CA2410688C - A system for cell-based screening - Google Patents

Abstract

The present invention provides systems, methods, and screens for an optic al system analysis of cells to rapidly determine the distribution, environment, or activity of fluorescently labeled reporter molecules in cells for the purpos e 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 numerou s 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 microplate having cells in a micropaterned array of locations. The invention includes apparatu s and computerized method for processing, displaying and storing the data.

Description

A SYS'fEllR Ft)lt (:Ii,LL-I~ASEI) SCREENING
Field of The Invention This invention is in thc: held of fluorescence-based cell and molecular biochemical assays for drug discovery.
Backt=_round of the Invention Drug discovery, as currently pr~~rcticed in the art, is a long, multiple step process involving identification of specific riisease targets, development of an assay based on a specific target, validation of the a~:,s<~y, optimization and automation of the assay to produce a screen, hieh throughput ;screening of compound libraries using the assay to identify "lots", hit validation and hit compound optimization, The output of tlvs 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;. ~Eesting compound efficacy in living biological systems.

Historically, drug discovery is a slow arld costly process, spanning numerous years and consuming hundreds of millions of dollars per drug created.
Developments in the areas of genomics and hi;h throughput screening have resulted in increased capacity and efficiency in the are,:l.s of target identification and volume of compounds screened. Significant advances in automated DNA sequencing, PCR application, positional cloning, hybridizati<7n ~tr~ays, and b101Ilformatlcs have greatly increased the number of genes (and gene fra~rrnents) encoding potential drug screening targets.
However, the basic scheme for dru; screening rernians the same.
Validation of genomic targets as points for therapeutic intervention using the to existing methods and protocol , ha; hecoltre a bottlelaeck in the drug discovery proce<.~s due to the slow, manual metho;_ls employed, such as ~n uvo functional models, functional analysis of recombinant proteins, and stable cell line expression of candidate genes. Primary DN.~1 sequence data acquired through automated sequencing does not permit identification of gene function, hut can provide; inforn~ation about common t 5 "motifs" and specific gene homology when compared to known sequence databases.
Genomic methods such as subtrac~!ion hybridi-r,ation ;end It.,AL>1~ (rapid amplification of differential expression) can be used to identify genes that are up or down regulated in a disease state model. However, ider~cilication and validation still proceed down the same pathway. Some proteomic methods use protein ident5fication t~~lobal expression arrays, ?0 2D electrophoresis, combinatorial libraries) in combination with reverse genetics to identify candidate genes of interest. Such putative "~.iisrase associated sequences" or DAS isolated as intact cDNA arc a ;;real advarotage to these methods. but they are identified by the hundreds without providing any intorrrlation regarding type, activity, and distribution of the encoded prc:~teirZ. t'hoosing 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 pravide new technologies to rapidly screen DAS to establish biolagical function, thereby improving target validation and candidate optimization in dru,, discovery.
There are three major avenues for improving early drug discovery productivity.
First, there is a need for tools that l7rovide increased info>rmation handling capability.
Bioinformatics has blossomed with the rapid development of DNA sequencing systems and the evolution of the genomic:s clatahase. Genomics is beginning to play a critical role in the identification of potenvial new targets. Proteomics has become indispensible 1o in relating structure and function of Iv>rotein targets in order to predict drug interactions.
However, the next level of bialoc;ical complexity is the cell. ~hherefr~re, there is a need to acquire, manage and search rvulti-dimensional infcrrmatian from cells.
Secondly, there is a need for higher throu;_~hput tools. Automation is a key to improving productivity as has already been cicr77onstrated in DNA sequencing and high throughput n primary screening. The instant iia~ewtion provides for automated systems that extract multiple parameter information Irr>rr7 cells that meet the need for higher throughput tools. The instant invention else provides for miniaturizing the methods, thereby allowing increased throughput, w~~ile decreasing the volumes of reagents and test compounds requirecj in each assa~r.
2o Radioactivity has been tl~e uominant read-out in e<rriv drug discovery assays.
However, the need for more inl~~rm~rtion, higher throughput and miniaturization has caused a shift towards using tluorescence detectior~_ Flr,wrc cence-based reagents c:an yield more powerful, multiple parameter assays that are higher rn throughput and information content and require lower volumes of reagents and test compounds.
Fluorescence is also safer and less expensive than radioactivity-based methods.
Screening of cells treated ~rith dyes and fluorescent reagents is well known in the art. There is a considerable body of literahtre 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 GF'P are disclosed by Morise et al.
(Biochemistry 13 (1974), p. 2656-~2fa62), and Ward et al. (Photochem.
Photobiol. 31 (1980), p. 611-615). The GFP of.' the jellyfish .4eguorea victoria has an excitation maximum at 395 nm and an emission maximum at 510 nm, and does not require an exogenous factor for fluorescence ~rctivity.. 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. 1501-12504)), as a tool for visualizing subcellular organelles (ltizzuto et al., (.,urr. Bi.;alc~y ~ (1995), p. ti35-fi42)), visualization of protein transport along the secretory pathv,~a;y (Kaether and Gerdes, FEES Letters 369 (1995), p. 267-271)), expression in plant cells (I-lu and Cheng, FEBS Letters 369 (1995), p.
331-334)) and Drosophila embryos (Davis et al., f~ev. Bialo~ry 170 (1995), p.

729)), and as a reporter molecule; hosed to another protein of" interest (U.
S. Patent 5,491,084 issued February 13,199t~). Similarly, W096/2:3898 published August 8,1996, relates to methods of detecting l~ialogically active substances affecting intracellular processes by utilizing a GF'P constmct having a protein kinase activation site.
Numerous references are :related to GFl' proteins in biological systems. For example, WO 96/09598 published March 28,1996, describes a system for isolating cells of interest utilizing the expression caf a UFP like protein. WO 96/27675 published September 12, 1996, describes tho expression of GI~F' in plants. WO 95/21191 published August 10, 1995, describes modified GFP protein expressed in transformed organisms to detect rnutagen Isis. U. S. Patents 5,401,629, issued March 28, 1995, and 5,4:6,128, issued Juty 2'.i, 1995, describe assays and compositions for detecting and evaluating the intracellular transduction of an extracellular signal using recombinant cells that cyxl>ress cell surface receptors and contain reporter gene constructs that include transcripoional regulatory elements that are responsive to the activity of the cell surface receptors.
Performing a screen on many thousands of compounds requires parallel handling and processing of many compounds and assay component reagents.
Standard high throughput screens ("I-iTS") tisc~ 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 ~.:ignal 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.
Science Applications Intennational Corporation (SAIC.'j 130 Fifth Avenue, Seattle, WA. 98109] describes arll inuaging 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.
rM
Molecular Devices, lnc. (S:mnyvale, CA) 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 backgroun.::l 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- vrc;ll l~or cell-based assays. Fluid delivery devices have also been incorporated into cell based screening systems, such as the FLIPRTM
system, in order to initiate a re~p<>rtse, which is then observed as a whole well population average response using .a macro-imaging system.
In contrast to high througtwput screens, various high-content screens ("HCS") have been developed to address th<; need for more detailed information about the temporal-spatial dynamics of cell constituents and processes. High-content screens automate the extraction of multicculor fluorescence information derived from specific fluorescence-based reagents incorp;>rated into cells (Ciiuliana and Taylor (1995), Curr.
Op. Cell Biol. 7:4; Criuliano et al. (1995) Ann. Rev. Biaphys. Biomol. Struct.
24:405).
Cells are analyzed using an optical system that can measure spatial, as well as temporal dynamics. (Farkas et al. {1993) Ar;~n. Rev. Physiol. :5:785; Ciiuliano et al.
(1990) In Optical Microscopy ,for Biology. 13. :Herman and K:, Jacobson (eds.), pp. 543-557.
Wiley-Liss, New York; Hahn et ,al ( 1992) Nature 359:73(i; Waggoner et al.
(1996) Hum. Pathol. 27:494). The concept: is to treat each toll as a "well" that has spatial and temporal information on the activities of the labeled canstit:uents.
The types of biochemical ;:jlld molecular information now accessible through fluorescence-based reagents applied to cells include ion concentrations, membrane potential, specific translocations, f~n~rytne activities, gene; expression, as well as the presence, amounts and patterns of o'etabolites, proteins, lipids, carbohydrates, and nucleic acid sequences (DeBiasio ~:~t ,:i1., ( 1996) Mol. Biol. (dell.
7:1259;Giuliano et al., (1995) Ann. Rev. Biaphys. Biomol. Stroct. 24:405:, Heirn and Tsien, ( 1996) Curr. Biol.
6:178).
(a High-content screens can be performed on either fixed cells, using fluorescently labeled antibodies, biological li~arus, and,%or nucleus acid hybridization probes, or live cells using multicolor fluorescent indicators and "biosensors." T'he choice of fixed or live cell screens depends on the :pec.ihc cell-based assay required.
Fixed cell assays are the: simplest, since an away of initially living cells in a microtiter plate for~tnat can be tr~f:ated with various corr~pounds and doses being tested, then the cells cart be fixed, lat:~eled with specific reagents, and measured.
No environmental control of the cells is required after tixation. Spatial information is acquired, but only at one time point. The availability of thousands of antibodies, 1ct ligands and nucleic: acid hybridisation probes that can ~~e 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.
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.
1. 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 fluoresca~n~c~~ measurements over tune. There is a growing list of fluorescent physiological im'.lic;ators and "biosensors" that can report changes in biochemical and nuolecular acm,.~itit~s within calls (Ciiuliaruo et al_, ( L'~95) .~fttn. Rev.
?C~ Biophys. Biomol. 5'truct. '~4:~(.)s., Ilahn et al., ( I993p In lvluur~esc~ent and Luminescent Probes for Biologiccil Activity. ',%V'I'. Mason, (eed.), pp 349-359, .academic Press, San Diego).
The availability and use of tluorescence-based reagents has helped to advance the development of both fixe~.l and live cell high-content screens. Advances in instrumentation to automatically extract multicolor, high-content information has recently made it possible to develc:~p I-1CS into an automated tool. An article by Taylor., et al. (American Sci~>~trist ~;0 (199::'j, ~~. s~?-33pj desr.ribes many of these methods and their applications. For example, Proffitt et. al. (C:ytometry 24: 204-213 (1996)) describe a semi-automated fluores~:ence digital imaging system for quantifying relative cell numbers in situ in a variety of tiss~.ie culture plate formats, especially 96-well microtiter plates. The system consists of an epifluorescence inverted microscope with a motorized stage, video camera, im~~ge intensifier, and a microcomputer with a PC-TM
Vision digitizer. Turbo Pascal software controls thc~ stage and scans the plate taking multiple images per well. 'the software calculates total fluorescence per well, provides for daily calibration, and configur~;s easily for a variety of tissue culture plate formats..
Thresholding of digital images arid reagents which fluoresce only when taken up by living cells are used to reduce background fluorescence without removing excess fluorescent reagent.
Scanning confocal microscope imaging (Go et al., (1997) Analytical Biochemistry 247:210-215; Cioldman et al., ( 1995) Erperimental C.'ell Research 221:311-319) and multiphoton minwrc>scope imaging (Denk et al., (1990) Science' 248:73; Gratton et al., (1994) Pro~r, ojthe Microscohical ,,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. F'or exarnl>le, it is possible to resolve internal cytoplasmic features of adherent cells from the features on the cell surface. Because scanning multiphoton imaging requires veryr 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. urrachem. ?02:316-330; ~:ierrittsen et al. ( 1997), J. of Fluorescence 7:1 l -15)), providing, additional capability for different detection med:s.
Small, reliable and relatively ins°xpensive laser systems, such as laser diode pumped lasers, are now available to allo~'N multiphoton confocal microscopy to be applied in a fairly routine fashion.
A combination of the biological heterogeneity of cells in populations (Bright, et al., (1989). J. Cell. Phvsiol. 141:41(1; CJiuliano, (1996) (,ell:'hfotil.
(:vtoskel. 35:237)} as well as the high spatial and temporal frequency of cherrucal and molecular information iC~ present within cells, makes it impossible to extract high-content information from populations of cells using existin:whale microtiter plate readers. No existing high-content screening platform ha.; been designed for multicolor, fluorescence-based screens using cells that ,:rre ana:yrcci individually. Sirnilarly, no method is currently available that combines automatec.l fluid delivery t~:> arrays of cells for the purpose of 15 systematically screening compot~ncls for the ability to induce a cellular response that is identified by HCS analysis, ,a~aE.Liallv fi~orn rolls ~;ro~n in microtiter plates.
Furthermore, no method exists iru the art combining, high throughput well-by-well measurements to identify "hits" m one aissay tolowed by a socond high content cell-by-cell measurement on the same platc° of only those wells identified as hits.
2D The instant invention prow: ides systems, methods, and screens that combine high throughput screening (>-TTSI arid high content screening (HC'S1 that significantly improve target validation and candidate optimization by combining many cell screening formats with fluorescence-basa:d molecular reagents and computer-based feature extraction, data analysis, and automation, resulting in increased quantity and speed of c) data collection, shortened cycle tithes, and, ultimately, faster evaluation of promising drug candidates. 'the instant inv~~rttion also provides for miniaturizing the methods, thereby allowing increased throi.~.g.l7put, while decreasing the volumes of reagents and test compounds required in each assay.
S
SUMMARY OF THE INVEN'h'ION
In one aspect, the present invention relates to a method for analyzing cells comprising ~ providing cells containing fluorescent reporter molecules in an array of locations, ~ treating the cells in thc: array of locations with one or more reagents, ~ imaging. numerous cells io each location with fluorescence optics, ~ converting the optical irofi~rnoation into digital data, is ~ utilizing the digital data to determine thE: distribution, environment or activity of the tluore~,cc:r~t?y labeled rcp~irter molecules in the cells and the distribution of the cel';s, and ~ interpreting that inforrn~:~ticm in ternis of at positive, negative or null effect of the compound being tested on the biological function In this embodiment, the method rapidly determines the distribution, environment, or activity of Iluarescently labeled reporter molecules in cells for the purpose of screening large numbers of compounds for those that specifically affect particular biological functions. fhe array of locations may be a microtiter plate or a

2~ microchip which is a microplate leaving cells in art an;tv of locations. In a preferred embodiment, the method inclulc:., computcri.eed means for acquiring, processing, displaying and storing the data rc°ceived. In a preferred embodiment, the method further comprises automated fluid delivery to the array; of cells. In another preferred embodiment, the information oht;:~ined 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.
In another aspect of the present invention, a cell screening system is provided that comprises:
~ a high magnification fluorescence optical system having a microscope objective, ~ an XY' stage adapted for holding a plate containing an array of cells and having a means for proving the plate for proper alignment and focusing on the cell arrays;
~ a digital camera;
~ a light source having optical means liar directing excitation light to cell arrays and a means for directing flrtores;;ent light emitted from the cells to the digital camera; and ~ a computer means for receiving and processing digital data from the digital camera whercrn the c:c~rnputer mca,ns Includes a digital frame grabber for receiving the irnage~ fr(rnr the camera, a ilisplay for user interaction and display of assay resulta, digital storage media for data storage and archiving, and a means f~.~r control., :acduisition, pre>cessin::rnd display of results.
2o 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, f.te cc7mputer means for receiving and processing digital data from the digital camera store's tloe data in a bo>infonnatics data base.
In a further preferred embodiment, the cell. ;screening systen; further comprises a reader that 2.. measures a signal from many or all the walls in parallel. In another preferred embodiment, the cell screening system further comprises :i mechanical-optical means for changing the magnification ~:~f the system, to allow changing modes between high throughput and high content sr-reening. In ainottter preferred embodiment, the cell screening system further comprises a chamber and control system to maintain the 3n temperature, CO~ concentration ,.rnd humidity surrounding the plate at levels required to keep cells alive. In a further prefe°rred embodiment, the cell screening system utilizes a confocal scanning illumination arid detection system, In another aspect of the present invention, a machine readable storage medium comprising a program ci>ntaining a set of instnrctions 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 ~ high magnificatiot7 fluorescence optical system with a stage adapted for holding cells and a means far moving the stage, a digital camera, a light source for receiving and pro:=cessin~~ the digital Data from the digital camera, an<i a to computer means for receiving and processinri the di;~ital data from the digital camera.
Preferred embodiments of the nnachine readable storage medium comprise programs consisting of a set of instnretiors fir causing a cell screening system to execute the procedures set forth in Figures 9, I 1, 12, 13, 14 or 1 ~. Another preferred embodiment compr7ses a program consisting o1' a set of in;atrur~tions for causing a cell screening is system to execute procedures fir clc:tecting the distribution and activity of specific cellular constituents and processes In mast preferred embodiments, the cellular processes include, but are not limited to. nuclear translocation i~f a protein, cellular hypertrophy, apoptosis, and protcasa...induced translocation of a protein.
?o BRIEF DESCRI1'TION,UF TIH_E t)RAWINGS
Figure 1 shows a diagram of the ct~nlponents of the cell-based scanning system.
Figure 2 shows a schematic of the microscope subassembly.
Figure 3 shows the camera subasses~nbly.
?, Figure 4 illustrates cell scanning :~} stem process.

Figure 5 illustrates a user interfac:~ showing major functions to guide the user.
Figure 6 is a block diagram of the two platforn~ 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.
Figure 7 is a detail of an optical system for a single platform architecture of the Dual Mode System for Cell Based Scre°.~er~ing that uses a moveable 'telescope' lens to read all wells of a microtiter plate and a r~mveahle higher magnification lens to read individual cells in a well.
to Figure 8 is an illustration of the ~fluica delivery syster7o f«r acquiring kinetic data on the Cell Based Screening System.
Figure 9 is a flow chart of processinf:~, step for the cell-based scanning system.
Figure 10 A-J illustrates the strategy of the Nuclear 'Cransloc:ation Assay.
Figure 11 is a flow chart detinirrg the processing steps in the Dual Mode System for Cell Based Screening combining high throughput and high content screening of microtiter plates.
Figure 12 is a flow chart defining; tlac processing steps in the High Throughput mode of the System for Cell Based Screenin~~.
Figure 13 is a flow chart ~iefinin~~; tlr<v processing, steps in tire High C"ontent mode of the 2o System for Cell Based Screening.
Figure 14 is a flow chart. defining the processing steps required for acquiring kinetic data in the High Content mode of the System for t'eil BasE;d Screening.
Figure 15 is a flow chart definin,; the processinstops performed within a well during the acquisition of kinetic data.
l Figure 16 is an example of data from a known inhibitor oftranslocation.
Figure 17 is an example of data from a known stimulator oftrauslocation.
Figure 18 illustrates data presentation on a graphical display.
Figure 19 is an illustration of the data from the High Throughput mode of the System for Cell Based Screening. A) is an example of the data passed to the High Content mode, B) of the data acquired in the higl;~ content mode., and C.'} of the results of the analysis of the data.
Figure 20 shows the measuremerut <nf a drug-induced cytoplasm to nuclear translocation.
The localization of GFP--hGR within the cell before and after stimulation with dexamethasone is represented by (.~~.) and (B) respectivel~~.'1~he translocation of GFP-hGR
from the cytoplasm to the nucleus of a cell is depicted in a cell nc>t treated (C) and treated (D) with dexamethasone.
Figure 21 illustrates a graphical user interface of the measurement shown in Figure 20.
Figure 22 illustrates a graphical user interface, wil:h data presentation, of the measurement shown in Fig. 20.
Figure 23 is a graph representing; the kinetic data obtained from the measurements depicted in Fig. 20.
Figure 24 details a high-content screen of drug-induced apoptosrs.
DETAILED DESCRIPTION C>!F' ~l'HE INVENTION:
As used herein, the follcnwing terms have the specii'ic meaning:
Markers of cellrxlar dorr~a~~~ns. Luminescent probes that have high affinity for specific cellular constituents including specific organelles or molecules.
These probes can either be small luminescent moln~<:ul~s or fluorescent 1y tagged rnacromolecules used as "labeling reagents", "environnnental indicators", or "Iiosensors".

Labeling r~ugen~.s. L.,;ribtvling reagents include, but are not limited to, lumineseently labeled n~acrorrnvlccules incluifin~; fluoresec:nt protein analogs and biosensors, luminescent macromolecular chimera: including those formed with 'the green fluorescent protein and mutants thereof, luntinescently labeled primary or secondary antibodies that react v~uth cellular antigens involved in a physiological response, luminescent stains, dyes, and other small molecules.
Markers of cellular tran:~loeatic>ns. Lumiruescently tagged macromolecules or organelles that move from one ccrli oaomain to another during some cellular process or physiological response. Transl.oc:ation marke°rs can either simply report location to relative to the marl'ers of' eel lular olomains or they can also be "biosensors" that report some biochemical or molecular a~;~ti a ity as well.
Biosensors. 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. ~'~ class of luminescently labeled macromolecules designed to sense and report tl~eae changes lrav~: been termed "'fluorescent-protein biosensors". The protein comt7onent of the biosensor provides a highly evolved molecular recognition moiety. .~~. fluorescent molecule attached to the protein component in the proximity of ,an active site transciuces environmental changes into fluorescence signals that are detcv~tcd using a system s~ ith an appropriate temporal and spatial resolution such as the coal scanning system of the present invention.
Because the modulation of native protein ;~ctiviy within the living cell is reversible, and because fluorescent-protein biosensors can he designed to sense reversible changes in protein activity, these biosensors are esscwnt~ally reusable.

Disease associute~ seyuernce.s f"l~.~l~"j- This tf;rtr~ refers to nucleic acid sequences identified by standard techniqu~a, :,uch as primary UN.A sequence data, genomic methods such as subtracaion hvb~ndization and I~:ACtE, and proteomic methods in combination with reverse geneti~rs, as being of drug candidate compounds. The term does not mean that the sequence is only associated with a disease state.
High content screening (Iwi(_'S1 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 limned to, apoptosis, cell division, cell adhesion, locomotion, exocytosis, and cell-~ccll communication. h9ulticolor fluorescence permits t0 multiple targets aril cell procc,sst:~s to be assayed in a single screen.
Cross-correlation of cellular responses will yield 4~ mcalth of information required for target validation and lead optimization.
In one aspect of the present invention. a cell screening system is provided comprising a high magnification fluorescence optical s;vstem having a microscope to objective, an XY stage adapted fc~r holding a plate with an array of locations for holding cells and having a mean's~ for moving the plKrte to align the locations with the microscope objective and a mean=; for movin' the plate in the direction to effect focusing; a digital camera; a light source having opmal means for directing excitation light to cells in the array of~loc:aticm~~ and a means far directing i~luoreseent light emitted '0 from the cells to the digital catnertt; and a computer means for receiving and processing digital data from tle digital canu~~ra wherein the c<>mputer means includes: a digital frame grabber for receiving the inrrges from the camera, a display for user interaction and display of assay results, cligitai storage media t:ur data storage and archiving, and means for control, acquisition, prixcmsing and display of results.
lb Figure 1 is a schematic dia~~,ram of a preferred embodiment of the cell scanning system. An inverted fluorescence microscope is used l, such as a Zeiss Axiovert inverted fluorescence microscope w-hich uses standard objectives with magnificatier, of 1-100x to the camera, and a white light source (e.g. 100W' mercury-arc lamp or xenon lamp) with power supply 2. 'lf'here is an XY stage ~ to move the plate 4 in the XY direction over the microscolne 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. 1i he printer 13 provides for printing of a hard copy record.
Figure 2 is a schematic of ~~nn embodiment ofathe 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 1 S and microscope 16, respectively, are provided. In addition, Figure 2 shows a 9G well microtiter plate 1 T
which is moved on the XY stage :3 in the XY direction. Light fiom the light source passes through the PC controlled shutter 18 to a. motorized filter wheel 19 with excitation filters 20. The light pa;scs into filter cube 25 which has a dichroic mirror 2fi and an emission fitter 27. Excitation light reflects off the diehroic 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.
Figure 3 shows a schematic drawing of a prefetTed camera assembly. The digital camera 7, which contains ;gin automatic shuttc;r for Exposure control and a power supply 31, receives fluorescent light ?8 from the microscope assembly. A
digital cable 30 transports digital signals to the cup?~puter.
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' rr~icr<nscopy. 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, includitzg, but not limited to, standard scanning confocal detection of a focused point or lure of illumination scanned over the specimen (Go et al.
1U 1997, supra), and multi-photork scannin g confocal microscopy (Denk et al., 1990, supra), both of which can forn-~ images on a C'CIJ detector or by synchronous digitization of the analog output o1 a photomultiplier tube.
In screening application's,, it is af~en necessary tea use a particular cell tine:, or primary cell culture, to take a~:lvantage of particular features of those cells. Those to skilled in the art of cell culture ~'vill recognize that sonic call lines are contact inhibited, meaning that they will stop ~ro~.vinY7 when they bei~ome 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 c3f su;.h a cell fine is the HEK. 293 (ATCC CRL.-15'?3) line. .An optical system that c:an ac~luire images of single cell 2O layers in multilayer preparations is required for nse~ a itft cell lines that tend to forni layers. The large depth of field or wide field microscopes produces an image that is a projection through the many layers of cells, making analysis of subcellular spatial distributions extremely difficr:Lt in layer-fomriry, cells :alternatively, the very shallow depth of field that can be achieved on a confbca! rr~icroscope. (about one micron), 1 ~;

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.
The output of a standard eonfocal 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 err~bodiments 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;. 'fhe optical roof guration of the confocal microscope system, is essentially the same as that described above, except for the illuminator and detectors. Illumination and detection systems required far confocal microscopy have been clesigned as accessories to be attached to standard microscope optical systems such as that of the present invention {Zeiss, Germany).
These alternative optical systen-~s therefore can be easily integrated into the system as described above.
Figure 4 illustrates an alternative embodiment of the invention in which cell arrays are in microwells 40 on a ~nicroplate 41. Typically the microplate is 20mm by 30mm as compared to a standard 9t~ well microtiter Plato which is $6mrn by 129mm. The higher density array of cells on ,~ microplate allows the. mic:roplate to be imaged at a low resolution of few microns per laixel for high throlrghput and particuhu 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 systt'm.
lc The microplate chamber 4::.' sewes as a microfluidic delivery system for the addition of compounds to cells. fI"ltc microplate 4~1 in the microplate chamber 42 is placed in an XY microplate reaclea 43. Digital data is pracessed as described above.
The small size of this micropl;ate 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 c>n a I'C screen 1 l and made part of a bioinformatics data base 44. 'Plus data base not only permits storage and retrieval of data obtained through the methods of this in wention, but also permits acquisition and storage of external data relating: to cells. Figure ~ is a P(_' display which illustrates the to operation of the software.
In an alternative ernbr~diment, a high throughput system (HTS) is directly coupled with the HGS either on the same platform or on tcvo separate platforms connected electronically (e.g. via a Local area nerivark). This embodiment of the invention, referred to as a dual mole optical system, has the advantage of increasing the throughput of a HCS by coul7linft it with a HT'S and thereby requiring slower high resolution data acquisition and analysis only an the small subset of wells that show a response in the coupled HTS.
High throughput 'who'ie plate° reader systems are well known in the art and are commonly used as a <,ampor:ent <>f an H I~S system used to screen large numbers of Zo compounds (Bef;gs (l~)97),.1. cal~Biomolcc~. Sc~rer~nin,~ ~:?1-78;
_vlacaffrey et al., (1996) J Btomolec. Scroerzing 1:187 -1 ~~()).
In one embodiment a9~ 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 a prirsided (Figure b).
Processing ~cv 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.
As illustrated in Figure 6, an exemplified two hlatforn~ dual mode optical system consists of two light optical. instruments, a high throughput platform 60 and a high content platform 6S which read fluorescent signals emitted from cells cultwed 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 TM
screening system (Topcount (hackard Instruments, Meriden, C'T); Spectramax,TM
Lumiskan (Molecular Devices, "~~unnyvale, CA); hluoroscan (Labsystems, Beverly, MA)). The high content platfornn fiS, as described above, scans from well to well and acquires and analyzes high resoluticm image data collected from individual cells within a well.
The HTS software, residing on the system's c:ornputer 62, controls the high throughput instrument, and results ,are displayed on the monitor 61. The HCS
sofWane, residing on it's computer system G7, controls the high content instrument hardware G5, optional devices (e.g. plate loader, environmental chamber, fluid dispenser), analyzes digital image data from the plan., displays results un the monitor 66 and manages data measured in an integrated database. The tvo 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 thr~:>ughput system tc~ the high content system 63 either '? 1 manually or by a robotic plate ~:ransfer~ device, as is well known in the art (Beggs (1997), supra; Mcaffrey ( 1996), ,~~~:Prcr).
In a preferred embodirn~nt, the dual mode optical system utilizes a single platform system (Figure 7). It consists of tw To 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 tiom the microtiter plate 201. The microtiter plate 201 is mounted irr a motorized X,Y stage so it can be positioned for imaging in either HTS or HC.'S mode. After collecting and analyzing the HTS
image data as described below, the HTS optical module ?09 is moved out of the optical path to and the HCS optical module 20~f i<; moved into place.
The optical module for 1-l I S 209 consists of a projection lens 214, excitation wavelength fitter 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 emissior~~ wavelength filter 21 1 by a lens 212 which forms an image on the camera 21 (~ with =~ertsor ,' 15.
The optical module far 1 1(.'S 203 consists oa' a projection lens 208, excitation wavelength filter 207 and dicnrcoic mirror 2t14 which are used to illuminate the back aperture of the microscope: objective 202, and thereby the. field of that objective, from a z0 standard microscope illuminatic>n system (not shown), he fluorescence emission is collected by the microscope of~j~:ctivc 202, pass;~s through the dichroic mirror 204 and emission wavelength filter 20°: ;rnd is focused by a tube lens 20G
which forms an image on the same camera 21 (~ with :,~er~~or ? 1 S.
'1 7 In an alternative ernbodirnent of the present invention, the cell screening system further comprises a fluid delivery device for usi° with the live cell embodiment of the method of cell screening (see below). Figure ~ exemptities 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. fach syringe 702 is sized according to the volume to be delivered to each well, typically bi;tween 1 and IOf) 11L. 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 ,rttached to a drive system so they can be lowered and raised relative to the microtiter Ivlate 706 to deliver fluid to each well.
The plate is mounted on an X,Y stage, allc~wi~~r~~ nnovement 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 I ~ wells simultaneously, or to fewer wells by removing some of the tips.
In another aspect, the prf~sc:nt invention tarovides a method for analyzing cells comprising providing an arra,,a ~~i locations which contain multiple cells wherein the cells contain one or more fluorescent reporter molecules; scanning multiple cells in each of the locations containi ng c<~lls to obtain ~luor~acent signals from the fluorescent reporter molecule in the cells; reconverting the fluorescent signals into digital data; and utilizing the digital data to c:~~twrZroine the distribution, environment or activity of the fluorescent reporter molecule v ithin the cells.

Cell Arrays Screening large numbers of c:ompaunds far 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 arc 86 nun by 129 mm, with 6mm diameter wells on a 9mm 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-:.'00 microns in dimension on a pitch of about 500 microns. Methods for making microplates are well known in the art. Microplates may consist of coplanar layers of matcri<;ls to which cells adhere, patterned with materials to which cells will not adhere, or tac~lued 3-dimensional surfaces of similarly patterned materials. For the purpose of the following discussion, the terms 'well" and 'microwell' refer to a location in an array of any construction to which cells adhere and within which the cells are imaged. Microplate:; may also include fluid delivery channels in the spaces between the wells. The smaller for~~aat 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 movi:n~c:nt required for the scanning operation.
In addition, the whole area of the microplate c:an be imaged more efficiently, allowing a second mode of operation for the microplate reader as described later in this document.
Fluorescence Reporter Molecarles A major connponent of the new drug discovery paradigm is a continually growing family of fluorescent and luminescent r~:agents that are used to measure the z~

temporal and spatia'I distribution, uc:~ntent, and activity of intracellular ions, metabolites, macromolecules, and organelles k.'lasses of these real;ents include labeling reagents that measure the distribution and amount of molecules in living and fi;;,:d cells, environmental indicators to repnrt srgrlal transduction events in time and space, and _°~ fluorescent protein biosensors to measure target, moiecular~
activities within living cells.
A multiparameter approach that corrtbines several reagents in a single cell is a powerful new tool for drug discovery.
The method of the present invention is baseii on the high affinity of fluorescent or luminescent molecules fe:~r specific cellular components. The affinity for specific to components is governed by phy:~iival forces such as ionic interactions, covalent bonding (which includes chimeric fusie;o v~ith protein-based chromophores, fluorophores, and lumiphores), as well as hydropluabic interactions, c~le~trical potential, and, in some cases, simple entrapment within a cellular component. The luminescent probes ca.n be small molecules, labeled nnacromolecules, or genetically engineered proteins, 1 ~ including, but not limited to gri:ern fluorescent protein chimeras.
Those skilled in this an mill recognize a wide variety of fluorescent reporter molecules that can be used in the present lrlventlon, includir»:. but not limited to, fluorescently labeled biomcrlei:rzles such as proteins, phosphoiipids and DNA
hybridizing probes. Similarl~~, fluorescent reagents specifically synthesized with ?o particular chemical properiie~ ~~t binding or ass~»iation have been used as fluorescent reporter molecules (Barak et u1.. ( I9971, J. Baol. ( 'hear. 272:27497-~7~00;
Southwick et al., ( 1990), Cyto»rety 11:41 ~-43t); 'Tsien ( 1980) ire :Merhoais~ in Cell Biology, Vol. 29 Taylor and Wang (eds.), pp. 12 .'-1507). Fluoresc-entlv labeled antibodies are particularly 2>

useful reporter molecules due to their high degree of'specificity for attaching to a single molecular target in a mixture of rnol~c.cules as complex as a cell or tissue.
The lumirpsc~:r~t probes catu 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; lfiright et al. ( 1996), Cytometry 24:226-233;
McI~'eil (1989) in Methods in Cell Biolo~v, Vol. 29, Taylor' and Wang (eds.), pp. 153-173).
These methods include electroporation and other mechanical methods such as scrape-loading, bead-loading, irr~pact-loading, syringe-loading, hypersonic 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. Patent No. 5,491,084 issued February 13, 1996;
Cubitt et al.
(1995.), Trends in Biochemical Science 20:448-455.
Once in the cell, the lumine~>cent 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-~~equence-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.Biophysi~~s and Biomolecular Structure 24:405-434;
Giuliano and Taylor (1995), Methods in Neurorcience 27: ! -16). 'fhe pH environment of the reporter can be determined (BOght et al. (1989), J: (:'ell Biology 104:1019-10:33;

Giuliano et al. (1987), ,~nczl. NracIrE~m. 167:3b2-371; Thomas et al. (1979), Biochernistrl' 18:2210-2218). It ~.ao be detennit+ed whcnher a reporter having a chelating group is bound to an ion, st.tch as (.'a-+-+, or not (Bright et al.
(1989), In Methods in Cell Biology, Vol. 30, Taylor and Wang (eds.}, pp. 157-192;
Shimoura et al.
(i988), J. of Biochemistry ('Toky«) 251:405-410; Tsien (1989) In Methods in Cell Biology, Wol. 30, Taylor and War-tg (eds.), pp. 12'7-156).
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 p~.~larized membrane components. 'That is, these cells lei asymmetrically distribute macrrvnoolecules along their plasma membrane.
Connective or supporting tissue cells often contain granules in ~y~hi~.h are trapped molecules specific to that cell type (e.g., heparin, histamine, serotonirt, etc.). V4ost muscular tissue cells contain a sarcoplasmic reticulurv, a specialized organelle whose function is to regulate the concentration of calcium forts ~~,~ithin the cell cytoplasm. Many nervous tissue cells contain secretocy granules arid vesicles in which are trapped neurohotmones or neurotransmitters. Therefore, fluorescent molecules can be designed to label not only specific components within spG:~cific cells, but also specific cells w ithin a population of mixed cell types.
Those skilled in the ;art will recognize ;~ reide variety of ways to measure ?o fluorescence. For example, s~,~nnc: tluorescent rlorter molecules exhibit a change in excitation or emissiotl spectra. some exhibit resonance energy transfer where one fluorescent reporter loses fluorescence, while a second gains in fluorescence, some exhibit a loss (quenching) or al:3pearance of fluorescence, while some report rotational ~ -.

movements (Giuliano et al. (1955), ,~lnn. Rev, o/~ Bio~ht:srcw and Biomol.
Structure 24:405-434; Giuliano et al. (19951, ~~lethods irr ~'~'ortru.scic:vc-a 2?:l-16).
Scanning cell arrays Referring to Figure 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 tile 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 tc) sample, specifies the filter settings and fluorescent channels to match the biological labels being used and the inforrllation 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 par~n.rrreter settings used to identify nuclei and cytoplasm, and selection of different tluorc~sc;erlt reagents, identification of cells of interest based is on morphology or brightness, anti cell numbers to be analyzed per well.
These parameters are stored irr the systc:rn's database fir easy retrieval f'or each automated run. The system's int'ractive 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 specitie,~~ which wells of the pl<rte the system will scan and how o many fields or how many cells to analyze in each well. Depending on the setup mode selected by the user at step 1 U I,_, 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-fiscuses 1U3 the scaruling region by selecting three "tag"
points which define the rectan,~;ular area to beg sca~lncd. A least-squares fit "focal plane ?8 model'" is then calculated from these tag points to estimate the focus of each well during an automated scat. The focus of each well is estimated by interpolating from the focal plane model during a sc:arr.
During an automated sc::ur, the software dynamically displays the scan status, including the number of cells anahrzed, 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 (Figure 1 ) is scanned in a serpentine style as the software automatically moves tire motorized microscope X~r' stage 3 tTOm well to well and field to field within each will of a 96-well plate. (hose skilled in the programming to art will recognize how to adapt sc~flware for scanning of other microplate formats such as 24, 48, and 384 well plates. '~l he scan pattern of the entire plate as well as the scan pattern of fields within each v~~ell are programmed. The system adjusts sample focus with an autofocus procedure l>4 lFigure 9) through the Z axis focus drive S, controls filter selection via a motorized fl Iter w heel 19, and acquires and analyzes images of up l5 to four different colors ("channels"' or "wavelengths'") The autofocus procedure i5 called at a user selected trequency, typically for the first field in each well and there c7nce every ~ =,o ~ tields within each well. The autofocus procedure calculates the starting. l-axis point b,~,~ interpolating from the pre-calculated plane focal model. Starting a t,r~»rar~lmable distance above or helwv this set point, the 2o procedure moves the mechanical l,-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 ~:~f each image. Tho t.. position of the image with the maximum focus score deterntines the best focus for a particular field. Those skilled in the art will recognize this as ~:r variant of automatic focusing algorithms as described in Harms et al. in C~~torrzetn.~ S (198=1), 2 36-243, Groen et al. in C'vtometrv 6 (1985), 81-91, and Firestone et al. in Cjuometn l a" 1.1991 ), 195-206 For image acquisition, tine camera's e~:posure 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. Backgroun~:l shading and uneven hlumination can also be corrected by the software using methods; known in the art (Bright et al. ( 1987), J.
Cell i'3iol.
to 104:1019-1033).
In one channel, imaga~ are acquired of a primary marker 105 (Figure 9) (typically cell nuclei counterstained with U.A.PI or PI fluorescent dyes) which are segmented ("identified") using an adaptive thresholding procedure. The adaptive thresholding procedure 106 is ~.ised to dynamically select the threshold of an image for t 5 separating cells from the background. The staining of cells with fluorescent dyes can vary to an unknown degree across calls 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 preparati~:in and-'or the dynamic nature of cells. A
global threshold is calculated for the complete image to separate tire cells from background and account ?.o for field to field variation. Clvese global ,rdaptive techniques are variants of those described in the art. (Kittler et al. in ( 'ornputer 1 'i.rion, Oruphics, and image Processing 30 ( 198 ), 125-1.47. Ridler et al in IF'E~' l fans ~Sp:stems, Man, and Cybernetics (1978), 630-632.1 An alternative adaptive tlurcsholding method utilizes local region thresholding in contrast to global image threshol~ding. Image an alysi:~ of local regions leads to better overall segmentation since staining oaf cell nuclei (as well as other labeled components) can vary across an image. Using; this globalilocal procedure, a reduced resolution v> 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 (oath using adaptive threshalding) for each region of interest.
I"he output of the segmentation proccdl.lre is a binary image wherein the objects are white and the background i~; black. 'This binary image, also called a mask in the art, is used to determine if the field contains objects 1i~7 T'he mask is labeled with a blob labeling algorithm whereby ea~:h object (or blab) has a unique number assigned to it.
Morphological features, such u.s area and shape, c~f the blobs are used to differentiate i.s blobs likely to be cells froth tlaost. 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 rnterest a.re found in the field, images are acquired far all ot'ncr active channel:, I(?~i, otherwise the stage is advanced to the next field 1()9 111 the current well. Earn oh~ect of interest is located in the image for further analysis 11,0. The s~:~ttware determll7es 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 staf;e location is recanted, a ;mall image of the cell is stored, and features are measured 1 l..?:.

The cell scanning method of the present invention can be used to perform many different assays on cellular satnl~Ies 1y applying a n umber of analyrtical methods simultaneously to measure features at multiple wavelengths. An example of one such assay provides for the following measurements:
,;
1. The total fluorescent vint~.nsity within the cell nucleus for colors 1-4 2. The area of the cell nuG:l~;us for color 1 (the primary marker)

3. The shape of the ccIl nu<;leus for color 1 is described by three shape features:

1 o a) perimeter sqr:~.ared area b) box area ration c) height width r-atr0

4. The average tluoresc:errt intensity within the cell nucleus for colors 1-4 (i.e.

#1 divided b~,-~ #? j

5. The total fluorescent intensity oi' a rind outside:
the nucleus (see Figure 10) that represents tluorescc:nce of the cell's cytoplasm (cytoplasmic mask;) for colors 2-4

6. The area of the cytor~lasmic mask

7. The average fluoresct:nt intensity of the c:ytoplasmic mask for colors 2-4 ?0 (i.e. #~ divided by #ti)

8. 'The ratio of the average fluorescent iraterusity of the cytoplasmic mask to average fluorescent intensity within the: cell nucleus for colors 2-4 (i.e. #7 divided by #4)

9. The difference of true average fluorescent intensity of the cytoplasmic mask 2, and the average fluor~~,cent intensity ~~=itlir~
the cell nucleus for colors 2-4 (i.e. #7 mimes #4)

10. The number of fluc~resc~;nt domairrs (also call spots, dots, or grains) within the cell nucleus for ~:~c>lc>c~s '-4 ;O Features 1 through 4 are general features of the different cell screening assays of the invention. Thescv ~t~ps art commonly caeci in a variety of image analysis applications and are we 11 knov.-n in art (Ross 1 l~)~i2) Tire lnra,~c~
Processing Handbook, CRC' Press Inc.; Gonzales et ai ( 1957), Digitu! Imus,<<° f'f-ocessing.
Addison-Vv'esley Publishing Co. pp. 391-448). i=eatures 5-9 have been developed specifically to provide 3~ measurements of a cell's i7uorescent molecules within the local c.y~toplasmic region of the cell and the transloc:aticm ( i.e. movement) oC tluorcacent molecules from the cyrtoplasm to the nucleus. ~l hes~ featurca (step:; s-9) are used for analyzing cells in ;1 microplates for the inhibition ol~ nuclear iranslocation. For example, inhibition of nuclear translocation of transcription factors provides a nc>vel approach to screening intact cells (detailed examples of other types of screens will be provided bca>v,). A
specific algorithm measures the ~unount of probe in the nuclear region (feature 4) > versus the local cytoplasmic region (feature 'l) of each e;ell.
Quantification of the difference between these two sub-cellular compartments provides a measure of cytoplasm-nuclear translocation (fc°,ature 9).
Feature 10 describes a screen used for counting; of DN.A or RNA probes within the nuclear region in colors 2-~. lvor example, probes ate commercially available for ti.i identifying chromosome-specific 9)N.~ sec~uerlce~ (1_ife hechnologies, Gaithersburg, MD; Genosys, Woodlands, ~T~; l:3iotechnologies. Inc.. Richmond, ('.4; Bio 101, lnc., Vista, CA) Cells are three-d,mensicmal 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 deli screening method of the present invention provides for detecting three-dimension.il probes in nuclei by acquiring images from multiple focal planes. The software nuw~°,'. the Z-axis motor drive 5 (Figure 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 foc.~.l steps, an image is ai:quired. The maximum gray-level intensity from each pixel in e=ach image is touro.i arrd stored in a resulting maximum 2o projection image. The maxim~.~.m projection imac;c i<< then used to count the probes. 'The above aigorithnn works well :n couratmg probes that: ;are not stacked directly above or below another one. 'I o acccurn for probes stacked on top of each other in the Z-direction, users can select an option to analyre probes in each of the focal planes acquired. In this mode, the ~:,c~~nning system perfor7ns 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.
After meu,urin~l cell features 112 (Figure ~)i, the, system checks if there are any unprocessed objects in the curr.vrG 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 detern~inea whether analysis of the current plate is complete 114;
if not, it determines the need to find more ce:Ils iii the current well I I5.
If the need exists, the system advances the X fZ stage to tire next field rwithin the current well 1U9 or advances the stage to the nest v>~;11 1 lh of~the pl;rte.
,After a plate scan is uornplete, images acrid data can be reviewed with the system's image review, data r~:vlC'l1-, and summar~° renew facilities, A11 images, data, and settings from a scan aue archived in the system's database for later review or for interfacing with a network infornvation management system. Data can also be exported t:~ to other third-party statistical packages to tabulate results and generate other reports.
Users can review the images alone of every rc:Il an.rlvred by the system with an interactive image review proec.dure 117. The user carr review data on a cell-by-cell basis using a combination cnf interactive f;raphs, a data spreadsheet of measured features, and images of all ilrt Ilur~rescence chanra els of a cell of interest with the interactive cell-by-cell. data r~.woev- procedure 1 I ~. t~.;~-apltical platting capabilities are provided in which data can h4 analyzed via interactive graphs such as histograms and scatter plots. Users can review ~ummary° data that are accumulated and summarized for all cells within each well of a plate with an interactive well-by-well data review 3~

procedure 119. Hard copies of graphs and images can be printed on a wide range of standard printers.
As a final pha ;, c,f ~a co'op(ete scan, repon.s can be generated on one or more statistics of the measured feat~.irt;s. Users care generate a graphical report of data a summarized on a well-by-well basis for the scanned region of the plate using an interactive report generation procedure 12(?. 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 ca.n be generated on many statistics and be printed with to the touch of one button. Reports can be previewed far placement and data before being printed.
The above-recited eminodiment of the metlroct 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 welt (on the order of 1 frm) to define the Is distribution of nnaterial withir7 thr: well, as well as within individual cells in the well.
The high degree of information c~~ntent accessible in that made, comes at the expense of speed and complexity ofthe rc:qr.tired signal processing.
In an alternative embo<ifnnent, a high throughput system (HTS) is directly coupled with the HCS either c>r7 the same platforni or on two separate platforms connected electronically (e.g. ~~ its a local ~irea net»~ork). This embodiment of the invention, referred to as a dual n~c.~cte optical system, has the advantage of increasing the throughput of an HCS by co~.r.pling it with an t-ITS .and thereby requiring slower high resolution data acquisition and analysis only on the small subset of wells that show a response in the coupled H rS.

High throughput 'whole l~~latr.' reader systems are well known in the art and are commonly used as a componenr of an HTS system used to screen large numbers of compounds (Beggs et al. (1997), .sr~pra; Mc('affrey et a!. (1996), supra ).
The HTS of the present invention is carried ol.rt on the microtiter plate or microwell array by reading many or all wells in the platen 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 IiTS (the 'hits') are flagged by the system. Then on the same micrc~titer plate or rrucrawell array, each well identified as a 1o hit is measured via HCS as described above.
Thus, the dual mode pro~.~e~ss involves:
1. Rapidly measuring numerous ~,~ ells of a microtirer plate or microwell array, 2. Interpreting the data to determine the overall activity of fluorescently labeled reporter molecules in the calls on a well-byr-will basis to identify ''hits"
(wells that 1 s exhibit a defined response), 3. Imaging numerous cells in ~ arh "hit" well, and 4. Interpreting tire digital im<rgi: data to deterrorinc the distribution, environment or activity of the f7uorescentl~.~ labeled reporter n~ol~culcs in the individual cells (i.e.
intracellular rneasurtments)~ ;rncl the distribution r~l~ the cells to test for specific biological functions In a preferred embodiment of dual mode prowsslng (1~igure 1 I), at the start of a run 3U1, the operator enters irnfornnatron 302 drat de~serib~s the plate and its contents, specifies the filter settings and tltrorescent channels to match the biological labels being 25 used, the information sought a:lnd the camera settings to match the sample brightness.
These parameters are stored in the system's database for easy retrieval for each automated run. 'I'he microtiter plate car microwell array is loaded into the cell screening system 303 either manually c:~r automatically by controlling a robotic loading device.
3 C:

An optional envircmmental chamber 3t>4 is controlled by the system to maintain the temperature, humidity and C(~~ '~ev-~:ls in the air surrounding live cello in the microt.iter plate or microwell array. Arn r,>lrtoonal fluid delivery ~~device 305 (see Figure 8) is controlled by the system to dispense fluids into the wells during the scan.
High throughput processing 30fi is first perf~nned on the microtiter plate or mierowell array by acquiring and analyzing the srgnal from each of the wells in the plate. The processing performec:l in high throughput mode 30 i is illustrated in Figure 12 and described below. Wells that exhibit some selected intensity response in this high throughput mode ("hits") ar~° identified by the system. The system performs a to conditional operation 308 that tests for hits. If hits ;ire found, those specific hit wells are further analyzed in high content (micro level ) mode ;409. The processing performed in high content mode 314 is iliuwtrated in Figure I ~. 'hhe system then updates 310 the informatics database 311 witr~ results of the measurements on the plate. If there are more plates to he analyzed 3_1 ?~_ the system loads the next plate 303;
otherwise the la analysis of the plates terminatt:s 314.
The following discus~~~ion describes the hi~~ll throughput mode illustrated in Figure 12. The preferred emioodiruent of the system, the single platfonm dual mode screening system, will be d~~si:ribed. Those skilled in the art will recognize that operationally the dual platforrn system simply involves moving the plate between two ?o optical systems rather than rn~:win~ the optics. C>nce the system has been set up and the plate loaded, the system begins tine Id 'CS acquisition and analysis 4U1. The HTS optical module is selected by controlling .~ motorized ~>ptical positioning device 402 on the dual mode system. Irr one fluorescence channel, data from a primary marker on the plate is acquired 403 and wc,lls are isolated from the plate background using a masking t _.

procedure 404. Images are also ac~ltrired in other fluorescence channels being used 405.
The region in each image come:>p~;~nding to each ~'vell 4i~(i is measured 407.
A feature calculated from the measurements for a particular :veil is compared with a predefined threshold or intensity response ~~013, and based on the result the well is either flagged as a "hit" 409 or not. T he locatior7s of the wells flagged as hits are recorded for subsequent high content mode processing. If then. are wells remaining to be processed 410 the program Ioops back 4~:aG until all the wells have been processed 411 and the system exits high throughput mode.
Following HTS analysis, the system starts i~°~e high content mode processing to 501 defined in Figure 13. '1"he system selects the Hl"S optical module 50z by controlling the motorized posetic,ning system. l~or each "hit" well identified in high throughput mode, the ;~Y star;e 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 5(~4 is called for the first hold in each hit well and then once every 5 to 8 fields within each well. In one channel, in~a'.=.es are acquired of' the primary marker 505 (typically cell nuclei counterstained witlo I:),4P1, Hoechst or P1 Iluorescent dye). The images are then segmented (separated irw;to regions of nuclei and non-nuclei) using an adaptive thresholding procedure 506. 'I he." output of the segmentation procedure is a binary mask wherein the objects are whit.:: aruii the background is black. This binary image, also 2o called a mask in the art, is usccl to determine if tine field contains objects 507. The mask is labeled with a blob labelirng .~lgor-ithm 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 advr~need to the: next field ~ 14 in the current well. Each object is Iocatec:i ire the image for further analysis 509.
Morphological features, such as area and shape oV~ the objects, are used to select objects likely to be cell nuclei 510, and discard (d<~ no further processing on 1 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 5 i i . 'The system then performs multiple tests on the cc~lfs 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 unproc~:~ssed objects in the current Held 512. if there are any unprocessed objects, it locates the next object 50~? and determines whether it meets the criteria for a valid cell nucleus 51 ii, and measure~5 its features. After processing all the objects in the current field, the system det~rernmos whether it needs to find more cells or fields in the current well Sl..'s. if it needs to find t~nore cells or fields in the current well it advances the XYZ 4tagc: to the next field within the current well 515.
Otherwise, the system checks whither it has any remaining hit wells to measure 515. If so, it advances to the next hit well 503 and pruceeds through another cycle of I > acquisition and analysis, otherwise the hIC'S mode is finished 51 U.
In an alternative embo~.:lirnent of the present v.nwmtion, a method of kinetic live cell screening is provided. Tyne prc;viousiy described embodiments crf the invention are used to characterize the spatial distribution of cellular c:ornponents at a specific point in time, the time of chemical fi~at~r~n. .As such. these ern6odimerrts 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 reqr;ire 3t) -- (~U minutes to road through all the wells, only very slow kinetic processes can be treasured by simply preparing a plate of live cells and then reading through all the 4vells more than once Faster kinetic processes can be 3y measured by taking multiple reacl.ings ot: each well before proceeding to the next well, but the elapsed time between the first arid last well w oi.Ud be too long, and fast kinetic processes would likely be compie~te before reaching the last well.
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 characterstics. 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 1~~ system in order to deliver reagents icE each well at a specific time in advance of reading the well. This embodiment trrereby 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 front sub-regions of the plate, allowing the t 5 system to read other wells between the time points required for an individual well.
Figure 8 describes an example of a fluid delivery device for use with the live cell embodiment of the invewtian and is described abcwe. Mhis set-up allows one set of pipette tips 70~, or even a sinf~,le: pipette tip, to deliver reagent to all the wells on the plate. The bank of syringe pumps ~Ol can Ire: t.i:~e~d to deliver fluid to 12 wells zo simultaneously, or to fewer wells by removing some of the tips iUS. The temporal resolution of the system can thcretore be adjusted, withocrt sacrificing data collection efficiency, by changing the nu,n~ber of tips and the scan pattern as follows.
Typically, the data collectiim and analysis from a single well tab;es about S seconds.
'Moving, from well to well and focusing in a moll requires about ~ seconds, so the overall cycle time :fit l for a well is about IU seconds. '( h~refore, 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 ueiwer fluids to 6 welts simultaneously, and the systerrr 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. Theretc~re, try adjusting the size of the sub-region being scanned on the plate, the temporal resolution can be adjusted without to having to insert u-ait times bet;vc:en acquisitions. Because the system is continuously scanning and aeyuiring data, th;: cwGrall time to collect a kinetic data set from the plate is then simply the time to perfcnrnt a single scan c7f the plate, multiplied by the number of time points required. Typically, t tune point before addition of compounds and 2 or 3 time points following additio:.n should be suf'ticient (or screening purposes.
Figure 14 shows the ac~.a,uvsitic>n sequence used fir kinetic analysis. The start of processing 801 is configurati~:n~ of the system. much of which is identical to the standard HC.'S configuration. 1n addition, the operator must enter information specific to the kinetic analysis being p~:vrio.rmed 8U2, ,ucch as the sub-region size, the number of time points required, and the rwquirecl time increment. ~ sub-region is a group of wells z0 that will be scanned repetitiwlv in order to accumulate kinetic data. The size of the sub-region is adjusted so that tlic: system can scan a whole sub-region once during a single time increment. thus nnirrimizing wait tunes. fhe optimum sub-region size is calculated from the setup parrrneters, and adjusted if necessary by the operator. The system then moves the plate t::~ the first sub-region 8U3. and to the first well in that sub-region 804 to acquire the prestin~ulation (time - 0) time points. The acquisition sequence performed in each well is. exactly the same as that required for the specific HCS being run ;n kine'.ic mode. Figure 15 details a flow chart for that processing. Alf of the steps between the start 90_l and the return 9_U? are identical to those described as s steps 504 - 514 in Figure; l3.
After processing each well in a sub-region, the system checks to see if all the wells in the sub-region have been processed 8(76 (Figure 14j, and cycles through all the Wells until the whole region has been processed. ~l"he: systmn then moves the plate into position for fluid addition, and controls fluidic system delivery of fluids to the entire sub-region 807. this may r~~qiaire multiple: additions for sub-regions which span several rows on the plate, with the system moving the plate on the X,l' stage between additions. Once the fluids hav:~ ioeen added, the systenn moves to the first well in the sub-region 808 to begin acquisit.ican of~time points. The data is acquired from each well 809 and as before the system cycles through all the ~~~ells in the sub-region 810. After t s each pass through the sub-region, the system checks whether all the time points have been collected 811 and if not, f~atyses X13 if necess~uy 812 to stay synchronized with the requested time increment. Otlvervvise, the sy:,teni checks for additional sub-regions on the plate 814 and eith~:r movt,s t~-A the next sub-re~gi~c~n 5113 or tinishes 815. Thus, the kinetic analysis mode comprises operator identifn-anon of sub-regions of the mierotiter 2o plate or microwells to be scn::errecl, based on thc° kin~ai~:
response to be investigated, with data acquisitions within ; scrh-region prior ro data acquisition in subsequent sub-regrons.
=t.'r Specific Screens In another aspect of the present invention, a rrrac:hine readable storage medium comprising a program containinl:~ a set of instructions for causing a cell screening system to execute procedures far defining the distribution and activity of specific S 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 rr~oving the stage, a digital camera, a light source for receiving and processing the digital data fiom the digital camera, and a computer means for receiving ,:rnd processing they digits( data from the digital camera.
to This aspect of the invention comprises programs that instruct the cell screening system to define the distributian and ~zctivit~ of specitic cellular constituents and processes, using the luminescent probes.. th~~ optical imaging s~,~stem, and the pattern recognition software of the invention. Preferred embodimf~nts of the machine readable storage medium comprise programs consisting of ;a sees of instructions for causing a cell t5 screening system to execute the procedures set forth in Figures 9. 11, 12, 13, 1:1< ar 15.
Another preferred embodiment comprises a program consisting of a set of instructions for causing a cell screening systeru to execute procedures for detecting the distribution and activity of specific cellular constituents and processes. In most preferred embodiments, the collular ;~ro~:csscs include:. but. are not limited to, nuclear ?0 translocation of a protein, ~<llular hypertrolrly, apc~plosis, and protease-induced translocation of a protein.
The following examlvles are intended for purposes of illustration only and should not be construed to lin~rit the scope of the invention, as defined in the claims appended heretu.
4s The various chemical con npounds, reagents.. dyes, and antibodies that are referred to in the following frxanthlcs are commercially available from such sources as Sigma Chemical (St. Louis, M~J}, Molecular Probes (Eugene, OR}, Aldrich Chemical Company (Milwaukee, WI}, A.ecuratc Chemical C.'omparty (Westbury, NY), Jackson Immunoresearch Laboratories ('Vest Cirove, P.4), and C.'lontech (Palo .Alto, CA).
Example l Automated Serc:~err ,,for Compounds that Induce or Inhibit Nuclear Translocation of a DNtf Transcvrr,ntio~r Factor l0 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:c 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 cuf a particular gene or group of genes.
A general description c>f the screen is given fc>Ilov~ed by a specific example.
The distribution of the transcription factv.~r is detc;tmined by labeling the nuclei with a DNA specific fluoroph;>rc° like Hoechst i ~?3 and the transcription factor with a specific fluorescent antibody :otter autofocusing on the Hoechst labeled nuclei, an 2o image of the nuclei is acquired in the cell-based scr~enir~g system at ?Ox magnification and used to create a mask by ~.~m of several optional thresholcling methods, as described supru. The mocpholo~~ical descriptors of the r~~;ions defined by the mask are compared with the user defined parameters and valid nuclear masks are identified and used with the following algorithm to extr;tct transcription factor distributions. Each valid nuclear ?5 mask is eroded to define a sli;~,l~t9y smaller nuclear region. fhe original nuclear mask is then dilated in two steps tc; define a ring shaloed region around the nucleus, which ,~a represents a cytoplasmic region:. °fhe average antibody fluorescence in each of these two regions is determined, and the difference between these averages is defined as the NucCyt Difference. Twc:r exam~:rl~°,s of determining nuclear translocation are discussed below and illustrated in Figure lt)A-J. Figure 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. Figure IUB illustrates the nuclear mask 202 derived by the cell-'based screening system. Figure IUC illustrates the cytoplasm 203 of the unstimr~latc:d cell imaged at a green wavelength. Figure lUD
illustrates the nu~:fear mask 20? s eroded (reduced) once to define a nuclear sampling to region 204 with minimal cy~tolrlasrpic distribution. The nucleus boundary 202 is dilated (expanded) several times to form a ring that is 2-> pixels wide that is used to define the cytoplasmic sampling region ~:O°} for the same cell. Figure lUE further illustrates a side view which shows the nuclear sartrpling region 204 and the cytoplasmic sampling region 205. Using these two sampling regions. data on nuclear translocation can be t ~ automatically analyzed by tlne c a ll-based screc:nin~; system on a cell by cell basis.
Figure IOF-J illustrates the strategy for determining nuclear translocation in a stimulated cell. Figure l OF illi.istrates a stimulated cell vvnth its nucleus 206 labeled with a blue fluorophore and a transcoption factor in the cytoplasm 207 labeled with a green tiuorophore. The nuclear mask ?~~$ ira i-igurc: 10(~ is derived by the cell based 2o screening system. Figure IOFI illustrates the c;,noplasm 2U~) ofa stimulated cell imaged at a green wavelength. Fignru 101 illustrates the nuclear sampling region 211 and cytoplasmic sampling region 21_2 of~ the stimulated cell. Figure 1 OJ further illustrates a side view w which shov~.~s the nuc: lean sampling region 21 l and the c~~toplasmic sampling region 212.
:) A specific application of this cnethod has been used to validate this method as a screen. A human cell line was lrlated in 9b well n=icrotiter plates. Some rows of wells were titrated with agonist, a l:n~~wn 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 fi:~ctor, and Hoechst ~,4<'?3. The cell-based screening system was used to acquire anti, analyze images from this plate and the NucCyt Difference was found to be stror~~glv correlated with the amount of agonist added to the wells as illustrated in Figure 1 (,. In a second c:xpc~riment, 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 NucCyc Difference was found to strongly correlate with this inhibition of translocation, as illustrated in Figure 17.
Additional experiments have shown that the Nuc(.'vt Difference gives consistent results over a wide range of cel'I densities and reagent con centrattons, and can therefore be routinely used to screen compound libraries fc~r specific nuclear translocation t s activity. Furthermore, the ~~.arne method can be used writh antibodies to other transcription faca.ors, or GFP-transcription factor chimeras, in living and fixed cells, to screen for effects on the regulation of transcrilotion of this and other genes.
Figure 18 is a representative display on a PC' <~crecn of data which was obtained in accordance with Example 1. Graph 1 18C! plots the dcfference between the average 2o antibody fluorescence in the n~.rcl~;ar ~amplinle reunion and cytoplasmic sampling region, NucCyt Difference verses ~Vr:~il :~. (traph 2 18i_ plots the average tluorescence of the antibody in the nuclear sampling region, NPR average, vt.rsus the Well #.
Graph 3 182 plots the average antibody iluoresc:eoce in the ~ytoplasmic sampling region, average, versus Well f#. 'fh;v sofRware permits displaying data from each cell. For 4~ >

example, Figure t 8 shows a s;:rc~i~n display t 83, the nuclear image 184, and the fluorescent antibody image 13~ f~~r c~:ll X26.
NucCyt Difference referred to in graph 1 180 of Figure l8 is the difference between the average cytoplasmic probe (fluorescent reporter molecule) intensity and a the average nuclear probe (flut:~rt~scent reporter molecule) intensity. NPl average referred to in graph 2 t81 of Figure 18 is the average of cyloplasmic probe (fluorescent reporter molecule) intensity within the nuclear sampling region. 1.1 P 1 average referred to in graph 3 1$2 of Figure la is the average prube (fluorescent reporter molecule) intensity within the cytoplasmic sampling region.
to E.rample 2 Automated ScrE>cvn tf>r (~ompourrds that Irrcluce or Inhibit Hypertrophy in Cardiac Mvocvtes Hypertrophy in cardiac: myocytes has been associated with a cascade of t ~ alterations in gene expression artd can be characterir_ed in cell culture by an alteration in cell size, that is clearly visible irs adherent cells growing on a coverslip.
A screen is implemented using the followin~.T strategy. My~~cvlc: cell line QM7 (Quail muscle clone 7; ATCC C'RL-19G?) c~~ltured in g6 well plates, can be treated with various compounds and then fixed and labeled with a Iluorescent antibody to a cell surface ?o marker and a Dl'JA label like f-lcaLc.hst. After focnsin~; on the Hoechst labeled nuclei, two images are acquired, one ~:~t tl7e 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 detined around the nuclei. The limits of the cells in a> those regions are them 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 cell, and a second set ofmorphological descriptors is used to identify single cells. The areas crf thc: individual cell" os tabulated in order to def ne the distribution of cell sizes for comparison 4~ ittt size data Pram normal and hypertrophic cells. In addition, c:~ ~~~.~cond fluorescf;nt arntibody to a particular cellular protein, such as one of the major muscle proteins actin or myosin can be included.
Images of this second antibody ~,. an be acquired and sicared 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.
t0 Example 3 Dual Moele Higlr T~tr-r~iighput and flyh-Content Screen The following example is a screen for activation of'a G-protein coupled receptor is (GPCR) as detected by the tran,~;location of the; <,;PCR from the plasma membrane to a proximal nuclear location. This e~:ample illustrates ho~~- a high throughput screen can be coupled with a high-content screen in the dual mode System for Cell Based Screening.
G-protein coupled recep~tc~rs arc a large class of ? traps-membrane domain cell :!o surface receptors. Ligands for tltese receptors stimulate a cascade of secondary signals in the cell, which may includ;:v, I~ut are not limited to. C guy transients, cyclic AMP
production, inositol triphosphate ( 1 P, ) produCa~on an<i phosphorylation.
Each of these signals are rapid, occuring in a matter of second. to minutes, but are also generic. For example, many different CJPC'l~s produce a sea.ondat-v C'a" signal when activated.
2~ Stimulation of a GPCR also re=suits in the transport of that tiPCR from the cell surface membrane to an internal, proxim,:rl nuclear compartment. ~fhis internalization is a much more receptor-specific indicator oi' activation of a particular receptor than are the secondary signals described above.
Figure 19 illustrates a dual mode screen for activation of a GPCR. Cells carrying a stable chimera of the G:PC'R with a blue fluorescent protein (BFP) would be loaded with the acetoxymethylester form of Fluo-~, a :ell permeable calcium indicator (green fluorescence) that is trapped in living cells by the hvdroiysis of the esters. They would then be deposited into th~r ~4-ells 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 to sequence of Fluo-3 images ou' the whole microtiter plate would be acquired and analyzed for wells exhibiting ..r. calcium response (t e., high throughput mode). The images would appear like the :,llusiration of the rrricrotit~r plate 601 in Figure 1ST. A
small number of wells, such a.; wells C4 and Eti in the illustration, would fluoresce more brightly due to the Ca~+ ri:leased upon stimulation oiT the receptors.
The locations of wells containing compound;~c tRiat induced ;:r reslaonse 6t)?, would then be transferred to the HCS program and the of~tica sv itched for detailed cell by cell analysis of the blue fluorescence for evidence of Gla(K translocation to the perinuclear region.
The bottom of Figure 19 illustrates the twi;~ possible outcomes of the analysis of the high resolution cell data. The camera images a Sub-region 6(J4 of the well area 603, producing images of the fluorescent cells 60s. In well (:'4, the i.jnif~i,rm distribution of the fluorescence in the cells indicates that the receptor has not internalized. implying that the Ca+' response seen was the result of the stimulation of some other si'naliing 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 :1~3 only a few hit wells have to be analyzed with high resolution, the overall throughput of the dual mode system can be quoits; high, comparable to th-~ high Lhroughput systE;m alone.
Example 4 Kinetic High Content Screen The following is an ~:::X~irIlplc; Of a screen to measure the kinetics of internalization of a receptor. As described above, the stirrrulation of a GPCR, results in the internalization of the recelvtc:rr, with a time course of about I S min.
Simply detecting the endpoint as internalir_ed or not, may nc5t he sufficient for defining the to potency of a compound as a (~F'CR agonist or antagonist. However, 3 time points at ~
min intervals would provide inf~;:>n~~~ation 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-regaon would be defined as two rows, the sampling interval as 5 minLtte~~, ~:um1 the total number of time points 3. The system t s would then start by scirnning tv,o> rows, and then adding reagent to the two rows, establishing the time=0 referenc::e <After reagent addition, the system would again scan the two row sub-region acquirving the first time point data. Since this process would take about 250 seconds. includm~~ scanning back to the beginning. of the sub-region, the system would wait 50 seconds to fyegin acquisition of the second time point.
Two more 2c~ cycles would praduce the thr~~ue rime points and thc: systen n would move on to the second 2 row sub-region. The final two 2-row sub-re~ior~s 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 pi,~int~; for the wrens woul~3 be offset slightly relative to time=(), the spacing of the tune l~oirtts wcould be very elc5se to the reguired S minutes, 5 (:' and the actual acquisition times a.nd results recorded with much greater precision than in a fixed-cell screen.
Example S High-content sere~:~n of human ~IucocworW ~oicl receptor trunslocation One class of H~C'S involves the drug-induced dynamic redistribution of intracellular constituents. The hunuan glucocorticoid receptor (hGR), a single "sensor"
in the complex environmental response machinery ~.~f the cell, binds steroid molecules that have diffused into the cei 1. The ligand-re~:eptor complex transloeates to the nucleus where transcr-iptional o.etivation occurs 11-Itun et al., Proc. Natl.
Acacl. ,Sci.
to 93:4845, 1996).
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 ~ 5 screening system of the presen, invention permits the screen to contain two additional parameters in parallel, such as outer receptors. other distinct targets or other cellular processes.
Plas»iid construct. :~ ~:~ukaryotic expression plasmid containing a coding sequence for a green fluorescent protein - human glucocorticoid receptor (GFP-hGR) 20 chimera was prepared using C~FIa mutants (Palm ~a <r1., :Vat. .Strcco!.
Biol. 4:361 ( 1997).
The construct was used to t.ranv>f~:oa a human cewical carcinoma cell fine (HeLa).
Cell preparation and transfection. Hel.a cells (.A'I'(.'(' C:t=I_-?) were trypsinized and plated using DM1M containing 5°io charcoal'dextran-treated fetal bovine serum (FBS) (HyGlone) and 1°,~o p~:nicillin-streptomycin ~C'-L~MEM) 12-24 hours prior to >i transfection and incubated at 37"(: and 5% (_'O~ Transfections were performed by calcium phosphate co-precipitat.~ur~ (Graham a~~d ~'an der Eb,, 6'iroloy 52:456, 1973;
Sambrook et al., ('1989). :~LTolerr~lc~r Cloning: ~1 Luhcrratow ,'t~unuul, Second ed. Cold Spring Harbor Laboratory Press, C'.~:~ld Spring Harbor, 1989) or with Lipofectamine (Life Technologies, Gaithersburg, MD)- For the calcium phosphate transfections, the medium was replaced, prior to transfecaion, with DMEM containing 5%
charcoal/dextran-treated FBS. (;.'.ells were incubated with the calcium phosphate-DNA
precipitate for 4-5 hours at 3~"°(' and 5% C'C),, w~uhed 3-4 times with DMEM to remove the precipitate, followed by the addition ol'C-DI~t>~.M.
:.0 Lipofectamine transfections were performed in serum-f=ree DMEM without antibiotics according to the nrar~ufacturer'~. instructions (Life Technologies, Gaithersburg, MD). Followings a 2-a 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 ~ 3"'(:'. and 5°,%
CO~ for 24-48 hours 15 prior to drug treatment. Experiments were performed with the receptor expressed transiently in HeLa cells.
Dexamethasone indur~tion of GFP-hGrR' translorution. 'fo obtain receptor-ligand translocation kinetic dat,~, nuclei of transfected cells were tirst labeled with 5 pg/ml Hoechst 63342 (Molecul;~r Probes) in C-~WII~M for ?0 minutes at 33"C and 5°,%
LO COz. Cells were w ~asl~ed oncw~ in Hzrnk's Balanced Salt Solution (HI3SS) followed by the addition of 100 nM dexa~°c~ethasone irr HBSS ~~~ith I%
charcoal/dextran-treated FBS. To obtain fixed time point dcxamethasorle titration data, transfected I-feLa cells were first washed with DML~:!-1 and then incubated at 3v3°<~ and 5°'o CO, for 1 h in the presence of 0 -- 1000 nM drxamethasone in ZrMEM containing f°,~o charcoalidextran-a ', treated FBS. Cells were analyrew:l live or they were rinsed with HBSS, fixed for 15 min with 3.7°,% foonaldehyde in 1-ll3;p~, stained with 1-loec;hst 33342, and washed before analysis. The intracellular GFP-h(.iR fluorescence signal was not diminished by this fixation procedure.
Image acquisition and analysis. Kinetic. data were collected by acquiring fluorescence image pairs (GFP-ilCUR and Hoechst 3332-labeled nuclei) from fields of living cells at 1 min interval,, for :30 min after the addition of dexamethasone.
Likewise, image pairs were obt,:3.ir~~ed from each veil of the fixed time point screening plates 1 h after the addition of clc~aamethasone. In hoth cases, the image pairs obtained to at each time point were used tc:~~ t.tefine nuclear adld cyloplasnlic regions in each cell.
Translocation of GFP-hGR w~a~s 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 t, from at least 200 cells at each ~-orlcentration of dexarnethasone tested.
Drug-induced translocation of GFP-h(R from the cytoplasm to the nucleus was therefore correlated with an increase in the transloctrti~n ratio.
Results. Figure 20 schematically dispiay~, the drub:-induced cytoplasm 253 to nucleus 252 translocation of tile human glucocorticoid receptor. The upper pair of U schematic diagrams depicts the localisation of GEP-h(~R wrthnl the cell before 25(.1 (A) and after 2~ 1 ~B) stimulation r~~ith dexamethasone. ~lnder these experimental conditions, the drug induces a ',urge portion of the cytoplasmic CJFP-hGR to translocate into the nucleus. This redi~>tribution is quantitied by determining the integrated intensities ratio of the cvtopla~mic and nuclear fluorescence in treated 255 and untreated 254 cells. The lower pair of' fluorescence micrographs show the dynamic redistribution of G:FP-hGR in a :,in~;le cell, before Z~4 arld after 2S5 treatment. The HCS is performed on wells con~.~ainirrg hundreds to thousands of transfected cells and the translocation is quantified fc~r each cell in the tieLd exhibiting GFP
fluorescence.
Although the use of a stably tr~:rnsfected 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.
To execute the screen, the cell screening system scans each well of the plate, to images a population of cells in each, and anal.~ros cf~lls individually.
Here, two channels of fluorescence are used to define the cyrtoplasmic and nuclear distribution of the GFP-hGR within each cell. Depicted in Fi;~ur~ 21 is the graphical user interface of the cell screening system near tine end of a GFF'-hGR sc:reert. The user interface depicts the parallel data collection and analysis capability i~f the system. The windows labeled t 5 "Nucleus" 261 and "GFP-hCaR" ?6? show the pair of fluorescetlce images being obtained and analyzed in a sin~_=fe freld. The window labeled "(dolor Overlay"
260 is formed by pseudocotoring th~:° ,above images and mer:;ing them so the user can immediately identify cellular ch'4ryes. Within the "~~tc~red Object Regions"
window 265, an image containcng oacu ;tnalvred cell arrd its neighbors is presented as it is a0 archived. Furthermore, as the l-lt:'S data are being coIlectecf, they aro analyzed, in this case for GFP-hGR translocation, and translated into an mumediate "hit"
response. The 96 welt plate depicted in the livver window of the screen 2G l shows which wells have met a set of user-defined scrc:et7.in~~ c:nteria. For exan~ple~, a whito-colored well 269 indicates that the drug-induce:cl translocation has exceeded a predetermined threshold j4 value of 50%. On the other hand, a black-colored well 2'70 indicates that the drug being tested induced less than 10°-r translcucation. Gray--colored walls 26$
indicate "hits"
where the translocation value fell between 10°~o and 50°,-0. low "E" on the 96 well plate being analyzed 266 show.<~ a titration with a drug known to activate GFP-h(sR
translocation, dexamethasone. This examplf: screen used only two fluorescence channels. Two additional chatrnels (Channels 3 263 and 4 264) are available for parallel analysis of other specific targets, cell processes, or cytotoxicity to create multiple parameter screens.
There is a link between the image database and the information database that is to a powerful tool during the valic9ation process of new screens At the completion of a screen, the user has total accvs~~ to image and calculated data (Figure 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 ~,eiewed separately, car summary data can be plotted. For t, example, the calculated results of a single parameter for each cell in a 96 well plate are shown in the panel labeled Gr~xp1 1 275. By selecting a single point in the graph, the user can display the entire data sca for a particular cell that is recalled from an existing database. Shown here are the image pair 276 and detailed fluorescence and morphometric data frorn a sin~ale cell (C'ell # l ~ 8, gray Line '~?7:9. hhe large graphical insert 278 shows the results of dcaamethasone concentration on the translocation of GFP-hGR. Each point is the average of data from at le~~st 200 cells. The calculated EC;~, for dexamethasone in thi~~ assay is 2 nM.
A powerful aspect of I~(-'S with the cell screening system is the capability of kinetic measurements using r7oulticolor fluorescence arid rnorphometric parameters in 'i 5 living cells. Temporal and spatial measurements can be made on single cells within a population of cells in a field. Figure 2 3 shows kinetic data for the dexamethasone-induced translocation of GFP-hGEZ i,n 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. -Chese data also illustrate the ability to analyze cells with different expression levels.
1G E.rample 6 High-content scrce~r, ohdrug-inclucec! apo,~to.sis Apoptosis is a complex c~ellawlar program that involves myriad molecular events and pathways. To understand th~~ mechanisms <af~ drug action on this process, it is essential to measure as many of vhese events within cells as possible with temporal and spatial resolution. Therefore, an apoptosis screen that requires little cell sample i 5 preparation yet provides an auto>nuated readout of several apoptosis-related parameters would be ideal. ,A cell-based as5av designed for the cell screening system has been used to simultaneously quantify sc:verai of ~he morphological, organellar, and macromolecular hallmarks of p;:nclitaxel-induced apoptosis.
Cell preparation. The cells chosen for this study were mouse connective tissue 'o fibroblasts (L-92~>; ATf_'C CCI_-'t y and a highly mva~~i~re glioblastoma cell line (~NB-19; ATC'C C_'RL-2219] (Welclt et al., l~r Y~itro ('ell. DEw Hiol. 31:610, 1995). 'The day before treatment with an apoptcasis inducing drug, 3500 cells were placed into each well of a 96-well plate and incubated ovemig,ht at ~i7"C' in a humidified 5% COs atmosphere. The following day. the culture medium was removed fiom each well and replaced with fresh medium containing various concentrations of paclitaxel (0 -SO
1M) from a 20 mM stock made in TaMiS(7. The maxin-ral 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 lner-iod, each well received flesh medium containing 7S0 nM MitoTracker Red (Molecular Probes; Eugene, (UR) and 3 Irg/ml Hoechst 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 1 S min at room temperature. The formaldehyde was washed out with HBSS
and the cells were permeabilized for CIO s with 0.5% lulu) 'Triton X-1()~, washed with HBSS, incubated with 2 U ml-' Bwdipy FL phallacidin (Molecular Probes) for 30 min, and washed with HBSS. °fhe wells on the plate were then filled with 200 ftl HBSS, sealed, and the plate stored at 4°C.' ifi necessary. T'he fluorescence signals from plates stored this way were stable for at Yeast 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.
Image acquisition and analysis on the .4rrayScan System. The fluorescence intensity of intracellular Mito'I'ras~ker 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., ~ ells and nuclei), and to calculate its size, shape, and integrated intensity.
Calculations and outpua. A total of 50-2S0 cells were measured per image field. For each field of cells, the following calculations were performed: (1 ) The average nuclear area (urnz) was calculated by dividing; the total nuclear area in a field by the number of nuclei detected. ('?) The average nuclear perimeter (Irm) was calculated by dividing the sum of thi; perimeters of all nuclei in a field by the number of nuclei detected it that field. Ijighly convoluted apoptotic nuclei. had the largest nuclear perimeter values. (~) The avt.rage 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 tire integrated intensity of an entire field of cells stained with Iv!lito"T racker dye by the number of nuclei in that field.
Because the amount of MitoTrac:l~er dye that accumulates within the mitochondria is proportional to the mitochondria) potential, an increase in the average cell brightness is consistent with an increase in mitochondria) potential. (5) T'he average cellular brightness was also calculated bar 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 TM
amount of Bodipy FL phallacidirr dye that accumulates within the cell is proportional to actin polymerization state. An increarse in the average cell brightness is consistent with an increase in actin polymerization..
Results. Figure 24 (top panels) shows the changes paclitaxel induced in the nuclear morphology of L-929 cells. Increasing arr~ounts of paclitaxel caused nuclei to enlarge and fragment 293; a hallmark oh apoptosis. (2uantitative 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 tharu were SNB-19 cells 297. At higher concentrations though, the L-929 cells shovve:d 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, ~~nghtness, and fragmentation of the nucleus 298 and actin polymerization vaiu~s 294 rx.<~ched a maximum value when SNB-19 cells wore treated with 10 nM paclitaxel (Figure 24; top and bottom graphs). However, mitochondria) potential 295 was minimal at the same concentration of paclitaxel (Figure 24; middle graph). The fact that all the parameters measured approached control levels at increasing paclitaxel concentrations ( >10 nM) suggests that cells have low affinity drug metabolic or clearance pathways that are compensatory at sufficiently high levels of the dnt,g. ( ontrasting the drug sensitivity of SNB-19 cells t0 297, L-929 showed a different response to paclitaxevl 296. These fibroblastic cells showed a maximal response in many parameters at S feM paclitaxel, a S00-fold higher dose than SNB-1~) cells. Furth;:rrnore, the L-929 cells did not show a sharp decrease in mitochondria) potential 295 at any of the paclitaxcf concentrations tested.
This result is consistent with the presence of unique apoptosis pathways between a normal and t5 cancer cell line. Therefore, tE~.ese results indicate that a relatively simple fluorescence labeling protocol can be coupl,~d with the cell scr~,enin~; system of the present invention to produce a high-content screen cri key events involved in programmed cell death.
E.ranrple 7, Protease indu~rect trurrslococtior~ i~/~ rr si~rrraling cnt~me contuin~ing a 2() disease-ussociclrc~d sequcjtoe fi~or~t <yuoBlusrn t<.> uuclcus.
Plasmid con.sr'ruct. ;1 eukaryotic a xpression plasmid containing a coding sequence for a green fluorescent protein - caspase f C'ohen ( 1997 ), Biochemical J.
326:1-15; Liang et al. (19t)7). J o;_,~folE~c. Biol. 2?4:~?~)1-3()2) chimera is prepared using 2, GFP mutants. 'the construct is used to traps°ect eukan~otic cells.
5~) Cell preparution and trans~''ectian. Cells are trysinized and plated 24 h prior to transfection and incubated at 3~" t' acrd ~~~~ C'O~' 'I'ransfe;ctions are performed by methods including, but not limited to calcium phosphate: coprecipitation or lipofection.
Cells are incubated with the calcium phosphate-DNr'~ precipitate for 4-S hours at 37°C
and 5% COz, washed 3-4 times ~nrith DMEM to remove the precipitate, followed by the addition of C-DMEM. Lipofectamine transfections are performed in serum-flee DMEM without antibiotics according to the manufacturer's instructions.
Following a 2-3 hour incubation with the DN,~-liposome complexes, the medium is removed and replaced with C-DMEM.
t0 .4popototic induction a~ ('aspase-GFP trarrslacatiarr. To obtain Caspase-GFP
translocation kinetic data, nuclei of transfected cells are first labeled with 5 Ilg/ml Hoechst 33342 (Molecular Prows') in C-DMEM fiar ?() minutes at 37°C and 5% CO2.
Cells are washed once in Hank's Balanced Salt Solution (HBSS) followed by the addition of compounds that insauce apoptosis. These compounds include, but are not limited to paclitaxel, staurospnrinc, ccramide, a~~d tumor necrosis factor. To obtain fixed time point titration data, tr;~nsfeeted cells are first washed with DMEM
and then incubated at 37°C and 5°,% C(:~, for I h in the presenco of (-'~
-- l c>00 n~NI compound in DMEM. Cells are analyzed lm~car thev are rinsed with HBSS. fixed for 1_S min with 3.7% fotmaldelrvde in I-1BSS, ~~ta~ined with Hoechst 33s~:', and washed before analysis.
?o Image acquisition arid analysis. Kinetic data are collected by acquiring fluorescence image pairs (C.'a~sp;rse-CxFP and Hoechst 38342-labeled nuclei) from tields of living cells at 1 min interv;:~is for 3U min after the addition of compound. Likewise, image pairs are obtained fro.r each well of the fixed tune point screening plates I h after the addition of compound. In both cases, the image pairs obtained at each time 6t) 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 int<f~rated fluorescence intensity of the chimera in the cytoplasm or as a nuclear-cytoplasmic difference of C:ifP fluorescence. In the fixed time point screen this translocatrorn 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 cvtoplasrn to the nucleus is therefore correlated with an increase in the translocation ratio. Mole~Luiar interaction libraries including, but not limited to those comprising putative activators or inhibitors of apoptosis-activated enzymes are 1~ use to screen the indicator cell ~i~ms and identify ~~ specafie ligand for the DAS, and a pathway activated by compound a<:tivity.
E.rumple 8. Identification of ~~u?~-ol steroid r47oeptors fi~orn D:4S
Two sources of materiw~l and/or information are required to make use of this t ~ embodiment, which allows as;es~;ment of the iimction of an uncharacterized gene.
First, disease associated segue-rvce banks) c:.antaining cDNA sequences suitable for transfection into mammalian c~eils can be used. Because every kZADE or differential expression experiment generntt~s up to several hundred sequences, it is possible to generate an ample supply caf 1=t.AS. S<<coi7d. information from primary sequence database searches can be use~.l. to r I<.ice DAS into broad categories, including, but not limited to, those that contain signal sequcr~ces, seven traps-membrane motifs, conserved protease active site ciamains, or outer identifiable motifs. Based on the information acquired from these sources. al;~oritlm,: types and indicator cell lines to be transfected are selected. A largo number of motifs are already will characterized and encoded in the linear sequences ce~ntainod within the large number genes in existing genomic databases.
In one embodiment, the fn~ll-owing steps are taken:
l ) Information from the' I)AS identification experiment (including database searches) is used as the basic: fttr selecting the relevant biological processes. (for example, look at the DAS fronn ,~ tumor line for cell c_,~cle modulation, apoptosis, metastatic proteases, etc. ) ?) Sorting of DNA sequences or DAS by identifiable motifs (ie. signal sequences, 7- transmembrane domarns, conser~~e~I protease active site domains, etc.) 1~~ This initial grouping will dete~rroine fluorescent tagging strategies, host cell lines, indicator cell linca, and banks o1~ hioactive molecules tea be screened, as described supra.
3) Using well established molecular biology methods, ligate DAS into an expression vector designed for this purpose. (.ieneralized expression vectors contain 1, promoters, enhanccrs, and ternzinators for which td> deliver target sequences to the cell for transient expression. Such vectors may also contain antibody tagging sequences, direct association sequences, clorcvroophore fusiG~r~ ;;eq~ae;nces like (:.iFP, etc. to facilitate detection when expresse~.i k~sv the: host.
4) Transiently transfec:t cells with DAS c:~ntaining vectors using standard transfeetion protocols ir~cluriin~;: calcium phosphate co-precipitation, liposome mediated, DEAF dexiran nrecfiated, polycatiunic mediated, viral mediated, or electroporation, arid plate intiv :rrii:raciter plates or mrcrowell arrays.
Alternatively, transfection can be done direct'~y is~ the microtiter ;>late. itself.
2, 5) Carry out the cell screening methods rrs described srsln"Cl.
In this embodiment, DAS shown to possess a motifs) suggestive of transcr-iptional activation potential f for example, ONA binding domain, amino terminal modulating domain, hinge r..~gri~n, or carh~a::~; ternrit7al ligand binding domain) are utilized to identify novel steroid receptors.
Defining the fluorescent tags for this experiment involves identification of the nucleus through staining, and tagging the DAS by creatrng a (iFP chimera via insertion of DAS into an expressiorv vr:ctor, proximally fcrsecf to the gene encoding GFP.
t l ., Alternatively, a single chain antibody fragment with high affinity to some portion of the expressed DAS could be constru;~tc;~t using technolGyy available in the art (Cambridge Antibody Technologies) and linked to a iluorophore (Fl'fC) to tag the putative transcriptional activator!receptor~ in the cells. This alternative would provide an external tag requiring no DNA tr,ansfection and therefore would be useful if distribution data were to be gathered from th~:~ ~:ariginal primary cultures used to generate the DAS.
Plasmid construct. A eukaryotie expression plasmid containing a coding sequence for a green fluorescent protein -- I~AS chimera is prepared using GFP
mutants. The construct is used t.o transfect HeLa cells ~('he plasmid, when transfected to into the host cell, produces a GFI" fused to the DAS pr~~tein product, designated GFP-DASpp.
Cell preparation and transfeetion. HcLa cells <~re trypsinized and plated using DMEM containing S°o charcoal dextrin-treated fetal bovine serum (FBS) (Hyclone) and 1°~° penicillin-streptomycin (('-l)MEM) 14"-:.'4 hours prior to transfection and t5 incubated at 37°C and 5°~o C.'('n . Transfections are performed by calcium phosphate coprecipitation or with Lipofe~.:t<rn~inc (Life 'I'ech~ol~.~'~ies). For the calcium phosphate transfections, the medium is rt:plai:ed, prior to trarjsfection, with DMEM
containing 5%
charcoal/dextran-treated FBS. ::'ells are incubated with the calcium phosphate-DNA
precipitate for ~-5 hours at 3 ~'°~' and ~°,-o Ct:)~, ;md wasi~ed 3-~ times with DMEM to 2o remove the precipitate, follm4~~.:d by the adciiticn~ of C-DMEM.
Lipofectamine transfections are perfc~r~ned i~u senrm-free DMEM withc>ut antibiotics according to the manufacturer's instructions. Fcalluwing a 2-3 h«ur incubation with the DNA-liposome complexes, the rnediurn is rernc>ved and replaced with C'-DMEM. All transfected cells in 96-well microtiter plates a.re incubated at s3''(' end 5°it> C.'O~
Cor 24-48 hours prior to (, _, drug treatment. Experiments arc pexforrned with the receptor expressed transiently in HeLa cells.
Localization of cpxpress~~d GF'P-DASpp inside cells. To obtain cellular distribution data, nuclei of transfected cells are first labeled with 5 llg/ml Hoechst 33342 (Molecular Probes) in C-L?~"11M for 20 minutes at 33°C_' and 5%
CO~. Cells are washed once in Hank's Balanced ;salt Solution (HBSS). I~he cells are analyzed live or they are rinsed with HBSS, fixed fc>r 15 min with 3_''°~ forrmldehyde in HBSS, stained with Hoechst 33342, and washed bef«re analysis.
In a prefewed embodiment, image acquisition and analysis are performed using the cell screening syste~rn of tl.~e present inv~ertti~n. The intracellular GFP-DA Spp fluorescence signal is collected by acquiring fluorescence image pairs (GFP-DASpp and Hoechst 33342-labeled nucteil Iiom field cells. The image pairs obtained at each time point are used to define nui:lear and cy~toplasrr~ir regions in each cell. Data demonstrating dispersed signal rn the cvtoplasrn would be consistent with known steroid receptors that are DNA trans~,riptional ;~caivators.
Screening for inductiurt of GFP-D.9Spp translocation. t.Jsing the above construct, confirnted for approlvriate expression ol~the (_iF~f-DASpp, as an indicator cell line, a screen of various ligands is performed using a series of steroid type ligands including, but riot limited to: estrogen, hro~~,est~rone. retinoids, growrth factors, 2~ androgens, and many other stcu~oid and steroid ba,.sed »~c~lecules. Image acquisition and analysis are performed usini; the cell screening system of the invention. The intracellular GFP-DASpp flu~::~rcvscTence signal Is CC~Ile:cted by acquiring fluorescence image pairs (CII~P-Dr'~Spp ancj llc:5echst 33342~1abeled nuclei) from fields cells. The image pairs obtained at each time: point aruae:d tc> c etine nuclear and cytoplasmic 6~1 regions in each cell. Translocation of GFP-I)ASpp is calculated by dividing the integrated fluorescence intensity- 01 GFP-DASpp in the nucleus by the integrated fluorescence intensity of the chimera in the cynoplasm or as a nuclear-cy~topiasmic difference of GFP fluorescence. ~'~ translocation fTOrrr the cytoplasm into the nucleus indicates a ligand binding activ;anon of the DASpp thus identifying the potential receptor class and action. C'om'bining this data with other data obtained in a similar fashion using known inhibitors a.nci modifiers of steroid receptors, would either validate the DASpp as a target, or more iiata would be generated from various sources.
E.rample 9 .9dditivnal .fcf~ecet.s Tran.slocation between the plamrtca mernbrano and the <~t.~t<ylc~.s~n~:
Profilactin complex dissociation and binding of profilin to the plasma rs membrane. In one embodiment, a fluorescent prc7tein hiosensor of profilin membrane binding is prepared by Dabelin~~ Ivurified profilin (Federov et al.( 1 r)94), J. Moles. Biol.
241:480-482; Lanbrechts et ai ; 1995), F.ur. .1. l~iaahenr. 2~i0:?81-?86} with a probe possessing a fluorescence lifetims~ iti the range of ?--aC)tl ns. The labeled profilin is introduced into living indicatoe~ cells using bulk leaading methodology and the indicator 2o cells are treated with test cornp«unds. Fluorescence :rrcisotropy imaging microscopy (cough and Taylor ( L x)93), .l. c: a.7ll f3ioL 1 ~! 1 :1095-1 10 -') is used to measure test-compound dependent moveno.a~t of the fluorescent derivative of protilin between the cytoplasm and membrane for a taeriod of time after treatment ranging from O.I
s to 10 h.
zs Rho-RhoGDI comple:~ translocation to the membrane. In another embodiment, indicator cells are treated with test compounds and then fixed, washed, and permeabilized. The indicator c~evll plasma rnembrant,, cytoplasm, and nucleus are ail labeled with distinctly colored rnarlcers followed by immunolocali2ation of Rho protein (Self ca al. ,1995), A~eti~u~is i» Enzymolo~v 256:3-lU; Tanaka et al.
(1995), Methods in Enzymologv 25b:41-=19) with antibodies labeled with a fourth color.
Each s of the four labels is imaged separately using the cell screening system, and the images used to calculate the amount of inhibition or activation o~ translocation effected by the test compound. To do this caculation, the imaes 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 protean. 7 he integrated brightness per to unit area under each mask is .~s~~c:l tea form a tr~Lnsloeation quotient by dividing the plasma membrane integrated bric;htness~'area by tl7e cytoplasmic integrated brightness/area. By comparing the. translocation quotient values from control and experimental wells, the percent translocation i_s calculated for each potential lead compound.
is ~3-Arrestin translocatio» to tire plciarno mernhrcu»e upon (r-protein receptor activation.
In another embodiment of a cytoplasm to membrane translocation high-content screen, the translocation of ~i-arrestin protein from the cytoplasm to the plasma membrane is measured in response to cell treatment. 'l ~r measure the translocation, 20 living indicator cells containint, luminescent vlomain markers are treated mith test compounds and the movemer_t c>t~ the (3-arre~stin marker is measured in time and space using the cell screening s-vsteon c>r the present irtventitm. In a preferred embodiment, the indicator cells contain lunuinescent markers consisting of a green fluorescent protein (3-arrestin (GFl'-~3-arrestin) protein chimera (~3arak et al. 1,19 97), J.
Biol. Chern.
2s 272:27497-275!70; Daaka et 3.1. ) 1998), J. Btc~l. c'~yenn '73:685-d88) that is expressed (a c, by the indicator cells through th<_ use o1~ transient oa- stable cell transfection and other reporters used to mark cytoplasn~ic ;u~d membrane dort7aios. Vfhen the indicator cells are in the resting state, tl.e dorr~ain marker molecules partition predominately in the plasma membrane or in the i;ytcrplasm. In the high-content screen, these markers are s used to delineate the cell cytolvlasm and plasma membrane in distinct channels of fluorescence. When the indicat.~r cells are treated with a test compound, the dynamic redistribution of the GFP-~i-arrestin is recorded as a series of images over a time scale ranging from 0.1 s to li> h. In a preferred embodiment, the time scale is 1 h.
Each image is analyzed by a metho:l float quantifies the movement of the GFP-~3-arrestin to 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-~3-arrestin probe marking the location of intracellular GFP-~-arrestin prc~teir~. 'l~he integrated E>rightness per unit area under each mask is used to foml a translocation quotient by dmiding the plasma membrane t5 integrated brightnessiarea by tl:.e cytoplasmic inte~.~;rat~~ct brightnesslarea. By comparing the translocation quotient values from control arnd experimental wells, the percent translocation is calculated for each potential lead compound. The output of the high-content screen relates quantita.tiw~ data describing ttte ma~;nit2~d~ of the translocation within a large number of individual cells that hay ~: bf:en treated with test compounds of 2o interest.
Translocation tretrl~een the em,io,,nlasmic reticulunr arre~ the holgi In one embodiment ~:~f an endoplasmic reticulurn to (Jolgi translocation high-content screen, the translocation of a VW'G protein from the ts045 mutant strain of vesicular stomatitis virus (Ellenbc°rg et al. ( i ~19~; j, .,r. t oTl Biol. 13f;:1193-1206; Presley et al, (1997) Nature 389:81-85) frorrr the endoplasmic retuculum to the Golgi domain is measured in response to cell tre,rtrnent. To nn_asrjre ~:he translucation, indicator cells containing luminescent reporters are treated with test compounds and the movement of the reporters is measured in spare and time using the cell screening system of the present invention. The indicatc>r 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 transfec:tiort and other domain markers used to measure the localization of the endoplasmic reticulum and Golg,i domains. When the indicator cells are in their resting state at 4U'(', the (~FP-VS~'t~; protein chimera molecules are partitioned predominately in ilne cndoplasmic reticulurn. In this high-content screen, domain markers of distinct color's used to delineate the endoplasmic reticulum and the Golgi domains in distinct channels c3f fluorescence V%hen the indicator cells are treated with a test compound and thc: temperature is .=~imultanoously lowered to 3~'''C, the dynamic redistribution of the (:iFP-VSVG protein <;himera is recorded as a series of to images over a time scale ranching from 0,1 s to 10 h. Each image is analysed by a method that quantifies the mcw~c:nrent of the CaFP-V:~'v G protein chimera between the endoplasmic retrculum and the ~Tolgi domains. fc dc> this calculation, the images of the probes used to mark the enclaplasmic reticu'um and the Ciolgi domains are used to mask the image of the GFP-'~'f>~-G prohe rrrark~ng the location of intracellular GFP-2o VSVG protein. The integrated brightness her unit area under each mask is used to form a translocation quotient by dividing the: endoplasmic reticulum integrated brightnessiarea by the Golgi integrated brightnessiarea By comparing the translocation quotient values from contr~::~i ~md c~xperirnental wells, the percent translocation is calculated for each potential land compound. The output of the high-content screen c, relates quantitative data describirng tl~e magnitude c~f the translocation within a large number of individual cells that h,a~~r; been treated with test compounds of interest at final concentrations ranging fronu 10 '" M to 10 ; M for a period ranging from 1 min to h.
Induction and inhibition of organellar function:
Intracellular microtubol~e stabilitw. In one embodiment of an organellar function high-content screen, the assembly state of i~rtracellular microtubules is 1o measured in response to cell treatment. T~> measure microtubule assembly state, indicator cells containing luminescent reporters are treated with test compounds and the distribution of the reporters is rrrc:asured in space and tin-re using the cell screening system of the present invention.
In a preferred embodin-cent, the reporter of inirtrcellular nricrotubule assembly is i> MAP 4 (Bulinski et al. (l'~)~~~')~ J. Cell Scimcn 110::3455-30<=Ij, a ubiqtutous microtubule associated protein tlaa.t is known to interact with microtubules in interphase and mitotic cells. Tho indic;itur cells contain luminescent reporters consistinv~ of a GFP-MAP 4 chimera that is e~,;pr~:ssed by the incficator cells through the use of transient or stable cell transfection and other reporters are used to measure the localization of the 2o cytoplasmic and membrane ~~c:~noponents. :'~ t rFf~~tAP 4 construct is prepared as follows: PCR amplification of native or mutant (pFP molecules using primers to introduce restriction enzyme sites is performed. The PCR product is ligated into the MAP 4 cDNA within a c:uf;arw>tic expression vector. Indicator cells are then a~>

transfected with the expression v~;ec~or to produce esther transiently or stably transfected indicator cells.
Lndieator cells are treate~;i ~,~~ith test compounds at final concentrations ranging from IO~iz M to 10-; M for a loeriod ranging from 1 min to 10 h. Growth medium a containing labeling reagent to rvark the nucleus and the cytoplasm are added. After incubation, the cells are washed with Hank's balani;ed salt solution (HBSS), fixed with 3.7% formaldehyde for 10 min ,:rt ~~oom temperature, and washed and stored in HBSS.
Image data are obtained fi-orn both fixed and living indicator cells. To extract morphometric data from each cyf the images obtained thc: following method of analysis i0 is used:
1. Threshold each nucleus acrd cytoplasmic image to product a mask that has value = 0 for each pixel outside a nucleus or cell boundary 2. Overlay the mask on h~: original image, detect each object in the field (i.e., nucleus or cell), and calculate its size, shape, and integrated intensity.
1 a 3. Overlay the whole cell rrarsk obtained above on the corresponding GFP-image and use an automa~.e~i measurement o(~ edge strength routine (Kolega et al.
(1993). Biolrnagin~,y 1:13h-1 ~t)1 to calculate the total edge strength within each cell.
To normalize for cell size, tlic total edge streo~;th is ~tivided by the cell area to ~uive a "fibrousnc:ss" v;rlue. I ar-~w i'ibrousness . aluf:~ arc associated with strong edge ?o strength values and are tl~~~refor~ maximal ire cc°Lls f~ontaining distinct microtubule structures. Likewise, srn~ll fihrousness ~' clues are associated with weal';
edge strength and are minimal in cells ~sitir depol~rnerized rnicrotubules. The physiological range of fihorursness valu~:,s is set by treating cells with either the microtubule stabilizing drug pavlitarel (1U yM) or the microtubule depolymerizing drug nocodazole ( 1U pg,~ml).
High-content screens involving t~h~ functional localisation o~~mucromoleeules Within this class of hif~h-content screen, the functional localization of macromolecules in response to extor~~al Stimuli is measured within living cells.
Glycolytic enzyme activity regulation, In a preferred embodiment of a cellular enzyme activity high-c~ontc.nt screen, the activity of key glycolytic regulatory enzymes are measured in treated cells. To measure enzyme activity, indicator cells '. o 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.
In one embodinnent, th~:weporter of intracellular enzyme activity is fructose-phosphate, 2-kinase/fn~ctose-2,6--bisphosphatase (I'FK-~'), a regulator's enzyme whose 15 phosphorylation state indicates intracellular carhohydrate anabolism or catabolism (Deprez et al. ( 1997) J Bio~. l 'hcerrr. 27?: l ?2(~t~-l r 2~'S; Kealer et al. ( 1996) hEBS
Letters 395:225-227; 1-ee et al. t; t 996), l3iochernist;ri' 3s:6U l U-6019).
The indicator cells contain luminescent relyorters consistin~~ ~~f .a t~uorescerV protein biosensor of PFK-2 phosphorylation. 'l he fluorescent protein biosensor is constructed by zo introducing an environmentall~~ _~cnsitme fli~oresc;ent dye near to the known phosphorylation site of the enzyX~~ l l:)eprez et al ( 19~)~), .supra;
~iutiano et al. (1995), supra). The dye can be of the ketocyanine class ( Kessler and VVolfbeis ( 1991 ), Spectrochimica ..dicta 47A:18'T-1 ~)2 ) car any class that contains a protein reactive moiety and a tluorochrome whose excitation or emission spectrum is sensitive to solution polarity. The fluorescent protein biosensor is introduced into the indicator cells using bulk loading methodology.
Living indicator cells are treated with test canipounds, at final concentrations ranging from 10-'2 M to 10'' M fc?r times ranging from u.1 s to 10 h. In a preferred embodiment, ratio image data are obtained from licking treated indicator cells by collecting a spectral pair of f'.uorescence 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 tuba spectral images at each time point, pixel by pixel.
Each pixel value is then used to calculate the fractional phasphorylation of PFK-2. At tt~ small fractional values of~ phosl~horvlation, PFK-~ stimulates carbohydrate catabolism.
At high fractional values c~f phosphorylatio~~, PFK-7 stimulates carbohydrate anabolism.
Protein lcinase A ac ivity and Iocalrlation of subunits. In another 1 > embodiment of a high-content :,cretin, bath the datt2ain localization and activity of protein kinase A (PKA) within indicator cells are measured in response to treatment with test compounds.
The indicator eclls contain lun~inesceot rE°l>orters including a fluorescent protein biosensor of PKA activation. 1'he fluorcaeent protein biasensor is constructf:d by introducing an environmentally ~~nstive fluorescent ~3y~~ into the catalytic subunit of PKA near the site known to !ncertict with th. re4.~ulatory subunit of PKA
(Harootunian et al. ( 1993), AToI. Biol. of thc- ('ell 4:993-100: Ji7hnson et al. ( 1996), Cell 85:149-158;
Giuliano et al. ( 1995), supru). -1 he dye can be ~.nf the ketocyanine class (Kessler, and Wolfbeis (1991), Sp~~rtroeh:mic~a :lcta 47.~:1~7-1~)2) or any class that contains a ,.

protein reactive moiety and a flu.orochrome whose excitation or emission spectrum is sensitive to solution polarity. Thc, tluoresccnt protein l~iosc;nsor of PKA
activation is introduced into the indicator cell: using bulk loading methodology.
In one embodiment, livirug indicator cells are treated with test compounds, at final concentrations ranging from l()'' M to 10' 1~9 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 bioseT~isc>r data from each time point, a ratio is made between each pair of images, and each l~i:~el value is then used to calculate the fractional activation of PKA (e.g., separation of the catalytic and regulatory subunits after cAMP
1o binding). At high fractional valuca i>f activity, f'Fk-2 stimulates biochemical cascades within the living cell.
To measure the transloiaiion 4rf the catalytic. subunit of PKA, indicator cells containing luminescent reporters ~rre treated with test compounds and the movement of the reporters is measured in spare and time using the cell screening system.
The indicator cells contain iumine:,cent reporters consisting of domain markers used to measure the localization of the cs.~toplasmic acrd nuclear domains. When the indicator cells are treated with a test ~.uympounds, tire dynamic redistribution of a PKA
fluorescent protein biosensor i:s rvcoc-ded intracellularly as ~t series of images over a time scale ranging from 0.1 s to 1(t h. Eac-h irnat?;e is analyzed by a method that 2o quantifies the movement of the PK.~~ between the cytoplasmic and nuclear domains. To do this calculation, the images o1~ the probes used to mark the cynoplasmic and nuclear domains are used to rr~ask the i~oag~, of the PKA fluorescent protein biosensor. The integrated brightness per unit area under each mask is used to torn a translocation quotient by dividing the c ~,~toplasmic integrated bri;~htness~=area by the nuclear ., ., integrated brightness/area. By comparing the translocation quotient values from control and experimental wells, the l:rercent translocation is G;rlculated for each potential lead compound. ~:'hc <output of the high-content screen relates yuantitative data describing the magnitude of the translocation within a large number of individual cells that have been treated with test compound in the concentratian range of l~-'' M to 103 M.
Nigh-content scrc.~ens involving !hE~ induction or inlubitron cy~gene erpression RNA-based flttorcascent hic~s~nsors to Cytoskeletal protein trarrscriptiorr and message localization. Regulation of the general classes of cell physiological responses including cell-substrate adhesion, cell-cell adhesion, signal transd~r<:tion, cell-cyl~~ events, intermediary and signaling molecule metabolism, cell locornoticm, cell-fi~cll corumunication, and cell death can involve the alteration of gene e:~pression. High-cinrrteut Screens can also be designed to f , measure this class of physio(o~ictrl re>ponse.
In one embodiment, tl'ne reporter cat intracellular gene expression is an oligonucleotide that cart hybridize with the tar~~tt rnl2NA and alter its fluorescence signal. In a preferred embodivment, the oiigunucleotide is a molecular beacon (Tyagi and Kramer ( 1906) ,'Vut. Brote~<~In~r~~:~l. 14:3()3- >t?~ 1, a itirt~inescence-based reagent whose ?o fluorescence signal is depene?ent on intermolecular an,l intramolecular interactions.
The fluorescent biosensor is c orastructed by introelucin~ a fluorescence energy transfer pair of fluorescent dyes such that there is one at each end (S' and 3') of the reagent.
The dyes can be of-any class that cornains a protein reactive moiety and fluorochromes whose excitation and erniss~on ~sp4ctra ovcrlah sufficiently to provide fluorescence energy tra~tsfer between the dytas in the resting state. including, but not limited to, fluorescein and rhodamine (Molecular Probes, lnc ). In a preferred embodiment, a portion of the message :;oding for ~3-actin (Kislauskis et al.. (1994), J.
Cell Biol.
127:441-451; McC'ann et al, ( 1997), Proc. ~'Vcrti' Acu~. Sci. 94:5679-5684;
Sutoh (1982), Biochemistry 21:3654-3f:.61 ) is inserted into the. loop region of a hairpin-shaped oligonucleotide with the ends tethered together due to intrarnolecular hybridization. At each end of the biosensor a flui>rescence donor (fluorescein) and a fluorescence acceptor (rhodamine) are covaletrtly bound. In the tethered state, the fluorescence energy transfer is maximal and therefore indicative of an unhybridized molecule.
t~~ When hybridized with the r~rR1'~d~~ coding for ~-actin, the other is broken and energy transfer is lost. The complete fluorescent biosensor is introduced into the indicator cells using bulk loading methodolcyy.
In one embodiment, living indicator cells are treated with test compounds, at ftnal concentrations ranging from 10~~' M to 117' "~~1 far tunes ranging from 0.1 s to 10 l 5 h. In a preferred embodiment, ratio image data are obtained from living treated indicator cells. ~I~o extract mcrrphometric data from each time point, a ratio is made between each pair of images, anif each pixel aaiue is then used to calculate the fractional hybridization of the: labeled nucleotide. .At small fractional values of hybridization little expression c:~f (3-actin is indicated. ~t hid=.h fractional values of ?o hybridization, maximal expres~;sion of G3-actin ns iruclicated.
Furthermore, the distribution of hybridized molecules within the cytoplasm c~f~the indicator cells is also a measure of the physiological response of the indicator cells.
Cell surface binding of a likancl 2>
7:

Labeled insulin binding to its cell surface receptor in living cells. Cells whose plasma membrane dom;:~irr has been labeled wOh a labeling reagent of a particular color are incubated with a solution contarning insulin molecules (Lee et al.
(1997), Biochemistn~ 36:2701-2a'0~, Martinet-Zaguilan et al. (1996), Am. J.
Physlol.
s 270:C1438-C144fi) 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 ,.ells fixed and the distribution and concentration of the insulin on the plasma membranc.~ is measured. '1'o do this, the cell membrane image is used as a mask for the insulin in-age. 'The integrated intensity from the masked insulin tt) image is compared to a set of images containing known ,:amounts of labeled insulin.
The amount of insulin bound tc~ the cell is deterzriined from the standards and used in conjunction with the tote! concentration of insulin incubated with the cell to calculate a dissociation constant or insulin to its cell surface rcc:eptor 1 s Labeling of cellukrr com~artmc~rrt,s Whole cell labeling Whole cell labeling is accomplished by labeling cellular components such that dynamics of cell shape and molilit~ of the cell earl be measured over time by analyzing fluorescence images of cells.
>o In one embodiment, sunall resrctive: fluorLseent molecules are introduced into living cells. These membrane-pi>erroeant molecules both diffuse through and react with protein components in the pla:~noa membrane. 1.)ye molecules react with intracellular molecules to both increase the fluorescence signal emitted from each molecule and to entrap the fluorescent dve w ittnrn living cells 7,h~ae rnoleeules include reactive ;6 chloromethyl derivatives of arninocoumarins, hydroxycoumarins, eosin diacetate, fluorescein diacetate, some l3odipy dye derivatives, and tetramethylrhodamine.
The reactivity of these dyes toward macromolecules includes free primary amino groups and free sulfhydryl groups.
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 flue cell of intereat that contain a green fluorescent protein, or mutant thereof, component can also be used to tluorescently label the entire W cell surface. Once the entire cell is labeled, images of I:he 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.
Plasma membrane iabceling to In one embodiment, labnlinthe whole plasma membrane employs some of the same methodolo;y described albove for labclntg the entire cells. Luminescent molecules that label the entire c:11 surface act 1o delineate tire plasnna membrane.
In a second embodiment subdomains oi~ the plasma membrane, the extracellular surface, the lipid bilayc:r, and ~:l~e sntracellular sr~rfac;e can bc; labeled separately and '0 used as components of high content screens. In floe first cmbodirnent, the extracellular surface is labeled using a brief treatment with a reactive fluorescent molecule such as the succinimidyl ester or iod~:~ac;etamde clcnvatives of fluorescent dyes such as the fluoresceins, rhodamines, cyanincs, and Bodipys.
~i In a third embodiment, the: c:xtracellular surface is labeled using fluorescently labeled macromolecules with a hii;~h ailinity for cell surface molecules.
These include fluorescently labeled Iectins such as the fluorcscein, rhodamine, and cyanine derivatives of lectins derived fiom jack boar (Con A), red kidney bean (erythroagglutinin PHA-E), or wheat germ.
In a fourth embodiment, fluarescently 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.
In a fifth embodiment, the lipid bilayer of the plasma membrane is labeled with fluorescent molecules. 'I'hese 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. Patents Nos. 4,7;E3:i,4t~l issued November 8, 1988; 4,762,701 issued August 9, 1988; and 4,8'.79,584 us~ued .August 22, 1989; available commercially from Sigma Chemical Company, SI. Louis, MO), fluorescent phospholipids such as nitrobenzoxadiazole glycerol:>hosplioethanolarn in L and fluorscein-derivatized dihexadecanoylglycerophospha<;tha-nolamine, flui>resc:ent fatty acids such as 5-butyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-nonamoic acid and 1-pyrenedecanoic acid (Molecular Probes, Inc.), fluorescent sterols including cholesteryl 4,4-difluoro-5,7-dimethyl-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, f:A).
-rs In another embodiment, tine intracellular component of the plasma membrane is labeled with fluorescent molecul~a. Examples of these molecules are the intracellular components of the trimeric (r-protein receptor, adenylyl cyclase, and ionic transport proteins. These molecules can bc; Labeled as a result of tight binding to a fluorescently S labeled specific antibody or by thf: incorporation of a fluorescent protein chimera that is comprised of a membrane-associated protein and the green fluorescent protein, and mutants thereof.
Endosome fluorescence labeling 1o In one embodiment, ligands that are transported into cells by receptor-mediated endocytosis are used to trace the dynamics of endosornal organelles. Examples of labeled ligands include Bodily FL-labeled lo~wv density lipoprotein complexes, tetramethylrhodamine transferrin analogs, and lluorescently labeled epidermal growth factor (Molecular Probes, lne.) t~ In a second embodiment, Iluorescently labeled primary or secondary antibodies (Sigma Chemical C o. St. Lou:s, !~i0; Molecular Probes, Inc. Eugene, OR;
Caltag Antibody Co.) that specifically lab~:l endosomal ligands are used to mark the endosomal compartment in cells In a third embodiment, ~;n~Iosomes are fluoresc:ently labeled in cells expressing 2~a protein chimeras formed 'by fus~n~~ a green fluorescent protein, or mutants thereof, with a receptor whose internalization labels endosome~~ C'Iiimeras of the EGF, transferrin, and low density lipoprotein receptors are examples of these molecules.
c~

Lysosome labeling In one embodirrrent, membrane permeant lysosome-specific luminescent reagents are used to label the lysosonral compartment of living and fixed cells. These reagents include the luminescent molecules neutral red, N-(3-((2,4-dinitrophenyl)amino)propyl)-N-{?~-trrninopropyl)methylamine, and the LysoTracker probes which report intralysos~on 7a1 pII as well as the dynamic distr7bution of lysosomes (Molecular Probes, Irn::. ) In a second embodimfvnt, antibodies against lysosomal antigens (Sigma 1o Chemical Co.; Molecular Prorr.es, lm.; C'altag "~ntil>ody t-'o.) are used to label lysosomal components drat are localized in specific ly:;osomal domains.
Examples of these components are the degrad,:rtcve enzymes inv<Oved in cholesterol ester hydrolysis, membrane protein proteases, anti nucleases as well as the A fI'-driven lysosomal proton pump.
In a third embodiment, prcvtein chimer,:ts consisting of a lysosomal protein genetically fused to an intrinsically luminescent protein such as the green fluorescent protein, or mutants thereof, are ns<.cl to label the lysoscan-ral domain.
Examples of these components are the degradative enzv~mes in volvt°d in cholesterol ester hydrolysis, membrane protein prote,.rses. arn~;i ocizcleases as well as t'nc: !~-I-P-driven iysosomal proton 2o pump.
Cytoplasmic fluarescer~~ck~ labeling In one embodiment, cell pern~eant fluorescent dyes (Molecular Probes, Ine.) with a reactive group are reacr.c~d with living cf:ll5. Reactrve dyes including '; CI

monobrornobimane, S-chloromethylfluorescein diacetate, carboxy fluorescein diacetate succinimidyl ester, and chloroi°n~~tln~~l tetramethylrhodarrnne are examples of cell permeant fluorescent dyes that art: used for long term labeling of the cytoplasm of cells.
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 Ure also used for cytoplrsmic labeling.
In a third embodiment, antibodies against cytoplasmic components (Sigma Chemical Co.; Molecular Probes., lnc.; ~'altag Antibody C~o.) are used to fluorescently label the cytoplasm. Example<.: ~>f cytoplasmic antigens are rrrany of the enzymes ~C~ involved in intermediary metabc;~lisrn. Enolase. phosphofi-uctokinase, and acetyl-CoA
dehydrogenase are examples of uniformly distributed cytoplasmic antigens.
In a fourth embodiment, protein chimeras consisting of a cytoplasmic protein genetically fused to an intrinsicaally luminescent protein such as the green fluorescent protein, or mutants thereof, are a sed to label the' .: ~,~toplasm.
Fluorescent chimeras of t ~ uniformly distributed proteins arc: used to label the entire cytopiasmie domain.
Examples of these proteins ~:rr roany of the proteins involved in intermediary metabolism and include enolase, la:~<aat~ dehydr~7ge:nas~~, and hexokinase.
In a fifth emrrodimer~u, antibodies agarzns~ cytoplasmic antigens (Sigrrra Chemical Co.; Molecular E'rev°.sc::s, lnc.; Calta'= .4ntihodw Co.) are used to label <.0 cytoplasmic components that are localized in specific cytoplasmic sub-domains.
Examples of these componer7ts are the cytoskelei.al proteins actin, tubulin, and cytokeratin. A population of there proteins ~~ithin cells is assembled into discrete structures, which in this case, are fibrous. Fluorescence labeling of these proteins with antibody-based reagents therefc~r~ labels a specific: sub-domain of the cytoplasm.
hl In a sixth embodiment, non-,~ruibody-based iluorescently labeled molecules that interact strongly with cytoplasm~ic: proteins are toned to label specific cytoplasmic components. (Une example is a t'luorescent analag of the enzyme DNAse 1 (Molecular Probes, Inc.) Fluc:rrescent analv:rgs of this en;~ynte bind tightly and specifically to s cytoplasmic actin, thus labeling a st b-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 comp:>nents of the acain- and microtubule-cytoskeletons, respectively.
In a seventh embodiment, prc.,tein chimeras oansisting of a cytoplasmic protein to genetically fused to an ir"ttrirrsic:.~lly lurtzinESCerot protein such as the green fluorescent protein, or mutants thereof, ar. used to label specific domains of the cytoplasm.
Fluorescent chimeras of highly localized proteins are used to label cytoplasmic sub-domains. Examples of these pr;~teiras sire many of the prcsteins involved in regulating the cytoskeleton. 'They include tha stnzctural protc;ins actin, tubulin, and cytokeratin as t vs well as the regulatory proteins nticrotubule associated l7rotein 4 and a-actinin.
Nuclear labeling In one embodiment, nnembrane pern~eant nucleic-acid-specific luminescent reagents (Molecular Probes, Inc.) arv used to label ~hr nucleus of living and fixed cells.
2o These reagents iroclude cyanint:-h~:tsed dyes (e g . TOTOr-. ~'C)YO~'. and BOBOT"), phenanthidines and acridines Ia:'.r-., ethidium hror-nide, pr~.~pidiurrt iodide, and acridine orange), indoles and irnidazole; (e_~., Hoechst y~t253. E-loechst 33342, and 4',6-diamidino-2-phcnylindole), an~a ~,otlter similar re;.yents (t~.,sr., 7-aminoactinomycin D, hydroxystilbanudine, and the psaral~ns).
h-, In a second embodiment, antibodies against nuclear antigens (Sigma Chemical Co.; Molecular Probes, Inc.; ( 'a9tag Antibody ~:o.) are used to label nuclear components that are localized in specific nuclear domains. Examples of these components are the macromolecules involved in maintaining I)NA structure and function. DNA, ItNA, histone;n, DNA polymor~ase, RNA polymerise, lamins, and nuclear variants of cytoplasmic proteins such as actin are examples of nuclear antigens.
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 holymerase, I2.NA polymerise, lamins, and nuclear variants of cytoplasmic proteins such as actin are examples of nuclear proteins.
Mitochondria) labeling In one embodiment, mc;mbrane penneant mitochondria)-specif c luminescent reagents (Molecular Probes, Inc.) .are used to label the mitochondria of living and fixed cells. These reagents include rl~adamine 123, tetralncthyl rosamine, JC-I, and the MitoTracker eactive dyes.
In a second embodiment, antibodies against mitochondria) antigens (Sigma Chemical Co.; Molecular Prr>bes, Inc.; C'a'itag Antibody Co.) are used to label mitochondria) components tl~.at are localized in specific mitochondria) domains.
Examples of these component:o are the macromolec:ulcs involved in maintaining mitochondriat DNA structure and function. DN.A, ItNA, histones, I)NA
polymerise, RNA polymerise, and mitochandrial variants of ~ytoplasmic macromolecules such as mitochondria) tRNA and rRNA vre examples mitochondria) antigens. Other examples of mitochondria) antigens are the cc>roponents o1 the oxiciativc:
phosphorylation system found in the mitoehondr-ia (e.g., c~.nochxome c, cyt«chrorne c oxidise, and succinate dehydrogenase).
In a third embodiment, protein chimeras consisting of a mitochondria) protein genetically fused to an intrinsically luminescent protein such as the green fluorescent protein, or mutants thereof, are usk:<i to label the rr~itoc;hondrial domain.
Examples of these components are the macromolecules involved in maintaining mitochondria) DNA
structure and function. Examples include histories, DNA polymerise, RNA
to polymerise, and the components oi~ the oxidati~;~ phosphorylation system found in the mitochondria (e.K., cvtochrorne c, cytochrorne ~ oxidise, and succinate dehydrogenase).
Endoplasmic reticulum iaheling t_> In one ernbodirnent, membrane penoeant c:ndoplasmic reticulum-specific luminescent reagents (Molecular Probes, Inc.) are used to label the endoplasmic reticulum of living and fixed ec-lls. These reagents include short chain carbocyanine dyes (e.~., DiOC,, and DiOC~), loro~! chain carbocyanirn. dyes (e.y , DiIC'», and DiIC~s), and luminescently labeled leetins such as concanav alin :~.
2o In a second embodiment, antibodies <rgatnst ~~ndoplasmic reticulum antigens (Sigma Chemical <.'o.; ~Molecul~r (robes, Inc.; (.'all.ae, :Antibody ('o.) are used to label endoplasmic reticulum compom:nts that are localised xn specific endopiasmic reticulum domains. Examples of these components are the macromolc;cules involved in the fatty acid elongation systems, glucose-t:~-phc>sphitase, and HMCz C:'uA-reductase.

In a third embodiment, pr~;~tc::in chimeras consisting c~f a endaplasmic reticulum protein genetically fused to an intrinsically lurrliztescent protein such as the green fluorescent protein, or mutants i.hc~r~eof, are used to Iabel the endoplasmic reticulum domain. Examples of these comz~onents are the macromolecules involved in the fatty acid elongation systems, glucose ~6-~phosphatase. and 1-(MG C'oA-roductase.
Golgi labeling to In one embodiment, membrane permeant Ciolgi-specific luminescent reagents (Molecular Probes, Inc.) are used to lahel the CTolgi ~~f living and fixed cells. These reagents include luminescently labeled macromolecules such as wheat germ agglutinin and Brefeldin A as well as luminescently labeled c;~ramidt~.
In a second embodiment, antibodies against t_iolgr antigens (Sigma Chemical Co.; Molecular Probes, lnc.; Caitag Antibody <'o.) are' used to label Golgi components that are localized in specific Ciol,~,i di>mains. Examples of these components are N-acetylglucosamine phc.~sphotr~~nsferase, G~:olgi-specific phosphodiesterase, and mannose-6-phosphate receptor l~r~ntein, In a third embodirne~U, protein ch~meE-~~s c~~nyisting of a Golgi protein genetically fused to an intrinsically lutninescc:nt protein such as the green fluorescent protein, or mutants thereof arc used to label thc~ Goigi domain. Examples of these components are N-acetylgfucosamine ph osphotransferase, Golgi-specific phosphodiesterase, and manno:.~:-~~~-phosphate rccehtor protein.
~5 While many of the exarnpies presented involve the measurement of single cellular processes, this is again i,~ intended for purposes . f illustration only. Multiple parameter high-content screens c,~.rt ho produced by combining several single parameter screens into a multlparameter hia;h-e:ontent screen or b,y adding cellular parameters to s any existing high-content screen. Furthermore, while each example is described as being based on either live or fixers ~~~,lls, each high-content screen can be designed to be used with both live and fixed cel3s.
Those skilled in the art w ill recognize a wide variety of distinct screens that can be developed based on the discla~siarc provided herein. 'Chore is a large and growing list to of known biochen a seal acid rool~:~c,ilar processes in :ells that involve translocations or reorganizations of specific com~:~oiients within cells. -hhe signaling pathway from the cell surface to target sites w,itloir~ the cell involves the translocation of plasma membrane-associated proteins tc> the cytoplasm. lvor r~xample, it is known that one of the src family of protein tyrosine: kinases, ppti0c-src ( Walker et al ( 1993), J. Biol.
to C'hem. ?68:19552-19558) translo~.:ate;s from the plasma tt~embrane to the cytoplasm upon stimulation of fibrobl;tsts with platelet-derived growth factor (PDGF) Additionally, the targets for screening can themselves be converted into fluorescence-based reagents that report mo)c~ctilar changes including ligand-binding and post-trans(ocational modifications.
:'o RG

Claims (10)

THE EMBODIMENTS OF THE INVENTION FOR WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for acquisition, storage, and retrieval of cell screening data on a computer system, comprising the steps of:

a) providing a plate containing wells, wherein the wells contain cells possessing one or more luminescent reporter molecules on or in the cells;

b) storing input parameters used for screening of the plate in a database;

c) automatically repeating steps (i)-(ix) for a desired arrays of wells:

i) selecting an individual well on the plate;

ii) collecting image data from the well;

iii) storing the image data in the database;

iv) collecting feature data from the image data, wherein the feature data comprise luminescent signals from the one or more luminescent reporter molecules on or in the cells;

v) storing the feature data in the database, wherein the feature data provide information on distribution, environment, and/or activiy of the one or more luminescent reporter molecules in individual cells;

vi) calculating well summary data using the image data and the feature data collected from the well;

vii) storing the well summary data in the database;

viii) calculating plate summary data using the well summary data from the database; and ix) storing the plate summary data in the database.

2. A computer readable medium having stored therein instructions for causing a computer to execute the method of claim 1.

3. The method of claim 1, wherein the wells include cells treated with a test compound.

4. The method of claim 1, wherein the plate comprises a microplate.

5. The method of claim 1, wherein the database includes microplate data.

6. The method of claim 1, wherein the database includes photographic image data.

7. The method of any one of claims 1 to 6, wherein the one or more luminescent reporter molecules on or in the cells are fluorescent reporter molecules.

8. The method of any one of claims 1 to 7 wherein the input parameters used for screening of the plate include parameters selected from the group of identifying nuclei, identifying cytoplasm, identifying fluorescent reagents, cell selection settings, number of cells to be analyzed per well, and range of size, shape, and intensity of cells to be analyzed.

9. The method of any one of claims 1 to 8, wherein the feature data are selected from the group of size, shape, intensity, location, area, perimeter, height, width, total fluorescence intensity, average fluorescent intensity, ratio of fluorescent intensities, difference in fluorescent intensities, and number.

10. The method of any one of claims 1 to 9, wherein the step of collecting well summary data includes calculating data selected from the group of size, shape, intensity, location, area, perimeter, height, width, total fluorescence intensity, average fluorescent intensity, ratio of fluorescent intensities, difference in fluorescent intensities, and number.