqueous environment can be determined (Giuliano et al.
(1995), Ann. Rev. of Biophysics and Biomolecular Structure
24:405-434; Giuliano and Taylor (1995), Methods in Neuroscience
27:1-16). The pH environment of the reporter can be determined
(Bright et al. (1989), J. Cell Biology 104:1019-1033; Giuliano et
al. (1987), Anal. Biochem. 167:362-371; Thomas et al. (1979),
Biochemistry 18:2210-2218). It can be determined whether a reporter
having a chelating group is bound to an ion, such as Ca++, or not
(Bright et al. (1989), In Methods in Cell Biology, Vol. 30, Taylor
and Wang (eds.), pp. 157-192; Shimoura et al. (1988), J. of
Biochemistry (Tokyo) 251:405-410; Tsien (1989) In Methods in Cell
Biology, Vol. 30, Taylor and Wang (eds.), pp. 127-156).
[0102] Furthermore, certain cell types within an organism may
contain components that can be specifically labeled that may not
occur in other cell types. For example, epithelial cells often
contain polarized membrane components. That is, these cells
asymmetrically distribute macromolecules along their plasma
membrane. Connective or supporting tissue cells often contain
granules in which are trapped molecules specific to that cell type
(e.g., heparin, histamine, serotonin, etc.). Most muscular tissue
cells contain a sarcoplasmic reticulum, a specialized organelle
whose function is to regulate the concentration of calcium ions
within the cell cytoplasm. Many nervous tissue cells contain
secretory granules and vesicles in which are trapped neurohormones
or neurotransmitters. Therefore, fluorescent molecules can be
designed to label not only specific components within specific
cells, but also specific cells within a population of mixed cell
types.
[0103] Those skilled in the art will recognize a wide variety of
ways to measure fluorescence. For example, some fluorescent
reporter molecules exhibit a change in excitation or emission
spectra, some exhibit resonance energy transfer where one
fluorescent reporter loses fluorescence, while a second gains in
fluorescence, some exhibit a loss (quenching) or appearance of
fluorescence, while some report rotational movements (Giuliano et
al. (1995), Ann. Rev. of Biophysics and Biomol. Structure
24:405-434; Giuliano et al. (1995), Methods in Neuroscience
27:1-16).
[0104] Scanning Cell Arrays
[0105] Referring to FIG. 9, a preferred embodiment is provided to
analyze cells that comprises operator-directed parameters being
selected based on the assay being conducted, data acquisition by
the cell screening system on the distribution of fluorescent
signals within a sample, and interactive data review and analysis.
At the start of an automated scan the operator enters information
100 that describes the sample, specifies the filter settings and
fluorescent channels to match the biological labels being used and
the information sought, and then adjusts the camera settings to
match the sample brightness. For flexibility to handle a range of
samples, the software allows selection of various parameter
settings used to identify nuclei and cytoplasm, and selection of
different fluorescent reagents, identification of cells of interest
based on morphology or brightness, and cell numbers to be analyzed
per well. These parameters are stored in the system's database for
easy retrieval for each automated run. The system's interactive
cell identification mode simplifies the selection of morphological
parameter limits such as the range of size, shape, and intensity of
cells to be analyzed. The user specifies which wells of the plate
the system will scan and how many fields or how many cells to
analyze in each well. Depending on the setup mode selected by the
user at step 101, the system either automatically pre-focuses the
region of the plate to be scanned using an autofocus procedure to
"find focus" of the plate 102 or the user interactively pre-focuses
103 the scanning region by selecting three "tag" points which
define the rectangular area to be scanned. A least-squares fit
"focal plane model" is then calculated from these tag points to
estimate the focus of each well during an automated scan. The focus
of each well is estimated by interpolating from the focal plane
model during a scan.
[0106] During an automated scan, the software dynamically displays
the scan status, including the number of cells analyzed, the
current well being analyzed, images of each independent wavelength
as they are acquired, and the result of the screen for each well as
it is determined. The plate 4 (FIG. 1) is scanned in a serpentine
style as the software automatically moves the motorized microscope
XY stage 3 from well to well and field to field within each well of
a 96-well plate. Those skilled in the programming art will
recognize how to adapt software for scanning of other microplate
formats such as 24, 48, and 384 well plates. The scan pattern of
the entire plate as well as the scan pattern of fields within each
well are programmed. The system adjusts sample focus with an
autofocus procedure 104 (FIG. 9) through the Z axis focus drive 5.
controls filter selection via a motorized filter wheel 19, and
acquires and analyzes images of up to four different colors
("channels" or "wavelengths").
[0107] The autofocus procedure is called at a user selected
frequency, typically for the first field in each well and then once
every 4 to 5 fields within each well. The autofocus procedure
calculates the starting Z-axis point by interpolating from the
pre-calculated plane focal model. Starting a programmable distance
above or below this set point, the procedure moves the mechanical
Z-axis through a number of different positions, acquires an image
at each position, and finds the maximum of a calculated focus score
that estimates the contrast of each image. The Z position of the
image with the maximum focus score determines the best focus for a
particular field. Those skilled in the art will recognize this as a
variant of automatic focusing algorithms as described in Harms et
al. in Cytometry 5 (1984), 236-243, Groen et al. in Cytometry 6
(1985), 81-91, and Firestone et al. in Cytometry 12 (1991),
195-206.
[0108] For image acquisition, the camera's exposure time is
separately adjusted for each dye to ensure a high-quality image
from each channel. Software procedures can be called, at the user's
option, to correct for registration shifts between wavelengths by
accounting for linear (X and Y) shifts between wavelengths before
making any further measurements. The electronic shutter 18 is
controlled so that sample photo-bleaching is kept to a minimum.
Background shading and uneven illumination can be corrected by the
software using methods known in the art (Bright et al. (1987), J.
Cell Biol. 104:1019-1033).
[0109] In one channel, images are acquired of a primary marker 105
(FIG. 9) (typically cell nuclei counterstained with DAPI or PI
fluorescent dyes) which are segmented ("identified") using an
adaptive thresholding procedure. The adaptive thresholding
procedure 106 is used to dynamically select the threshold of an
image for separating cells from the background. The staining of
cells with fluorescent dyes can vary to an unknown degree across
cells in a microtiter plate sample as well as within images of a
field of cells within each well of a microtiter plate. This
variation can occur as a result of sample preparation and/or the
dynamic nature of cells. A global threshold is calculated for the
complete image to separate the cells from background and account
for field to field variation. These global adaptive techniques are
variants of those described in the art. (Kittler et al. in Computer
Vision, Graphics, and Image Processing 30 (1985), 125-147, Ridler
et al. in IEEE Trans. Systems, Man, and Cybernetics (1978),
630-632.)
[0110] An alternative adaptive thresholding method utilizes local
region thresholding in contrast to global image thresholding. Image
analysis of local regions leads to better overall segmentation
since staining of cell nuclei (as well as other labeled components)
can vary across an image. Using this global/local procedure, a
reduced resolution image (reduced in size by a factor of 2 to 4) is
first globally segmented (using adaptive thresholding) to find
regions of interest in the image. These regions then serve as
guides to more fully analyze the same regions at full resolution. A
more localized threshold is then calculated (again using adaptive
thresholding) for each region of interest.
[0111] The output of the segmentation procedure is a binary image
wherein the objects are white and the background is black. This
binary image, also called a mask in the art, is used to determine
if the field contains objects 107. The mask is labeled with a blob
labeling algorithm whereby each object (or blob) has a unique
number assigned to it. Morphological features, such as area and
shape, of the blobs are used to differentiate blobs likely to be
cells from those that are considered artifacts. The user pre-sets
the morphological selection criteria by either typing in known cell
morphological features or by using the interactive training
utility. If objects of interest are found in the field, images are
acquired for all other active channels 108, otherwise the stage is
advanced to the next field 109 in the current well. Each object of
interest is located in the image for further analysis 110. The
software determines if the object meets the criteria for a valid
cell nucleus 111 by measuring its morphological features (size and
shape). For each valid cell, the XYZ stage location is recorded, a
small image of the cell is stored, and features are measured
112.
[0112] The cell scanning method of the present invention can be
used to perform many different assays on cellular samples by
applying a number of analytical methods simultaneously to measure
features at multiple wavelengths. An example of one such assay
provides for the following measurements:
[0113] 1. The total fluorescent intensity within the cell nucleus
for colors 1-4
[0114] 2. The area of the cell nucleus for color 1 (the primary
marker)
[0115] 3. The shape of the cell nucleus for color 1 is described
three shape features:
[0116] a) perimeter squared area
[0117] b) box area ratio
[0118] c) height width ratio
[0119] 4. The average fluorescent intensity within the cell nucleus
for colors 1-4 (i.e. #1 divided by #2)
[0120] 5. The total fluorescent intensity of a ring outside the
nucleus (see FIG. 10) that represents fluorescence of the cell's
cytoplasm (cytoplasmic mask) for colors 2-4
[0121] 6. The area of the cytoplasmic mask
[0122] 7. The average fluorescent intensity of the cytoplasmic mask
for colors 2-4 (i.e. #5 divided by #6)
[0123] 8. The ratio of the average fluorescent intensity of the
cytoplasmic mask to average fluorescent intensity within the cell
nucleus for colors 2-4 (i.e. #7 divided by #4)
[0124] 9. The difference of the average fluorescent intensity of
the cytoplasmic mask and the average fluorescent intensity within
the cell nucleus for colors 2-4 (i.e. #7 minus #4)
[0125] 10. The number of fluorescent domains (also call spots,
dots, or grains) within the cell nucleus for colors 2-4
[0126] Features 1 through 4 are general features of the different
cell screening assays of the invention. These steps are commonly
used in a variety of image analysis applications and are well known
in art (Russ (1992) The Image Processing Handbook, CRC Press Inc.;
Gonzales et al. (1987), Digital Image Processing. Addison-Wesley
Publishing Co. pp. 391-448). Features 5-9 have been developed
specifically to provide measurements of a cell's fluorescent
molecules within the local cytoplasmic region of the cell and the
translocation (i.e. movement) of fluorescent molecules from the
cytoplasm to the nucleus. These features (steps 5-9) are used for
analyzing cells in microplates for the inhibition of nuclear
translocation. For example, inhibition of nuclear translocation of
transcription factors provides a novel approach to screening intact
cells (detailed examples of other types of screens will be provided
below). A specific algorithm measures the amount of probe in the
nuclear region (feature 4) versus the local cytoplasmic region
(feature 7) of each cell. Quantification of the difference between
these two sub-cellular compartments provides a measure of
cytoplasm-nuclear translocation (feature 9).
[0127] Feature 10 describes a screen used for counting of DNA or
RNA probes within the nuclear region in colors 2-4; For example,
probes are commercially available for identifying
chromosome-specific DNA sequences (Life Technologies, Gaithersburg,
Md.; Genosys, Woodlands, Tex.; Biotechnologies, Inc., Richmond,
Calif.; Bio 101, Inc., Vista, Calif.) Cells are three-dimensional
in nature and when examined at a high magnification under a
microscope one probe may be in-focus while another may be
completely out-of-focus. The cell screening method of the present
invention provides for detecting three-dimensional probes in nuclei
by acquiring images from multiple focal planes. The software moves
the Z-axis motor drive 5 (FIG. 1) in small steps where the step
distance is user selected to account for a wide range of different
nuclear diameters. At each of the focal steps, an image is
acquired. The maximum gray-level intensity from each pixel in each
image is found and stored in a resulting maximum projection image.
The maximum projection image is then used to count the probes. The
above algorithm works well in counting probes that are not stacked
directly above or below another one. To account for probes stacked
on top of each other in the Z-direction, users can select an option
to analyze probes in each of the focal planes acquired. In this
mode, the scanning system performs the maximum plane projection
algorithm as discussed above, detects probe regions of interest in
this image, then further analyzes these regions in all the focal
plane images.
[0128] After measuring cell features 112 (FIG. 9), the system
checks if there are any unprocessed objects in the current field
113. If there are any unprocessed objects, it locates the next
object 110 and determines whether it meets the criteria for a valid
cell nucleus 111, and measures its features. Once all the objects
in the current field are processed, the system determines whether
analysis of the current plate is complete 114; if not, it
determines the need to find more cells in the current well 115. If
the need exists, the system advances the XYZ stage to the next
field within the current well 109 or advances the stage to the next
well 116 of the plate.
[0129] After a plate scan is complete, images and data can be
reviewed with the system's image review, data review, and summary
review facilities. All images, data, and settings from a scan are
archived in the system's database for later review or for
interfacing with a network information management system. Data can
also be exported to other third-party statistical packages to
tabulate results and generate other reports. Users can review the
images alone of every cell analyzed by the system with an
interactive image review procedure 117. The user can review data on
a cell-by-cell basis using a combination of interactive graphs, a
data spreadsheet of measured features, and images of all the
fluorescence channels of a cell of interest with the interactive
cell-by-cell data review procedure 118. Graphical plotting
capabilities are provided in which data can be analyzed via
interactive graphs such as histograms and scatter plots. Users can
review summary data that are accumulated and summarized for all
cells within each well of a plate with an interactive well-by-well
data review procedure 119. Hard copies of graphs and images can be
printed on a wide range of standard printers.
[0130] As a final phase of a complete scan, reports can be
generated on one or more statistics of the measured features. Users
can generate a graphical report of data summarized on a
well-by-well basis for the scanned region of the plate using an
interactive report generation procedure 120. This report includes a
summary of the statistics by well in tabular and graphical format
and identification information on the sample. The report window
allows the operator to enter comments about the scan for later
retrieval. Multiple reports can be generated on many statistics and
be printed with the touch of one button. Reports can be previewed
for placement and data before being printed.
[0131] The above-recited embodiment of the method operates in a
single high resolution mode referred to as the high content
screening (HCS) mode. The HCS mode provides sufficient spatial
resolution within a well (on the order of 1 .mu.m) to define the
distribution of material within the well, as well as within
individual cells in the well. The high degree of information
content accessible in that mode, comes at the expense of speed and
complexity of the required signal processing.
[0132] In an alternative embodiment, a high throughput system (HTS)
is directly coupled with the HCS either on the same platform or on
two separate platforms connected electronically (e.g. via a local
area network). This embodiment of the invention, referred to as a
dual mode optical system, has the advantage of increasing the
throughput of an HCS by coupling it with an HTS and thereby
requiring slower high resolution data acquisition and analysis only
on the small subset of wells that show a response in the coupled
HTS.
[0133] High throughput `whole plate` reader systems are well known
in the art and are commonly used as a component of an. HTS system
used to screen large numbers of compounds (Beggs et al. (1997),
supra; McCaffrey et al. (1996), supra ). The HTS of the present
invention is carried out on the microtiter plate or microwell array
by reading many or all wells in the plate simultaneously with
sufficient resolution to make determinations on a well-by-well
basis. That is, calculations are made by averaging the total signal
output of many or all the cells or the bulk of the material in each
well. Wells that exhibit some defined response in the HTS (the
`hits`) are flagged by the system. Then on the same microtiter
plate or microwell array, each well identified as a hit is measured
via HCS as described above. Thus, the dual mode process
involves:
[0134] 1. Rapidly measuring numerous wells of a microtiter plate or
microwell array,
[0135] 2. Interpreting the data to determine the overall activity
of fluorescently labeled reporter molecules in the cells on a
well-by-well basis to identify "hits" (wells that exhibit a defined
response),
[0136] 3. Imaging numerous cells in each "hit" well, and
[0137] 4. Interpreting the digital image data to determine the
distribution, environment or activity of the fluorescently labeled
reporter molecules in the individual cells (i.e. intracellular
measurements) and the distribution of the cells to test for
specific biological functions
[0138] In a preferred embodiment of dual mode processing (FIG. 11),
at the start of a run 301, the operator enters information 302 that
describes the plate and its contents, specifies the filter settings
and fluorescent channels to match the biological labels being used,
the information sought and the camera settings to match the sample
brightness. These parameters are stored in the system's database
for easy retrieval for each automated run. The microtiter plate or
microwell array is loaded into the cell screening system 303 either
manually or automatically by controlling a robotic loading device.
An optional environmental chamber 304 is controlled by the system
to maintain the temperature, humidity and CO.sub.2 levels in the
air surrounding live cells in the microtiter plate or microwell
array. An optional fluid delivery device 305 (see FIG. 8) is
controlled by the system to dispense fluids into the wells during
the scan.
[0139] High throughput processing 306 is first performed on the
microtiter plate or microwell array by acquiring and analyzing the
signal from each of the wells in the plate. The processing
performed in high throughput mode 307 is illustrated in FIG. 12 and
described below. Wells that exhibit some selected intensity
response in this high throughput mode ("hits") are identified by
the system. The system performs a conditional operation 308 that
tests for hits. If hits are found, those specific hit wells are
further analyzed in high content (micro level) mode 309. The
processing performed in high content mode 312 is illustrated in
FIG. 13. The system then updates 310 the informatics database 311
with results of the measurements on the plate. If there are more
plates to be analyzed 313 the system loads the next plate 303;
otherwise the analysis of the plates terminates 314.
[0140] The following discussion describes the high throughput mode
illustrated in FIG. 12. The preferred embodiment of the system, the
single platform dual mode screening system, will be described.
Those skilled in the art will recognize that operationally the dual
platform system simply involves moving the plate between two
optical systems rather than moving the optics. Once the system has
been set up and the plate loaded, the system begins the HTS
acquisition and analysis 401. The HTS optical module is selected by
controlling a motorized optical positioning device 402 on the dual
mode system. In one fluorescence channel, data from a primary
marker on the plate is acquired 403 and wells are isolated from the
plate background using a masking procedure 404. Images are also
acquired in other fluorescence channels being used 405. The region
in each image corresponding to each well 406 is measured 407. A
feature calculated from the measurements for a particular well is
compared with a predefined threshold or intensity response 408, and
based on the result the well is either flagged as a "hit" 409 or
not. The locations of the wells flagged as hits are recorded for
subsequent high content mode processing. If there are wells
remaining to be processed 410 the program loops back 406 until all
the wells have been processed 411 and the system exits high
throughput mode.
[0141] Following HTS analysis, the system starts the high content
mode processing 501 defined in FIG. 13. The system selects the HCS
optical module 502 by controlling the motorized positioning system.
For each "hit" well identified in high throughput mode, the XY
stage location of the well is retrieved from memory or disk and the
stage is then moved to the selected stage location 503. The
autofocus procedure 504 is called for the first field in each hit
well and then once every 5 to 8 fields within each well. In one
channel, images are acquired of the primary marker 505 (typically
cell nuclei counterstained with DAPI, Hoechst or PI fluorescent
dye). The images are then segmented (separated into regions of
nuclei and non-nuclei) using an adaptive thresholding procedure
506. The output of the segmentation procedure is a binary mask
wherein the objects are white and the background is black. This
binary image, also called a mask in the art, is used to determine
if the field contains objects 507. The mask is labeled with a blob
labeling algorithm whereby each object (or blob) has a unique
number assigned to it. If objects are found in the field, images
are acquired for all other active channels 508, otherwise the stage
is advanced to the next field 514 in the current well. Each object
is located in the image for further analysis 509. Morphological
features, such as area and shape of the objects, are used to select
objects likely to be cell nuclei 510:, and discard (do no further
processing on) those that are considered artifacts. For each valid
cell nucleus, the XYZ stage location is recorded, a small image of
the cell is stored, and assay specific features are measured 511.
The system then performs multiple tests on the cells by applying
several analytical methods to measure features at each of several
wavelengths. After measuring the cell features, the systems checks
if there are any unprocessed objects in the current field 512. If
there are any unprocessed objects, it locates the next object 509
and determines whether it meets the criteria for a valid cell
nucleus 510, and measures its features. After processing all the
objects in the current field, the system deteremines whether it
needs to find more cells or fields in the current well 513. If it
needs to find more cells or fields in the current well it advances
the XYZ stage to the next field within the current well 515.
Otherwise, the system checks whether it has any remaining hit wells
to measure 515. If so, it advances to the next hit well 503 and
proceeds through another cycle of acquisition and analysis,
otherwise the HCS mode is finished 516.
[0142] In an alternative embodiment of the present invention, a
method of kinetic live cell screening is provided. The previously
described embodiments of the invention are used to characterize the
spatial distribution of cellular components at a specific point in
time, the time of chemical fixation. As such, these embodiments
have limited utility for implementing kinetic based screens, due to
the sequential nature of the image acquisition, and the amount of
time required to read all the wells on a plate. For example, since
a plate can require 30-60 minutes to read through all the wells,
only very slow kinetic processes can be measured by simply
preparing a plate of live cells and then reading through all the
wells more than once. Faster kinetic processes can be measured by
taking multiple readings of each well before proceeding to the next
well, but the elapsed time between the first and last well would be
too long, and fast kinetic processes would likely be complete
before reaching the last well.
[0143] The kinetic live cell extension of the invention enables the
design and use of screens in which a biological process is
characterized by its kinetics instead of, or in addition to, its
spatial characteristics. In many cases, a response in live cells
can be measured by adding a reagent to a specific well and making
multiple measurements on that well with the appropriate timing.
This dynamic live cell embodiment of the invention therefore
includes apparatus for fluid delivery to individual wells of the
system in order to deliver reagents to each well at a specific time
in advance of reading the well. This embodiment thereby allows
kinetic measurements to be made with temporal resolution of seconds
to minutes on each well of the plate. To improve the overall
efficiency of the dynamic live cell system, the acquisition control
program is modified to allow repetitive data collection from
sub-regions of the plate, allowing the system to read other wells
between the time points required for an individual well.
[0144] FIG. 8 describes an example of a fluid delivery device for
use with the live cell embodiment of the invention and is described
above. This set-up allows one set of pipette tips 705, or even a
single pipette tip, to deliver reagent to all the wells on the
plate. The bank of syringe pumps 701 can be used to deliver fluid
to 12 wells simultaneously, or to fewer wells by removing some of
the tips 705. The temporal resolution of the system can therefore
be adjusted, without sacrificing data collection efficiency, by
changing the number of tips and the scan pattern as follows.
Typically, the data collection and analysis from a single well
takes about 5 seconds. Moving from well to well and focusing in a
well requires about 5 seconds, so the overall cycle time for a well
is about 10 seconds. Therefore, if a single pipette tip is used to
deliver fluid to a single well, and data is collected repetitively
from that well, measurements can be made with about 5 seconds
temporal resolution. If 6 pipette tips are used to deliver fluids
to 6 wells simultaneously, and the system repetitively scans all 6
wells, each scan will require 60 seconds, thereby establishing the
temporal resolution. For slower processes which only require data
collection every 8 minutes, fluids can be delivered to one half of
the plate, by moving the plate during the fluid delivery phase, and
then repetitively scanning that half of the plate. Therefore, by
adjusting the size of the sub-region being scanned on the plate,
the temporal resolution can be adjusted without having to insert
wait times between acquisitions. Because the system is continuously
scanning and acquiring data, the overall time to collect a kinetic
data set from the plate is then simply the time to perform a single
scan of the plate, multiplied by the number of time points
required. Typically, 1 time point before addition of compounds and
2 or 3 time points following addition should be sufficient for
screening purposes.
[0145] FIG. 14 shows the acquisition sequence used for kinetic
analysis. The start of processing 801 is configuration of the
system, much of which is identical to the standard HCS
configuration. In addition, the operator must enter information
specific to the kinetic analysis being performed 802, such as the
sub-region size, the number of time points required, and the
required time increment. A sub-region is a group of wells that will
be scanned repetitively in order to accumulate kinetic data. The
size of the sub-region is adjusted so that the system can scan a
whole sub-region once during a single time increment, thus
minimizing wait times. The optimum sub-region size is calculated
from the setup parameters, and adjusted if necessary by the
operator. The system then moves the plate to the first sub-region
803, and to the first well in that sub-region 804 to acquire the
prestimulation (time=0) time points. The acquisition sequence
performed in each well is exactly the same as that required for the
specific HCS being run in kinetic mode. FIG. 15 details a flow
chart for that processing. All of the steps between the start 901
and the return 902 are identical to those described as steps
504-514 in FIG. 13.
[0146] After processing each well in a sub-region, the system
checks to see if all the wells in the sub-region have been
processed 806 (FIG. 14), and cycles through all the wells until the
whole region has been processed. The system then moves the plate
into position for fluid addition, and controls fluidic system
delivery of fluids to the entire sub-region. 807. This may require
multiple additions for sub-regions which span several rows on the
plate, with the system moving the plate on the X,Y stage between
additions. Once the fluids have been added, the system moves to the
first well in the sub-region 808 to begin acquisition of time
points. The data is acquired from each well 809 and as before the
system cycles through all the wells in the sub-region 810. After
each pass through the sub-region, the system checks whether all the
time points have been collected 811 and if not, pauses 813 if
necessary 812 to stay synchronized with the requested time
increment. Otherwise, the system checks for additional sub-regions
on the plate 814 and either moves to the next sub-region 803 or
finishes 815. Thus, the kinetic analysis mode comprises operator
identification of sub-regions of the microtiter plate or microwells
to be screened, based on the kinetic response to be investigated,
with data acquisitions within a sub-region prior to data
acquisition in subsequent sub-regions.
[0147] Specific Screens
[0148] In another aspect of the present invention, a machine
readable storage medium comprising a program containing a set of
instructions for causing a cell screening system to execute
procedures for defining the distribution and activity of specific
cellular constituents and processes is provided. In a preferred
embodiment, the cell screening system comprises a high
magnification fluorescence optical system with a stage adapted for
holding cells and a means for moving the stage, a digital camera, a
light source for receiving and processing the digital data from the
digital camera, and a computer means for receiving and processing
the digital data from the digital camera. This aspect of the
invention comprises programs that instruct the cell screening
system to define the distribution and activity of specific cellular
constituents and processes, using the. luminescent probes, the
optical imaging system, and the pattern recognition software of the
invention. Preferred embodiments of the machine readable storage
medium comprise programs consisting of a set of instructions for
causing a cell screening system to execute the procedures set forth
in FIGS. 9, 11, 12, 13, 14 or 15. Another preferred embodiment
comprises a program consisting of a set of instructions for causing
a cell screening system to execute procedures for detecting the
distribution and activity of specific cellular constituents and
processes. In most preferred embodiments, the cellular processes
include, but are not limited to, nuclear translocation of a
protein, cellular hypertrophy, apoptosis, and protease-induced
translocation of a protein.
[0149] The following examples are intended for purposes of
illustration only and should not be construed to limit the scope of
the invention, as defined in the claims appended hereto.
[0150] The various chemical compounds, reagents, dyes, and
antibodies that are referred to in the following Examples are
commercially available from such sources as Sigma Chemical (St.
Louis, Mo.), Molecular Probes (Eugene, Oreg.), Aldrich Chemical
Company (Milwaukee, Wis.), Accurate Chemical Company (Westbury,
N.Y.), Jackson Immunolabs, and Clontech (Palo Alto, Calif.).
EXAMPLE 1
Automated Screen for Compounds that Induce or Inhibit Nuclear
Translocation of a DNA Transcription Factor
[0151] Regulation of transcription of some genes involves
activation of a transcription factor in the cytoplasm, resulting in
that factor being transported into the nucleus where it can
initiate transcription of a particular gene or genes. This change
in transcription factor distribution is the basis of a screen for
the cell-based screening system to detect compounds that inhibit or
induce transcription of a particular gene or group of genes. A
general description of the screen is given followed by a specific
example.
[0152] The distribution of the transcription factor is determined
by labeling the nuclei with a DNA specific fluorophore like Hoechst
33423 and the transcription factor with a specific fluorescent
antibody. After autofocusing on the Hoechst labeled nuclei, an
image of the nuclei is acquired in the cell-based screening system
at 20.times. magnification and used to create a mask by one of
several optional thresholding methods, as described supra. The
morphological descriptors of the regions defined by the mask are
compared with the user defined parameters and valid nuclear masks
are identified and used with the following algorithm to extract
transcription factor distributions. Each valid nuclear mask is
eroded to define a slightly smaller nuclear region. The original
nuclear mask is then dilated in two steps to define a ring shaped
region around the nucleus, which represents a cytoplasmic region.
The average antibody fluorescence in each of these two regions is
determined, and the difference between these averages is defined as
the NucCyt Difference. Two examples of determining nuclear
translocation are discussed below and illustrated in FIGS. 10A-J.
FIG. 10A illustrates an unstimulated cell with its nucleus 200
labeled with a blue fluorophore and a transcription factor in the
cytoplasm 201 labeled with a green fluorophore. FIG. 10B
illustrates the nuclear mask 202 derived by the cell-based
screening system. FIG. 10C illustrates the cytoplasm 203 of the
unstimulated cell imaged at a green wavelength. FIG. 10D
illustrates the nuclear mask 202 is eroded (reduced) once to define
a nuclear sampling region 204 with minimal cytoplasmic
distribution. The nucleus boundary 202 is dilated (expanded)
several times to form a ring that is 2-3 pixels wide that is used
to define the cytoplasmic sampling region 205 for the same cell.
FIG. 10E further illustrates a side view which shows the nuclear
sampling region 204 and the cytoplasmic sampling region 205. Using
these two sampling regions, data on nuclear translocation can be
automatically analyzed by the cell-based screening system on a cell
by cell basis. FIGS. 10F-J illustrates the strategy for determining
nuclear translocation in a stimulated cell. FIG. 10F illustrates a
stimulated cell with its nucleus 206 labeled with a blue
fluorophore and a transcription factor in the cytoplasm 207 labeled
with a green fluorophore. The nuclear mask 208 in FIG. 10G is
derived by the cell based screening system. FIG. 10H illustrates
the cytoplasm 209 of a stimulated cell imaged at a green
wavelength. FIG. 10I illustrates the nuclear sampling region 211
and cytoplasmic sampling region 212 of the stimulated cell. FIG.
10J further illustrates a side view which shows the nuclear
sampling region 211 and the cytoplasmic sampling region 212.
[0153] A specific application of this method has been used to
validate this method as a screen. A human cell line was plated in
96 well microtiter plates. Some rows of wells were titrated with
agonist, a known inducer of a specific nuclear transcription
factor. The cells were then fixed and stained by standard methods
with a fluorescein labeled antibody to the transcription factor,
and Hoechst 33423. The cell-based screening system was used to
acquire and analyze images from this plate and the NucCyt
Difference was found to be strongly correlated with the amount of
agonist added to the wells as illustrated in FIG. 16. In a second
experiment, an antagonist to the receptor for the agonist was
titrated in the presence of agonist, progressively inhibiting
agonist-induced translocation of the transcription factor. The
NucCyt Difference was found to strongly correlate with this
inhibition of translocation, as illustrated in FIG. 17.
[0154] Additional experiments have shown that the NucCyt Difference
gives consistent results over a wide range of cell densities and
reagent concentrations, and can therefore be routinely used to
screen compound libraries for specific nuclear translocation
activity. Furthermore, the same method can be used with antibodies
to other transcription factors, or GFP-transcription factor
chimeras, in living and fixed cells, to screen for effects on the
regulation of transcription of this and other genes.
[0155] FIG. 18 is a representative display on a PC screen of data
which was obtained in accordance with Example 1. Graph 1 180 plots
the difference between the average antibody fluorescence in the
nuclear sampling region and cytoplasmic sampling region, NucCyt
Difference verses Well #. Graph 2 181 plots the average
fluorescence of the antibody in the nuclear sampling region, NP1
average, versus the Well #. Graph 3 182 plots the average antibody
fluorescence in the cytoplasmic sampling region, LIP1 average,
versus Well #. The software permits displaying data from each cell.
For example, FIG. 18 shows a screen display 183, the nuclear image
184, and the fluorescent antibody image 185 for cell #26.
[0156] NucCyt Difference referred to in graph 1 180 of FIG. 18 is
the difference between the average cytoplasmic probe (fluorescent
reporter molecule) intensity and the average nuclear probe
(fluorescent reporter molecule) intensity. NP1 average referred to
in graph 2 181 of FIG. 18 is the average of cyloplasmic probe
(fluorescent reporter molecule) intensity within the nuclear
sampling region. L1P1 average referred to in graph 3 182 of FIG. 18
is the average probe (fluorescent reporter molecule) intensity
within the cytoplasmic sampling region.
EXAMPLE 2
Automated Screen for Compounds that Induce or Inhibit Hypertrophy
in Cardiac Myocytes
[0157] Hypertrophy in cardiac myocytes has been associated with a
cascade of alterations in gene expression and can be characterized
in cell culture by an alteration in cell size, that is clearly
visible in adherent cells growing on a coverslip. A screen is
implemented using the following strategy. Myocyte cell line QM7
(Quail muscle clone 7; ATCC CRL-1962) cultured in 96 well plates,
can be treated with various compounds and then fixed and labeled
with a fluorescent antibody to a cell surface marker and a DNA
label like Hoechst. After focusing on the Hoechst labeled nuclei,
two images are acquired, one of the Hoechst labeled nuclei and one
of the fluorescent antibody. The nuclei are identified by
thresholding to create a mask and then comparing the morphological
descriptors of the mask with a set of user defined descriptor
values. Local regions containing cells are defined around the
nuclei. The limits of the cells in those regions are then defined
by a local dynamic threshold operation on the same region in the
fluorescent antibody image. A sequence of erosions and dilations is
used to separate slightly touching cells and a second set of
morphological descriptors is used to identify single cells. The
area of the individual cells is tabulated in order to define the
distribution of cell sizes for comparison with size data from
normal and hypertrophic cells. In addition, a second fluorescent
antibody to a particular cellular protein, such as one of the major
muscle proteins actin or myosin can be included. Images of this
second antibody can be acquired and stored with the above images,
for later review, to identify anomalies in the distribution of
these proteins in hypertrophic cells, or algorithms can be
developed to automatically analyze the distributions of the labeled
proteins in these images.
EXAMPLE 3
Dual Mode High Throughput and High-Content Screen
[0158] The following example is a screen for activation of a
G-protein coupled receptor (GPCR) as detected by the translocation
of the GPCR from the plasma membrane to a proximal nuclear
location. This example illustrates how a high throughput screen can
be coupled with a high-content screen in the dual mode System for
Cell Based Screening.
[0159] G-protein coupled receptors are a large class of 7
trans-membrane domain cell surface receptors. Ligands for these
receptors stimulate a cascade of secondary signals in the cell,
which may include, but are not limited to, Ca.sup.++ transients,
cyclic AMP production, inositol triphosphate (IP.sub.3) production
and phosphorylation. Each of these signals are rapid, occuring in a
matter of seconds to minutes, but are also generic. For example,
many different GPCRs produce a secondary Ca.sup.++ signal when
activated. Stimulation of a GPCR also results in the transport of
that GPCR from the cell surface membrane to an internal, proximal
nuclear compartment. This internalization is a much more
receptor-specific indicator of activation of a particular receptor
than are the secondary signals described above.
[0160] FIG. 19 illustrates a dual mode screen for activation of a
GPCR. Cells carrying a stable chimera of the GPCR with a blue
fluorescent protein (BFP) would be loaded with the
acetoxymethylester form of Fluo-3, a cell permeable calcium
indicator (green fluorescence) that is trapped in living cells by
the hydrolysis of the esters. They would then be deposited into the
wells of a microtiter plate 601. The wells would then be treated
with an array of test compounds using a fluid delivery system, and
a short sequence of Fluo-3 images of the whole microtiter plate
would be acquired and analyzed for wells exhibiting a calcium
response (i.e., high throughput mode). The images would appear like
the illustration of the microtiter plate 601 in FIG. 19. A small
number of wells, such as wells C4 and E9 in the illustration, would
fluoresce more brightly due to the Ca.sup.++ released upon
stimulation of the receptors. The locations of wells containing
compounds that induced a response 602, would then be transferred to
the HCS program and the optics switched for detailed cell by cell
analysis of the blue fluorescence for evidence of GPCR
translocation to the perinuclear region. The bottom of FIG. 19
illustrates the two possible outcomes of the analysis of the high
resolution cell data. The camera images a sub-region 604 of the
well area 603, producing images of the fluorescent cells 605. In
well C4, the uniform distribution of the fluorescence in the cells
indicates that the receptor has not internalized, implying that the
Ca.sup.++ response seen was the result of the stimulation of some
other signalling system in the cell. The cells in well E9 606 on
the other hand, clearly indicate a concentration of the receptor in
the perinuclear region clearly indicating the full activation of
the receptor. Because only a few hit wells have to be analyzed with
high resolution, the overall throughput of the dual mode system can
be quite high, comparable to the high throughput system alone.
EXAMPLE 4
Kinetic High Content Screen
[0161] The following is an example of a screen to measure the
kinetics of internalization of a receptor. As described above, the
stimulation of a GPCR, results in the internalization of the
receptor, with a time course of about 15 min. Simply detecting the
endpoint as internalized or not, may not be sufficient for defining
the potency of a compound as a GPCR agonist or antagonist. However,
3 time points at 5 min intervals would provide information not only
about potency during the time course of measurement, but would also
allow extrapolation of the data to much longer time periods. To
perform this assay, the sub-region would be defined as two rows,
the sampling interval as 5 minutes and the total number of time
points 3. The system would then start by scanning two rows, and
then adding reagent to the two rows, establishing the time=0
reference. After reagent addition, the system would again scan the
two row sub-region acquiring the first time point data. Since this
process would take about 250 seconds, including scanning back to
the beginning of the sub-region, the system would wait 50 seconds
to begin acquisition of the second time point. Two more cycles
would produce the three time points and the system would move on to
the second 2 row sub-region. The final two 2-row sub-regions would
be scanned to finish all the wells on the plate, resulting in four
time points for each well over the whole plate. Although the time
points for the wells would be offset slightly relative to time=0,
the spacing of the time points would be very close to the required
5 minutes, and the actual acquisition times and results recorded
with much greater precision than in a fixed-cell screen.
EXAMPLE 5
High-Content Screen of Human Glucocorticoid Receptor
Translocation
[0162] One class of HCS involves the drug-induced dynamic
redistribution of intracellular constituents. The human
glucocorticoid receptor (hGR), a single "sensor" in the complex
environmental response machinery of the cell, binds steroid
molecules that have diffused into the cell. The ligand-receptor
complex translocates to the nucleus where transcriptional
activation occurs (Htun et al., Proc. Natl. Acad. Sci. 93:4845,
1996).
[0163] In general, hormone receptors are excellent drug targets
because their activity lies at the apex of key intracellular
signaling pathways. Therefore, a high-content screen of hGR
translocation has distinct advantage over in vitro ligand-receptor
binding assays. The availability of up to two more channels of
fluorescence in the cell screening system of the present invention
permits the screen to contain two additional parameters in
parallel, such as other receptors, other distinct targets or other
cellular processes.
[0164] Plasmid Construct.
[0165] A eukaryotic expression plasmid containing a coding sequence
for a green fluorescent protein--human glucocorticoid receptor
(GFP-hGR) chimera was prepared using GFP mutants (Palm et al., Nat.
Struct. Biol. 4:361 (1997). The construct was used to transfect a
human cervical carcinoma cell line (HeLa).
[0166] Cell Preparation and Transfection.
[0167] HeLa cells (ATCC CCL-2) were trypsinized and plated using
DMEM containing 5% charcoal/dextran-treated fetal bovine serum
(FBS) (HyClone) and 1% penicillin-streptomycin (C-DMEM) 12-24 hours
prior to transfection and incubated at 37.degree. C. and 5%
CO.sub.2. Transfections were performed by calcium phosphate
co-precipitation (Graham and Van der Eb, Virology 52:456, 1973;
Sambrook et al., (1989). Molecular Cloning: A Laboratory Manual,
Second ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
1989) or with Lipofectamine (Life Technologies, Gaithersburg, Md.).
For the calcium phosphate transfections, the medium was replaced,
prior to transfection, with DMEM containing 5%
charcoal/dextran-treated FBS. Cells were incubated with the calcium
phosphate-DNA precipitate for 4-5 hours at 37.degree. C. and 5%
CO.sub.2, washed 3-4 times with DMEM to remove the precipitate,
followed by the addition of C-DMEM.
[0168] Lipofectamine transfections were performed in serum-free
DMEM without antibiotics according to the manufacturer's
instructions (Life Technologies, Gaithersburg, Md.). Following a
2-3 hour incubation with the DNA-liposome complexes, the medium was
removed and replaced with C-DMEM. All transfected cells in 96-well
microtiter plates were incubated at 33.degree. C. and 5% CO.sub.2
for 24-48 hours prior to drug treatment. Experiments were performed
with the receptor expressed transiently in HeLa cells.
[0169] Dexamethasone Induction of GFP-hGR Translocation.
[0170] To obtain receptor-ligand translocation kinetic data, nuclei
of transfected cells were first labeled with 5 .mu.g/ml Hoechst
33342 (Molecular Probes) in C-DMEM for 20 minutes at 33.degree. C.
and 5% CO.sub.2. Cells were washed once in Hank's Balanced Salt
Solution (HBSS) followed by the addition of 100 nM dexamethasone in
HBSS with 1% charcoal/dextran-treated FBS. To obtain fixed time
point dexamethasone titration data, transfected HeLa cells were
first washed with DMEM and then incubated at 33.degree. C. and 5%
CO.sub.2 for 1 h in the presence of 0-1000 nM dexamethasone in DMEM
containing 1% charcoal/dextran-treated FBS. Cells were analyzed
live or they were rinsed with HBSS, fixed for 15 min with 3.7%
formaldehyde in HBSS, stained with Hoechst 33342, and washed before
analysis. The intracellular GFP-hGR fluorescence signal was not
diminished by this fixation procedure.
[0171] Image Acquisition and Analysis.
[0172] Kinetic data were collected by acquiring fluorescence image
pairs (GFP-hGR and Hoechst 33342-labeled nuclei) from fields of
living cells at 1 min intervals for 30 min after the addition of
dexamethasone. Likewise, image pairs were obtained from each well
of the fixed time point screening plates 1 h after the addition of
dexamethasone. In both cases, the image pairs obtained at each time
point were used to define nuclear and cytoplasmic regions in each
cell. Translocation of GFP-hGR was calculated by dividing the
integrated fluorescence intensity of GFP-hGR in the nucleus by the
integrated fluorescence intensity of the chimera in the cytoplasm
or as a nuclear-cytoplasmic difference of GFP fluorescence. In the
fixed time point screen this translocation ratio was calculated
from data obtained from at least 200 cells at each concentration of
dexamethasone tested. Drug-induced translocation of GFP-hGR from
the cytoplasm to the nucleus was therefore correlated with an
increase in the translocation ratio.
[0173] Results.
[0174] FIG. 20 schematically displays the drug-induced cytoplasm
253 to nucleus 252 translocation of the human glucocorticoid
receptor. The upper pair of schematic diagrams depicts the
localization of GFP-hGR within the cell before 250(A) and after
251(B) stimulation with dexamethasone. Under these experimental
conditions, the drug induces a large portion of the cytoplasmic
GFP-hGR to translocate into the nucleus. This redistribution is
quantified by determining the. integrated intensities ratio of the
cytoplasmic and nuclear fluorescence in treated 255 and untreated
254 cells. The lower pair of fluorescence micrographs show the
dynamic redistribution of GFP-hGR in a single cell, before 254 and
after 255 treatment. The HCS is performed on wells containing
hundreds to thousands of transfected cells and the translocation is
quantified for each cell in the field exhibiting GFP fluorescence.
Although the use of a stably transfected cell line would yield the
most consistently labeled cells, the heterogeneous levels of
GFP-hGR expression induced by transient transfection did not
interfere with analysis by the cell screening system of the present
invention.
[0175] To execute the screen, the cell screening system scans each
well of the plate, images a population of cells in each, and
analyzes cells individually. Here, two channels of fluorescence are
used to define the cytoplasmic and nuclear distribution of the
GFP-hGR within each cell. Depicted in FIG. 21 is the graphical user
interface of the cell screening system near the end of a GFP-hGR
screen. The user interface depicts the parallel data collection and
analysis capability of the system. The windows labeled "Nucleus"
261 and "GFP-hGR" 262 show the pair of fluorescence images being
obtained and analyzed in a single field. The window labeled "Color
Overlay" 260 is formed by pseudocoloring the above images and
merging them so the user can immediately identify cellular changes.
Within the "Stored Object Regions" window 265 an image containing
each analyzed cell and its neighbors is presented as it is
archived. Furthermore, as the HCS data are being collected, they
are analyzed, in this case for GFP-hGR translocation, and
translated into an immediate "hit" response. The 96 well plate
depicted in the lower window of the screen 267 shows which wells
have met a set of user-defined screening. criteria For example, a
white-colored well 269 indicates that the drug-induced
translocation has exceeded a predetermined threshold value of 50%.
On the other hand, a black-colored well 270 indicates that the drug
being tested induced less than 10% translocation. Gray-colored
wells 268 indicate "hits" where the translocation value fell
between 10% and 50%. Row "E" on the 96 well plate being analyzed
266 shows a titration with a drug known to activate GFP-hGR
translocation, dexamethasone. This example screen used only two
fluorescence channels. Two additional channels (Channels 3 263 and
4 264) are available for parallel analysis of other specific
targets, cell processes, or cytotoxicity to create multiple
parameter screens.
[0176] There is a link between the image database and the
information database that is a powerful tool during the validation
process of new screens. At the completion of a screen, the user has
total access to image and calculated data (FIG. 22). The
comprehensive data analysis package of the cell screening system
allows the user to examine HCS data at multiple levels. Images 276
and detailed data in a spread sheet 279 for individual cells can be
viewed separately, or summary data can be plotted. For example, the
calculated results of a single parameter for each cell in a 96 well
plate are shown in the panel labeled Graph 1 275. By selecting a
single point in the graph, the user can display the entire data set
for a particular cell that is recalled from an existing database.
Shown here are the image pair 276 and detailed fluorescence and
morphometric data from a single cell (Cell #118, gray line 277).
The large graphical insert 278 shows the results of dexamethasone
concentration on the translocation of GFP-hGR. Each point is the
average of data from at least 200 cells. The calculated EC.sub.50
for dexamethasone in this assay is 2 nM.
[0177] A powerful aspect of HCS with the cell screening system is
the capability of kinetic measurements using multicolor
fluorescence and morphometric parameters in living cells. Temporal
and spatial measurements can be made on single cells within a
population of cells in a field. FIG. 23 shows kinetic data for the
dexamethasone-induced translocation of GFP-hGR in several cells
within a single field. Human HeLa cells transfected with GFP-hGR
were treated with 100 nM dexamethasone and the translocation of
GFP-hGR was measured over time in a population of single cells. The
graph shows the response of transfected cells 285, 286, 287, and
288 and non-transfected cells 289. These data also illustrate the
ability to analyze cells with different expression levels.
Example 6
High-Content Screen of Drug-Induced Apoptosis
[0178] Apoptosis is a complex cellular program that involves myriad
molecular events and pathways. To understand the mechanisms of drug
action on this process, it is essential to measure as many of these
events within cells as possible with temporal and spatial
resolution. Therefore, an apoptosis screen that requires little
cell sample preparation yet provides an automated readout of
several apoptosis-related parameters would be ideal. A cell-based
assay designed for the cell screening system has been used to
simultaneously quantify several of the morphological, organellar,
and macromolecular hallmarks of paclitaxel-induced apoptosis.
[0179] Cell Preparation.
[0180] The cells chosen for this study were mouse connective tissue
fibroblasts (L-929; ATCC CCL-1) and a highly invasive glioblastoma
cell line (SNB-19; ATCC CRL-2219) (Welch et al., In Vitro Cell.
Dev. Biol. 31:610, 1995). The day before treatment with an
apoptosis inducing drug, 3500 cells were placed into each well of a
96-well plate and incubated overnight at 37.degree. C. in a
humidified 5% CO.sub.2 atmosphere. The following day, the culture
medium was removed from each well and replaced with fresh medium
containing various concentrations of paclitaxel (0-50 .mu.M) from a
20 mM stock made in DMSO. The maximal concentration of DMSO used in
these experiments was 0.25%. The cells were then incubated for 26 h
as above. At the end of the paclitaxel treatment period, each well
received fresh medium containing 750 nM MitoTracker Red (Molecular
Probes; Eugene, Oreg.) and 3 .mu.g/ml Hoechst 33342 DNA-binding dye
(Molecular Probes) and was incubated as above for 20 min. Each well
on the plate was then washed with HBSS and fixed with 3.7%
formaldehyde in HBSS for 15 min at room temperature. The
formaldehyde was washed out with HBSS and the cells were
permeabilized for 90 s with 0.5% (v/v) Triton X-100, washed with
HBSS, incubated with 2 U m1.sup.-1 Bodipy FL phallacidin (Molecular
Probes) for 30 min, and washed with HBSS. The wells on the plate
were then filled with 200 .mu.l HBSS, sealed, and the plate stored
at 4.degree. C. if necessary. The fluorescence signals from plates
stored this way were stable for at least two weeks after
preparation. As in the nuclear translocation assay, fluorescence
reagents can be designed to convert this assay into a live cell
high-content screen.
[0181] Image Acquisition and Analysis on the ArrayScan System.
[0182] The fluorescence intensity of intracellular MitoTracker Red,
Hoechst 33342, and Bodipy FL phallacidin was measured with the cell
screening system as described supra. Morphometric data from each
pair of images obtained from each well was also obtained to detect
each object in the image field (e.g., cells and nuclei), and to
calculate its size, shape, and integrated intensity.
[0183] Calculations and Output.
[0184] A total of 50-250 cells were measured per image field. For
each field of cells, the following calculations were performed: (1)
The average nuclear area (.mu.m.sup.2) was calculated by dividing
the total nuclear area in a field by the number of nuclei detected.
(2) The average nuclear perimeter (.mu.m) was calculated by
dividing the sum of the perimeters of all nuclei in a field by the
number of nuclei detected in that field. Highly convoluted
apoptotic nuclei had the largest nuclear perimeter values. (3) The
average nuclear brightness was calculated by dividing the
integrated intensity of the entire field of nuclei by the number of
nuclei in that field. An increase in nuclear brightness was
correlated with increased DNA content. (4) The average cellular
brightness was calculated by dividing the integrated intensity of
an entire field of cells stained with MitoTracker dye by the number
of nuclei in that field. Because the amount of MitoTracker dye that
accumulates within the mitochondria is proportional to the
mitochondrial potential, an increase in the average cell brightness
is consistent with an increase in mitochondrial potential. (5) The
average cellular brightness was also calculated by dividing the
integrated intensity of an entire field of cells stained with
Bodipy FL phallacidin dye by the number of nuclei in that field.
Because the phallotoxins bind with high affinity to the polymerized
form of actin, the amount of Bodipy FL phallacidin dye that
accumulates within the cell is proportional to actin polymerization
state. An increase in the average cell brightness is consistent
with an increase in actin polymerization.
[0185] Results.
[0186] FIG. 24 (top panels) shows the changes paclitaxel induced in
the nuclear morphology of L-929 cells. Increasing amounts of
paclitaxel caused nuclei to enlarge and fragment 293, a hallmark of
apoptosis. Quantitative analysis of these and other images obtained
by the cell screening system is presented in the same figure. Each
parameter measured showed that the L-929 cells 296 were less
sensitive to low concentrations of paclitaxel than were SNB-19
cells 297. At higher concentrations though, the L-929 cells showed
a response for each parameter measured. The multiparameter approach
of this assay is useful in dissecting the mechanisms of drug
action. For example, the area, brightness, and fragmentation of the
nucleus 298 and actin polymerization values 294 reached a maximum
value when SNB-19 cells were treated with 10 nM paclitaxel (FIG.
24; top and bottom graphs). However, mitochondrial potential 295
was minimal at the same concentration of paclitaxel (FIG. 24;
middle graph). The fact that all the parameters measured approached
control levels at increasing paclitaxel concentrations (>10 nM)
suggests that SNB-19 cells have low affinity drug metabolic or
clearance pathways that are compensatory at sufficiently high
levels of the drug. Contrasting the drug sensitivity of SNB-19
cells 297 L-929 showed a different response to paclitaxel 296.
These fibroblastic cells showed a maximal response in many
parameters at 5 .mu.M paclitaxel, a 500-fold higher dose than
SNB-19 cells. Furthermore, the L-929 cells did not show a sharp
decrease in mitochondrial potential 295 at any of the paclitaxel
concentrations tested. This result is consistent with the presence
of unique apoptosis pathways between a normal and cancer cell line.
Therefore, these results indicate that a relatively simple
fluorescence labeling protocol can be coupled with the cell
screening system of the present invention to produce a high-content
screen of key events involved in programmed cell death.
EXAMPLE 7
Protease Induced Translocation of a Signaling Enzyme Containing a
Disease-Associated Sequence from Cytoplasm to Nucleus.
[0187] Plasmid Construct.
[0188] A eukaryotic expression plasmid containing a coding sequence
for a green fluorescent protein--caspase (Cohen (1997), Biochemical
J. 326:1-16; Liang et al. (1997), J. of Molec. Biol. 274:291-302)
chimera is prepared using GFP mutants. The construct is used to
transfect eukaryotic cells.
[0189] Cell Preparation and Transfection.
[0190] Cells are trypsinized and plated 24 h prior to transfection
and incubated at 37.degree. C. and 5% CO.sub.2. Transfections are
performed by methods including, but not limited to calcium
phosphate coprecipitation or lipofection. Cells are incubated with
the calcium phosphate-DNA precipitate for 4-5 hours at 37.degree.
C. and 5% CO.sub.2, washed 3-4 times with DMEM to remove the
precipitate, followed by the addition of C-DMEM. Lipofectamine
transfections are performed in serum-free DMEM without antibiotics
according to the manufacturer's instructions. Following a 2-3 hour
incubation with the DNA-liposome complexes, the medium is removed
and replaced with C-DMEM.
[0191] Apopototic Induction of Caspase-GFP Translocation.
[0192] To obtain Caspase-GFP translocation kinetic data, nuclei of
transfected cells are first labeled with 5 .mu.g/ml Hoechst 33342
(Molecular Probes) in C-DMEM for 20 minutes at 37.degree. C. and 5%
CO.sub.2. Cells are washed once in Hank's Balanced Salt Solution
(HBSS) followed by the addition of compounds that induce apoptosis.
These compounds include, but are not limited to paclitaxel,
staurosporine, ceramide, and tumor necrosis factor. To obtain fixed
time point titration data, transfected cells are first washed with
DMEM and then incubated at 37.degree. C. and 5% CO.sub.2 for 1 h in
the presence of 0-1000 .mu.M compound in DMEM. Cells are analyzed
live or they are rinsed with HBSS, fixed for 15 min with 3.7%
formaldehyde in HBSS, stained with Hoechst 33342, and washed before
analysis.
[0193] Image Acquisition and Analysis.
[0194] Kinetic data are collected by acquiring fluorescence image
pairs (Caspase-GFP and Hoechst 33342-labeled nuclei) from fields of
living cells at 1 min intervals for 30 min after the addition of
compound. Likewise, image pairs are obtained from each well of the
fixed time point screening plates 1 h after the addition of
compound. In both cases, the image pairs obtained at each time
point are used to define nuclear and cytoplasmic regions in each
cell. Translocation of Caspase-GFP is calculated by dividing the
integrated fluorescence intensity of Caspase-GFP in the nucleus by
the integrated fluorescence intensity of the chimera in the
cytoplasm or as a nuclear-cytoplasmic difference of GFP
fluorescence. In the fixed time point screen this translocation
ratio is calculated from data obtained from at least 200 cells at
each concentration of compound tested. Drug-induced translocation
of Caspase-GFP from the cytoplasm to the nucleus is therefore
correlated with an increase in the translocation ratio. Molecular
interaction libraries including, but not limited to those
comprising putative activators or inhibitors of apoptosis-activated
enzymes are use to screen the indicator cell lines and identify a
specific ligand for the DAS, and a pathway activated by compound
activity.
EXAMPLE 8
Identification of Novel Steroid Receptors from DAS
[0195] Two sources of material and/or information are required to
make use of this embodiment, which allows assessment of the
function of an uncharacterized gene. First, disease associated
sequence bank(s) containing cDNA sequences suitable for
transfection into mammalian cells can be used. Because every RADE
or differential expression experiment generates up to several
hundred sequences, it is possible to generate an ample supply of
DAS. Second, information from primary sequence database searches
can be used to place DAS into broad categories, including, but not
limited to, those that contain signal sequences, seven
trans-membrane motifs, conserved protease active site domains, or
other identifiable motifs. Based on the information acquired from
these sources, algorithm types and indicator cell lines to be
transfected are selected. A large number of motifs are already well
characterized and encoded in the linear sequences contained within
the large number genes in existing genomic databases.
[0196] In one embodiment, the following steps are taken:
[0197] 1) Information from the DAS identification experiment
(including database searches) is used as the basis for selecting
the relevant biological processes. (for example, look at the DAS
from a tumor line for cell cycle modulation, apoptosis, metastatic
proteases, etc.)
[0198] 2) Sorting of DNA sequences or DAS by identifiable motifs
(ie. signal sequences, 7-transmembrane domains, conserved protease
active site domains, etc.) This initial grouping will determine
fluorescent tagging strategies, host cell lines, indicator cell
lines, and banks of bioactive molecules to be screened, as
described supra.
[0199] 3) Using well established molecular biology methods, ligate
DAS into an expression vector designed for this purpose.
Generalized expression vectors contain promoters, enhancers, and
terminators for which to deliver target sequences to the cell for
transient expression. Such vectors may also contain antibody
tagging sequences, direct association sequences, chromophore fusion
sequences like GFP, etc. to facilitate detection when expressed by
the host.
[0200] 4) Transiently transfect cells with DAS containing vectors
using standard transfection protocols including: calcium phosphate
co-precipitation, liposome mediated, DEAE dextran mediated,
polycationic mediated, viral mediated, or electroporation, and
plate into microtiter plates or microwell arrays. Alternatively,
transfection can be done directly in the microtiter plate
itself
[0201] 5) Carry out the cell screening methods as described
supra.
[0202] In this embodiment, DAS shown to possess a motif(s)
suggestive of transcriptional activation potential (for example,
DNA binding domain, amino terminal modulating domain, hinge region,
or carboxy terminal ligand binding domain) are utilized to identify
novel steroid receptors.
[0203] Defining the fluorescent tags for this experiment involves
identification of the nucleus through staining, and tagging the
DAS-by creating a GFP chimera via insertion of DAS into an
expression vector, proximally fused to the gene encoding GFP.
Alternatively, a single chain antibody fragment with high affinity
to some portion of the expressed DAS could be constructed using
technology available in the art (Cambridge Antibody Technologies)
and linked to a fluorophore (FITC) to tag the putative
transcriptional activator/receptor in the cells. This alternative
would provide an external tag requiring no DNA transfection and
therefore would be useful if distribution data were to be gathered
from the original primary cultures used to generate the DAS.
[0204] Plasmid Construct.
[0205] A eukaryotic expression plasmid containing a coding sequence
for a green fluorescent protein--DAS chimera is prepared using GFP
mutants. The construct is used to transfect HeLa cells. The
plasmid, when transfected into the host cell, produces a GFP fused
to the DAS protein product, designated GFP-DASpp.
[0206] Cell Preparation and Transfection.
[0207] HeLa cells are trypsinized and plated using DMEM containing
5% charcoal/dextran-treated fetal bovine serum (FBS) (Hyclone) and
1% penicillin-streptomycin (C-DMEM) 12-24 hours prior to
transfection and incubated at 37.degree. C. and 5% CO.sub.2.
Transfections are performed by calcium phosphate coprecipitation or
with Lipofectamine (Life Technologies). For the calcium phosphate
transfections, the medium is replaced, prior to transfection, with
DMEM containing 5% charcoal/dextran-treated FBS. Cells are
incubated with the calcium phosphate-DNA precipitate for 4-5 hours
at 37.degree. C. and 5% CO.sub.2, and washed, 3-4 times with DMEM
to remove the precipitate, followed by the addition of C-DMEM.
Lipofectamine transfections are performed in serum-free DMEM
without antibiotics according to the manufacturer's instructions.
Following a 2-3 hour incubation with the DNA-liposome complexes,
the medium is removed and replaced with C-DMEM. All transfected
cells in 96-well microtiter plates are incubated at 33.degree. C.
and 5% CO.sub.2 for 24-48 hours prior to drug treatment.
Experiments are performed with the receptor expressed transiently
in HeLa cells.
[0208] Localization of Expressed GFP-DASpp Inside Cells.
[0209] To obtain cellular distribution data, nuclei of transfected
cells are first labeled with 5 .mu.g/ml Hoechst 33342 (Molecular
Probes) in C-DMEM for 20 minutes at 33.degree. C. and 5% CO.sub.2.
Cells are washed once in Hank's Balanced Salt Solution (HBSS). The
cells are analyzed live or they are rinsed with HBSS, fixed for 15
min with 3.7% formaldehyde in HBSS, stained with Hoechst 33342, and
washed before analysis.
[0210] In a preferred embodiment, image acquisition and analysis
are performed using the cell screening system of the present
invention. The intracellular GFP-DASpp fluorescence signal is
collected by acquiring fluorescence image pairs (GFP-DASpp and
Hoechst 33342-labeled nuclei) from field cells. The image pairs
obtained at each time point are used to define nuclear and
cytoplasmic regions in each cell. Data demonstrating dispersed
signal in the cytoplasm would be consistent with known steroid
receptors that are DNA transcriptional activators.
[0211] Screening for Induction of GFP-DASpp Translocation.
[0212] Using the above construct, confirmed for appropriate
expression of the GFP-DASpp, as an indicator cell line, a screen of
various ligands is performed using a series of steroid type ligands
including, but not limited to: estrogen, progesterone, retinoids,
growth factors, androgens, and many other steroid and steroid based
molecules. Image acquisition and analysis are performed using the
cell screening system of the invention. The intracellular GFP-DASpp
fluorescence signal is collected by acquiring fluorescence image
pairs (GFP-DASpp and Hoechst 33342-labeled nuclei) from fields
cells. The image pairs obtained at each time point are used to
define nuclear and cytoplasmic regions in each cell. Translocation
of GFP-DASpp is calculated by dividing the integrated fluorescence
intensity of GFP-DASpp in the nucleus by the integrated
fluorescence intensity of the chimera in the cytoplasm or as a
nuclear-cytoplasmic difference of GFP fluorescence. A translocation
from the cytoplasm into the nucleus indicates a ligand binding
activation of the DASpp thus identifying the potential receptor
class and action. Combining this data with other data obtained in a
similar fashion using known inhibitors and modifiers of steroid
receptors, would either validate the DASpp as a target, or more
data would be generated from various sources.
EXAMPLE 9
Additional Screens
[0213] Translocation between the plasma membrane and the
cytoplasm:
[0214] Profilactin Complex Dissociation and Binding of Profilin to
the Plasma Membrane.
[0215] In one embodiment, a fluorescent protein biosensor of
profilin membrane binding is prepared by labeling purified profilin
(Federov et al.(1994), J. Molec. Biol. 241:480-482; Lanbrechts et
al. (1995), Eur. J. Biochem. 230:281-286) with a probe possessing a
fluorescence lifetime in the range of 2-300 ns. The labeled
profilin is introduced into living indicator cells using bulk
loading methodology and the indicator cells are treated with test
compounds. Fluorescence anisotropy imaging microscopy (Gough and
Taylor (1993), J. Cell Biol. 121:1095-1107) is used to measure
test-compound dependent movement of the fluorescent derivative of
profilin between the cytoplasm and membrane for a period of time
after treatment ranging from 0.1 s to 10 h.
[0216] Rho-RhoGDI Complex Translocation to the Membrane.
[0217] In another embodiment, indicator cells are treated with test
compounds and then fixed, washed, and permeabilized. The indicator
cell plasma membrane, cytoplasm, and nucleus are all labeled with
distinctly colored markers followed by immunolocalization of Rho
protein (Self et al. (1995), Methods in Enzymology 256:3-10; Tanaka
et al. (1995), Methods in Enzymology 256:41-49) with antibodies
labeled with a fourth color. Each of the four labels is imaged
separately using the cell screening system, and the images used to
calculate the amount of inhibition or activation of translocation
effected by the test compound. To do this calculation, the images
of the probes used to mark the plasma membrane and cytoplasm are
used to mask the image of the immunological probe marking the
location of intracellular Rho protein. The integrated brightness
per unit area under each mask is used to form a translocation
quotient by dividing the plasma membrane integrated brightness/area
by the cytoplasmic integrated brightness/area. By comparing the
translocation quotient values from control and experimental wells,
the percent translocation is calculated for each potential lead
compound.
[0218] .beta.-Arrestin Translocation to the Plasma Membrane upon
G-Protein Receptor Activation.
[0219] In another embodiment of a cytoplasm to membrane
translocation high-content screen, the translocation of
.beta.-arrestin protein from the cytoplasm to the plasma membrane
is measured in response to cell treatment. To measure the
translocation, living indicator cells containing luminescent domain
markers are treated with test compounds and the movement of the
.beta.-arrestin marker is measured in time and space using the cell
screening system of the present invention. In a preferred
embodiment, the indicator cells contain luminescent markers
consisting of a green fluorescent protein .beta.-arrestin
(GFP-.beta.-arrestin) protein chimera (Barak et al. (1997), J.
Biol. Chem. 272:27497-27500; Daaka et al. (1998), J. Biol. Chem.
273:685-688) that is expressed by the indicator cells through the
use of transient or stable cell transfection and other reporters
used to mark cytoplasmic and membrane domains. When the indicator
cells are in the resting state, the domain marker molecules
partition predominately in the plasma membrane or in the cytoplasm.
In the high-content screen, these markers are used to delineate the
cell cytoplasm and plasma membrane in distinct channels of
fluorescence. When the indicator cells are treated with a test
compound, the dynamic redistribution of the GFP-.beta.-arrestin is
recorded as a series of images over a time scale ranging from 0.1 s
to 10 h. In a preferred embodiment, the time scale is 1 h. Each
image is analyzed by a method that quantifies the movement of the
GFP-.beta.-arrestin protein chimera between the plasma membrane and
the cytoplasm. To do this calculation, the images of the probes
used to mark the plasma membrane and cytoplasm are used to mask the
image of the GFP-.beta.-arrestin probe marking the location of
intracellular GFP-.beta.-arrestin protein. The integrated
brightness per unit area under each mask is used to form a
translocation quotient by dividing the plasma membrane integrated
brightness/area by the cytoplasmic integrated brightness/area. By
comparing the translocation quotient values from control and
experimental wells, the percent translocation is calculated for
each potential lead compound. The output of the high-content screen
relates quantitative data describing the magnitude of the
translocation within a large number of individual cells that have
been treated with test compounds of interest.
[0220] Translocation between the Endoplasmic Reticulum and the
Golgi:
[0221] In one embodiment of an endoplasmic reticulum to Golgi
translocation high-content screen, the translocation of a VSVG
protein from the ts045 mutant strain of vesicular stomatitis virus
(Ellenberg et al. (1997), J. Cell Biol. 138:1193-1206; Presley et
al. (1997) Nature 389:81-85) from the endoplasmic reticulum to the
Golgi domain is measured in response to cell treatment. To measure
the translocation, indicator cells containing luminescent reporters
are treated with test compounds and the movement of the reporters
is measured in space and time using the cell screening system of
the present invention. The indicator cells contain luminescent
reporters consisting of a GFP-VSVG protein chimera that is
expressed by the indicator cell through the use of transient or
stable cell transfection and other domain markers used to measure
the localization of the endoplasmic reticulum and Golgi domains.
When the indicator cells are in their resting state at 40.degree.
C., the GFP-VSVG protein chimera molecules are partitioned
predominately in the endoplasmic reticulum. In this high-content
screen, domain markers of distinct colors used to delineate the
endoplasmic reticulum and the Golgi domains in distinct channels of
fluorescence. When the indicator cells are treated with a test
compound and the temperature is simultaneously lowered to
32.degree. C., the dynamic redistribution of the GFP-VSVG protein
chimera is recorded as a series of images over a time scale ranging
from 0.1 s to 10 h. Each image is analyzed by a method that
quantifies the movement of the GFP-VSVG protein chimera between the
endoplasmic reticulum and the Golgi domains. To do this
calculation, the images of the probes used to mark the endoplasmic
reticulum and the Golgi domains are used to mask the image of the
GFP-VSVG probe marking the location of intracellular GFP-VSVG
protein. The integrated brightness per unit area under each mask is
used to form a translocation quotient by dividing the endoplasmic
reticulum integrated brightness/area by the Golgi integrated
brightness/area. By comparing the translocation quotient values
from control and experimental wells, the percent translocation is
calculated for each potential lead compound. The output of the
high-content screen relates quantitative data describing the
magnitude of the translocation within a large number of individual
cells that have been treated with test compounds of interest at
final concentrations ranging from 10.sup.-12 M to 10.sup.-3 M for a
period ranging from 1 min to 10 h.
[0222] Induction and Inhibition of Organellar Function:
[0223] Intracellular microtubule stability. In one embodiment of an
organellar function high-content screen, the assembly state of
intracellular microtubules is measured in response to cell
treatment. To measure microtubule assembly state, indicator cells
containing luminescent reporters are treated with test compounds
and the distribution of the reporters is measured in space and time
using the cell screening system of the present invention.
[0224] In a preferred embodiment, the reporter of intracellular
microtubule assembly is MAP 4 (Bulinski et al. (1997), J. Cell
Science 110:3055-3064), a ubiquitous microtubule associated protein
that is known to interact with microtubules in interphase and
mitotic cells. The indicator cells contain luminescent reporters
consisting of a GFP-MAP 4 chimera that is expressed by the
indicator cells through the use of transient or stable cell
transfection and other reporters are used to measure the
localization of the cytoplasmic and membrane components. A GFP-MAP
4 construct is prepared as follows: PCR amplification of native or
mutant GFP molecules using primers to introduce restriction enzyme
sites is performed. The PCR product is ligated into the MAP 4 cDNA
within a eukaryotic expression vector. Indicator cells are then
transfected with the expression vector to produce either
transiently or stably transfected indicator cells.
[0225] Indicator cells are treated with test compounds at final
concentrations ranging from 10.sup.-12 M to 10.sup.-3 M for a
period ranging from 1 min to 10 h. Growth medium containing
labeling reagent to mark the nucleus and the cytoplasm are added.
After incubation, the cells are washed with Hank's balanced salt
solution (HBSS), fixed with 3.7% formaldehyde for 10 min at room
temperature, and washed and stored in HBSS.
[0226] Image data are obtained from both fixed and living indicator
cells. To extract morphometric data from each of the images
obtained the following method of analysis is used:
[0227] 1. Threshold each nucleus and cytoplasmic image to produce a
mask that has value=0 for each pixel outside a nucleus or cell
boundary.
[0228] 2. Overlay the mask on the original image, detect each
object in the field (i.e., nucleus or cell), and calculate its
size, shape, and integrated intensity.
[0229] 3. Overlay the whole cell mask obtained above on the
corresponding GFP-MAP 4 image and use an automated measurement of
edge strength routine (Kolega et al. (1993). BioImaging 1:136-150)
to calculate the total edge strength within each cell. To normalize
for cell size, the total edge strength is divided by the cell area
to give a "fibrousness" value. Large fibrousness values are
associated with strong edge strength values and are therefore
maximal in cells containing distinct microtubule structures.
Likewise, small fibrousness values are associated with weak edge
strength and are minimal in cells with depolymerized microtubules.
The physiological range of fibrousness values is set by treating
cells with either the microtubule stabilizing drug paclitaxel (10
.mu.M) or the microtubule depolymerizing drug nocodazole (10
.mu.g/ml).
[0230] High-Content Screens Involving the Functional Localization
of Macromolecules
[0231] Within this class of high-content screen, the functional
localization of macromolecules in response to external stimuli is
measured within living cells.
[0232] Glycolytic Enzyme Activity Regulation.
[0233] In a preferred embodiment of a cellular enzyme activity
high-content screen, the activity of key glycolytic regulatory
enzymes are measured in treated cells. To measure enzyme activity,
indicator cells containing luminescent labeling reagents are
treated with test compounds and the activity of the reporters is
measured in space and time using cell screening system of the
present invention.
[0234] In one embodiment, the reporter of intracellular enzyme
activity is fructose-6-phosphate,
2-kinase/fructose-2,6-bisphosphatase (PFK-2), a regulatory enzyme
whose phosphorylation state indicates intracellular carbohydrate
anabolism or catabolism (Deprez et al. (1997) J. Biol. Chem.
272:17269-17275; Kealer et al. (1996) FEBS Letters 395:225-227; Lee
et al. (1996), Biochemistry 35:6010-6019). The indicator cells
contain luminescent reporters consisting of a fluorescent protein
biosensor of PFK-2 phosphorylation. The fluorescent protein
biosensor is constructed by introducing an environmentally
sensitive fluorescent dye near to the known phosphorylation site of
the enzyme (Deprez et al. (1997), supra; Giuliano et al. (1995),
supra). The dye can be of the ketocyanine class (Kessler and
Wolfbeis (1991), Spectrochimica Acta 47A:187-192) or any class that
contains a protein reactive moiety and a fluorochrome whose
excitation or emission spectrum is sensitive to solution polarity.
The fluorescent protein biosensor is introduced into the indicator
cells using bulk loading methodology.
[0235] Living indicator cells are treated with test compounds, at
final concentrations ranging from 10.sup.-12 M to 10.sup.-3 M for
times ranging from 0.1 s to 10 h. In a preferred embodiment, ratio
image data are obtained from living treated indicator cells by
collecting a spectral pair of fluorescence images at each time
point. To extract morphometric data from each time point, a ratio
is made between each pair of images by numerically dividing the two
spectral images at each time point, pixel by pixel. Each pixel
value is then used to calculate the fractional phosphorylation of
PFK-2. At small fractional values of phosphorylation, PFK-2
stimulates carbohydrate catabolism. At high fractional values of
phosphorylation, PFK-2 stimulates carbohydrate anabolism.
[0236] Protein Kinase A Activity and Localization of Subunits.
[0237] In another embodiment of a high-content screen, both the
domain localization and activity of protein kinase A (PKA) within
indicator cells are measured in response to treatment with test
compounds.
[0238] The indicator cells contain luminescent reporters including
a fluorescent protein biosensor of PKA activation. The fluorescent
protein biosensor is constructed by introducing an environmentally
sensitive fluorescent dye into the catalytic subunit of PKA near
the site known to interact with the regulatory subunit of PKA
(Harootunian et al. (1993), Mol. Biol. of the Cell 4:993-1002;
Johnson et al. (1996), Cell 85:149-158; Giuliano et al. (1995),
supra). The dye can be of the ketocyanine class (Kessler, and
Wolfbeis (1991), Spectrochimica Acta 47A:187-192) or any class that
contains a protein reactive moiety and a fluorochrome whose
excitation or emission spectrum is sensitive to solution polarity.
The fluorescent protein biosensor of PKA activation is introduced
into the indicator cells using bulk loading methodology.
[0239] In one embodiment, living indicator cells are treated with
test compounds, at final concentrations ranging from 10.sup.-12 M
to 10.sup.-3 M for times ranging from 0.1 s to 10 h. In a preferred
embodiment, ratio image data are obtained from living treated
indicator cells. To extract biosensor data from each time point, a
ratio is made between each pair of images, and each pixel value is
then used to calculate the fractional activation of PKA (e.g.,
separation of the catalytic and regulatory subunits after cAMP
binding). At high fractional values of activity, PFK-2 stimulates
biochemical cascades within the living cell.
[0240] To measure the translocation of the catalytic subunit of
PKA, indicator cells containing luminescent reporters are treated
with test compounds and the movement of the reporters is measured
in space and time using the cell screening system. The indicator
cells contain luminescent reporters consisting of domain markers
used to measure the localization of the cytoplasmic and nuclear
domains. When the indicator cells are treated with a test
compounds, the dynamic redistribution of a PKA fluorescent protein
biosensor is recorded intracellularly as a series of images over a
time scale ranging from 0.1 s to 10 h. Each image is analyzed by a
method that quantifies the movement of the PKA between the
cytoplasmic and nuclear domains. To do this calculation, the images
of the probes used to mark the cytoplasmic and nuclear domains are
used to mask the image of the PKA fluorescent protein biosensor.
The integrated brightness per unit area under each mask is used to
form a translocation quotient by dividing the cytoplasmic
integrated brightness/area by the nuclear integrated
brightness/area. By comparing the translocation quotient values
from control and experimental wells, the percent translocation is
calculated for each potential lead compound. The output of the
high-content screen relates quantitative data describing the
magnitude of the translocation within a large number of individual
cells that have been treated with test compound in the
concentration range of 10.sup.-12 M to 10.sup.-3 M.
[0241] High-content Screens Involving the Induction or Inhibition
of Gene Expression RNA-based-fluorescent Biosensors
[0242] Cytoskeletal Protein Transcription and Message
Localization.
[0243] Regulation of the general classes of cell physiological
responses including cell-substrate adhesion, cell-cell adhesion,
signal transduction, cell-cycle events, intermediary and signaling
molecule metabolism, cell locomotion, cell-cell communication, and
cell death can involve the alteration of gene expression.
High-content screens can also be designed to measure this class of
physiological response.
[0244] In one embodiment, the reporter of intracellular gene
expression is an oligonucleotide that can hybridize with the target
mRNA and alter its fluorescence signal. In a preferred embodiment,
the oligonucleotide is a molecular beacon (Tyagi and Kramer (1996)
Nat. Biotechnol. 14:303-308), a luminescence-based reagent whose
fluorescence signal is dependent on intermolecular and
intramolecular interactions. The fluorescent biosensor is
constructed by introducing a fluorescence energy transfer pair of
fluorescent dyes. such that there is one at each end (5' and 3') of
the reagent. The dyes can be of any class that contains a protein
reactive moiety and fluorochromes whose excitation and emission
spectra overlap sufficiently to provide fluorescence energy
transfer between the dyes in the resting state, including, but not
limited to, fluorescein and rhodamine (Molecular Probes, Inc.). In
a preferred embodiment, a portion of the message coding for
.beta.-actin (Kislauskis et al. (1994), J. Cell Biol. 127:441-451;
McCann et al. (1997), Proc. Natl. Acad. Sci. 94:5679-5684; Sutoh
(1982), Biochemistry 21:3654-3661) is inserted into the loop region
of a hairpin-shaped oligonucleotide with the ends tethered together
due to intramolecular hybridization. At each end of the biosensor a
fluorescence donor (fluorescein) and a fluorescence acceptor
(rhodamine) are covalently bound. In the tethered state, the
fluorescence energy transfer is maximal and therefore indicative of
an unhybridized molecule. When hybridized with the mRNA coding for
.beta.-actin, the tether is broken and energy transfer is lost. The
complete fluorescent biosensor is introduced into the indicator
cells using bulk loading methodology.
[0245] In one embodiment, living indicator cells are treated with
test compounds, at final concentrations ranging from 10.sup.-12 M
to 10.sup.-3 M for times ranging from 0.1 s to 10 h. In a preferred
embodiment, ratio image data are obtained from living treated
indicator cells. To extract morphometric data from each time point,
a ratio is made between each pair of images, and each pixel value
is then used to calculate the fractional hybridization of the
labeled nucleotide. At small fractional values of hybridization
little expression of .beta.-actin is indicated. At high fractional
values of hybridization, maximal expression of .beta.-actin is
indicated. Furthermore, the distribution of hybridized molecules
within the cytoplasm of the indicator cells is also a measure of
the physiological response of the indicator cells.
[0246] Cell Surface Binding of a Ligand
[0247] Labeled Insulin Binding to its Cell Surface Receptor in
Living Cells.
[0248] Cells whose plasma membrane domain has been labeled with a
labeling reagent of a particular color are incubated with a
solution containing insulin molecules (Lee et al. (1997),
Biochemistry 36:2701-2708; Martinez-Zaguilan et al. (1996), Am. J.
Physiol. 270:C1438-C1446) that are labeled with a luminescent probe
of a different color for an appropriate time under the appropriate
conditions. After incubation, unbound insulin molecules are washed
away, the cells fixed and the distribution and concentration of the
insulin on the plasma membrane is measured. To do this, the cell
membrane image is used as a mask for the insulin image. The
integrated intensity from the masked insulin image is compared to a
set of images containing known amounts of labeled insulin. The
amount of insulin bound to the cell is determined from the
standards and used in conjunction with the total concentration of
insulin incubated with the cell to calculate a dissociation
constant or insulin to its cell surface receptor.
[0249] Labeling of Cellular Compartments
[0250] Whole Cell Labeling
[0251] Whole cell labeling is accomplished by labeling cellular
components such that dynamics of cell shape and motility of the
cell can be measured over time by analyzing fluorescence images of
cells.
[0252] In one embodiment, small reactive fluorescent molecules are
introduced into living cells. These membrane-permeant molecules
both diffuse through and react with protein components in the
plasma membrane. Dye molecules react with intracellular molecules
to both increase the fluorescence signal emitted from each molecule
and to entrap the fluorescent dye within living cells. These
molecules include reactive chloromethyl derivatives of
aminocoumarins, hydroxycoumarins, eosin diacetate, fluorescein
diacetate, some Bodipy dye derivatives, and tetramethylrhodamine.
The reactivity of these dyes toward macromolecules includes free
primary amino groups and free sulfhydryl groups.
[0253] In another embodiment, the cell surface is labeled by
allowing the cell to interact with fluorescently labeled antibodies
or lectins (Sigma Chemical Company, St. Louis, Mo.) that react
specifically with molecules on the cell surface. Cell surface
protein chimeras expressed by the cell of interest that contain a
green fluorescent protein, or mutant thereof, component can also be
used to fluorescently label the entire cell surface. Once the
entire cell is labeled, images of the entire cell or cell array can
become a parameter in high content screens, involving the
measurement of cell shape, motility, size, and growth and
division.
[0254] Plasma Membrane Labeling
[0255] In one embodiment, labeling the whole plasma membrane
employs some of the same methodology described above for labeling
the entire cells. Luminescent molecules that label the entire cell
surface act to delineate the plasma membrane.
[0256] In a second embodiment subdomains of the plasma membrane,
the extracellular surface, the lipid bilayer, and the intracellular
surface can be labeled separately and used as components of high
content screens. In the first embodiment, the extracellular surface
is labeled using a brief treatment with a reactive fluorescent
molecule such as the succinimidyl ester or iodoacetamde derivatives
of fluorescent dyes such as the fluoresceins, rhodamines, cyanines,
and Bodipys.
[0257] In a third embodiment, the extracellular surface is labeled
using fluorescently labeled macromolecules with a high affinity for
cell surface molecules. These include fluorescently labeled lectins
such as the fluorescein, rhodamine, and cyanine derivatives of
lectins derived from jack bean (Con A), red kidney bean
(erythroagglutinin PHA-E), or wheat germ.
[0258] In a fourth embodiment, fluorescently labeled antibodies
with a high affinity for cell surface components are used to label
the extracellular region of the plasma membrane. Extracellular
regions of cell surface receptors and ion channels are examples of
proteins that can be labeled with antibodies.
[0259] In a fifth embodiment, the lipid bilayer of the plasma
membrane is labeled with fluorescent molecules. These molecules
include fluorescent dyes attached to long chain hydrophobic
molecules that interact strongly with the hydrophobic region in the
center of the plasma membrane lipid bilayer. Examples of these dyes
include the PKH series of dyes (U.S. Pat. Nos. 4,783,401,
4,762,701, and 4,859,584; available commercially from Sigma
Chemical Company, St. Loius, Mo.), fluorescent phospholipids such
as nitrobenzoxadiazole glycerophosphoethanolamine and
fluorescein-derivatized dihexadecanoylglycerophosphoetha-nolamine,
fluorescent fatty acids such as
5-butyl-4,4-difluoro-4-bora-3a,4a-diaza-s- -indacene-3-nonanoic
acid and 1-pyrenedecanoic acid (Molecular Probes, Inc.),
fluorescent sterols including cholesteryl 4,4-difluoro-5,7-dimethy-
l-4-bora-3a,4a-diaza-s-indacene-3-dodecanoate and cholesteryl
1-pyrenehexanoate, and fluorescently labeled proteins that interact
specifically with lipid bilayer components such as the fluorescein
derivative of annexin V (Caltag Antibody Co, Burlingame,
Calif.).
[0260] In another embodiment, the intracellular component of the
plasma membrane is labeled with fluorescent molecules. Examples of
these molecules are the intracellular components of the trimeric
G-protein receptor, adenylyl cyclase, and ionic transport proteins.
These molecules can be labeled as a result of tight binding to a
fluorescently labeled specific antibody or by the incorporation of
a fluorescent protein chimera that is comprised of a
membrane-associated protein and the green fluorescent protein, and
mutants thereof.
[0261] Endosome Fluorescence Labeling
[0262] In one embodiment, ligands that are transported into cells
by receptor-mediated endocytosis are used to trace the dynamics of
endosomal organelles. Examples of labeled ligands include Bodipy
FL-labeled low density lipoprotein complexes, tetramethylrhodamine
transferrin analogs, and fluorescently labeled epidermal growth
factor (Molecular Probes, Inc.)
[0263] In a second embodiment, fluorescently labeled primary or
secondary antibodies (Sigma Chemical Co. St. Louis, Mo.; Molecular
Probes, Inc. Eugene, Oreg.; Caltag Antibody Co.) that specifically
label endosomal ligands are used to mark the endosomal compartment
in cells.
[0264] In a third embodiment, endosomes are fluorescently labeled
in cells expressing protein chimeras formed by fusing a green
fluorescent protein, or mutants thereof, with a receptor whose
internalization labels endosomes. Chimeras of the EGF, transferrin,
and low density lipoprotein receptors are examples of these
molecules.
[0265] Lysosome Labeling
[0266] In one embodiment, membrane permeant lysosome-specific
luminescent reagents are used to label the lysosomal compartment of
living and fixed cells. These reagents include the luminescent
molecules neutral red,
N-(3-((2,4-dinitrophenyl)amino)propyl)-N(3-aminopropyl)methylamine,
and the LysoTracker probes which report intralysosomal pH as well
as the dynamic distribution of lysosomes (Molecular Probes,
Inc.)
[0267] In a second embodiment, antibodies against lysosomal
antigens (Sigma Chemical Co.; Molecular Probes, Inc.; Caltag
Antibody Co.) are used to label lysosomal components that are
localized in specific lysosomal domains. Examples of these
components are the degradative enzymes involved in cholesterol
ester hydrolysis, membrane protein proteases, and nucleases as well
as the ATP-driven lysosomal proton pump.
[0268] In a third embodiment, protein chimeras consisting of a
lysosomal protein genetically fused to an intrinsically luminescent
protein such as the green fluorescent protein, or mutants thereof,
are used to label the lysosomal domain. Examples of these
components are the degradative enzymes involved in cholesterol
ester hydrolysis, membrane protein proteases, and nucleases as well
as the ATP-driven lysosomal proton pump.
[0269] Cytoplasmic Fluorescence Labeling
[0270] In one embodiment, cell permeant fluorescent dyes (Molecular
Probes, Inc.) with a reactive group are reacted with living cells.
Reactive dyes including monobromobimane, 5-chloromethylfluorescein
diacetate, carboxy fluorescein diacetate succinimidyl ester, and
chloromethyl tetramethylrhodamine are examples of cell permeant
fluorescent dyes that are used for long term labeling of the
cytoplasm of cells.
[0271] In a second embodiment, polar tracer molecules such as
Lucifer yellow and cascade blue-based fluorescent dyes (Molecular
Probes, Inc.) are introduced into cells using bulk loading methods
and are also used for cytoplasmic labeling.
[0272] In a third embodiment, antibodies against cytoplasmic
components (Sigma Chemical Co.; Molecular Probes, Inc.; Caltag
Antibody Co.) are used to fluorescently label the cytoplasm.
Examples of cytoplasmic antigens are many of the enzymes involved
in intermediary metabolism. Enolase, phosphofructokinase, and
acetyl-CoA dehydrogenase are examples of uniformly distributed
cytoplasmic antigens.
[0273] In a fourth embodiment, protein chimeras consisting of a
cytoplasmic protein genetically fused to an intrinsically
luminescent protein such as the green fluorescent protein, or
mutants thereof, are used to label the cytoplasm. Fluorescent
chimeras of uniformly distributed proteins are used to label the
entire cytoplasmic domain. Examples of these proteins are many of
the proteins involved in intermediary metabolism and include
enolase, lactate dehydrogenase, and hexokinase.
[0274] In a fifth embodiment, antibodies against cytoplasmic
antigens (Sigma Chemical Co.; Molecular Probes, Inc.; Caltag
Antibody Co.) are used to label cytoplasmic components that are
localized in specific cytoplasmic sub-domains. Examples of these
components are the cytoskeletal proteins actin, tubulin, and
cytokeratin. A population of these proteins within cells is
assembled into discrete structures, which in this case, are
fibrous. Fluorescence labeling of these proteins with
antibody-based reagents therefore labels a specific sub-domain of
the cytoplasm.
[0275] In a sixth embodiment, non-antibody-based fluorescently
labeled molecules that interact strongly with cytoplasmic proteins
are used to label specific cytoplasmic components. One example is a
fluorescent analog of the enzyme DNAse I (Molecular Probes, Inc.)
Fluorescent analogs of this enzyme bind tightly and specifically to
cytoplasmic actin, thus labeling a sub-domain of the cytoplasm. In
another example, fluorescent analogs of the mushroom toxin
phalloidin or the drug paclitaxel (Molecular Probes, Inc.) are used
to label components of the actin- and microtubule-cytoskeletons,
respectively.
[0276] In a seventh embodiment, protein chimeras consisting of a
cytoplasmic protein genetically fused to an intrinsically
luminescent protein such as the green fluorescent protein, or
mutants thereof, are used to label specific domains of the
cytoplasm. Fluorescent chimeras of highly localized proteins are
used to label cytoplasmic subdomains. Examples of these proteins
are many of the proteins involved in regulating the cytoskeleton.
They include the structural proteins actin, tubulin, and
cytokeratin as well as the regulatory proteins microtubule
associated protein 4 and .alpha.-actinin.
[0277] Nuclear Labeling
[0278] In one embodiment, membrane permeant nucleic-acid-specific
luminescent reagents (Molecular Probes, Inc.) are used to label the
nucleus of living and fixed cells. These reagents include
cyanine-based dyes (e.g., TOTO.RTM., YOYO.RTM., and BOBO.TM.),
phenanthidines and acridines (e.g., ethidium bromide, propidium
iodide, and acridine orange), indoles and imidazoles (e.g., Hoechst
33258, Hoechst 33342, and 4',6-diamidino-2-phenylindole), and other
similar reagents (e.g., 7-aminoactinomycin D, hydroxystilbamidine,
and the psoralens).
[0279] In a second embodiment, antibodies against nuclear antigens
(Sigma Chemical Co.; Molecular Probes, Inc.; Caltag Antibody Co.)
are used to label nuclear components that are localized in specific
nuclear domains. Examples of these components are the
macromolecules involved in maintaining DNA structure and function.
DNA, RNA, histones, DNA polymerase, RNA polymerase, lamins, and
nuclear variants of cytoplasmic proteins such as actin are examples
of nuclear antigens.
[0280] In a third embodiment, protein chimeras consisting of a
nuclear protein genetically fused to an intrinsically luminescent
protein such as the green fluorescent protein, or mutants thereof,
are used to label the nuclear domain. Examples of these proteins
are many of the proteins involved in maintaining DNA structure and
function. Histones, DNA polymerase, RNA polymerase, lamins, and
nuclear variants of cytoplasmic proteins such as actin are examples
of nuclear proteins.
[0281] Mitochondrial Labeling
[0282] In one embodiment, membrane permeant mitochondrial-specific
luminescent reagents (Molecular Probes, Inc.) are used to label the
mitochondria of living and fixed cells. These reagents include
rhodamine 123, tetramethyl rosamine, JC-1, and the MitoTracker
reactive dyes.
[0283] In a second embodiment, antibodies against mitochondrial
antigens (Sigma Chemical Co.; Molecular Probes, Inc.; Caltag
Antibody Co.) are used to label mitochondrial components that are
localized in specific mitochondrial domains. Examples of these
components are the macromolecules involved in maintaining
mitochondrial DNA structure and function. DNA, RNA, histones, DNA
polymerase, RNA polymerase, and mitochondrial variants of
cytoplasmic macromolecules such as mitochondrial tRNA and rRNA are
examples mitochondrial antigens. Other examples of mitochondrial
antigens are the components of the oxidative phosphorylation system
found in the mitochondria (e.g., cytochrome c, cytochrome c
oxidase, and succinate dehydrogenase).
[0284] In a third embodiment, protein chimeras consisting of a
mitochondrial protein genetically fused to an intrinsically
luminescent protein such as the green fluorescent protein, or
mutants thereof, are used to label the mitochondrial domain.
Examples of these components are the macromolecules involved in
maintaining mitochondrial DNA structure and function. Examples
include histones, DNA polymerase, RNA polymerase, and the
components of the oxidative phosphorylation system found in the
mitochondria (e.g., cytochrome c, cytochrome c oxidase, and
succinate dehydrogenase).
[0285] Endoplasmic Reticulum Labeling
[0286] In one embodiment, membrane permeant endoplasmic
reticulum-specific luminescent reagents (Molecular Probes, Inc.)
are used to label the endoplasmic reticulum of living and fixed
cells. These reagents include short chain carbocyanine dyes (e.g.,
DiOC.sub.6 and DiOC.sub.3), long chain carbocyanine dyes (e.g.,
DiIC.sub.16 and DiIC.sub.18), and luminescently labeled lectins
such as concanavalin A.
[0287] In a second embodiment, antibodies against endoplasmic
reticulum antigens (Sigma Chemical Co.; Molecular Probes, Inc.;
Caltag Antibody Co.) are used to label endoplasmic reticulum
components that are localized in specific endoplasmic reticulum
domains. Examples of these components are the macromolecules
involved in the fatty acid elongation systems,
glucose-6-phosphatase, and HMG CoA-reductase.
[0288] In a third embodiment, protein chimeras consisting of a
endoplasmic reticulum protein genetically fused to an intrinsically
luminescent protein such as the green fluorescent protein, or
mutants thereof, are used to label the endoplasmic reticulum
domain. Examples of these components are the macromolecules
involved in the fatty acid elongation systems,
glucose-6-phosphatase, and HMG CoA-reductase.
[0289] Golgi Labeling
[0290] In one embodiment, membrane permeant Golgi-specific
luminescent reagents (Molecular Probes, Inc.) are used to label the
Golgi of living and fixed cells. These reagents include
luminescently labeled macromolecules such as wheat germ agglutinin
and Brefeldin A as well as luminescently labeled ceramide.
[0291] In a second embodiment, antibodies against Golgi antigens
(Sigma Chemical Co.; Molecular Probes, Inc.; Caltag Antibody Co.)
are used to label Golgi components that are localized in specific
Golgi domains. Examples of these components are N-acetylglucosamine
phosphotransferase, Golgi-specific phosphodiesterase, and
mannose-6-phosphate receptor protein.
[0292] In a third embodiment, protein chimeras consisting of a
Golgi protein genetically fused to an intrinsically luminescent
protein such as the green fluorescent protein, or mutants thereof,
are used to label the Golgi domain. Examples of these components
are N-acetylglucosamine phosphotransferase, Golgi-specific
phosphodiesterase, and mannose-6-phosphate receptor protein.
[0293] While many of the examples presented involve the measurement
of single cellular processes, this is again is intended for
purposes of illustration only. Multiple parameter high-content
screens can be produced by combining several single parameter
screens into a multiparameter high-content screen or by adding
cellular parameters to any existing high-content screen.
Furthermore, while each example is described as being based on
either live or fixed cells, each high-content screen can be
designed to be used with both live and fixed cells.
[0294] Those skilled in the art will recognize a wide variety of
distinct screens that can be developed based on the disclosure
provided herein. There is a large and growing list of known
biochemical and molecular processes in cells that involve
translocations or reorganizations of specific components within
cells. The signaling pathway from the cell surface to target sites
within the cell involves the translocation of plasma
membrane-associated proteins to the cytoplasm. For example, it is
known that one of the src family of protein tyrosine kinases,
pp60c-src (Walker et al (1993), J. Biol. Chem. 268:19552-19558)
translocates from the plasma membrane to the cytoplasm upon
stimulation of fibroblasts with platelet-derived growth factor
(PDGF). Additionally, the targets for screening can themselves be
converted into fluorescence-based reagents that report molecular
changes including ligand-binding and post-translocational
modifications.
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