MXPA01002684A - A system for cell-based screening - Google Patents

Abstract

The present invention provides systems, methods, screens, and kits for optical system analysis of cells to rapidly determine the distribution, environment, or activity of fluorescently labeled reporter molecules in cells for the purpose of screening large numbers of compounds for those that specifically affect particular biological functions. The invention involves providing cells containing fluorescent reporter molecules in an array of locations and scanning numerous cells in each location with a high magnification fluorescence optical system, converting the optical information into digital data, and utilizing the digital data to determine the distribution, environment or activity of the fluorescently labeled reporter molecules in the cells. The array of locations may be an industry standard 96 well or 384 well microtiter plate or a microplate which is a microplate having cells in a micropatterned array of locations. The invention includes apparatus and computerized method for processing, displaying and storing the data.

Description

A SYSTEM OF ANALYSIS BASED ON CELLS FIELD OF THE INVENTION This invention is in the field of molecular and cellular biochemical tests based on fluorescence for the discovery of drugs.

BACKGROUND OF THE INVENTION Drug discovery, as currently practiced in the art, is a long multi-stage process involving the identification of targets of specific diseases, the development of a test based on a specific target, the validation of the test. , the optimization and automation of the test to produce a sieve, high research of the performance of the libraries of compounds using the test to identify "impacts", impact validation and optimization of impact compounds. The power of this process is a leading compound that works in pre-clinical tests and if it is validated, eventually in clinical trials. In this process, the analysis phase is different from the development phases of the test and involves the efficiency of the test compound in living biological systems. Historically, the discovery of drugs is a costly and slow process, many years in duration and the consumption of hundreds of millions of dollars per drug created. Developments in the areas of genomes and high performance research have resulted in increased capacity and efficiency in the areas of target identification and the volume of compounds analyzed. Significant advances in automatic DNA sequencing, PCR application, positional cloning, hybridization series and bio-informatics have greatly increased the number (and gene fragments) of target drug analysis potential coding j_ > - »__b_. ^ »_-¡_-_» ¡---. . < aw »MMÍC« __ ttj «genes. However, the basic scheme for drug analysis remains the same. The validation of target genomes as points for therapeutic intervention using existing methods and protocols has become an obstruction in the drug discovery process due to the slow manual methods employed, such as in vivo functional models, functional protein analysis recombinants and expression of stable cell lines of candidate genes. The main DNA sequence data acquired through automated sequencing does not allow for the identification of gene function, but can provide information about common "themes" and specific gene homology when compared to known sequence databases . Genome methods such as subtraction hybridization and RADE (rapid differential expression amplification) can be used to identify genes that are more or less regulated in a disease state model. However, identification and validation still proceed under the same path. Some proteomic methods use protein identification (combinatorial libraries, 2D electrophoresis, global expression series) in combination with inverse genetics to identify candidate genes of interest. Such "putative disease-associated sequences" or DAS isolated as intact cDNA are a great advantage for these methods but are identified by the hundreds without providing any information, without considering the type, activity and distribution of the encoded protein. The selection of a subset of DAS as drug analysis targets is "random" and therefore extremely inefficient, with no functional data to provide a mechanized link with disease. If necessary, therefore to provide new technologies to quickly analyze the DAS to establish the biological function, thereby providing the improvement of target validation and optimizing the candidate in drug discovery.

There are three main routes to improve the productivity of early drug discovery. First, there is a need for tools that provide improved information handling capacity. Bioinformatics has emerged with the rapid development of DNA sequencing systems and the evolution of genome databases. The genome is initiated to play a critical role in the identification of potential new targets. Proteomics have become indispensable in relation to the structure and function of target proteins to predict drug interactions. However, the next level of biological complexity is the cell. Therefore, there is a need to acquire, manage and search multi-dimensional information of the cells. Second, there is a need for high performance tools. Automation is a key to improving productivity as demonstrated recently in DNA sequencing and high performance core analysis. The present invention provides automated systems that extract information from multiple parameters of cells that meet the need for high performance tools. The present invention also provides miniaturization methods, thereby allowing for increased yield, while decreasing the volumes of reagents and test compounds required in each test. Radioactivity has been a dominant reporting extraction in the evidence of anticipated drug discovery. However, the need for more information, high performance and miniaturization have caused a shift towards the use of fluorescent detection. Fluorescence-based reagents can produce more efficient multi-parameter tests that are higher in yield and information content and require less volumes of reagents and test compounds. Fluorescence is also safer and less expensive than methods based on radioactivity.

The analysis of cells treated with dyes and fluorescent reagents is well known in the art. There is a considerable body of literature related to the genetic engineering of cells to produce fluorescent proteins, such as the modified green fluorescent protein (GFP) as a reporter molecule. Some properties of the GFP of the non-cultivated type are described by Morise et al. (Biochemistry 13 (1974), p 2656-2662) and Ward et al. (Photochem, Photobiol, 31 (1980), pp. 611-615). The GFP of the jellyfish Aequorea victoria has a maximum excitation at 395 nm and a maximum emission at 510 nm and does not require an exogenous factor for the fluorescence activity. The uses of GFP described in the literature are generalized and include the study of gene expression and protein localization (Chalfie et al., Science 263 (1994), p.12501-12504)), as a tool for visualizing subcellular organelles (Rizzuto et al., Curr. Biology 5 (1995), pp. 635-342)), visualization of protein transport along the secretory pathway (Kaether and Gerdes, FEBS Letters 369 (1995) , p 267-271), expression in the plantar cells (Hu and Cheng, FEBS Letters 369 (1995), p.331-334)) and the Drosophila embryo (Davis et al., Dev. Biology 170 (1995 ), pp. 726-729)) and as a reporter molecule fused to another protein of interest (US Patent 5,491, 084). Similarly, WO96 / 23898 relates to methods of detecting biologically active detection substances by affecting intracellular processes by utilizing a GFP construct that has a protein kinase activation site. This patent and other patents referenced in this application are incorporated herein by reference in their entirety. Numerous references are related to GFP proteins in biological systems. For example, WO 96/09598 describes a system for the isolation of cells of interest using the expression of a protein such as GFP. The publication WO 96/276675 describes the expression of GFP in plants. The publication WO 95/21191 describes the modified GFP protein expressed in transformed organisms to detect mutagenesis. U.S. Patent Nos. 5,401, 128 and 5,436,128 describe tests and compositions for detecting and evaluating the intracellular transduction of an extracellular signal using recombinant cells that express cell surface receptors and contain reporter gene constructions that include transcriptional regulatory elements that are responsible for the activity of cell surface receptors. Performing an analysis on many thousands of compounds requires the parallel handling and processing of many compounds and testing of component reagents. High-throughput assays ("HTS") use mixtures of biological compounds and reagents together with some indicator compounds loaded in the series of wells in standard micro-concentration plates with 96 or 384 wells. The signal measured by each well, emission of fluorescence, optical density or radioactivity integrates the signal of all the material in the well, giving an average of the total population of all the molecules in the well. The International Scientific Applications Corporation (SAIC) 130 Fifth Avenue, Seattle, WA. 98109) describes an image plate reader. This system uses a CCD camera to project the entire area of a 96-well plate. The image is analyzed to calculate the total fluorescence per well for all the material in the well. Molecular Devices, Inc. (Sunnyvale, CA) describes a system (FLIPR) that uses low-angle laser scanning illumination and a mask to selectively excite fluorescence at approximately 200 microns from the bottoms of wells in standard 96-well plates to reduce the background when the cellular monolayers are represented. This system uses a CCD camera to project the entire area of the bottom of the well. Although this system measures signals originating from a cell monolayer at the bottom of the well, the measured signal is averaged over the well area and therefore is still considered a measure of the average response of a population of cells. The image is analyzed to calculate the total fluorescence per well for cell-based tests. Fluid release devices have been incorporated into cell-based analysis systems, such as the FLIPR system to initiate a response, which is subsequently observed as a response of the total well population average using a micro-representation system. In contrast to high-throughput analyzes, several high-content analyzes ("HCS") have been developed to address the need for more detailed information about the temporal spatial dynamics of cellular processes and constituents. High content analyzes automate the extraction of multicolored fluorescence information derived from reagents based on specific fluorescence incorporated in the cells (Giuliano and Taylor (1995), Curr. Op. Cell Biol. 7: 4; Guiliano et al. (1995) Ann. Rev. Biophys. Biomol. Struct. 24: 405). Cells are analyzed using an optical system that can measure space, as well as temporal dynamics. (Farkas et al. (1993) Ann. Rev. Physiol. 55: 785; Giuliano et al. (1990) In Optical Microscopy for Biology. B. Herman and K. Jacobson (eds.) Pp. 543-557. Liss, New York; Hahn et al (1992) Nature 359: 736; Wagoner et al. (1996) Hum. Pathol. 27: 494). The concept is to treat each cell as a "well" that has space and temporary information in the activities of the marked constituents. The types of molecular and biochemical information currently accessible through fluorescence-based reagents applied to cells include ion concentrations, membrane potential, specific translocations, enzyme activities, gene expression as well as the presence, amounts and templates of metabolites , proteins, lipids, carbohydrates and nucleic acid sequences (DeBiasio et al., (1996) Mol. Biol. Cell 7: 1259; Giuliano et al., (1995) Ann. Rev. Biophys. Biomol. Struct. 24: 405; Heim and Tsien, (1996) Curr. Biol .. 6: 178).

PBP ^ H | Sj High-content assays can be performed on fixed cells using fluorescently labeled antibodies, biological ligands and / or nucleic acid hybridization tests or live cells using multicolored fluorescent indicators and "biosensors". The selection of the analysis of living or fixed cells depends on the test based on specific cells required. Fixed cell tests are the simplest, since a series of cells initially alive in a micro-concentration well format can be treated with several compounds and the doses being tested, subsequently the cells can be fixed, labeled with specific reagents and measured . No environmental control of the cells after fixation. Spatial information is acquired, but only in a lapse of time. The availability of thousands of antibodies, ligands and nucleic acid hybridization tests that can be applied to cells makes this an attractive access for many types of cell-based assays. The fixing and marking stages can be automated, allowing efficient processing of the tests. 15 Tests with living cells are more sophisticated and effective, since a series of living cells containing the desired reagents can be analyzed in time, as well as in space. The environmental control of the cells (temperature, humidity and carbon dioxide) is required during the measurement, since the physiological health of the cells must be maintained for multiple measurements of fluorescence in time. There is a list growth of the fluorescent physiological indicators and "biosensors" that can report changes in the molecular and biochemical activities in the cells (Giuliano et al., (1995) Ann. Rev. Biophys., Biomol. Struct. 24: 405; Hahn et al. al., (1993) In Fluorescent and Luminescent Probes for Biological Activity, WT Mason, (ed), pp. 349-359, Academic Press, San Diego). 25 The bioavailability and use of cell-based reagents have helped advance the development of analyzes of high content of living and fixed cells. The Advances in instrumentation to automatically extract information from high multicolored content have recently made possible the development of HCS in an automated tool. An article by Taylor, et al. (American Scientist 80 (1992), pp. 322-335) describes many of these methods and their applications. For example, Proffitt et al. (Cytometry 24: 204-213 (1996)) discloses a semi-automated fluorescence digital display system for rating the relative numbers of cells in situ in a variety of tissue culture plate formats, especially microwell plates. 96 wells The system consists of an inverted epifluorescence microscope with a motorized state, video camera, image intensifier and a microcomputer with a PC-Vision digitizer. The Turbo Pascal program controls the state and explores the plate by taking multiple images per well. The program calculates total fluorescence per well, provides daily calibration and easily configures a variety of tissue culture plate formats. The threshold of digital images and reagents that emit light only when absorbed by living cells are used to reduce background fluorescence without removing excess fluorescent reagent. The presentation with a confocal scanning microscope (Go et al., (1997) Analytical Biochemistry 247: 210-215; Goldman et al., (1995) Experimental Cell Research 221: 311-319) and the presentation with a multi-photon microscope ( Denk et al., (1990) Science 248: 73; Gratton et al., (1994) Proc. Of the Microscopical Society of America, pp. 154-155) are also well established methods for acquiring high resolution images of the microscopic samples. The main advantage of these optical systems is the very low depth of the focus, which allows limited axial extension characteristics to be resolved against the background. For example, it is possible to resolve the internal cytoplasmic characteristics of the adherent cells from the characteristics on the cell surface. Because the exploration of multi-photon representations requires laser systems of very short duration to achieve the high flow of photons required, the fluorescence life cycles can also be measured in these systems (Lakowicz et al., (1992) Anal. Biochem. 202: 316-330; Gerrittsen et al., (1997), J. of Fluorescence 7: 11-15)), providing additional capacity for different modes of detection. Small, safe and relatively inexpensive laser systems such as lasers pumped with laser diodes are now available to allow the multi-photon confocal microscope to be applied in a completely routine model. A combination of the biological heterogeneity of the cells in the populations (Bright, et al., (1989) J. Cell, Physiol.141: 410, Giuliano, (1996) Cell Motil, Cytoskel, 35: 237)) as well as the High temporal and spatial frequency of the molecular and chemical information present in the cells makes it possible to extract high content information from cell populations using all existing micro-concentration plate readers. A non-existent high-content scanning platform has been designed for multicolored fluorescence-based assays using cells that are analyzed individually. Similarly, currently no method is available that combines automated fluid delivery for cell series for the purpose of systematically screening compounds for their ability to induce a cellular response that is identified by HCS analysis, especially of cells grown on micro plates. -concentration. In addition, no method exists in the art combining high-performance well-to-well measurements to identify "impacts" in a test followed by a second high-cell-by-cell measurement in the same well of only those wells identified as impacts. The present invention provides systems, methods and analyzes that combine high performance analysis (HTS) and high content analysis (HCS) that significantly improve target validation and candidate optimization by combining many formats of cellular analysis with molecular reagents based on fluorescence and the extraction of computer-based features, analysis of data and automation, resulting in increased quantity and speed of data collection, shortened cycle times and lately more rapid evaluation of promising drug candidates . The present invention also provides miniaturization methods, thereby allowing for increased yield, while decreasing the volumes of reagents and test compounds required in each test.

SUMMARY OF THE INVENTION In one aspect, the present invention relates to a method for analyzing cells comprising • providing cells containing fluorescent reporter molecules at a number of locations, • treating the cells in the series of locations with one or more reactive, • present numerous cells at each location with fluorescent optics, • convert optical information into digital data, • use digital data to determine the distribution, environment or activity of fluorescently labeled reporter molecules in cells and the distribution of the cells and • interpret that information in terms of a null, negative or positive effect of the compound that is being tested in the biological function. In this embodiment, the method quickly determines the distribution, environment or activity of fluorescently labeled reporter molecules in cells for the purpose of analyzing large numbers of compounds for those that specifically affect particular biological functions. The series of locations can be a micro-concentration plate or a microchip which is a micro-plate having cells in a number of locations. In a preferred embodiment, the method includes computerized means to acquire, process, deploy and store the received data. In a preferred embodiment, the method also includes the sending of automated fluids to the series of cells. In another preferred embodiment, the information obtained from the high-throughput measurements on the same plate is used to selectively perform the high-content analysis on only one subject of the cell locations on the plate. In another aspect of the present invention, there is provided a cellular analysis system comprising: • a high magnification fluorescent optical system having a target microscope, • an XY state adapted to hold a plate containing a series of cells and having a means to move the plate to the proper alignment and focus it on the cell series; • a digital camera; • a light source that has optical means to direct the excitation light to the cell series and a means to detect the fluorescent light emitted from the cells to the digital camera and • a computerized means to receive and process the digital data of the camera digital where the computerized means include a digital frame recorder to receive the images from the camera, an image display apparatus for the user interaction and the display of the results of the series, digital storage media for the file and the storage of data and a means for the control, acquisition, processing and deployment of results. In a preferred embodiment, the cellular analysis system further comprises a computational analysis operatively associated with the computer to display the * f. -J ^ S data. In another preferred embodiment, the computational means for receiving and processing digital data from the digital camera store the data in a bioinformatics database. In a further preferred embodiment, the cellular analysis system further comprises a reader that measures a signal from many or all of the wells in parallel. In another preferred embodiment, the cellular analysis system further comprises mechanical optical means for changing the amplification of the system to allow the modes of change between high throughput and high content analyzes. In another preferred embodiment, the cellular analysis system further comprises a camera and the control system for maintaining the temperature, the concentration of CO2 and the humidity around the plate at levels required to keep the cells alive. In a preferred additional mode, the cellular analysis system uses a confocal scanning detection and illumination system. In another aspect of the present invention, a readable storage medium for machines comprising a program containing a set of instructions for cause the cellular analysis system to execute procedures to define the distribution and activity of specific cellular constituents and processes are provided. In a preferred embodiment, the cellular analysis system comprises a high amplification fluorescence optical system with a state adapted to maintain the cells and a means to move the state, a digital camera, a source of light to receive and process the digital data of the digital camera and a computerized means to receive and process the digital data of the digital camera. Preferred embodiments of the machine readable storage medium comprise programs consisting of a set of instructions for causing the cellular analysis system to perform the procedures set forth in Figures 9, 11, 12, 13, 14 or . Another preferred embodiment comprises a program consisting of a set of instructions for causing the cellular analysis system to execute the procedures for detecting the distribution and activity of the specific cellular constituents and processes. In most preferred embodiments, cellular processes include, but are not limited to, the nuclear translocation of a protein, cell hypertrophy, apoptosis and the induced translocation of a protein protease. In another preferred embodiment, a variety of automated cellular analysis methods are provided including analyzes to identify compounds that affect the activity of the transcription factor, protein kinase activity, cell morphology, microtubule structure, apoptosis, the hospitalization of the receptor and the induced translocation of the protease of a protein.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a diagram of the components of the cell-based scanning system. Figure 2 shows a schematic diagram of the sub-assembly of the microscope. Figure 3 shows the sub-assembly of the camera. Figure 4 illustrates the process of the cell scanning system. Figure 5 illustrates a user interface showing the main functions to guide the user. Figure 6 is a block diagram of two platform architectures of the Dual Mode System for Cell-Based Analysis in which one platform uses telescopic lenses to read all the wells of a micro-concentration plate and a second platform that use a higher magnification lens to read the individual cells in each well, where: A = HTS reader mode B = HCS reader mode a '= mechanical optics b' = acquisition of data control. Figure 7 is a detailed view of an optical system for an individual platform architecture of the Dual Mode System for Cell-Based Analysis that uses movable "telescopic" lenses to read individual cells in a well, where: A = HTS reader mode B = HCS reader mode Mo = move Figure 8 is an illustration of the fluid delivery system for acquiring the kinetic data in the Cell Based Analysis System. Figure 9 is a flow chart of the processing step for the cell-based scanning system, where: Au = Automatic Co = Conditional In = Interactive. Figure 10 A-J illustrates the strategy of the Nuclear Translocation Test, where: C = unstimulated cells D = Stimulated cells. Figure 11 is a flowchart that defines the processing steps in the Dual Mode System for Cell-Based Analysis combining high-throughput analysis and high-content analysis of micro-concentration plates, where: Ma = Manual input Ap = Automatic Process De = Decision With = Connector. Figure 12 is a flowchart that defines the processing steps in the High Performance mode of the System for Cell-Based Analysis. Figure 13 is a flow diagram defining the processing steps in the High System Content mode for Cell Based Analysis. Figure 14 is a flow chart defining the processing steps required to acquire the kinetic data in the High System Content mode for Cell Based Analysis. Figure 15 is a flowchart that defines the processing steps performed in a well during the acquisition of the kinetic data. Figure 16 is an example of data of a known inhibitor of translocation, where: E = Difference NucCyt F = Concentration of the inhibitor (pM). Figure 17 is an example of the data of a known translocation stimulator, where: E = Difference NucCyt G = Concentration of the stimulator. Figure 18 illustrates the presentation of data in a graphical display. Figure 19 is an illustration of the High Performance mode data of the Cell-based Analysis System, an example of the data goes to the High Content mode, the data acquired in the high content mode and the results of the analysis of that data, where: h = Well data in low resolution i = Well data in high resolution j = Intensity analysis k = Spatial analysis I = No translocation m = Translocation. Figure 20 shows the measurement of a cytoplasm induced by drugs for nuclear translocation, where: n = Pre-treatment m = Translocation or = Post-treatment. Figure 21 illustrates a graphical user interface of the measurement shown in Figure 20. Figure 22 illustrates a graphical user interface with the display of measurement data shown in Fig. 20. Figure 23 is a graph that represents the kinetic data obtained from the measurements represented in Fig. 20, where: p = relative response q = time (min) r = transfected cells s = non-transfected cells. Figure 24 details an analysis of high content of drug-induced apoptosis, wherein: PA = polymerization of actin PM = mitochondrial potential p = relative response t = paclitaxel μM DETAILED DESCRIPTION OF THE INVENTION All patents, patent applications and other cited references are incorporated herein in their entirety as a reference. As used in this document, the following terms have specific meaning: Markers of cellular domains. Luminescent tests that have high affinity for specific cellular constituents including specific organelles or molecules. These tests can be luminescent molecules or fluorescently labeled macromolecules used as "labeling reagents", "indicators Environmental "or" biosensors. "Marking Reagents: Labeling reagents include, but are not limited to, luminescently labeled macromolecules including fluorescent and biosensing protein analogues, luminescent macromolecular chimeras including those formed with green fluorescent protein and mutants of the same, the 15 luminiscently labeled secondary or primary antibodies that react with cellular antigens involved in a physiological response, luminescent spots, dyes and other small molecules, markers of cellular translocations, luminescently marked macromolecules or organelles that move from a domain of cells to another during some cellular process or physiological response.Translocation markers can simply report the location relative to cell domain markers or they can also be "biosensors" that report some molecular or biochemical activity Also, Biosensors. The macromolecules that consist of a biological functional domain and a test or luminescent tests that report environmental changes that occur internally or on its surface. A class of luminescent macromolecules - • - • < - & _-- - * > -i- ... «ii fei-.a» Efea¡. & fce, »*. _, ^ ¿. marked designated to observe and report these changes that have been called "fluorescent protein biosensors". The protein component of the biosensor provides a widely developed portion of molecular recognition. A fluorescent molecule attached to the protein component in the vicinity of an active site transduces environmental changes into fluorescence signals that are detected using a system with appropriate spatial and temporal resolution such as the cellular scanning system of the present invention. Because the modulation of the activity of the natural protein in living cells is reversible and because the fluorescent protein biosensors can be designed to undergo reversible changes in the activity of the proteins, these biosensors are essentially reusable. Sequences associated with diseases ("DAS"). This term refers to nucleic acid sequences identified by standard techniques, such as data from the main DNA sequence, genome methods such as subtraction hybridization and RADE, and proteomic methods in combination with inverse genetics, such as the candidate compounds of the drug. The term does not mean that the sequence is only associated with a disease state. High content analysis (HCS) can be used to measure the effects of drugs on complex molecular events such as signal transduction pathways, as well as cellular functions including but not limited to apoptosis, cell division, cell adhesion, locomotion, exocytosis and cell to cell communication. Multicolour fluorescence allows multiple targets and cellular processes to be tested in a simple analysis. The cross-correlation of cellular responses will produce abundant information for target validation and leads to optimization. In one aspect of the present invention, there is provided a cellular analysis system comprising a high amplification fluorescence optical system having a target microscope, an XY state adapted to maintain a plate with a series of locations to support the cells and which has means for moving the plate to align the locations with the objective microscope and means for moving the plate in the direction to effect the focus, a digital camera, a light source having optical means to direct the excitation of light to the cells in the series of locations and means for directing the fluorescent light emitted from the cells to the digital camera and computerized means for receiving and processing the digital data of the digital camera wherein the computerized means include: a digital frame recorder for receiving the images of the camera, a deployment device for user interaction and for the deployment of the results of the tests, means of digital storage for the storage and archiving of the data and means for the control, acquisition, processing and deployment of the results. Figure 1 is a schematic diagram of a preferred embodiment of the cellular scanning system. An inverted fluorescence microscope i is used, such as a Zeiss Axiovert inverted fluorescence microscope that uses standard lenses with 1-1 OOx amplification in the camera and a white light source (ie, 100W mercury arc lamp). a 75W xenon lamp) with a power supply 2. There is an XY 3 state to move plate 4 in the XY direction in the objective microscope. A Z-axis focusing device 5_ moves the lens in the Z direction for focusing. A joystick 6 provides manual movement of the state in the XYZ direction. A high resolution digital camera 7 acquires images of each well or location on the plate. There is a power supply for the camera 8, an automation controller 9 and a central processing unit 10. The PC 1 1 provides a display 12 and has associated programs. The printer 13 provides the printing of a record.

Figure 2 is a schematic diagram of one embodiment of the microscope assembly of the invention, showing in more detail the XY 3 state, the Z-axis focusing device 5, the joystick 6, the light source 2 and the controller of automation 9. Cables are provided to computer 15 and microscope 16, respectively. In addition, Figure 2 shows a 96-well micro concentration plate 17 that moves in the 3 XY state in the XY direction. The light from the light source 2 passes through the camera shutter 18 controlled by the PC to a motorized filter wheel 19 with excitation filters 20. The light passes in the filter hub 25 having a dichroic mirror 26 and a emission filter 27. The excitation light is reflected off the dichroic mirror to the wells in the micro-concentration plate and the fluorescent light 28 passes through the dichroic mirror 26 and the emission filter 27 and the digital camera 7. The Figure 3 is a schematic drawing of a preferred camera assembly. The digital camera 7, which contains an automatic camera shutter for exposure control and a power supply 31, receives the fluorescent light 28 from the microscope assembly. A digital cable 30 transports the digital signals to the computer. The standard optical configurations described above use microscopic optics to directly produce an enlarged image of the specimen in the camera sensor to capture a high resolution image of the specimen. This optical system is commonly referred to as a "wide field" microscopy. Those skilled in the art of microscopy will recognize that a high resolution image can be created by a variety of other optical systems, including, but not limited to, the standard scanning confocal detection of a focused point or line of explored specimen illumination (Go. et al., 1997, supra) and confocal scanning microscopy of multiple photons (Denk et al., 1990, supra), both of which can be imaged in a CCD detector or by synchronous scanning of the analogue output of a photomultiplier tube.

In the analysis applications, it is usually necessary to use a particular cell line or main cell culture to take advantage of the particular characteristics of those cells. Those skilled in the cell culture art will recognize that some cell lines are inhibited on contact, meaning that they will stop growing when they become surrounded by other cells, while other cell lines will continue to grow under those conditions and the cells will literally be They will pile up forming many layers. An example of said cell line is the HEK 293 line (ATCC CRL-1573). An optical system is required that can acquire images of single cell layers in multi-layer preparations for use with cell lines that tend to form layers. The greater depth of wide-field microscopes produces an image that projects through many layers of cells, making the analysis of subcellular spatial distributions extremely difficult in layer-forming cells. Alternatively, the lowest depth of the field can be achieved in a confocal microscope (approximately one micron), allowing the discrimination of a simple cellular layer in high resolution, simplifying the determination of the subcellular spatial distribution. Similarly, confocal representation is preferable when detection modes such as the fluorescence life cycle representation are required. The output of a standard confocal representation annex for a microscope is a digital image that can be converted to the same format as the images produced by the modalities of the cell analysis system described above and can therefore be processed in exactly the same way as those images. The total control, the acquisition and analysis in this modality are essentially the same. The optical configuration of the confocal microscope system is essentially the same as that described above except for the illuminator and the detectors. Lighting and detection systems require for microscopy confocal that have been designed as accessories to be attached to standard optical systems such as the one of the present invention (Zeiss, Germany). These alternate optical systems can therefore be easily integrated into the system as described above. Figure 4 illustrates an alternative embodiment of the invention in which the cell series in the micro-wells 40 in a micro-plate 4. ion co-pending is described in the application of E.U.A. S / N 08 / 865,341 incorporated herein by reference in its entirety. Typically the microplate is 20mm by 30mm as compared to the 96-well standard micro-concentration plate which is 86mm per 129 mm. The high density series of the cells in a microplate allows microplate to be presented in a low resolution of a few microns pro pixel for high performance and particular locations in the microplate to be presented in a high resolution of less than 0.5 microns per pixel. These two resolution modes help improve the overall performance of the system. The chamber of the microplate 42 serves as a microfluidic delivery system for the addition of compounds to the cells. The micro-plate 4. in the microplate chamber 42 it is placed in an XY 43 microplate reader. The digital data is processed as described above. The small amount of this micro-plate system increases the performance, minimizes the volume of the reagent and allows the control of the distribution and displacement of cells for rapid and accurate cell-based analysis. The processed data can be displayed on a PC H screen and be part of a bioinformatics database 44. This database not only allows the storage and retrieval of the data obtained through the methods of this invention, but also allows the acquisition and storage of external data related to cells. Figure 5 is a display on the PC that illustrates the operation of the program.

Z? ».« »-; In an alternative mode, a high performance system (HTS) is directly coupled to the HCS on the same platform or on two separate platforms connected electronically (ie, via a local area network). This embodiment of the invention refers to a dual-mode optical system, which has the advantage of increasing the performance of an HCS by coupling it with an HTS and therefore requiring less acquisition of high resolution data and analyzing only the small subsets of wells that show a response in the coupled HTS. High-throughput "total plate" reader systems are known in the art and are commonly used as a component of an HTS system to analyze large numbers of compounds (Beggs (1997), J. Of Biomolec Screening 2: 71-78 Macaffrey at al., (1996) J. Biomolec, Screening 1: 187-190). In a dual-mode cell-based analysis mode, a two-platform architecture is provided in which high-performance acquisition occurs on one platform and high-content acquisition occurs on a second platform (Figure 6). The processing occurs on each platform independently, with the results going through a network interface or a simple controller is used to process the data from both platforms. As illustrated in Figure 6, an exemplified two-platform dual mode optical system consists of two light optical instruments, a high performance platform 60 and a high content platform 65, which read the fluorescent signals emitted from the cell culture in the micro-concentration plates or in the series of micro-wells in a micro-plate and which communicate with each other via an electronic connection 64. The high-performance platform 60 analyzes all the wells in all the plates in rapid serial form or parallel. Those skilled in the art will recognize that there are many commercially available high performance reader systems that could be integrated into the dual-mode cell-based analysis system (Topcpunt (Packard Instruments, Meriden CT); Spectramax Lumiskan (Molecular Devices, Sunnyvale, CA); Fluoroscan (Labsystems, Beverly, MA)). The high content platform 65, as described above, explores well by well and acquires and analyzes the high resolution image data collected from the individual cells in a well. The HTS program, resident in the computer system 62 controls the high performance instrument and the results are displayed on the monitor 61. The HCS program, resident in its computer system 62 controls the equipment of the high content instrument 65, the devices Optionals (ie, the plate charger, the environmental chamber, the fluid distributor) analyze the data of the digital images, display the results on the monitor 66 and manage the measured data in an integrated database. The two systems can also share a single computer, in which case all the data could be collected, processed and deployed on that computer, without the need for a local area network to transfer the data. The micro-concentration plates are transferred from the high performance system to the high content system 63 either manually or by means of a robotic plate transfer device, they are well known in the art (Beggs (1997), supra; Mcaffrey (1996) , supra). In a preferred embodiment, the dual mode optical system uses a simple platform system (Figure 7). This consists of two separate optical modules, an HCS module 203 and an HTS module 209 that can be independently or collectively moved so that only in a moment it is used to collect the data from the micro-concentration plate 201. The micro plate -concentration 201 is mounted in an X, Y motorized state so that it is placed for presentation in the HTS or HCS mode. After collecting and analyzing the HTS image data as described below, the HTS 209 optical module moves out of the optical path and the HCS 203 optical module moves into place.

The optical module for the HTS 209 consists of unps projection lenses 214. excitation wavelength filters 213 and dichroic mirror 210 that are used to illuminate the entire background of the plate with a specific wavelength band of a laser system. conventional microscope lamp (not shown). The fluorescence emission is collected through the dichroic mirror 210 and the wavelength emission filter 211 by lenses 212 which form an image in the camera 216 with the I sensor 215. The optical module for the HCS 203 consists of a few lenses of projection 208, a wavelength excitation filter 207 and a dichroic mirror 204 which are used to illuminate the rear opening of the objective microscope 202 and therefore the field of that objective of a standard microscope illumination system (not shown ). The fluorescence emission is collected by the objective microscope 202, passes through the dichroic mirror 204 and the wavelength emission filter 205 and is focused by tube lenses 206 which form an image in the same camera 216 with the sensor 215. In an alternative embodiment of the present invention, the cellular analysis system further comprises a fluid delivery device for use with the live cell mode of the cell analysis method (see below). Figure 8 exemplifies a fluid delivery device for use with the system of the invention. It consists of a bank of 12 syringe pumps 701 driven by a simple motor device. Each syringe 702 is dimensioned according to the volume to be sent to each well, typically between 1 and 100 μl. Each syringe is attached via a flexible tube 703 to a similar bank of accepting connectors} standard pipette tips 705. The bank of pipette tips is attached to a conduction system so that they are lowered and raised relative to the micro-concentration plate 706 to send the fluids to each well. The plate is mounted in a state X, Y, allowing movement relative to the -E- -wagaai &MMtMdap =. 707 optical system for data collection purposes. This installation allows a set of pipette tips or even a simple pipette tip to send the reagent to all wells on the plate. The bank of the syringe pumps can be used to send the fluid to the 12 wells simultaneously or for fewer wells by removing some of the limbs. In another aspect, the present invention provides a method for analyzing the cells comprising providing a series of locations containing multiple cells wherein the cells contain one or more fluorescent reporter molecules, scanning multiple cells in each of the locations containing cells for obtain fluorescent signals from the fluorescent reporter molecule in the cells, converting the fluorescent signals into digital data and using the digital data to determine the distribution, environment or activity of the fluorescent reporter molecule in the cells.

Cellular Series The analysis of several numbers of compounds for activity with respect to a particular biological function requires the preparation of cell series for the parallel handling of cells and reagents. Standard 96-well micro-concentration plates that are 86 mm by 129 mm with 6 mm diameter wells at a 9 mm incline were used for compatibility with the current automatic loading and robotic handling systems. The microplate is typically 20mm by 30mm with cell locations that are 100-200 microns in dimension at an inclination of approximately 500 microns. Methods for marking microplates are described in the U.S. Patent Application. Serial No. 08 / 865,341 incorporated herein by reference in its entirety. The microplates consist of coplanar layers of materials to which the cells adhere, designed with materials to which the cells do not adhere or three-dimensional surfaces pickled of similarly designed materials. For the purpose of the following description, the terms "well" and "micro-well" refer to a location in the series of any construction to which the cells adhere and in which the cells present themselves. The microplates may also include channels for fluid delivery in the spaces between the wells. The smaller format of a microplate increases the overall efficiency of the system by reducing the quantities of reagents, storage and handling of the duration of the preparation and the total movement required for the scanning operation. In addition, the total area of the microplate can be presented more efficiently, allowing a second mode of operation for the microplate reader as described later in this document.

Fluorescent Reporting Molecules A major component of the new drug discovery paradigm is a family of continuous growth of luminescent and fluorescent reagents that are used to measure the spatial and temporal distribution, content and activity of intracellular ions, metabolites, macromolecules and organelles. Classes of these reagents include labeling reagents that measure the distribution and number of molecules in the fixed and living cells, the environment indicators to report the events of signal transduction in time and space and the biosensors of fluorescent proteins to measure white molecular activities in living cells. A multi-parameter approach that combines several reagents into a single cell is a powerful new tool for drug discovery. The method of the present invention is based on the high affinity of the molecules luminescent or fluorescent for specific cellular components. The affinity for specific components is governed by physical forces such as ionic interactions, covalent bond (which includes chimeric fusion with chromophores based on proteins, fluorophores and lumifors) as well as hydrophobic interactions, electrical potential and in some cases, simple entrapment in a cellular component. Luminescent tests can be small molecules, labeled macromolecules or genetically constructed proteins, including but not limited to green fluorescent protein chimeras. Those skilled in the art will recognize a wide variety of green reporter molecules that can be used in the present invention, including but not limited to fluorescently labeled biomolecules such as proteins, phospholipids and tests of DNA hybridization. Similarly, fluorescent reagents specifically synthesized with particular chemical binding or association properties have been used as fluorescent reporter molecules (Barak et al., (1997J, J. Biol. Chem. 272: 27497-27500; Southwick et al., (1990 ), Cytometry 11: 418-430, Tsien (1989) in Methods in Cell Biology, Vol. 29 Taylor and Wangs (eds.), Pp. 127-156. fluorescently labeled antibodies are particularly useful reporter molecules due to their high degree of specificity to be added to a single molecular target in a mixture of molecules as a complex such as a cell or tissue. Luminescent tests can be synthesized in the living cell or can be transported in the cell via several non-mechanical modes including diffusion, transport active or facilitated, mediated transport of signal sequence and uptake of pinocytotes or endocytotes. Mechanical volume loading methods, which are well known in the art, can also be used for luminescent loading tests in living cells (Barber et al., (1996), Neuroscience Letters 207 ': 17' -20; Bright et al. al., (1996), Cytometry 24: 226-233; McNeil (1989) in Methods in Cell Biology, Vol. 29, Taylor and Wang (eds.), Pp. 153-173). These methods include electroporation and other mechanical methods such as waste-loading, bed loading, impact loading, load with syringe, hypotonic and hypertonic load. Additionally, the cells can be genetically engineered to express reporter molecules such as GFP, coupled to a protein of interest as previously described (Chalfie and Prasher US Patent: No. 5,491,084; Cubitt et al. (1995), Trends in Biochemical Science 20: 448-455). Once in the cell, the luminescent tests accumulate in their target domain as a result of high-affinity and specific interactions with the target domain or other modes of molecular targets such as signal-mediated transport of sequence. The fluorescently labeled reporter molecules are useful for determining the location, quantity, and chemical environment of the reporter. For example, they can be determined whether the reporter is in the environment of the lipophilic membrane or in a more aqueous environment (Giuliano et al. (1995), Ann. Rev. of Biophysics and Biomolecular Structure 24: 405-434; Giuliano and Taylor (1995), Methods in Neuroscience 27: 1-16). The pH environment of the reporter can be determined (Bright et al., (1989), J. Cell Biology 104: 1019-1033, Giulano et al. (1987), Anal Biochem 167: 362-371, Thomas et al. 1979), Biochemistry 18: 2210-2218.It can be determined whether a reporter having a chelation group is linked to an ion, such as Ca ++ or not (Bright et al. (1989), In Methods in Cell Biology, Vol. , Taylor and Wang (eds.), Pp. 157-192; Shimoura et al. (1988), J. of Biochemistry (Tokyo) 251: 405-410; Tsien (1989) In Methods in Cell Biology, Vol. 30, Taylor and Wangs (eds.), Pp. 127-156) In addition, certain cell types in an organism may contain components that may be specifically labeled that can not occur in other cell types, for example, epithelial cells commonly contain polarized membrane components, that is, these cells distribute asymmetrically macromolecules along their plasma membrane.The supporting or connective tissue cells regularly obtain in granules in their trapped molecules specific to that cell type (ie, heparin, histamine, serotonin, etc.). The majority of muscle tissue cells contain a sarcoplasmic reticulum, a specialized organelle whose function is to regulate the concentration of calcium ions in the cell cytoplasm. Many cells of nervous tissue contain secretory glands and vesicles in which neurohormones or neurotransmitters are trapped. Therefore, fluorescent molecules can be designed to mark not only specific components but also specific cells in a population of mixed cell types. Those skilled in the art will recognize a wide variety of means to measure fluorescence. For example, some fluorescent reporter molecules exhibit a change in excitation or emission of spectrum, some exhibit energy transfer resonance in which one fluorescent reporter loses fluorescence, while a second gains in fluorescence, some exhibit a (temperate) loss or appearance of fluorescence, while some report rotational movements (Giuliano et al. (1995), Ann.Rev.of Biophysics and Biomol. Structure 24: 405-434; Giuliano et al. (1995), Methods in Neuroscience 27: 1 -16).

Exploration of cellular series With reference to Figure 9, a preferred embodiment is provided to analyze the cells comprising parameters directed by the operator being selected based on the test being conducted, the acquisition of the data by means of the analysis system of cells in the distribution of fluorescent signals in a sample and the review and analysis of interactive data. At the start of an automated scan the operator enters the information 100 that describes the sample, specifies the filter configuration and the fluorescence channels to match the biological markers used and the recorded information and then adjusts the camera settings to match the brightness of the sample. For flexibility, to handle a range of , - A, ~. .. J¿B £ _ .. samples, the program allows the selection of several configuration parameters used to identify the nucleus and the cytoplasm and the selection of different fluorescent reagents of cells of interest based on the morphology and brightness and the number of cells to be analyzed by well. These parameters are stored in the system for quick recovery for each automated run. The system's interactive cell identification mode simplifies the selection of limits of morphological parameters such as the range of size, shape and intensity of the cells to be analyzed. The user specifies what wells on the system board he will explore and how many fields or how many cells he will analyze in each well. Depending on the configuration mode selected by the user in step 101, the system will automatically pre-focus the region of the plate to be scanned using a self-focusing procedure to "find the focus" of plate 102 or the user will pre-focus interactively 103 the exploration region by selecting three "mark" points that define the rectangular area to be scanned. A "focal plane model" of at least square fit is subsequently calculated from these mark points to estimate the focus of each well during an automated scan. The focus of each well is estimated by interpolation of the focal plane model during a scan. During an automated scan, the program automatically displays the state of the scan including the number of cells analyzed, the current being analyzed, the images of each independent wavelength as acquired, and the result of the analysis of each well as determined. Plate 4 (Figure 1) is scanned in a serpentine style as the program automatically moves the XY 3 status of the powered microscope from each well to well and field to field in each well of a 96-well plate. Those skilled in the art of programming will recognize how to adapt the scanning program of other micro-plate formats such as plates of 24, 48 and 384 wells. The exploration design of the total plate as the exploration design of the fields in each well is programmed. The system adjusts the focus of the sample with a self-focusing procedure 104 (Figure 9) through the Z-axis device 5, controls the selection of the filter via a motorized filter wheel 19 and acquires and analyzes the images in more than four different colors ("channels" or "wavelengths"). The auto-approach procedure is called at a frequency selected by the user, typically for the first field in each well and subsequently every 4 to 5 fields in each well. The auto-focus procedure calculates the start of the Z axis point by interpolating the focal model of the pre-calculated plane. Starting at a programmable distance above or below this point, the procedure moves the mechanical Z-axis through a number of different positions by acquiring an image in each position and finding the maximum of a calculated focus mark that estimates the contrast of each image . The Z position of the image with the maximum focus mark determines the best focus of a particular field. Those skilled in the art will recognize that this as a variant of automatic focusing methods as described in Harms et al. in Cytometry 5 (1984), 236-243, Groen et al. in Cytometry 6 (1985), 81-91, and Firestone et al. in Cytometry 12 (1991), 195-206. For image acquisition, the exposure time of the camera is adjusted separately for each dye to ensure a high quality image of each channel. Program procedures can be called at the user's option, to correct the registration changes between the wavelengths by counting the linear changes (X and Y) between the wavelengths before marking any additional measurements. The electronic camera shutter 18 is controlled so that the photo bleaching of the sample is kept to a minimum. Background shading and uneven lighting can be corrected by the program using methods known in the art (Bright et al. (1987), J. Cell Biol. 104: 1019-1033). -. - - -, > i, *? -. "*» '^ ¡. »jt» aiw In a channel, images are acquired from a primary marker 105 (Figure 9) (the cell nucleus typically counter-stained with fluorescent dyes DAPI or Pl) that are segmented ("identified") using an adaptive threshold procedure The adaptive threshold procedure 106 is used to dynamically select the threshold of an image to separate the cells from the background Marking of cells with fluorescent dyes may vary by a degree unknown through the cells in a sample in the micro-concentration plate as well as in the images of a cell field in each well of a micro-concentration plate. This variation can occur as a result of the preparation of the sample and / o the organic nature of the cells.A global threshold is calculated for the complete image to separate the cells from the background and the count of field variation in field.These global adaptive techniques are variants of here they are described in the art. (Kittler et al., In Computer Vision, Graphics and Image Processing 30 (1985), 125-147, Ridler et al., In IEEE Trans. Systems, Man and Cybernetics (1978), 630-632). An alternate adaptive threshold method uses local region thresholds in contrast to the overall image threshold. The image analysis of the local regions leads to a better total segmentation since the staining of the nucleus of the cell (as well as other marked components) can vary through an image. Using this local / global procedure, a reduced resolution image (reduced in size by a factor of 2 to 4) is globally segmented (using the adaptive threshold) to find regions of interest in the image. These regions later serve as guides to more broadly analyze the same regions in full resolution. A further localized threshold is calculated (again using an adaptive threshold) for each region of interest. The output of the segmentation procedure is a binary image where the objects are white and the background is black. This binary image, also called a mask in the art, is used to determine if the field contains objects 107. The mask is marked with a bubble marking method where each object (or bubble) has a unique number assigned to it. The morphological characteristics, such as the area and shape of the bubbles are used to differentiate the bubbles probably to be cells of those that are considered artifacts. The user pre-establishes the morphological selection criterion by writing on the known cellular morphological characteristics or by using the interactive preparation utility. If the objects of interest are in the field, the images are acquired for all other channels 108, otherwise the state advances to the next field 109 in the current well. Each object of interest is located in the image for subsequent analysis 110. The program determines whether the object meets the criteria for a valid cell nucleus 111 by measuring its morphological characteristics (size and shape). For each valid cell, the location of the XYZ state is recorded, a small image of the cell is stored and features 112 are measured. The cell scanning method of the present invention can be used to perform many different tests on cell samples by applying a number of analytical methods simultaneously by measuring the characteristics of the multiple wavelengths. An example of one such test provides the following measurements: 1. The total fluorescent intensity in the cell nucleus for 1-4 colors. 2. The area of the cell nucleus for color 1 (the primary marker). 3. The shape of the cell nucleus for color 1 is described by three shape characteristics: a) square area of the perimeter. b) radius of the section area. c) radius of the amplitude of the height. 4. The average fluorescent intensity in the cell nuclei for colors 1-4 (ie, # 1 divided by # 2). 5. The total fluorescent intensity of an outer ring of the nucleus (see Figure 10) that represents the fluorescence of the cytoplasm of the cell (mask cytoplasmic) for colors 2-4. 6. The area of the cytoplasmic mask. 7. The average fluorescent intensity of the cytoplasmic mask for colors 2-4 (ie # 5 divided by # 6). 8. The radius of the average fluorescent intensity of the cytoplasmic mask 10 to average the fluorescent intensity in the cell nuclei for the colors 2-4 (ie # 7 divided by # 4). 9. The difference of the average fluorescence intensity of the cytoplasmic mask and the average fluorescent intensity in the cell nucleus for the colors 2-4 (ie # 7 minus # 4). 15 10. The number of fluorescence domains (also called sites, spots or grains) in the cell nucleus for colors 2-4. The characteristics from 1 to 4 are general characteristics of the different cellular analysis tests of the invention. These stages are commonly used in a variety of image analysis applications and are well known in the art (Russ. (1992) The Image Processing Handbook, CRC Press Inc., Gonzalez et al., (1987), Digital Image Processing. Addison-Wesley Publishing Co. pp. 391-448). Characteristics 5-9 have been specifically developed to provide measurements of fluorescent molecules of a cell in the local cytoplasmic region of the cell and the translocation (ie, movement) of fluorescent molecules from the cytoplasm to the nucleus. These characteristics (stages 5-9) are used to analyze the cells in the micro-plates for the inhibition of the nuclear translocation of the transcription factors provide a novel approach for the analysis of intact cells (detailed examples of other types of analysis are provided below). A specific method measures the amount of the test in the nuclear region (characteristic 4) against the local cytoplasmic region (characteristic 7) of each well. The quantification of the differences between these two sub-cellular compartments provides a measure of nuclear translocation-cytoplasm (characteristic 9). Feature 10 describes an analysis used to count DNA or RNA tests in the nuclear region in colors 2-4. For example, tests are commercially available for the identification of chromosome-specific DNA sequences (Life Technologies, Gaitherburg, MD; Genosys, Woodlands, TX; Biotechnologies, Inc., Richmond, CA; Bio 101, Inc., Vista, CA) Cells are three-dimensional in nature and when examined at high magnification under a microscope, one test may be in focus while another may be completely out of focus. The cell analysis method of the present invention provides three-dimensional detection tests in the nucleus by acquiring multiple focal planes. The program moves the Z-axis motor device 5 (Figure 1) in small stages where the distance of the stage is selected by the user to have a wide range of different nuclear diameters. In each of the focal stages, an image is acquired. The maximum gray level intensity of each pixel in each image is found and stored in a resulting maximum projection image. The maximum projection image is subsequently used to count the tests. The above method works well in counting tests that are not stacked directly above or below one another. To count the tests stacked on top of each of the other Z-direction, users can select an option to analyze the acquired focus-plane tests. In this mode, the scanning system performs the maximum plane projection method as described above, it detects the test regions of interest in this image, later it also analyzes these regions in all the focal plane images. After measuring the cellular features 112 (Figure 9), the system checks whether any object not processed in the current field 113 exists. If there is any object not processed, it is located in the continuous object 110 and determines whether it meets the criteria for a core valid cellular 111 and measures its characteristics. Once all the objects in the current field are processed, the system determines if the analysis of the current board is completed 114; or not, the need to find more cells in the current well 115 is determined. If the need exists, the system advances the XYZ state to the next field in the current well 109 or advances the state to the next well H6 of the plate. After the scan is complete, images and information can be reviewed by reviewing system images, reviewing data, and reviewing the summary. All images, data and settings of a scan are stored in the system database for later review or for interfacing with a network information management system. The data can also be exported to another third part of statistical packages to tabulate the results and generate other reports. Users can review the images alone in each cell analyzed by the system with an interactive image review procedure 117. The user can review the data on a cell-by-cell basis using a combination of interactive graphs, an electronic spreadsheet of measured characteristics and images of all the fluorescence channels of a cell of interest with the cell-per-cell interactive data review procedure 118. The graphical tracing capabilities are provided in whose data they can be analyzed via interactive graphs such as histograms and frames dispersion. Users can review the summary of the data that was accumulated and summarized for all cells in each well of a plate with a data review procedure. well per interactive well 119. Copies of graphics and images can be printed on a wide range of standard printers. As a final phase of a complete exploration, the reports can be generated in one or more statistics of the measured characteristics. The user can generate a graphical report of the data summarized in one well base per well for the region explored by the plate using an interactive report generation procedure 120. This report includes a summary of the statistics by well in graphic format and by columns and identifying information in the sample. The report window allows the operator to enter comments about the scan for later retrieval. Multiple reports can be generated in many statistics and printed at the touch of a button. The reports can be pre-visualized for the placement of the data before being printed. The aforementioned modality of the method operates in a simple high resolution mode referred to as the high content analysis mode (HCS). The HCS mode provides sufficient spatial resolution in a well (in the order of 1 μm) to define the distribution of the material in the well as well as in the individual cells in the well. The high degree of information content accessible in that mode comes at the expense of the speed and complexity of the signal processing required. In an alternative mode, a high performance system (HTS) is coupled directly to the HCS on the same platform or on two separate electronically connected platforms (ie, via a local area network). This embodiment of the invention is referred to as a dual-mode optical system, has the advantage of increasing the performance of an HCS by coupling it with an HTS and therefore requiring the slower acquisition of high resolution data and analysis only in the small subset of the wells that show a response in the coupled HTS.

High performance "full plate" reader systems are well known in the art and are commonly used as a component of an HTS system to analyze large numbers of compounds (Beggs et al. (1997), supra; McCaffrey et al. 1996), supra). The HTS of the present invention is carried out in the micro-concentration or micro-well plate test by reading many or all of the wells in the plate simultaneously with sufficient resolution to perform the determinations on a basis of well per well. That is, calculations are made by averaging the total signal output of many or all of the cells or the volume of material in each well. Wells that exhibit some definite response in the HTS (the "impacts") are blocked by the system. Later in the same test of the micro-concentration or micro-well plate, each well defined as an impact is measured via HCS as described above. Therefore, the dual-mode process involves: 1. Rapid measurement of numerous wells of a micro-concentration plate or a series of micro-wells; 2. Interpretation of the data to determine the total activity of the reporter molecules fluorescently labeled in the cells in a well-by-well basis to identify the "impacts" (wells that exhibit a definite response), 3. The representation of numerous cells in each well "impact" and 4. The interpretation of the image data digital to determine the distribution, environment or activity of the fluorescently labeled reporter molecules in the individual cells (ie, intracellular measurements) and the distribution of the cells to test the specific biological functions.

«.».,. -'- In a preferred embodiment of the dual mode processing (Figure 11) at the start of a run 301. the operator enters the information 302 that describes the plane and its contents, specifies the filter configuration and the fluorescent channels to match the marks biological materials being used, the information requested and the configuration of the camera to match the brightness of the sample. These parameters are stored in the system database for easy retrieval by each automatic run. The micro-concentration plate or the micro-well series is loaded into the cellular analysis system 303 manually or automatically by controlling a robotic loading device. An optional environmental chamber 304 is controlled by the system to maintain the temperature, humidity and CO2 levels in the air surrounding the living cells in the micro-concentration plate or series of micro-wells. An optional fluid delivery device 305 (see Figure 8) is controlled by the system for the distribution of fluids in the wells during the exploration. High-throughput processing 306 is first performed on the micro-concentration plate or series of micro-wells by acquiring and analyzing the signal from each of the wells on the plate. The processing performed in the high throughput mode 307 is illustrated in Figure 12 and is described below. Wells that exhibit some selected response in this high performance mode ("impacts") are identified by the system. The system performs a conditional operation 308 that tests the impacts. If the impacts are not found, those specific impact wells are also analyzed in the high content 309 (micro level) mode. The processing performed in the high content mode 312 is illustrated in Figure 13. Subsequently the system updates 310 the computer databases 311 with the results of the measurements on the board. If there are more plates to be analyzed 313, the system loads the next plate 303; otherwise the analysis of the plates ends 314.

The following description describes the high performance mode illustrated in Figure 12. The preferred embodiment of the system, the simple platform dual mode analysis system is described. Those skilled in the art will recognize that the optional dual platform system simply involves the movement of the plate between two optical systems rather than the movement of the optics. Once the system has been configured and the plate loaded, the system initiates HTS acquisition and analysis 401. The HTS optical module is selected by controlling a motorized optical positioning device 402 in the dual mode system. In a fluorescence channel, 403 is acquired from a primary label in the plate and the wells are isolated from the bottom of the plate using a 404 masking method. The images are also acquired in other fluorescence channels 405 being used. each image corresponding to each well 406 is measured 407. A calculated characteristic of the measurements for a particular well is compared to a pre-defined threshold or intensity response 408 and based on the result, the well is blocked as an "impact" 409 or not. The locations of wells blocked as impacts are recorded for processing of high subsequent content mode. If there are remaining wells to be processed 410, the program feeds 406 until all the wells have been processed 4H and the system exits the high performance mode. Following the HTS analysis, the system starts the high content mode processing 501 defined in Figure 13. The system selects the HCS optical module by controlling the motorized positioning system. For each "impact" well identified in the high performance mode, the location of the XY state of the well is retrieved from the memory or disk and the state is subsequently moved to the selected state location 503. The 504 auto-approach procedure is called for the first field in each impact well and subsequently once every 5 to 8 fields in each well. In a channel, the images are acquired from the primary marker 505 (? * »R *» ¡> *. Fj »t- 4 zZ a» .-, s.cM5.JÍ < tfe > ft j »-_ ___ jj_ -Zi -y (typically the cell nucleus against -stained with DAPI, Hoechst or fluorescent dye Pl.) The images are subsequently segmented (separated into core and non-core regions) using an adaptive threshold procedure 506. The output of the segmentation procedure is a binary mask where the objects They are white and the background is black.This binary image, also called a mask in the technique, is used to determine if the field contains 507 objects. The mask is marked with a drop-mark method where each object (or spot) has a Unique number assigned to it If the objects are in the field, the images are acquired for all other active channels 508, otherwise the status is advanced to the next field 214 in the current well.Each object is located in the image for additional analysis 509. The morphological characteristics Thus, such as the area and shape of the objects are used to select the objects likely to be the cell nucleus 510 and to discard (without further processing) those that are considered artifact. For each valid cell nucleus, the location of the XYZ state is recorded, a small image of the cell is stored and the specific test characteristics are measured 511. The system subsequently performs multiple tests on the cells by applying several analytical methods to measure the characteristics in each of the various wavelengths. After the measurement of the cellular characteristics, the systems verify if there is any object not processed in the current field 512. If there is any object not processed, the next object 509 is located and it is determined if it meets the criteria for a valid cell nucleus 510 and measures its characteristics. After processing all the objects in the current field, the system determines whether more cells or fields need to be found in the current well 513. If more cells or fields need to be found in the current well, the XYZ state is advanced to the next field in the current well 515. Otherwise, the system verifies if it has any remaining impact to measure 515. If it exists, it advances to the next 503 impact well and proceeds through another acquisition and analysis cycle, otherwise the HCS is terminated 516. In an alternate embodiment of the present invention, a method of kinetic analysis of living cells is provided. The previously described embodiments of the invention are used to characterize the spatial distribution of cellular components at a specific point of time, the time of chemical fixation. As such, these modalities have limited utility for the implementation of kinetic-based analyzes, due to the sequential nature of the image acquisition and the amount of time required to read all the wells in a plate. For example, since a plate requires 30-60 minutes to read through all the wells, only the kinetic process can be measured very slowly by simply preparing a plate of living cells and then reading through all the wells more than in a. The fastest kinetic processes can be measured by taking multiple readings from each well before proceeding to the next well, but the time between the first and the last well would be very long and the rapid kinetic process would be complete before reaching the last well. The kinetic extension of living cells of the invention enables the design and use of analyzes in which a biological process is characterized by its kinetics instead of or in addition to its spatial characteristics. In many cases, a response in living cells can be measured by adding a reagent to a specific well and making multiple measurements in that well with appropriate regulation. This dynamic mode of living cells of the invention therefore includes an apparatus for sending Fluids to individual wells of the system to send the reagents to each well at a specific time by reading the well. This modality also allows the kinetic measurements to be made with temporal resolution from seconds to minutes in each well of the plate. To improve the full efficiency of the dynamic system of living cells, the acquisition control program is modified to allow the collection of repetitive data from the sub-regions of the plate allowing the system to read other wells between the time points required for an individual well. Figure 8 describes an example of a fluid delivery apparatus for use with the live cell mode of the invention and is described below. This configuration allows an installation of pipette tips 705 or even a single pipette tip to send the reagent to all the wells in the plate. The side of syringe pumps 701 can be used to send the fluid to the 12 wells simultaneously or to fewer wells by removing some of the limbs 705. The temporal resolution of the system can therefore be adjusted without sacrificing the efficiency of the collection of data by changing the number of limbs and the scan design as follows. Typically, the collection of data and analysis of an individual well takes approximately 5 seconds. The movement from well to well and focus on a well requires approximately 5 seconds, so the complete cycle of time for each well is approximately 10 seconds. Therefore, if a single pipette tip is used to send fluids to a single well and data is collected repetitively from that well, measurements can be made with approximately 5 seconds of temporal resolution. If 6 pipette tips are used to send fluids to 6 wells simultaneously and the system repeatedly scans all 6 wells, each scan will require 60 seconds, thus setting the temporal resolution. For slower processes in which only data collection is required every 8 minutes, fluids can be sent to one half of the plate by moving the plate during the fluid delivery phase and then repetitively scanning that half of the plate . Therefore, by adjusting the size of the sub-region being scanned on the board, the time resolution can be adjusted without having to insert wait times between acquisitions. Because the system continuously scans and acquires the data, the total time to collect a set of kinetic data from the plate later simplifies the time to perform a simple plate scan, multiplied by the number of time points required. Typically, 1 point of time before the addition of the compounds and 2 or 3 time points following the addition should be sufficient for the purposes of analysis. Figure 14 shows the acquisition sequence used for the kinetic analysis. The start of 801 processing is the system configuration, much of which is identical to the standard HCS configuration. In addition, the operator must enter the information specific to the kinetic analysis being made 802, such as the size of the sub-region, the number of time points required and the increase in time required. A sub-region is a group of wells that will be explored repetitively to accumulate kinetic data. The size of the sub-region is adjusted so that the system can scan a complete sub-region once during a single increment of time, thus reducing waiting times. The optimum size of the sub-region is calculated from the configuration parameters and the adjustment if necessary by the operator. The system subsequently moves the plate to the first sub-region 803 and to the first well in which the 804 sub-region acquires the pre-stimulation time points (time = 0). The acquisition sequence performed in each well is exactly the same as that required for the specific HCS being run in the kinetic mode. Figure 15 details a flow chart for that purpose. All stages between start 901 and return 902 are identical as those described in steps 504-514 in Figure 13. After the processing of each well in a sub-region, the system checks to see if all the wells in the sub-region have been processed 806 (Figure 14) and running through all the wells until the entire region has been processed. Subsequently, the system moves the plate in position for the addition of the fluid and controls • ¿& ßJtt &S-t sending fluids from the fluidic system to enter sub-region 807. This may require multiple additions for the sub-regions that scan several columns on the plate, with the system moving the plate in state X, And among the additions. Once the fluids have been added, the system moves to the first well in sub-region 808 5 to initiate the acquisition of time points. The data acquired from each well 809 and as the system traverses all wells in sub-region 810. After each step through the sub-region, the system checks whether all time points have been collected 811 and if no, it stops 813 if 812 is necessary to remain synchronized with the requested time increment. Otherwise, the system checks the sub-10 additional regions on the plate 814 and moves to the next sub-region 803 and ends 815. Thus, the kinetic analysis mode comprises the identification of the operator of the sub-regions of the micro-concentration plate or micro-wells to be analyzed, based on the kinetic response to be investigated with the data acquisitions in a sub-region before the acquisition of the data in the subsequent sub-regions. Specific Analyzes In another aspect of the present invention, cell analysis methods and machine readable storage means comprise a program containing a set of instructions for causing a cellular analysis system to execute the procedures to define the distribution and activity of the specific cellular constituents and the processes are provided. In a preferred embodiment, the cellular analysis system comprises a high amplification fluorescent optical system with a state adapted to maintain the cells and a means to move the state, a digital camera, a light source to receive and process the digital data of the digital camera and a computerized means to receive and process the digital data of the digital camera. This aspect of the invention comprises programs that instruct the analysis system < «» < < -Jfcttt ~ * te «- *» ».. - -. . -jt _? - .. «e-a a- ¿¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡. < A.1"v-rs» - »tu» "cellular to define the distribution and activity of the specific cellular constituents and processes, using the luminescent tests, the optical presentation system and the pattern recognition program of the invention The preferred embodiments of the machine-readable storage medium comprising programs of a set of instructions for causing a cellular analysis system to execute the procedures set forth in Figures 9, 11, 12, 13, 14 or 15. Another preferred embodiment comprises a program consisting of a set of instructions for causing a cellular analysis system to execute procedures for the detection of the distribution and activity of specific cellular constituents and processes.

Preferred, cellular processes include but are not limited to the nuclear translocation of a protein, cell morphology, apoptosis, the admission of receptors and the protease-induced translocation of a protein. In a preferred embodiment, cell analysis methods are used to identify compounds that modify the various cellular processes. The cells can contact a compound test and the effect of the test compound on a particular cellular process can be analyzed. Alternatively, the cells can be contacted with a test compound and a known agent that modifies the particular cell process to determine if the test compound can inhibit or improve the effect of the known agent. Therefore, the methods can be used to identify test compounds that increase or decrease a particular cellular response as well as to identify test compounds that affect the ability of other agents to increase or decrease a particular cellular response. In another preferred embodiment, the locations containing the cells are analyzed using the above methods at a low resolution in a high performance mode and only a subset of the locations containing the cells is analyzed in a high content mode to obtain luminescent signals from the reporter molecules luminescently marked in the subcellular compartments of the cells that are being analyzed. The following examples are for purposes of illustration only and are not to limit the scope of the invention as defined in the accompanying claims. The various chemical compounds, reagents, dyes and antibodies referred to in the following Examples are commercially available from those sources such as Sigma Chemical (St. Louis, MO), Molecular Probes (Eugene, OR), Aldrich Chemical Company (Milwaukee, Wl) , Achúrate Chemical Company (Westbury, NY), Jackson Immunolabs and Clontech (Palo Alto, CA). Example 1 Translocation Analysis of the Nucleus to the Cytoplasm a. Transcription Factors The regulation of the transcription of some genes involved in the activation of a transcription factor in the cytoplasm, resulting in the factor being transported in the nucleus where the transcription of a particular gene or genes can be initiated. This change in the distribution of the transcription factor is the basis of an analysis for the cell-based analysis system to detect compounds that inhibit or induce the transcription of a particular gene or group of genes. A general description of the analysis is given followed by a specific example. The distribution of the transcription factor is determined by labeling the nucleus with a specific DNA fluorophore such as Hoechst 33423 and the transcription factor with a specific fluorescent antibody. After self-focus on the Hoechst-marked core, a core image is acquired in the cell-based analysis system and used to create a mask by one of several optional threshold methods, as described above. The morphological descriptors of the regions defined by the mask are compared with the parameters defined by the user and _-____ & _., Valid nuclear masks are identified and used with the following method to extract the distributions of the transcription factor. Each valid nuclear mask wears out to define a slightly smaller nuclear region. The original nuclear mask is subsequently dilated in two stages to define a ring-shaped region around the nucleus that represents a cytoplasmic region. The fluorescence of the average antibody is determined in each of these two regions and the difference between these averages is defined as the NucCyt Difference. Two examples for determining nuclear translocation are described below and are illustrated in Figure 10A-J. Figure 10A illustrates an unstimulated cell with its core 200 labeled with a fluorophore blue and a transcription factor in cytoplasm 201 marked with a green fluorophore. Figure 10B illustrates the nuclear mask 202 derived by the cell-based analysis system. Figure 10C illustrates cytoplasm 203 of an unstimulated cell depicted at a green wavelength. Figure 10D illustrates the nuclear mask 202 wears (reduces) once to define a nuclear sampling region 204 with distribution minimal cytoplasmic. The edge of the core 202 expands (expands) several times to form a ring having an amplitude of 2-3 pixels which is used to define the cytoplasmic sampling region 205 for the same cell. Figure 10E further illustrates a side view showing the nuclear sampling region 204 and the cytoplasmic sampling region 205. Using these two sampling regions, the data in the translocation Nuclear can be analyzed automatically by the system of analysis based on cells in a cell through the cellular basis. Figure 10F-J illustrates the strategy for determining nuclear translocation in a stimulated cell. FIG. 10F illustrates a stimulated cell with its nucleus 206 labeled with a blue fluorophore and a transcription factor in cytoplasm 207 labeled with a green fluorophore. The nuclear mask 208 in Figure 10G is derived by the cell-based analysis system. Figure 10H illustrates the cytoplasm 209 of a stimulated cell represented in a green wavelength. Figure 101 illustrates the nuclear sampling region 211 and the cytoplasmic sampling region 212 of the stimulated cell. Figure 10J further illustrates a side view showing the nuclear sampling region 211 and the cytoplasmic sampling region 212. A specific application of this method has been used to validate this method as an analysis. A human cell line was placed in 96-well micro-concentration plates. Some columns of the wells were titrated with IL-1, a known inducer of the transcription factor NF-KB. The cells are then fixed and labeled by standard methods with a fluorescein-labeled antibody to the transcription factor and Hoechst 33423. The cell-based analysis system was used to acquire and analyze the images of this plate and the NucCyt Difference was found to be strongly correlated with the amount of the agonist added to the wells as illustrated in Figure 16. In a second experiment, a receptor antagonist for IL-1, IL-1 RA was titrated in the presence of IL-1a, inhibiting progressively the translocation induced by IL-1a. The NucCyt Difference was found to strongly correlate with this inhibition of translocation, as illustrated in Figure 17. Additional experiments have shown that the NucCyt Difference, as well as the NucCyt ratio gives consistent results over a wide range of cell densities and concentrations of reagents and which can therefore be routinely used for the libraries of the test compounds for the specific nuclear translocation activity. Therefore, the same method can be used with antibodies to other transcription factors or the transcription factor GFP chimeras or fluorescently labeled transcription factors introduced into the fixed or live cells to analyze the effects on the regulation of factor activity. of transcription. Figure 18 is a representative display on a PC monitor of the data obtained in accordance with Example 1. Graph 1 180 traces the difference && £ * - > ~. * ± gt? between the average fluorescence of the antibody in the nuclear sampling region and the cytoplasmic sampling region, the Difference NucCyt vs Well Graph 2 181 plots the average fluorescence of the antibody in the nuclear sampling region, average NP1, against Well Graph 3 182 plots the average fluorescence of the antibody in the cytoplasmic sampling region, the average LIP1. against del Pozo The program allows to display the data of each well. For example, Figure 18 shows an on-screen display 183, nuclear image 184 and fluorescent image of antibody 185 for cell # 26. The NucCyt Difference referred to in a graph 1 180 of Figure 18 is the difference between the average of the intensity of the cytoplasmic test (fluorescent reporter molecule) and the intensity of the average nuclear test (fluorescent reporter molecule). The average NP1 referenced in graph 2 181 of Figure 18 is the average of the intensity of the cytoplasmic test (fluorescent reporter molecule) in the nuclear sampling region. The average L1 P1 referred to in a graph 3 182 of Figure 18 is the intensity of the average test (fluorescent reporter molecule) in the cytoplasmic sampling region. One skilled in the art will understand that this aspect of the invention can be realized using other transcription factors that are transferred from the cytoplasm to the nucleus in activation. In another specific example, the activation of the c-fos factor Transcription is evaluated by defining its spatial position in the cells. The activated c-fos is found only with the nucleus while the c-fos residues inactive in the cytoplasm. The 3T3 cells were placed in 5000-10000 cells per well in a 96-well Polyfiltronics plate. The cells were added and grew overnight. The cells are rinsed twice with 100 μl of serum-free medium incubated for 24-30 hours in serum-free MEM culture medium and subsequently stimulated with platelet-derived growth factor (PDGF-BB) (Sigma Chemical Co., St. Louis, MO) diluted directly in serum-free medium in concentrations ranging from 1-50 ng / ml for an average time of 20 minutes. Following the stimulation, the cells are fixed for 20 minutes in 3.7% formaldehyde solution in 1 H Hanks stabilized saline solution (HBSS). After fixation, the cells are washed with HBSS to remove the residual fixative, permeabilized for 90 seconds with 0.5% Triton X-100 solution in HBSS and washed twice with HBSS to remove the residual detergent. The cells are subsequently blocked for 15 minutes with a 0.1% solution of BSA in HBSS and further washed with HBSS before adding the diluted primary antibody solution.

The rabbit polyclonal c-Fos antibody (Calbiochem, PC05) was diluted 1: 50 in HBSS and 50 μl of the dilution was applied to each well. The cells were incubated in the presence of the primary antibody for one hour at room temperature and subsequently for one hour at room temperature in a closed container with light with goat anti-rabbit secondary antibody conjugated to ALEXA ™ 488 (Molecular Probes), diluted 1 : 500 of an existence of 100 μg / ml in HBSS. Subsequently, Hoechst DNA dye (Molecular Probes) was added in a 1: 1000 dilution of the manufacturer's solution (10 mg / ml). The cells are then washed with HBSS and the plate sealed before analysis with the cellular analysis system of the invention. The data from these experiments demonstrate that the methods of the invention could be used to measure the transcriptional activation of c-fos by defining their spatial position in the cells. One skilled in the art will recognize that while the following method is applied to the detection of c-fos activation, it can be applied to the analysis of any transcription factor that transfers from the cytoplasm to the nucleus in activation. Examples of such transcription factors include, but are not limited to fos and homologs jun, NF-KB (kappa nuclear factor of B cells), NFAT (nuclear factor of activated T lymphocytes) and STATs factors (signal transducer and transcription activator) (For example, see Strehlow I. and Schindler, C. 1998. J. Biol. Chem. 273: 28049-28056; Chow, et al., 1997, Science 278: 1638-1641; Ding et al., 1998 J. Biol. Chem. 273: 28897-28905; Baldwin, 1996. Annu Rev Immunol., 14: 649. -83; Kuo, CT and JM Leiden 1999. Annu Rev Immunol., 15: 707-47, Masuda et al., 1998. Cell Signal, 10: 599-611, Hoey T and Schindler, 1998. Curr Opin Immunol., 10: 271 -8). Therefore, in this aspect of the invention, the cellular indicators are treated with test compounds and the distribution of the luminescently labeled transcription factor is measured in space and time using a cellular analysis system, such as one of those described above. The luminescently labeled transcription factor can be expressed by or added to the cells before, together with or after contacting the cells with a test compound. For example, the transcription factor can be expressed as a luminescently-labeled protein chimera by the transfected indicator cells. Alternatively, the luminescently labeled transcription factor can be expressed, isolated and loaded in volume into the indicator cells as described above or the transcription factor can be luminescently labeled after isolation. As a further alternative, the transcription factor is expressed by the cell indicator which is subsequently contacted with a luminescent label such as an antibody, which detects the transcription factor. In a further aspect, kits are provided for analyzing the activation of the transcription factor, comprising an antibody that specifically recognizes a transcription factor of interest and instructions for using the antibody and carrying out the methods described above. In a preferred embodiment, the specific antibodies of the transcription factor or a secondary antibody that detects the - - »- '- -' transcription factor antibody is luminescently labeled. In additional preferred embodiments, the kit contains cells expressing the transcription factor of interest and / or the kit contains a compound that is known to modify the activation of the transcription factor of interest, including but not limited to platelet-derived growth factor. (PDGF) and serum, which modify the activation of fos and interleukin 1 (1 L-1) and tumor necrosis factor (TNF) that modify NFKB activation. In another embodiment, the kit comprises a recombinant expression vector comprising a nucleic acid encoding a transcription factor of interest that transfers from the cytoplasm to the nuclei in activation and instructions for using the expression vector to identify compounds that modify the activation of the transcription factor in a cell of interest. Alternatively, the kits contain a luminescently labeled and purified transcription factor. In a preferred embodiment, the transcription factor is expressed as a fusion protein with a luminescent protein including but not limited to green luminescent protein, luceriferase or mutants or fragments thereof. In several preferred embodiments, the kit also contains cells that are transfected with the expression vector, an antibody or fragment that specifically binds to the transcription factor of interest and / or a compound that is known to modify the activation of the transcription factor of interest (as described). b. Protein kinases Nuclear analysis methods can also be used for the cytoplasm to analyze the activation of any protein kinase that is present in an inactive state in the cytoplasm and is transported to the nucleus under activation or that phosphorylates a substrate that transfers from the cytoplasm to the nucleus under phosphorylation Examples of appropriate protein kinases include but are not limited to protein kinases regulated extracellular signals (ERKs), amino terminal c-Jun kinases (JNKs), protein regulatory kinases Fos (FPKs), protein kinase activated with mitogen p38 (p38MAPK), protein kinase A (PKA) and protein kinase kinases activated with mitogen (MAPKKs). (For example, see Hall, et al., 1999, J. Biol. Chem. 274: 376-83, Han et al., 1995. Biochim. Biophys. Acta. 1265: 224-227; Jarro et al., 1997. Proc. Nati, Acad Sci USA 94: 3742-3747, Taylor, et al 1994, J. Biol. Chem. 269: 308-318, Zhao, Q., and FS Lee, 1999. J. Biol. Chem. 274: 8355-8, Paolilloet et al 1999. J. Biol. Chem. 274: 546-52, Coso et al., 1995. Cell 81: 1137-1146, Tibbles, LA, and JR Woodgett, 1999. Cell Mol Life Sci. 55: 1230-54; Schaeffer, HJ and MJ Weber, 1999. Mol Cell Cell Biol .. 19: 2435-44). Alternatively, the protein kinase activity is tested by monitoring the translocation of a luminescently labeled protein kinase substrate from the cytoplasm to the nucleus after being phosphorylated by the protein kinase of interest. In this embodiment, the substrate is not phosphorylated and cytoplasmic prior to phosphorylation and is transferred to the nucleus in phosphorylation by the protein kinase. There is no requirement that the protein kinase itself transfer from the cytoplasm to the nucleus in this modality. Examples of such substrates (and the corresponding protein kinase) include, but are not limited to c-jun (JNK substrate); fos (FRK substrate) and p38 (p38 MAPK substrate). Thus, in these embodiments, the cellular indicators are treated with test compounds and the distribution of the luminescently labeled protein kinase or the protein kinase substrate is measured in space and time using a cell analysis system, as described previously. The luminescently labeled protein kinase or the kinase protein substrate can be expressed by or added to the cells before, in conjunction with, or after contacting the cells with a test compound. For example, the protein kinase or the protein kinase substrate can be expressed as a protein chimera luminescently labeled by transfected cell indicators. Alternatively, the luminescently labeled protein kinase or protein kinase substrate can be expressed, isolated and loaded in volume into the cell indicators as described above or the protein kinase or the protein kinase substrate can be luminescently labeled after isolation. As a further alternant, the protein kinase or protein kinase substrate is expressed by the cell indicator which is subsequently contacted with a luminescent label such as a labeled antibody that detects the protein kinase or protein kinase substrate. In a further embodiment, the protein kinase activity is tested by monitoring the phosphorylation state (i.e., phosphorylated or non-phosphorylated) of a protein kinase substrate. In this modality, there is no requirement that the protein kinase or substrate protein kinase transfer from the cytoplasm to the nucleus in activation. In a preferred embodiment, the phosphorylation state is monitored by contacting the cells with an antibody that binds only to the phosphorylated form of the protein kinase substrate of interest (eg, as described in U.S. Patent No. 5,599,681). In another preferred embodiment, a phosphorylation biosensor is used. For example, a luminescently labeled protein or fragment thereof can be fused to the protein that has been transformed to contain (a) a phosphorylation site that is recognized by a protein kinase of interest and (b) a nuclear location signal that is demarcated by phosphorylation. Said biosensor will therefore be transferred to the nucleus in the phosphorylation and its translocation can be used as a measure of the activation of the protein kinase. In another aspect, kits are provided to analyze activation of the protein kinase comprising a primary antibody that specifically binds to a protein kinase, a protein kinase substrate or a phosphorylated form of the protein kinase substrate of interest and instructions for use the primary antibody to identify compounds that modify the activation kinase protein in a cell of interest. In a preferred embodiment, the primary antibody or a secondary antibody that detects the primary antibody is luminescently labeled. In another preferred embodiment, the kit further comprises cells expressing the protein kinase of interest and / or a compound that is known to modify the activation of the protein kinase of interest, including but not limited to cAMP (modifies PKA), forskolin (PKA) ) and anisomycin (p38MAPK). Alternatively, the kits comprise an expression vector encoding a protein kinase or a protein kinase substrate of interest that transfers from the cytoplasm to the nucleus in the activation and instructions for using the expression vector to identify compounds that modify protein activation kinase in a cell of interest. Alternatively, the kits contain a labeled and purified luminescent protein kinase or protein kinase substrate. In a preferred embodiment, the protein kinase or protein kinase substrate of interest is expressed as a fusion of the protein with a luminescent protein. In a further preferred embodiment, the kit further comprises cells that are transfected with the expression vector, an antibody or fragment thereof that specifically binds to the protein kinase or protein kinase substrate of interest and / or compound that is known to modify the activation of the protein kinase of interest, (as described). In another aspect, the present invention comprises a machine-readable storage medium comprising a program containing a set of instructions for causing a cellular analysis system to execute the methods described for the analysis of the transcription factor or the activation of the protein kinase, wherein the cellular analysis system comprises an optical system with a state adapted for ^^? _______? ___ ¡______ Í M? t hold a plate containing cells, a digital camera, a means to detect the fluorescence or luminescence emitted from the cells to the digital camera and a computational means to receive and process digital data from the digital camera.

Example 2 Automated Analysis for Compounds that Modify Cell Morphology Changes in cell size are associated with a number of cellular conditions such as hypertrophy, cell binding and propagation, differentiation, growth and division, necrotic and programmed cell death, cellular mobility, morphogenesis , tubular formation and formation of colonies. For example, cell hypertrophy has been associated with a cascade of alterations in gene expression and can be characterized in cell cultures by alteration in cell size, which is clearly visible in adherent cells in a glass cap. Cell size can also be measured to determine the binding or propagation of adherent cells. Cellular propagation results from the selective binding of cell surface receptors to the substrate ligands and the subsequent activation of the signaling pathways for the cytoskeleton. The cellular link and the propagation to the substrate molecules is an important step for the metastasis of the cancer cells, the activation of leukocytes during the inflammatory response, the keratinocyte movement during the healing of a wound and the endothelial cell movement during angiogenesis. Compounds that affect these surface receptors, signaling pathways or the cytoskeleton will affect cell propagation and can be analyzed by measuring cell size. The total cell area can be monitored by marking the entire cell body or the cell cytoplasm using cytoskeletal markers, markers - • * Mfc ** »« ^ cytosolic volume or cell surface markers together with a DNA marker. Examples of such markers (many available from Molecular Probes (Eugene, Oregon) and Sigma Chemical Co. (St. Louis, Missouri) include the following: CELLULAR SIZE AND AREA MARKERS CYXESQUETIC MARKERS ALEXA rr'w M 488 phalloidin (Molecular Probes, Oregon) Green Fluorescent Protein Chimeras-Tubulin Green Fluorescent Protein Chimeras- Cytokeratin Cytoskeletal Protein Antibodies Cytosolic Volume Markers Green Fluorescent Proteins Chloromethylfluorescein Diacetate (CMFDA) Green Calcein BCECF / AM Ester Rodamine Dextran Cell Surface Markers for Lipods, Proteins o Oligosaccharides Dihexadecyl perchlorate tetramethylindocarbocyanine lipid dyes (DMCI6) Triethylammonium propyl dibutylamino stiril pyridine lipid dyes (FM 4-64, FM 1-43) MICROTRACKER TI WM .FM green Lectins for oligosaccharides such as fluorescein concanavalin A or agglutinin Wheat germ SYPR Or non-specific red protein markers Antibodies to several surface proteins such as epidermal growth factor Biotin labeling of surface proteins followed by fluorescent estrepavidin labeling The protocols for cell labeling with these various agents are well known to those skilled in the art. Live cells labeled or after fixation and cell area can be measured. For example, living cells labeled with DÍIC16 have homogenously labeled plasma membranes and the projected cross-sectional area of the cell is discriminated uniformly from the background by the fluorescence intensity of the dye. Live cells labeled with cytosolic spots such as CMFDA produce a fluorescent intensity that is proportional to the cell thickness. Although cell marking is dark in the thin regions of the cell, the total cell area can be discriminated against the background. The fixed cells can be labeled with cytoskeletal markers such as ALEXA ™ phalloidin 488 which labels the polymerized actin. Phalloidin does not homogenously mark the cytoplasm, but still allows discrimination of the total cellular area of the background.

Cellular Hypertrophy An analysis to analyze cell hypertrophy is implemented using the following strategy. Primary myocytes from rats can be cultured in 96-well plates, treated with various compounds and subsequently fixed and labeled with a fluorescent marker for cell membrane or cytoplasm or cytoskeleton, such as an antibody for a cell surface marker or a fluorescent marker for the cytoskeleton such as rhodamine-phalloidin in combination with DNA marker as Hoechst. After focusing on the core marked with Hoechst, two images were acquired, one of the nucleus marked with Hoechst and one of the image of the fluorescent cytoplasm. The cores were identified by the start to create a mask and then to compare the morphological descriptions of the mask with a set of user-defined description values. Each non-core image (or "cytoplasmic image") is subsequently processed separately. The original cytoplasmic image can be initiated, creating a cytoplasmic mask image. Local regions containing cells are defined around the nucleus. The boundaries of the cells in these 20 regions are subsequently defined by a local dynamic initial operation in the same region in the fluorescent antibody image. A sequence of erosions and dilatations is used to slightly separate the contact cells and a second set of morphological descriptors is used to identify the single cells. The area of individual cells is tabulated to define the distribution of cell sizes to be compared with the size of the hypertrophic and normal cell data. ^ gg¡ ^^^ ¡m - ^^^^^^^ - ^^^^^^^^^^^^^^^^^^^^^^^^ P ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ 96 well plates (measured as average cytoplasmic area / cell) were analyzed by the above methods and the results showed that the test will do the same on a day by day basis, plate by plate and well by well (below 15% cov for the maximum signal). The data shown showed very good correlation for each day and there was no variability due to the good position in the plate. The following totals can be computed for the field. The aggregate of the total core area is the number of non-zero pixels in the nuclear mask. The average of the total area of the nucleus is the aggregate of the total area of the nucleus divided by the total number of the nucleus. For each cytoplasm image, several values are computed. These are the total cytoplasmic area which is the counting of the non-zero pixels in the cytoplasmic mask. The intensity of the aggregate of the cytoplasm is the sum of the intensities of all the pixels in the cytoplasmic mask. The cytoplasmic area per nucleus is the total cytoplasmic area divided by the total counting of the nucleus. The cytoplasmic intensity per nucleus is the intensity of the aggregate of the cytoplasm divided by the total counting of the nucleus. The average cytoplasmic intensity is the intensity of the cytoplasm aggregate divided by the cytoplasmic area. The radius of the nucleus of the cytoplasm is the total area of the cytoplasm divided by the total area of the nucleus. Additionally, one or more fluorescent antibodies to other cellular proteins such as the actin of the major muscle proteins or myosin may be included. Images of these labeled additional proteins can be acquired and stored with the above images for further review to identify abnormalities in the distribution and morphology of these proteins in hypertrophic cells. This example of a multi-parametric analysis allows the simultaneous analysis of cell hypertrophy and changes in the action or distribution of myosin.

! Z? I? E? ~ «- tSÁ-á iZ - A person skilled in the art will recognize that while the example examines myocyte hypertrophy, the methods can be applied to analyze hypertrophy or general morphological changes in any type of cell.

Cellular morphological tests for prostate carcinoma Cellular propagation is a measure of the response of cell surface receptors to substrate bound to ligands. Propagation is proportional to the concentration of ligand or to the concentration of compounds that reduce the function of the ligand-receptor. An example of selective cell-substrate binding is the adhesion of the prostate carcinoma cell to the collagen of the extracellular matrix protein. Prostate carcinoma cells undergo bone metastasis via selective adhesion of collagen. Compounds that interfere with the metastasis of prostate carcinoma cells were analyzed as follows. PC3 human prostate carcinoma cells were cultured in a medium with appropriate stimulants and transferred to 96-well plates covered with collagen. The concentration of ligands can vary or inhibitors of cell propagation can be added to the wells. Examples of compounds that can affect the propagation are receptor antagonists such as integrin- or preoteoglycan blocking antibodies, signaling inhibitors including phosphatidyl inositol-3 kinase inhibitors and cytoskeletal inhibitors such as cytochalasin D. After two hours, the cells were fixed and labeled with ALEXA ™ phalloidin 488 (Molecular Probes) and Hoechst 33342 as per the protocol for cell hypertrophy. The size of the cells under these various conditions, as measured by cytoplasmic labeling, can be distinguished above the background levels. The number of cells per field is determined by measuring the number of the core labeled with the Hoechst DNA dye. The area per cell is found by dividing the area * - '- ^ fftfl-fT cytoplasmic (phalloidin image) by cell number (Hoechst image). The size of the cells is proportional to the function of the receptor-ligand. Since the area is determined by the concentration of ligand and by the resulting function of the cell, the efficacy of the drug as well as the potency of the drug can be determined by this cell-based test. Other measures may be performed as described above for cellular hypertrophy. Methods for analyzing cell morphology can be used in a high-content-combined analysis. In one example, the high performance mode scans the entire well for an increase in fluorescent phalloidin intensity. A start is established at which the core (Hoechst) and the cells (phalloidin) were measured in a high content mode. In another example, an ambient biosensor (examples include but are not limited to, those biosensors that are sensitive to changes in pH and calcium) was added to the cells and the cells contacted with a compound. The cells are screened in a high performance mode and those wells that exceed a p-determined re-determination for the luminescence of the biosensor are scanned in a high-content mode. In a further aspect, kits are provided to analyze cell morphology comprising a luminescent compound that can be used to specifically mark the cell cytoplasm, membrane or cytoskeleton (such as those described above) and instructions for using the luminescent compound to identify the test stimulus that induces or inhibits changes in cell morphology in accordance with the above methods. In a preferred embodiment, the kit further comprises a luminescent marker for the cell nucleus. In a preferred embodiment, the kit comprises at least one compound that is known to modify cell morphology including, but not limited to, integrin-blocking antibodies - - - ***** "* •> **. - ****. *. U - ~ ^^ _-_-,.,. proteoglycans, signaling inhibitors including inositol-3 kinase inhibitors and cytoskeletal inhibitors such as cytochalasin D. In another aspect, the present invention comprises a machine readable storage medium comprising a program containing a set of instructions 5 for causing a cellular analysis system to execute the described methods for analyzing cell morphology wherein the cellular analysis system comprises a system optical with a state adapted to hold a plate containing cells, a digital camera, a means for directing the fluorescence or luminescence emitted from the cells to the digital camera and a computerized means for receiving and processing the digital data of the digital camera.

Example 3 High Performance Dual Mode and High Content Analysis The following example is an analysis for the activation of a G-protein coupled receptor (GPCR) as detected by the translocation of the GPCR from the plasma membrane 15 to a nuclear location. next. This example illustrates how a high performance analysis can be coupled with a high content analysis in the Dual Mode System for Cell Based Analysis. The G protein-coupled receptors are a large class of 7-trans membrane domain cell surface receptors. The ligands for these receptors stimulate a cascade of secondary signals in the cell, which may include but are not limited to transient Ca ++, cyclic AMP production, inositol thiophosphate (IP3) production and phosphorylation. Each of these signals are fast occurring in a matter of seconds or minutes but they are also generic. For example, many different GPCRs produce a secondary Ca ++ signal when they are activated. Stimulation of a GPCR 25 also results in the transport of that GPCR from the cell surface membrane to an internal near nuclear compartment. This hospitalization is a much more indicator aJ-MM ^ .. - _.,. - ... J ,. z "_____. receptor-specific activation of a particular receptor which are the secondary signals previously described. Figure 19 illustrates a dual-mode analysis for the activation of a GPCR. Cells loading a stable GPCR chimera with a blue fluorescent protein (BFP) would be loaded with the acetoxymethyl ester form of Fluo-3, a cell-permeable calcium indicator (green fluorescence) that is trapped in living cells by hydrolysis of the esters. Subsequently, they were deposited in the wells of a micro-concentration plate 601. Subsequently, the wells would be treated with a series of test compounds using a fluid delivery system and a short sequence of Fluo-3 images of the complete micro-plate. Concentration would be acquired and analyzed for wells that exhibit a calcium response (ie, high performance mode). The images would appear as the illustration of the micro-concentration plate 601 in Figure 19. A small number of wells, such as C4 and E9 in the illustration, would be more brightly fluorescent due to the Ca ++ released in the stimulation of the receptors. The well locations containing compounds that induce a 602 response would subsequently be transferred to the HCS program and the optics exchanged by detailed cell-by-cell analysis of the blue fluorescence for evidence of the GPCR translocation to the perinuclear region. The lower part of Figure 19 illustrates the two possible outputs of high-resolution cellular data analysis. The chamber presents a sub-region 604 of the well area 603, producing images in the 605 fluorescent cells. In the C4 well, the uniform distribution of the fluorescence in the cells indicates that the receptor has not been internalized, implying that the Ca ++ response observed was the result of the stimulation of some other signaling system in the cell. The cells in well E9 606 on the other hand, clearly indicate a concentration of the receptor in the perinulear region clearly indicating the total activation of the receptor. Because only a few impact wells have been analyzed with high resolution, the t- -.

Total performance of the dual-mode system can be completely high, comparable with the high-performance system alone.

Example 4 High Content Kinetic Analysis 5 The following is an example of an analysis to measure the kinetics of a recipient's hospitalization. As described above, the stimulation of a GPCR results in the internment of the receiver, with a time period of approximately 15 min. Simply by detecting the endpoint as it entered or not, it might not be enough to define the potency of a compound as a GPCR agonist or antagonist. Nevertheless, 3 periods of time at 5 min intervals would provide information not only about the power over the time course or measurement, but would also allow extrapolation of much larger data over time periods. To perform this test, the sub-region could be defined as two columns, the sampling interval as 5 minutes and the total number of time periods 3. The system Subsequently, it would be started by scanning two columns and then adding the reagent to the two columns, establishing the reference time = 0. After the addition of the reagent, the system would again explore the sub-region of two columns acquiring the data of the first period of time. Since this process would take approximately 250 seconds, including the scan back to the beginning of sub-region, the system would wait 50 seconds to start the acquisition of the second period of time. Two more cycles would produce the three time periods and the system would move in the second sub-region of 2 columns. The two sub-regions of the final 2-columns would be scanned to finish all the wells in the plate, resulting in four time periods for each well in the total plate. Although the time periods for the wells would be compensated slightly relative to time = 0, the spacing of the time periods would be very close to the required 5 minutes and the times of Real acquisition and recorded results much more accurately than in a fixed cell analysis.

Example 5 Analysis of high content of translocation of the human glucocorticoid receptor One class of HCS involves the drug-induced dynamic redistribution of the intracellular constituents. The human glucocorticoid receptor (hGR), a simple "sensor" in the complex environmental response machinery of the cell, links the steroid molecules that have diffused into the cells. The ligand-receptor complex transfers to the nucleus where transcriptional activation occurs (Htun et al., Proc. Nati, Acad. Sci. 93: 4845, 1996). In general, hormone receptors are excellent drug targets because their activity tends at the apex of key intracellular signaling pathways. Therefore, a high content analysis of the hGR translocation has a distinct advantage in ligand-receptor binding tests in vitro. The availability above two more channels of fluorescence in the cellular analysis system of the present invention allows the analysis to contain two additional parameters in parallel, such as receptors, other different targets or other cellular processes. Construction of the plasmid. A eukaryotic expression plasmid containing a coding sequence for a green fluorescent protein-chimeric human glucocorticoid receptor (GFP-nGR) was prepared using GFP mutants (Palm et al., Nat. Struct. Biol. 4: 361 (1997) The construct was used to transfect a human cervical carcinoma cell line (HeLa), cell preparation and transfection, HeLa cells (ATCC CCL-2) were trypsinized and plated using DMEM containing 5% fetal bovine serum. treated with carbon / dextran (FBS) (HyClone) and 1% penicillin-streptomycin "tó ^^ jg ^^^ i ^ ^^.

(C-DMEM) 12-24 hours before transfection and incubation at 37 ° C and 5% CO2. Transfections were performed by co-precipitation of calcium phosphate (Graham and Van der Eb, Virology 52: 456, 1973; Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Second ed. Cold Spring Harbor Laboratory Press, Cold Spring 5 Harbor, 1989) or with Lipofectamine (Life Technologies, Gaithersburg, MD). For calcium phosphate transfections, the medium is replaced before the DMEM transfection containing 5% FBS treated with carbon / dextran. The cells were incubated with the DNA precipitate with calcium phosphate for 4-5 hours at 37 ° C and 5% CO2 washed 3-4 times with DMEM to remove the precipitate., followed by the addition of C-DMEM. 10 Lipofectamine transfections were performed in serum-free DMEM without antibiotics in accordance with manufacturers' instructions (Life Technologies, Gaithersburg, MD). Following a 2-3 hour incubation with the DNA-liposome complexes, the medium was removed and replaced with C-DMEM. All cells transfected in 96-well micro-concentration plates were incubated at 33 ° C and CO2 to 5% for 24-48 hours before drug treatment. The experiments were performed with the transiently expressed receptor on HeLa cells. Dexamethasone induction of GFP-hGR translocation. To obtain the kinetic data of the receptor-ligand translocation, the nucleus of the transfected cells was first labeled with 5 μg / ml of Hoechst 33342 (Molecular Probes) in C-DMEM by 20 minutes at 33 ° C and 5% CO2. The cells were washed once in Hank's Balanced Salt Solution (HBSS) following the addition of 100 nM dexamethasone in HBSS with 1% FBS treated with carbon / dextran. To obtain the dexamethasone titration data in the fixed period of time, the transfected HeLa cells were first washed with DMEM and subsequently incubated at 33 ° C and 5% CO2 for 1 hour in the presence of 0-1000 nM dexamethasone in DMEM containing 1% FBS treated with carbon / dextran. The cells were analyzed alive and rinsed with HBSS, they were fixed for 15 min with 37% formaldehyde in HBSS, labeled with Hoechst 33342 and washed before analysis. The intracellular GFP-hGR signal was not decreased by this fixation procedure. Acquisition of the image and analysis. Kinetic data were collected by acquiring pairs of fluorescent images (GFP-hGR and core labeled Hoechst 33342) from the fields of living cells at 1 min intervals for 30 min after the addition of dexamethasone. Likewise, pairs of images were obtained from each well of the analysis plates for the time periods set 1 h after the addition of dexamethasone. In both cases, the pairs of images obtained in each time period were used to define the cytoplasmic and nuclear regions in each cell. The translocation of GFP-hGR was calculated by dividing the integrated fluorescence intensity of GFP-hGR in the nucleus by means of the integrated fluorescence intensity of the chimera in the cytoplasm or as a cytoplasmic-nuclear difference of the GFP fluorescence. In the analysis of the fixed period of time this translocation ratio is calculated from the data obtained from at least 200 cells in each concentration of dexamethasone tested. The translocation induced with GFP-hGR drugs from the cytoplasm to the nucleus therefore correlated with an increase in the translocation rate. Results Figure 20 systematically displays the cytoplasm induced with drugs 253 to the translocation of nucleus 252 of the human glucocorticoid receptor. The top pair of the schematic diagrams represent the location of GFP-hGR in the cell before stimulation 250 (A) and after 251 (B) with dexamethasone. Under these experimental conditions, the drug induces a large portion of the cytoplasmic GFP-hGR to transfer into the nucleus. This redistribution is quantified by determining the radius of integrated intensities of the nuclear and cytoplasmic fluorescence in the untreated 254 and treated 255 cells. The lower pair of the micro- Fluorescence graphs show the dynamic redistribution of GFP-hGR in an individual cell, before 254 treatment and after 255. The HCS was performed in wells containing hundreds of thousands of transfected cells and the translocation was quantified by each cell in the field of fluorescence GFP. Although the use of a stably transfected cell line would produce the most consistently labeled cells, the heterogeneous levels of GFP-hGR expression induced by transient transfection did not interfere with the analysis by the cellular analysis system of the present invention. To perform the analysis, the cell analysis system scans each well of the plate, presents a population of cells in each and analyzes the cells individually. Here, two fluorescence channels are used to define the cytoplasmic and nuclear distribution of GFP-hGR in each well. Figure 21 shows the graphical user interface of the cellular analysis system near the end of a GFP-hGR analysis. The user interface represents the collection of parallel data and the ability to analyze the system. The windows marked "Nucleus" 261 and "GFP-hGR" 262 show the pair of fluorescence images being obtained and analyzed in an individual field. The window marked "Color Coating" 260 is formed by pseudo-coloring the previous images and merging them so that the user can immediately identify the cell changes. In the "Stored Objects Regions" window 265, an image that contains every cell analyzed and its neighbors are presented as if it were stored. In addition, since the HCS data are being collected, they are analyzed, in this case, for the GFP-hGR translocation and are transferred in an immediate "impact" response. The 96 well plate shown in the bottom window of Analysis 267 shows that the wells have met an establishment of the user defined analysis criteria. For example, a well 269 colored white indicates that the translocation induced with drugs has exceeded a predetermined start value - - - - * "- '- :: -" - t ^ * aafefc' - - • - ^ _ _. 50% On the other hand, a well 270 colored black indicates that the drug being tested induces less than 10% translocation. Wells 268 colored with gray indicate "impacts" where the value of the translocation falls between 10% and 50%. Column "E" in the 96 well plate analyzed 266 shows a titration with a known drug 5 to activate the GFP-hGR translocation, dexamethasone. This example of analysis was used only in two fluorescence channels. Two additional channels (Channels 3 263 and 4 264) are available for the parallel analysis of other specific targets, cellular processes or cytotoxicity to create multi-parameter analysis. There is a link between the image database and the database of the information that is a powerful tool during the validation process of new analyzes. At the end of an analysis, the user has full access to present and calculate the data (Figure 22). The complete data analysis package of the cellular analysis system allows the user to examine the HCS data at multiple levels. The images 276 and the detailed data in a spreadsheet 279 for the individual cells can observed separately or can be traced. For example, the calculated results of a single parameter for each cell in a 96-well plate are shown in Graph 1 marked panel 275. By selecting a single point in the graph, the user can display the complete data configuration for a cell particular that is called back from an existing database. Here the pair of images 276 and the detailed fluorescence and the morphometric data of a single cell (Cell # 118, gray line 277). The larger graphic insertion 278 shows the results of the dexamethasone concentration in the translocation of GFP-hGR. Each point is the average of the data of at least 200 cells. The EC50 calculated for dexamethasone in this test is 2 nM. 25 One powerful aspect of HCS with the cellular analysis system is the ability of kinetic measurements using multicolored fluorescence and morphometric parameters in -. < ¿M »» B-. t _ .....__ ..- - üj? ffft live cells. Spatial and temporal measurements can be made on individual cells in a population of cells in a field. Figure 23 shows the kinetic data for the dexamethasone-induced translocation of GFP-hGR in several cells in an individual field. Human HeLa cells transfected with GFP-hGR are treated with 100 nM dexamethasone and the GFP-hGR translocation was measured in time in a population of individual cells. The graph shows the response of transfected cells 285, 286, 287 and 288 and untransfected cells 289. These data also illustrate the ability to analyze cells with different levels of expression.

Example 6 Analysis of high content of apoptosis induced with drugs Apoptosis is a complex cellular program involving innumerable events and molecular pathways. To understand the mechanisms of action of drugs in this process, it is essential to measure as many as possible these events in cells with spatial and temporal resolution. Therefore, the analysis of apoptosis that requires preparation of the small cell sample still provides an automatic report extraction of several parameters related to apoptosis, which would be ideal. A cell-based test for the cell analysis system has been used to simultaneously quantitate several macromolecular, organelle and morphological markers of paclitaxel-induced apoptosis. Cellular preparation The cells selected for this study were connective tissue fibroblasts from mice (L-929; ATCC CCL-1) and a highly invasive glioblastoma cell line (SNB-19; ATCC CRL-2219) (Welch et al., In Vitro Cell. Dev. Biol. 31: 610, 1995). The day before treatment with a drug inducing apoptosis, 3500 cells were placed in each well of a 96-well plate and incubated overnight at 37 ° C in a humidified atmosphere with 5% CO2. The next day, the culture medium was removed from each well and replaced with fresh medium containing various concentrations of paclitaxel (0-50 μM) from a 20 mM stock in DMSO. The maximum concentration of DMSO used in these experiments was 0.25%. The cells were subsequently incubated for 26 h as mentioned. At the end of the paclitaxel treatment period, each well received fresh medium containing 750 nM of Red Tracer Myth (Molecular Probes).; Eugene, OR) and 3 μg / ml Hoechst 33342 DNA-binding dye (Molecular Probes) and incubated as mentioned for 20 minutes. Each well in the plate was subsequently washed and fixed with 3.7% formaldehyde in HBSS for 15 min at room temperature. The formaldehyde was washed with HBSS and the cells were permeabilized for 90 seconds with 0.5% (v / v) Triton X-10 100, washed with HBSS with 2 U ml-1 of Bodipy FL phalladicin (Molecular Probes) for 30 min. and washed with HBSS. The wells in the plates were subsequently filtered with 200 μl of HBSS, sealed and the plate stored at 4 ° C, if necessary. The fluorescence signals of the plates stored in this manner were stable for at least two weeks after the preparation. As in the nuclear translocation test, the 15 fluorescent reagents can be designated to convert this test into an analysis of high content of living cells. Acquisition of image and analysis of the Series Exploration System. The intensity of the fluorescence of the intracellular Red Mito Tracker, Hoechst 33342 and Bodipy FL phallacidin was measured with the cell analysis system as described supra. The 20 morphometric data of each pair of images obtained from each well were also obtained to detect each object in the image field (ie cells and core) and to calculate its integrated size, shape and intensity. Calculations and output. A total of 50-250 cells were measured per image field. For each cell field, the following calculations were made: (1) The average nuclear area (μm2) was calculated by dividing the total nuclear area in a field by the area A «« hd _-__ «- > ^^ w- of the detected core. (2) The average nuclear perimeter (Pm) was calculated by dividing the sum of the perimeters of the entire core in a field by the number of nuclei detected in that field. The highly complicated apoptotic nucleus has the largest perimetric values. (3) The average nuclear brightness was calculated by dividing the integrated intensity of the total kernel field by the number of the nucleus in that field. An increase in nuclear brilliance correlated with the increased DNA content. (4) Average cell brightness was calculated by dividing the integrated intensity of a total field marked with Mito Tracker dye by the number of the nucleus in that field. Because the amount of Mito Tracker dye that accumulates in the mitochondria, 10 is proportional to the mitochondrial potential, an increase in average cellular brightness is consistent with an increase in mitochondrial potential. (5) The average cellular brightness was also calculated by dividing the total integrated field of cells Bodipy FL .Dyed phallacidin by the number of the core in the field intensity. 15 Because the binding of phalotoxins with high affinity to the polarized form of actin, the amount of Bodipy FL dye falacidin that accumulates with the cell is proportional to the polymerization state of the actin. An increase in average cell brightness is consistent with an increase in actin polymerization. Results Figure 24 (upper panels) shows paclitaxel 20 changes induced in the nuclear morphology of L-929 cells. The increased amounts of paclitaxel caused the nucleus to increase and fragment 293, a hallmark of apoptosis. The quantitative analysis of these and other images obtained by the cellular analysis system in the same figure. Each measured parameter showed that L-929 296 cells were less sensitive to low concentrations of paclitaxel than 25 were SNB-19 297 cells. At high concentrations although L-929 cells showed a response for each parameter measured. This multi-parameter approach of this G ^^ i ^ í ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^ ^^^^ e ^^^^^^ - j ^^^ ^^ ^^ í- ?? ^^ * fca¿_ test is useful in dissecting the mechanisms of drug action. For example, the ares, brightness and fragmentation of the nucleus 298 and polymerization values of actin 294 reached a maximum value when the SNB-19 cells were treated with 10 nM paclitaxel (Figure 24, lower and upper plots). However, mitochondrial potential 295 was minimal in the same concentration of paclitaxel (Figure 24, intermediate graph). The fact that all measured parameters approximated to control levels in the increase of paclitaxel concentrations (> 10 nM) suggests that SNB-19 cells have low affinity with the clear or metabolic pathways of the drug that are compensatory in sufficiently high levels of the drug. In contrast, drug sensitivity of SNB-19 297 cells, L-929 showed a different response to paclitaxel 296. These fibroblast cells showed a maximum response in many parameters in 5 μM paclitaxel, a 500-fold higher dose than SNB-cells. 19 In addition, L-929 cells did not show an acute decrease in mitochondrial potential 295 at any of the paclitaxel concentrations tested. East result is consistent with the presence of the unique apoptotic pathways between a cancerous and normal cell line. Therefore, these results indicate that a relatively simple fluorescence labeling protocol can be coupled with the cellular analysis system of the present invention to produce an analysis of high content of the key events involved in programmed cell death. Example 7 The protease-induced translocation of a signaling enzyme by contacting a sequence associated with disease of the cytoplasm to the nucleus. Construction of the plasmid. A eukaryotic expression plasmid containing a coding sequence for a green-caspase fluorescent protein (Cohen) (1997), Biocehmical J. 326: 1-16; Liang et al. (1997), J. of Molec. Biol. 274: 291-302) 274: 291-302) the chimera was prepared using GFP mutants. The construct is used to transfect eukaryotic cells. Cell preparation and transfection. The cells were triplicated and plated 24 h before transfection and incubated at 37 ° C and 5% CO2. Transfections were performed by methods including but not limited to coprecipitation of calcium phosphate or lipofection. The cells were incubated with the calcium-calcium phosphate precipitate for 4-5 hours at 37 ° C and 5% CO2 washed 3-4 times with DMEM to remove the precipitate, followed by the addition of C-DMEM. Lipofectamine transfections were performed in serum-free DMEM without antibiotics in accordance with the manufacturer's instructions. After a 2-3 hour incubation with liposome DNA complexes, the medium was removed and replaced with C-DMEM. Apoptotic induction of the GFP-Caspase translocation. To obtain the kinetic data of the translocation of the GFP-Caspase, the nucleus of the transfected cells was first labeled with 5 μg / ml Hoechst 33342 (Molecular Probes) in C-DMEM for 20 minutes at 37 ° C and 5% CO2 . The cells were washed once in Hank's Balanced Salt Solution (HBSS) followed by the addition of the apoptotic inducing compounds. These compounds include, but are not limited to paclitaxel, staurosporine, ceramide and tumor necrosis factor. To obtain the titration data of the fixed period of time, the transfected cells were first washed with DMEM and subsequently incubated at 37 ° C and 5% CO2 for 1 h in the presence of 0-100 nM of the compound in DMEM. The cells were analyzed alive or rinsed with HBSS, fixed for 15 min with 3.7% formaldehyde in HBBS, labeled with Hoechst 33342 and washed before analysis. Acquisition of image and analysis. Kinetic data were collected by acquiring pairs of fluorescence images (GFP-Caspasa and Hoechst 33342-labeled nucleus) from the fields of living cells at 1 min intervals for 30 minutes ^^ ^ ^ ^ ^ ^ after the addition of the compound. Likewise, the image pairs are obtained from each well of the analysis plates of the fixed period of time 1 h after the addition of the compound. In both cases, the pairs of images obtained in each time period are used to define the cytoplasmic and nuclear regions in each well. The translocation of the GFP-Caspase is calculated by dividing the integrated fluorescence intensity of GFP-Caspase in the nucleus by the integrated fluorescence intensity of the chimera in the cytoplasm or as a cytoplasmic-nuclear difference of the GFP fluorescence. In the analysis of the fixed period of time, this translocation radius is calculated from the data obtained from the minus 200 cells in each concentration of tested compounds. The translocation induced with GFP-Caspase drugs from the cytoplasm to the nucleus was therefore correlated with an increase in the translocation radius. Molecular interaction libraries include, but are not limited to, those that comprise putative activators or inhibitors of activated apoptosis enzymes used to analyze the indicator of cell lines and identify a specific ligand for DAS and a pathway activated by the activity of the compound.

Example 8. Identification of novel steroid receptors of the DAS Two sources of material and / or material are required to make use of this modality, which allows the evaluation of the function of an uncharacterized gene. First, the sequence-associated bank (s) may be used which contains appropriate cDNA sequences for transfection in mammalian cells. Because each RADE or differential expression experiment generates more than several hundred sequences, it is possible to generate a broad supply of DAS. Second, information from searches of the primary sequence database can be used to place DAS in broad categories, including but not limited to those that contain ¿^ ^ ^ ^ ^ ^ ^ Signal sequences, seven trans-membrane subjects, protected protease active site domains, or other identifiable motifs. Based on the information acquired from these sources, the types of methods and indicators of the cell lines to be transfected are selected. A large number of motifs are well characterized and encoded in the linear sequences contained in the large numbers of genes in the existing genomic databases. In one modality, the following steps are taken: 1) The information from the DAS identification experiment (including database searches) is used as the basis for selecting the relevant biological processes (for example, to see the DAS of a line tumor for the modulation of the cell cycle, apoptosis, metastatic proteases, etc.). 2) Classification of the DNA or DAS sequences by identifiable motifs (ie, signaling sequences, 7-transmembrane domains, active site domains of conserved protease, etc.). This initial grouping will determine fluorescent identification strategies, host cell lines, indicators of cell lines and banks of bioactive molecules to be analyzed, as described above. 3) The well-established use of molecular biology methods, link DAS into an expression vector designated for this purpose. The generalized expression vectors contain promoters, enhancers and terminators to which the target sequences are sent to the cell for transient expression. Such vectors may also contain antibody identification sequences, direct association sequences, chromophoric fusion sequences such as GFP, etc. to facilitate detection when expressed by the host. 4) Transiently transfected cells with DAS containing vectors using standard transfection protocols including: co-precipitation of calcium phosphate, mediated liposome, dextran DEAE mediated, mediated polycation, viral mediated or ^^^^^^^^^^^^ ^^^ rffe ^ jfe ^ electrophoration and plate in the plates of micro-concentration or series of micro-wells. Alternatively, the transfection can be done directly on the micro-concentration plate itself. 5) Carry out the cell analysis methods as described above. In this embodiment, the DASs demonstrate having a motif (s) suggestive of transcriptional activation potential (eg, DNA binding domain, amino terminal modulation domain, binding region or carboxy terminal ligand binding domain), are used to identify novel steroid receptors. Defining the fluorescent labels for this experiment involves the identification of the nucleus through the labeling and naming of DAS by creating a GFP chimera via the insertion of the DAS into an expression vector, closely fused to the GFP coding gene. Alternatively. A fragment of the single chain antibody with high affinity for some portion of the DAS expressed could be constructed using the technology available in the art (Cambridge Antibody Technologies) and linked to a fluorophore (FITC) to mark the putative transcriptional activator / receptor in the cells. This alternative would provide an external label that does not require DNA transfection and would therefore be useful if the distribution data were accumulated from the original primary cultures used to generate the DAS. Plasmid construction. A eukaryotic expression plasmid containing a coding sequence for a green fluorescent protein - the DAS chimera is prepared using GFP mutants. The construction is used to transfect HeLa cells. The plasmid, when transfected into the host cell, produces a GFP fused to the DAS protein product, designated GFP-DASpp. j ^ jg ^ Cell preparation and transfection. HeLa cells were trisinized and plated using DMEM containing 5% fetal bovine serum dextran / charcoal treated (FBS) (Hyclone) and 1% penicillin-streptomycin (C-DMEM) 12-24 hours before transfection and incubated at 37 ° C and 5% CO2. Transfections were performed by co-precipitation of calcium phosphate or with Lipofectamine (Life Technologies). For calcium phosphate transfections, the medium was replaced, before transfection with DMEM containing 5% FBS treated with dextran / carbon. The cells were incubated with the calcium phosphate-DNA precipitate for 4-5 hours at 37 ° C and 5% CO2 and washed 3-4 times with MEM to remove the precipitate, followed by the addition of C-DMEM. Lipofectamine transfections were performed in serum-free DMEM without antibiotics in accordance with the manufacturer's instructions. After a 2-3 hour incubation with the DNA-liposome complexes, the medium was removed and replaced with C-DMEM. All transfected cells in 96-well microtiter plates were incubated at 33 ° C and 5% CO2 for 24-48 hours before the drug treatment. The experiments were performed with the transiently expressed receptor on HeLa cells. Location of the internal GFP-DASpp cells expressed. To obtain the cell distribution data, the core of the transfected cells was first labeled with 5 μg / ml Hoechst 33342 (Molecular Probes) in C-DMEM for 20 minutes at 33 ° C and CO2 at 5%. The cells were washed once in Hank's Balanced Salt Solution (HBSS). The cells were analyzed alive or rinsed with HBSS, fixed for 15 min with 3.7% formaldehyde in HBSS, labeled with Hoechst 33342 and washed before analysis. In a preferred embodiment, image acquisition and analysis were performed using the cellular analysis system of the present invention. The intracellular GFP-DASpp fluorescence signal was collected by acquiring pairs of images from tufar * -. zz .r.ia «gft_aat_r. fluorescence (GFP-DASpp and core labeled with Hoechst 33342) of the field cells. The pairs of images obtained in each time period are used to define the cytoplasmic and nuclear regions in each well. The dispersed signal of demonstration of data in the cytoplasm would be consistent with the known steroid receptors that are DNA transcriptional activators. Analysis for the induction of GFP-DASpp translocation. Using the above construct, confirmed for the appropriate expression of GFP-DASpp as a cell line indicator, an analysis of several ligands is performed using a series of steroid-type ligands including but not limited to: estrogens, progesterone, retinoids, factors of growth, androgens and many other steroids and steroid-based molecules. Image acquisition and analysis were performed using the cellular analysis system of the invention. The intracellular GFP-DASpp fluorescence signal was collected by acquiring pairs of fluorescence images (GFP-DASpp and core labeled Hoechst 33342) of the field cells. The pairs of images obtained in each time period were used to define the cytoplasmic and nuclear regions in each well. The translocation of GFP-DASpp is calculated by dividing the integrated fluorescence intensity of GFP-DASpp in the nucleus by the integrated fluorescence intensity of the chimera in the cytoplasm or as a cytoplasmic-nuclear difference of the GFP fluorescence. A translocation of the cytoplasm in the nucleus indicates an activation of the ligand binding of the DASpp in this way identified the class of the potential receptor and the action. Combining these data with other data obtained in a similar way using known inhibitors and steroid receptor modifiers would validate the DASpp as a target or more data would be generated from several sources.

M__i _______ li_íi _________ i______i_____? iajás-i ^ s-f -, ^. ^ Example 9. Additional Analysis Translocation between the plasma membrane and the cytoplasm: Dissociation of the profilactin complex and binding of profilin to the plasma membrane. In one embodiment, a profilin membrane-bound fluorescent protein biosensor was prepared by labeling purified profilin (Federov et al. (1994), J. Molec, Biol. 241 ¡480-482; Lanbrechts et al. (1995) , Eur. J. Biochem. 230: 281-286) with a probe with a fluorescence life cycle in the range of 2-300 ns. The labeled profilin was introduced into the live indicator cells using the bulk loading methodology and the indicator cells were treated with the test compounds. The fluorescence anisotropy imaging microscopy (Gough and Taylor (1993), J. Cell, Biol. 121: 1095-1107) was used to measure the dependent movement of the test compound of the fluorescent derivative of profilin between the cytoplasm and the membrane for a period of time after treatment ranging from 0.1 to 10 h.

Translocation of the Rho-RhoGDI complex to the membrane. In another embodiment, the indicator cells were treated with the test compounds and subsequently fixed, washed and permeabilized. The indicator of the cellular plasma membrane, the cytoplasm and the nucleus are all marked with markers of different colors followed by the immunolocalization of the Rho protein (Self et al. (1995), Methods in Enzymology 256: 3-10; Tanaka et al. . (1995), Methods in Enzymology 256: 41-49) with antibodies marked with a fourth color. Each of the four labels was presented separately using the cell analysis system and the images were used to calculate the amount of inhibition or activation of the translocation effected by the test compound. When doing this calculation, the images of the probes used to mark the plasma membrane and the cytoplasm were used to mask the image of ! if ____________? g ^ ^ | the immunological probe marking the location of the intracellular Rho protein. The integrated brightness per unit area under each mask was used to form a translocation quotient by dividing the brilliance / integrated area of the plasma membrane by the brightness / integrated cytoplasmic area. By comparing the values of the translocation quotient of the control and experimental wells, the translocation percent of each potential leader compound was calculated.

Translocation of β-arrestin to the plasma membrane in the activation of the G protein receptor In another cytoplasmic modality for the analysis of high content of membrane translocation, the translocation of the β-arrestin protein from the cytoplasm was measured to the plasma membrane in response to cellular treatment. To measure translocation, live indicator cells containing luminescent domain markers were treated with test compounds and movement of the β-arrestin marker was measured in time and space using the cell analysis system of the present invention. In a preferred embodiment, the indicator cells contain luminescent markers consisting of a protein chimeric, green fluorescent β-arrestin protein (GFP-β-arrestin) (Barak et al (1997), J. Biol. Chem. 272: 27497 -27500: Daaka et al. (1998), J. Biol. Chem. 273: 685-688) which is expressed by the indicator cells through the use of transient or stable cell transfection and other reporters used to mark the cytoplasmic domains and the membrane. When the indicator cells are in the resting state, the domain marker molecules are predominantly divided in the plasma membrane or in the cytoplasm. In the high content analysis, these markers are used to delineate the cell cytoplasm and the plasma membrane in different fluorescence channels.

When the indicator cells are treated with a test compound, the dynamic redistribution of GFP-β-arrestin is recorded as a series of images on a time scale ranging from 0.1 s to 10h. In a preferred embodiment, the time scale is 1 h. Each image is analyzed by a method that quantifies the movement of the protein GFP-β-arrestin chimera between the plasma membrane and the cytoplasm. To make this calculation, the images of the probes used to mark the plasma membrane and cytoplasm are used to mask the image of the GFP-β-arrestin probe by mapping the location of the intracellular GFP-β-arrestin protein. The integrated brightness per unit area under each mask is used to form a translocation ratio by dividing the brightness / integrated area of the plasma membrane by the brightness / integrated cytoplasmic area. By comparing the translocation quotient values of the control and experimental wells, the translocation percent is calculated for each potential leader compound. The capacity of the high content analysis is related to the quantitative data describing the magnitude of the translocation in a large number of individual cells that have been treated with the test compounds of interest.

Translocation between the endoplasmic reticulum and the Golgi apparatus: In a modality of an enodplasmic reticulum for the analysis of high translocation content of the Golgi apparatus, the translocation of a ts045 mutant filter of the vesicular stomatitis virus (Ellenberg et al., (1997), J. Cell. Biol. 138: 1193-1206; Presley et al. (1997) Nature 389: 81-85) from the endoplasmic reticulum to the domain of the Golgi apparatus in response to cell treatment. To measure the translocation, the reporter cells containing luminescent reporters were treated with test compounds and the movement of the reporters was measured in space and time using the cellular analysis system of the present invention. Indicator cells contain luminescent reporters consisting of a GFP-VSVG protein chimera that is expressed by the cell indicator through the use of stable or transient cell transfection and other domain markers used to measure the location of the endoplasmic reticulum and the device of Golgi. When the indicator cells are in their resting state at 40 ° C, the molecules of the GFP-VSVG protein chimera are predominantly divided in the endoplasmic reticulum. In this high-content analysis, domain markers of different colors are used to delineate the domain of the endoplasmic reticulum and the domain of the Golgi apparatus in different fluorescence channels. When the indicator cells are treated with a test compound and the temperature is simultaneously reduced to 32 ° C, the dynamic redistribution of the GFP-VSVG protein chimera is recorded as a series of images on a time scale ranging from 0.1 to 10 h . Each image is analyzed by a method that quantifies the movement of the protein chimera of GFP-VSVG between the domains of the endoplasmic reticulum and the Golgi apparatus. To make this calculation, the images of the probes used to mark the domains of the endoplasmic reticulum and the Golgi apparatus are used to mask the image of the GFP-VSVG probe that marks the location of the intracellular GFP-VSVG protein. The integrated brightness per unit area under each mask is used to form a translocation quotient by dividing the brightness / integrated area of the endoplasmic reticulum by the brightness / integrated area of the Golgi apparatus. By comparing the values of the translocation quotient of the control and experimental wells, the translocation percent of each potential leader compound is calculated. The capacity of the high content analysis is related to the quantitative data describing the magnitude of the translocation in a large number of individual cells that have been treated with the test compounds of interest in - • * _. & - > final concentrations ranging from 10"12 M to 10" 3 M during a period ranging from 1 minute to 10 hours.

Induction and inhibition of the organellar function: 5 Stability of the intracellular microtubule. In another aspect of the invention, an automated method is provided for the identification of compounds that modify the structure of the microtubule. In this embodiment, the indicator cells are treated with the test compounds and the distribution of the luminescent microtubule labeling molecules are measured in space and time using a cellular analysis system, such as that described above. The luminescent microtubule labeling molecules can be expressed by or added to the cells before, together with or after contacting the cells with a test compound. In one embodiment of this aspect of the invention, living cells express a luminescently labeled protein biosensor of microtubule dynamics, comprising a protein that labels the microtubules fused to a luminescent protein. Microtubule labeling proteins appropriate for this aspect of the invention include, but are not limited to, tubulin isoforms a and β and MAP4. Preferred embodiments of the luminescent protein include but are not limited to green fluorescent protein (GFP) and the GFP mutants. In a preferred embodiment, the method involves transfecting the cells with a luminescent microtubule labeling protein, wherein the microtubule labeling protein can be, but is not limited to a-tubulin, β-tubulin or protein 4 associated with the microtubule (MAP4). The approach established in this document enables those experts in the art make the measurements of the living cells to determine the effect of the leading compounds on tubulin activity and the stability of the microtubule in vivo. ^ g ^ gjgy In a preferred embodiment, MAP4 is fused to a modified version of the green fluorescent protein Aequorea victoria (GFP). A DNA construct consisting of a fusion between the EGFP coding sequence (available in Clontech) and the coding sequence for mouse MAP4 has been made. (Olson et al., (1995), J. 5 Cell. Biol. 130 (3): 639-650). MAP4 is a ubiquitous microtubule-associated protein that is known to interact with microtubules at the interface as well as mitotic cells (Olmsted and Murofushi, (1993), MAP4) in "Guidebook to the Cytoskeleton and Motor Proteins." Oxford University Press T. Kreis and R. Vale, Eds.). Its location can subsequently serve as an indicator of the location, organization and integrity of microtubules in living (or fixed) cells in all cell cycle states for cell-based HCS tests. Whereas MAP2 and tau (microtubule-associated proteins specifically expressed in neuronal cells) have been used to form GFP chimeras (Kaech et al., (1996) Neuron 17: 1189-1199; may et al., (1997 ), Proc. Nati. Acad. Sci. 94: 4733-4738) its restricted cellular type 5 distribution and the tendency of these proteins for the microtubule package when expressed above make these proteins less desirable as molecular reagents for the analysis in the living cells originated from various tissues and organs. Moderate overexpression of GFP-MAP4 does not interrupt microtubule function or integrity (Olson et al., 1995). Similar constructs can be made using β-0 tubulin or α-tubulin via standard techniques in the art. These chimeras will provide a means to observe and analyze microtubule activity in living cells during all cell cycle states. In another embodiment, the biosensor of the luminiscently labeled protein of the microtubule dynamics is expressed, isolated and added to the cells to be analyzed via the five loading techniques in volume, such as micro-injection, waste loading and mediated load of impacts. In this modality, there is no issue of surcharges "- * - - - * - - ^ - ^ _» d¿ ----... > ~~ £ ___--- ^ -_- -. ~ __. ... _. .___ ... sa? »Ag.á-i _-__. expression in the cell and therefore the isoforms of a and β-tubulin, MAP4, MAP2 and / or tau can all be used. In a further embodiment, the biosensor of the protein is expressed by the cell and the cell is subsequently contacted with a luminescent label, such as a labeled antibody, which detects the biosensor of the protein, the endogenous levels of a protein antigen or both. In this embodiment, a luminescent label that detects isoforms a and β-tubulin, MAP4, MAP2 and / or tau can be used. A variety of GFP mutants are available, all of which could be effective in this invention, including but not limited to GFP mutants that are commercially available (Clontech, California). The MAP4 construct has been introduced into several mammalian cell lines (BHK-21, Swiss 3T3, HeLa, HEK293, LLCPK) and the organization and location of tubulin has been visualized in living cells by virtue of the fluorescence GFP as a MAP4 location indicator. The construction can be expressed transiently or cell lines can be prepared by standard methods. Stable HeLa cell lines expressing the EGFP-MAP4 chimera have been obtained, indicating that the expression of the chimera is not toxic and does not interfere with mitosis. Possible selective markers for the establishment and maintenance of stable cell lines include but are not limited to I neomycin-resistant gene, zeocin-resistant gene 20, puromycin-resistant gene, bleomycin-resistant gene and gene resistant to blastacidin. The utility of this method for monitoring microtubule assembly, disassembly and rearrangement has been demonstrated by treating transfected cells stably and transiently with microtubule drugs such as paclitaxel, nocodazole, vincristine or vinblastine. and-ate, ------- _. . _ _ ^ __, - _ ^ - "- _ ........ ^^^ g .. ... ^ .. ^ ...

The present method provides high-content, high-content, high-content cell-based analyzes combined for anti-microtubule drugs, particularly as a parameter in a multi-parametric cancer target analysis. The EGFP-MAP4 construct used in this document can also be used as one of the components of a high content analysis that measures the multiple signaling pathways or physiological events. In a preferred embodiment, a high content and combined high performance analysis is employed, wherein multiple cells in each of the locations containing cells were analyzed in a high throughput mode and only a subset of locations containing cells were analyzed in a of content content. The high throughput analysis can be any analysis that could be useful to identify those locations that contain cells that should be further analyzed, including but not limited to the identification of locations with increased luminescence intensity, those exhibiting the expression of a reporter gene, those suffering calcium changes and those that suffer pH changes. In addition for drug analysis applications, the present invention can be applied to clinical diagnostics, the detection of biological warfare chemicals and weapons and the basic search market since they are fundamental cellular processes, such as cell division and mobility, they are highly dependent on the dynamics of the microtubule.

Analysis and Acquisition of Images Images can be obtained from live or fixed indicator cells. To extract morphometric data from each of the obtained images, the following analysis method was used: 1. The threshold of each cytoplasmic image and the nucleus to produce a mask that has a value of = 0 for a pixel outside the core or cell limit . ^ ._, z ~ .. ^ .. -. _-__ & m 2. The overlay of the mask in the original image, detects each object in the field (ie, the nucleus or cell), and calculates its integrated size, shape and intensity. 3. The superimposition of the complete cell mask obtained previously in the image of the corresponding luminescent microtubule and apply one or more of the following equipment of classifiers to determine the morphology of the microtubule and the effect of the drugs on the microtubule morphology. The morphology of the microtubule is defined using a set of classifiers to quantify the aspects or shape of the microtubule, size, state of aggregation and state of polymerization. These classifiers can be based on approximations that include co-occurrence matrices, texture measurements, spectral methods, structural methods, small wave conversions, statistical methods or combinations thereof. Examples of such classifiers are the following: 1. A classifier for quantifying microtubule length and extension using edge detection methods such as those described by Kolega et al. ((1993) Biolmaging 1: 136-150), which describes a non-automated method for edge resistance in individual cells) to calculate the total edge resistance in each cell. To normalize the size of the cell, the total resistance of the edge can be divided by the area of the cell to give a value of "morphology of the microtubulus". The higher values of the microtubule morphology are associated with the values of strong edge strength and therefore are maximum in cells that contain different microtubular structures. Similarly, small microtubule morphology values are associated with weak edge strength and are minimal in cells such as depolymerized microtubules. The physiological range of the values of the microtubule morphology is established by treating the cells with the drug paclitaxel (10 μM) that ^ ______ ______ E stabilizes microtubules or the drug nocodazole (10 μg / ml) that depolymerizes microtubules. 2. A classifier to quantify the aggregation of the microtubule in the accentuated spots or foci using the methodology of the methods of hospitalization of the receiver described above. 3. A classifier for quantifying the microtubule depolymerization using a measurement of the texture of the image. 4. A classifier to quantify the apparent interconnectivity or the branching (or both) of the microtubules. 5. Measurement of the kinetics of the microtubule rearrangement using the above classifiers in a time series of images of cells treated with the test compounds. In a further aspect, equipment for analyzing the stability of the microtubule is provided, comprising an expression vector comprising a nucleic acid encoding a microtubule labeling protein and instructions for using the expression vector to carry out the methods described above. In a preferred embodiment, the expression vector further comprises a nucleic acid encoding a luminescent protein, wherein the miRNAlobule binding protein and the luminescent protein thereof are expressed as a fusion protein. Alternatively, the The kit can contain an antibody that specifically binds to the microtubule labeling protein. In a further embodiment, the kit includes cells that express the microtubule labeling protein. In a preferred embodiment, the cells are transfected with the expression vector. In another preferred embodiment, the equipment also contains a compound that is known to interrupt the structure of the microtubule, including but not limited to cure, nocodazole, vincristine or vinblastine. In another preferred embodiment, the equipment further comprises a compound that is known to stabilize the microtubule structure, including but not limited to taxol (paclitaxel) and discodermolide. In another aspect, the present invention comprises a machine-readable storage medium comprising a program containing a set of instructions for causing a cellular analysis system to execute the methods described to analyze the stability of the microtubule, wherein the cellular analysis system it comprises an optical system with a state adapted to hold a plate containing cells, a digital camera, a means for directing the fluorescence or the luminescence emitted to the cells to the digital camera and a computerized means for receiving and processing the digital data of the digital camera.

High content analysis involving the functional localization of macromolecules Within this class of high content analysis, the functional localization of macromolecules in response to external stimuli within living cells is measured.

Regulation of the activity of glycolytic enzymes. In a preferred embodiment of a high content analysis of the cellular enzymatic activity, the activity of the key glycolytic regulatory enzymes in treated cells is measured. To measure the enzymatic activity, the indicator cells containing luminescent labeling reagents were treated with test compounds and the activity of the reporters was measured in space and time using the cellular analysis system of the present invention. In one embodiment, the reporter of the intracellular enzymatic activity is fructose-6-phosphate, 2-kinase / fructose-2,6-bisphosphatase (PFK-2), a regulatory enzyme whose phosphorylation status indicates the anabolism or catabolism of intracellular carbohydrates (Deprez et al (1997) J. Biol. Chem. 272: 17269-17275; Kealer et al. (1996) FEBS Letters 395: 225-227; Lee et al. (1996), Biochemistry 35: 6010-6019) . The indicator cells ^^^ H contain luminescent reporters consisting of a fluorescent protein biosensor of phosphorylation PFK-2. The fluorescent protein biosensor is constructed by introducing an environmentally sensitive fluorescent dye near the known phosphorylation site of the enzyme (Deprez et al. (1997), supra).; Giuliano et al. (1995), supra). The dye may be of the ketocyanin class (Kessier and Wolfbeis (1991), Spectrochimica Acta 47A: 187-192) or any class that contains a reactive portion with proteins and a fluorophore whose excitation or emission spectrum is polarity sensitive in solution. The fluorescent protein biosensor is introduced into the indicator cells using the volume loading methodology. The live indicator cells are treated with the test compounds, in final concentrations ranging from 10"12 M to 10 3 M for times ranging from 0.1 s to 10 h. In a preferred embodiment, the image data of the ratio is obtained of the living indicator cells treated by collecting a spectral pair of fluorescence images in each period of time To extract the morphometric data of each time period, a relationship between each pair of images is made by numerically dividing the two spectral images in each period of time, pixel by pixel.Each pixel value is subsequently used to calculate the fractional phosphorylation of PFK-2.A small fractional phosphorylation values, PFK-2 stimulates carbohydrate catabolism.A high fractional phosphorylation values , PFK-2 stimulates the anabolism of carbohydrates.

Protein kinase A activity and location of subunits. In another embodiment of a high content analysis, both the localization of the domain and the activity of the protein kinase A (PKA) in the indicator cells in response to treatment with test compounds were measured. i ^ a ^^^^ tl ^^^^^^^^^^ m? m ^ í átmmß J ^ & _ ..

Indicator cells contain luminescent reporters that include a fluorescent protein biosensor of PKA activation. The fluorescent protein biosensor is constructed by introducing an environmentally sensitive fluorescent dye into the catalytic subunit of PKA near the known site that interacts with the PKA regulatory subunit (Harootunian et al. (1993), Mol. Biol. Of the Cell 4 : 993-1002; Johnson et al. (1996), Cell 85: 149-158; Giuliano et al. (1995), supra). The dye may be of the ketocyanin class (Kessier, and Wolfbeis (1991), Spretrochimica Acta 47A: 187-192) or any class containing a reactive portion with proteins and a fluorophore whose excitation or emission spectrum is polarity sensitive in solution. The fluorescent protein biosensor of PKA activation is introduced into the indicator cells using the volume loading methodology. In one embodiment, living indicator cells are treated with test compounds, in final concentrations ranging from 10"12 M to 10" 3 M for times ranging from 0.1 s to 10 h. In a preferred embodiment, the image data of the ratio 15 are obtained from live indicator cells treated. To extract the biosensor data at each time period, a relationship is made between each pair of images, and each pixel value is subsequently used to calculate the fractional activation of PKA (ie, separation of the catalytic and regulatory subunits after the union of cAMP). At high fractional activity values, PFK-2 stimulates the biochemical cascades 20 in the living cell. To measure the translocation of the catalytic subunit of PKA, the reporter cells containing luminescent reporters are treated with test compounds and the movement of the reporters measured is measured in space and time using the cell analysis system. Indicator cells contain luminescent reporters that consist of domain markers used to measure the location of the cytoplasmic and nuclear domains. When the indicator cells are treated with a compound of - * «****» -? - - - -. ^, * .-, - .J jm - ^ -. . - ^ _ ", .. J _ ^^^^ _ a ^ .... ¿. . .-_ ^. ,, | ,. r. .. ^ _ fl £ j &jft | ¡g £ ^ test, the dynamic redistribution of a PKA fluorescent protein biosensor is recorded intracellularly with a series of images on an image scale ranging from 0.1 s to 10 h. Each image is analyzed by a method that quantifies the movement of PKA between the cytoplasmic and nuclear domains. To make this calculation, the images of the probes used to mark the cytoplasmic and nuclear domains are used to mask the fluorescent protein biosensor image of PKA. The integrated brightness per unit area under each mask is used to form a translocation quotient by dividing the brilliance / integrated cytoplasmic area by the brightness / integrated nuclear area. By comparing the values of the translocation quotient of the control and experimental wells, the translocation percent is calculated for each potential leader compound. The result of the high content analysis is related to quantitative data describing the magnitude of the translocation in a large number of individual cells that have been treated with test compounds in the concentration range of 10"12 M to 10" 3 M.

High content analysis involving the induction or inhibition of RNA-based fluorescent biosensing gene expression Cytoskeletal protein transcription and message localization. The regulation of the general classes of cellular physiological responses that include cell-substrate adhesion, cell-cell adhesion, signal transduction, cell cycle events, the metabolism of intermediary and signaling molecules, cellular locomotion, cell-cell communication and cell death may involve the alteration of gene expression. High content analyzes can also be designed to measure this kind of physiological response.

In one embodiment, the reporter of the expression of the intracellular gene is an oligonucleotide that can hybridize with the target mRNA and alter its fluorescence signal. In a preferred embodiment, the oligonucleotide is a molecular guide (Tyagi and Kramer (1996) Nat. Biotechnol 14: 303-308), a reagent based on luminescence 5 whose fluorescence signal is dependent on intermolecular or intramolecular interactions. The fluorescent biosensor is constructed by introducing a fluorescence energy transfer pair of fluorescent dyes as there is one at each end (5 'and 3') of the reagent. The dyes can be of any kind which contain a reactive portion with proteins and fluorophores whose excitation and emission spectra overlap sufficiently to provide fluorescence energy transfer between the dyes in the resting state, including but not limited to fluorescein and rhodamine (Molecular Probes, Inc.). In a preferred embodiment, a portion of the coding message for β-actin is inserted (Kislauskis et al (1994), J. Cell Biol. 127: 441-451; McCann et al (1997), Proc. Nati. Acad. Sci. 94: 5679-155884; Sutoh (1982), Biochemistry 21: 3654-3661) in the feedback region of a hairpin oligonucleotide with the ends threaded together due to intramolecular hybridization. At each end of the biosensor a fluorescence donor (fluorescein) and a fluorescence receptor (rhodamine) bind covalently. In the bound state, the fluorescence energy transfer is maximal and therefore indicative of a non-hybridized molecule. When it hybridizes with the mRNA encoding the β-acitine, the halter breaks and the energy transfer is lost. The complete fluorescent biosensor is introduced into the indicator cells using the volume loading methodology. In one embodiment, live indicator cells are treated with test compounds, at final concentrations ranging from 10"12 M to 10" 3 M for times ranging from 0.1 s to 10 h. In a preferred embodiment, the image data of the relationship - * í jU w ¿. R * ... ^. ^ _ s "-. ^ - ^: vjzfr & * ^ .. - .. ^ *, 9 + f * if ^ ^ nf ^ hf ^^ i are obtained from the live indicator cells treated. morphometrics of each time period, a relationship is made between each pair of images and each value of the pixel is subsequently used to calculate the fractional hybridization of the labeled nucleotide.An small fractional hybridization values, little ß-acythin expression is indicated. In addition, the distribution of the hybridized molecules in the cytoplasm of the indicator cells is also a measure of the physiological response of the indicator cells.

Linking the cell surface of a ligand Linking the labeled insulin to its cell surface receptor in living cells. Cells whose plasma membrane domain has been labeled with a particular color labeling reagent are incubated with a solution containing insulin molecules (Lee et al. (1997), Biochemistry 36: 2701-2708; Martinez-Zaguilan et al. al. (1996), Am. J. Physiol. 270: C1438-C1446) which are labeled with a luminescent probe of a different color for an appropriate time under the appropriate conditions. After incubation, the unbound insulin molecules are washed, the cells are fixed and the distribution and concentration of the insulin in the plasma membrane is measured. To do this, the image of the cell membrane is used as a mask for the insulin image. The integrated intensity of the masked insulin image is compared to a set of images containing known quantities of labeled insulin. The amount of insulin bound to the cell is determined by standards and is used in conjunction with the total concentration of insulin incubated with the cell to calculate a dissociation constant or insulin for its cell surface receptor.

Marking of cell compartments Marking of the complete cell The marking of the whole cell is carried out by marking the cellular components so that the dynamics of the cell shape and the mobility of the cell can be measured over time by analyzing the fluorescence images of the cells. cells In one embodiment, small reactive fluorescent molecules are introduced into living cells. The molecules that permeate the membrane diffuse and react with the protcomponents in the plasma membrane. The dye molecules react with intracellular molecules to increase the fluorescence signal emitted from each molecule and to trap the fluorescent dye in living cells. These molecules include reagents derived from chloromethyl, aminocumarins, hydroxycoumarins, eosin diacetate, fluorescdiacetate, some Bodipy dye derivatives, and tetramethylrhodamine. The reactivity of these dyes towards the macromolecules includes free primary amino groups and free sulfhydryl groups. In another embodiment, the cell surface is labeled allowing the cell to interact with fluorescently labeled antibodies or lecithins (Sigma Chemical Company, St. Louis, MO) that specifically react with molecules on the cell surface. Cell surface protchimeras expressed by the cell of interest that contains a green fluorescent protcomponent, or a mutant thereof, can also be used to fluorescently label the entire cell surface. Once the entire surface is marked, images of the entire cell or cell series can become a parameter in high-content analysis, involving the measurement of cell shape, mobility, size, growth and division. - "- - j" * lll, rt *, jM * Marking of the plasma membrane In one embodiment, the labeling of the entire plasma membrane employs some of the methodologies described above to label whole cells. The luminescent molecules that mark the entire cell surface act to delineate the plasma membrane. In a second embodiment, the subdomains of the plasma membrane, the extracellular surface, the lipid bilayer and the intracellular surface can be separately marked and used as high-content analysis components. In the first embodiment, the extracellular surface is labeled using a brief treatment with a reactive fluorescent molecule such as succinimidyl ester or iodoacetamide derivatives of fluorescent dyes such as fluoresc, rhodamines, harmful and Bodipys. In a third embodiment, the extracellular surface is labeled using macromolecules fluorescently labeled with a high affinity for the cell surface molecules. These include fluorescently-labeled lecithins such as fluoresc rhodamine and lecithin-derived yeast derivatives of bean (Con A), red bean (erythroagglutinin PHA-E) or wheat germ. In a fourth embodiment, fluorescently labeled antibodies with a high affinity for cell surface components are used to mark the extracellular region of the plasma membrane. The extracellular regions of cell surface receptors and ion channels are examples of prot that can be labeled with antibodies. In a fifth embodiment, the lipid bilayer of the plasma membrane is labeled with fluorescent molecules. These molecules include fluorescent dyes attached to hydrophobic long-chain molecules that strongly interact with the hydrophobic region at the center of the lipid bilayer of the plasma membrane. Examples of ^ ^ _-__., - - ,. .... __ _ .. ^., ^, .1, r ..- xsc ^ - ^. . .. .._, __ ^^ .. -Jte -Ó .-- »- * - these dyes include the PKH dye series (US 4,783,401, 4,762,701 and 4,659,584; commercially available from Sigma Chemical Company, St. Louis, MO), fluorescent phospholipids, such as nitrobenzoxadiazole glycerophosphoethanolamine and dihexadecanoylglycerophosphatha nolamine derived from fluorescein, fluorescent fatty acids such as 5-butyl-4,4-difluoro-4-bora-3a, 4a-diazo-s-indacen-3-nonanoic acid and 1-pirendecanoic acid (Molecular Probes, Inc.), sterols fluorescents including 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diazo-s-indacen-3-dodecanoate cholesterol and 1-pirenohexanoate cholesterol and fluorescently labeled proteins that specifically interact with components of lipid bilayer such as the annexin V fluorescein derivative (Caltag Antibody Co, Burlingame, CA). In another embodiment, the intracellular component of the plasma membrane is labeled with fluorescent molecules. Examples of these molecules are the intracellular components of the trimeric G protein receptor, adenylyl cyclase, and proteins that carry ions. These molecules can be labeled as a result of strong binding to a specific fluorescently labeled antibody or by the incorporation of a fluorescent protein chimera comprising a membrane-associated protein and green fluorescent protein and mutants thereof.

Endosome fluorescence labeling In one embodiment, ligands that are transported to cells by receptor-mediated endocytosis are used to map the dynamics of endosomal organelles. Examples of labeled ligands include low density lipoprotein complexes labeled with Bodipy FL, tetramethylrhodamine transferrin analogues and fluorescently labeled epidermal growth factor (Molecular Probes, Inc.). In a second embodiment, fluorescently labeled primary or secondary antibodies are used (Sigma Chemical Co. St. Louis MO; Molecular Probes Inc.

Eugene, OR; Caltag Antibody Co.) that specifically mark endosomal ligands that are used to mark the endosomal compartment in cells. In a third embodiment, the endosomes are fluorescently labeled in cells expressing protein chimeras formed by the fusion of a green fluorescent protein, mutants thereof, with a receptor whose entry marks the endosomes. EGF, transferin and low density lipoprotein receptor chimeras are examples of these molecules. Marking the lysosome In one embodiment, lysosome-specific luminescent reagents are used that permeate the membrane to mark the lysosomal compartment of living and fixed cells. These reagents include the luminescent molecules of neutral red, N- (3 - ((2,4-dinitrophenyl) amino) propyl) -N- (3-aminopropyl) methylamine and LysoTracker probes that report the intralysosomal pH as well as the dynamic distribution of the lysosomes (Molecular Probes, Inc.). In a second embodiment, antibodies against lysosomal antigens (Sigma Chemical Co., Molecular Probes, Inc., Caltag Antibody Co.) are used to label the lysosomal components that are located in specific lysosomal domains. Examples of these components are the degradative enzymes involved in the hydrolysis of cholesterol ester, proteases of the membrane protein and nucleases, as well as the lysosomal proton pump directed to ATP. In a third embodiment, protein chimeras are used which consist of a lysosomal protein genetically fused to an intrinsically luminescent protein such as the green fluorescent protein or mutants thereof, to mark the lysosomal domain. Examples of these components are the degradative enzymes involved in the hydrolysis of the cholesterol ester, the proteases of the membrane protein and nucleases, as well as the lysosomal proton pump directed at ATP. ^^^^^^ s ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ modality, the fluorescent dyes that permeate the cell (Molecular Probes, Inc.) with a reactive group are reacted with living cells. Reactive dyes including monobromobimene, 5-chloromethyl fluororescein diacetate, carboxy fluorescein diacetate succimidyl ester and chloromethyl tetramethylrhodamine are examples of fluorescent dyes that permeate the cell that are used for long-term cell cytoplasm labeling. In a second embodiment, polar marker molecules such as Lucifer's yellow and blue fluorescent dye cascade (Molecular Probes, Inc.) are introduced into the cells using the bulk loading methods and also used for cytoplasmic labeling. . In a third embodiment, antibodies against cytoplasmic components (Sigma Chemical Co., Molecular Probes, Inc., Caltag Antibody Co.) are used to fluorescently label the cytoplasm. Examples of cytoplasmic antigens are many of the enzymes involved in intermediary metabolism. Enolase, phosphofructokinase and acetyl-CoA dehydrogenase are examples of uniformly distributed cytoplasmic antigens. In a fourth embodiment, protein chimeras consisting of a cytoplasmic protein genetically fused to an intrinsically luminescent protein such as green fluorescent protein or mutants thereof are used to label the cytoplasm. Uniformly distributed fluorescent protein chimeras are used to label the entire cytoplasmic domain. Examples of these proteins are many of the proteins involved in intermediary metabolism and include enolase, lactate dehydrogenase and hexokinase. In a fifth embodiment, antibodies against cytoplasmic antigens are used (Sigma Chemical Co.; Molecular Probes, Inc .; Caltag Antibody Co.) to mark cytoplasmic components that are located in specific cytoplasmic sub-domains. Examples of these components are the cytoskeletal proteins actin, tubulin and cytokeratin. A population of these proteins in the cells is assembled in discrete structures, which in this case, are fibrous. The fluorescence labeling of these proteins with antibody-based reagents, therefore, marks a specific sub-domain of the cytoplasm. In a sixth embodiment, fluorescently labeled molecules not based on antibodies that strongly interact with cytoplasmic proteins are used to label specific cytoplasmic components. An example is a fluorescent analogue of the enzyme DNase I (Molecular Probes, Inc.). The fluorescent analogues of this invention bind strongly and specifically to cytoplasmic actin, thereby marking a sub-domain of the cytoplasm. In another example, fluorescent analogs of the phalloidin fungus toxin or the drug paclitaxel (Molecular Probes, Inc.) are used to label components of the actin and microtubule cytoskeletons, respectively. In a seventh embodiment, protein chimeras consisting of a cytoplasmic protein genetically fused to an intrinsically luminescent protein such as the green fluorescent protein, mutants thereof, are used to label specific domains of the cytoplasm. Fluorescent chimeras of highly localized proteins are used to mark cytoplasmic sub-domains. Examples of these proteins are many of the proteins involved in the regulation of the cytoskeleton. They include the structural proteins actin, tubulin and cytokeratin, as well as the microtubule of regulatory proteins associated with protein 4 and a-actinin.

Nuclear Marking In one embodiment, luminescent or specific nucleic acid reagents permeating the membrane (Molecular Probes, Inc.) are used to label the nucleus of living and fixed cells. These reagents include cyanine-based dyes (e.g., TOTO®, YOYO®, and BOBO ™), phenanthidines and acridines (ie, ethidium bromide, propidium iodide and acridine orange), Índoles and imidazoles (e.g. Hoechst 33258, Hoechst 33342, and 4 ', 6-diamidino-2-phenylindole) and other similar reagents (for example, 7-aminoactinomycin D, hydroxystilbamidine and psoralens). In a second embodiment, antibodies against nuclear antigens (Sigma Chemical Co., Molecular Probes, Inc., Caltag Antibody Co.) are used to label the nuclear components that are located in specific nuclear domains. Examples of these components are the macromolecules involved in maintaining the structure and function of DNA. DNA, RNA, histones, DNA polymerase, RNA polymerase, laminins and nuclear variants of cytoplasmic proteins such as actin are examples of nuclear antigens. In a third embodiment, protein chimeras are used which consist of a nuclear protein genetically fused to an intrinsically luminescent protein such as the green fluorescent protein or mutants thereof to mark the nuclear domain. Examples of these proteins are many of the proteins involved in maintaining the structure and function of DNA. Histones, DNA polymerase, RNA polymerase, laminins and nuclear variants of cytoplasmic proteins such as actin are examples of nuclear proteins.

Mitochondrial Marking In one modality, specific mitochondrial luminescent reagents that permeate the membrane (Molecular Probes, Inc.) are used to mark the mitochondria of cells ^^^^^^^ ~ alive and fixed. These reagents include rhodamine 123, tetramethyl rosamine, JC-1 and MitoTracker reagent dyes. In a second embodiment, antibodies against mitochondrial antigens (Sigma Chemical Co., Molecular Probes, Inc., Caltag Antibody Co.) are used to mark the mitochondrial components that are localized in specific mitochondrial domains. Examples of these components are the molecules involved in the maintenance of the structure and function of mitochondrial DNA. DNA, RNA, histones, DNA polymerase, RNA polymerase and mitochondrial variants of cytoplasmic molecules such as mitochondrial tRNA and rRNA are examples of antigens mitochondria. Other examples of mitochondrial antigens are the components of the oxidative phosphorylation system found in the mitochondria (eg, cytochrome c, cytochrome c oxidase and succinate dehydrogenase). In a third modality, protein chimeras are used that consist of a mitochondrial protein genetically fused to a protein intrinsically luminescent such as the green fluorescent protein, mutants thereof to mark the mitochondrial domain. Examples of these components are the macromolecules involved in maintaining the structure and function of mitochondrial DNA. Examples include histones, DNA polymerase, RNA polymerase and the components of the oxidative phosphorylation system found in the mitochondria (eg, cytochrome c, cytochrome c oxidase, and succinate dehydrogenase).

Marking the Endoplasmic Reticulum In one embodiment, luminescent reagents specific to the endoplasmic reticulum that permeate the membrane (Molecular Probes, Inc.) are used to mark the reticulum endoplasmic of living and fixed cells. These reagents include short-chain carbocyanine dyes (eg, DiOC6 and DiOC3), long-chain carbocyanin dyes (by example, DilC16 and DilC18) and luminescently labeled lecithins such as concanavalin A. In a second embodiment, antibodies are used against endoplasmic reticulum antigens (Sigma Chemical Co.; Molecular Probes, Inc .; Caltag Antibody Co.) 5 to mark the components of the reticulum that are located in specific endoplasmic reticulum domains. Examples of these components are the macromolecules involved in the prolongation system of the fatty acid, glucose-6-phosphatase and HMG CoA-reductase. In a third embodiment, protein chimeras are used which consist of an endoplasmic reticulum protein genetically fused to an intrinsically luminescent probe such as the green fluorescent protein or mutants thereof to mark the domain of the endoplasmic reticulum. Examples of these components are the macromolecules involved in the fatty acid, glucose-6-phosphatase and HMG CoA-reductase elongation systems. 15 Marking the Golgi Apparatus In one embodiment, luminescent reagents specific to the Golgi apparatus permeating the membrane (Molecular Probes, Inc.) are used to label the Golgi apparatus of living and fixed cells. These reagents include luminescently labeled macromolecules such as agglutinate the wheat germ and Brefeldia A as well as the luminescently marked ceramide. In a second embodiment, antibodies against antigens of the Golgi apparatus (Sigma Chemical Co, Molecular Probes Inc., Caltag Antibody Co.) are used to label components of the Golgi apparatus which are located in specific domains 25 of the Golgi Examples of these components are N-acetylglucosamine J? Ifí? F:? -? F- - - - - - - r -t II'I i -? ? »N ^ - ^^ 1 ^ l ^ t ^ e ^ ^ ^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Mannose-6-phosphate receptor protein. In a third embodiment, protein chimeras are used which consist of a protein of the Golgi apparatus genetically fused to an intrinsically luminescent protein such as the green fluorescent protein or mutants thereof to mark the domain of the Golgi apparatus. Examples of these components are N-acetylglucosamine phosphotransferase, phosphodiesterase specific to the Golgi apparatus and mannose-6-phosphate receptor protein. Although many of the examples presented involve the measurement of processes unique cell phones, it is intended to serve once again with illustrative only. High content analysis of multiple parameters can be produced by combining several single parameter analyzes in a high content multiparameter analysis or by adding cellular parameters for any existing high content analyzes. In addition, although each example was described as being based on cells live or fixed, each high content analysis can be designated for use with live or fixed cells. Those skilled in the art will recognize a wide variety of different analyzes that can be developed based on the description in this provided document. There is a large and growing list of molecular processes and known biochemicals in cells that involve translocations and rearrangements of specific components in cells. The signaling pathway from the cell surface to the target sites in the cell involves the translocation of proteins associated with the plasma membrane to the cytoplasm. For example, it is known that one of the protein tyrosine kinases of the src family, pp60c-src (Waiker et al (1993), J. Biol. Hem. 268: 19552-25 19558) is translocated from the plasma membrane to the cytoplasm after Stimulation of fibroblasts with platelet-derived growth factor (PDGF). Additionally, ^ ^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ to be converted into reagents based on fluorescence that report molecular changes including post-translocation modifications and ligand binding.

Claims (69)

1. An automated method for identifying compounds that modify the activation of the transcription factor comprising: a) providing a series of locations containing multiple cells to be treated with a compound test, wherein the cells possess a luminescently labeled transcription factor, wherein the luminescently labeled transcription factor translocates from the cytoplasm to the nucleus at activation and wherein the luminescently labeled transcription factor is present in the cells before, together with or after treatment with the test compound; b) contacting the cells with the test compound; c) scanning the multiple cells in each of the cell-containing locations to obtain luminescent signals of the luminescently labeled transcription factor in the subcellular compartments of the cells being analyzed, wherein the cellular compartments comprise the cell nucleus and the cell cytoplasm; d) convert the luminescent signals to digital data and e) use the digital data to make measurements automatically, where the measurements are used to automatically calculate the changes in the distribution of the luminescently labeled transcription factor in or between the cell nucleus and the cytoplasm Cellular cells being analyzed and where the change in the distribution correlates for the modification of the activity of the transcription factor induced by the test compound.

2. The method according to claim 1 further comprises scanning multiple cells from the locations containing cells in a high performance mode and selectively scanning only a subset of the locations containing cells in a high content mode to obtain luminescent signals of the luminescently labeled transcription factor in the subcellular compartments of the cells being analyzed.

3. The method according to claim 1, wherein the measurements comprise determining one or more of the following: a) a total or average luminescence intensity of the luminescently labeled transcription factor on or in the cell nucleus; b) a total or luminescence intensity average of the luminescently labeled transcription factor outside the cell nucleus, representing the cell cytoplasm; c) an area of the cell nucleus or d) an area of the cytoplasm.

4. The method according to claim 3, wherein the calculated changes comprise one or more of the following: a) changes in the total or average luminescence intensity of the luminescently labeled transcription factor on or in the cell nucleus of the cells being analyzed; b) changes in the total or average luminescence intensity of the luminescently labeled transcription factor in the cell cytoplasm of the cells being analyzed; ^ S ^^^^^ tíj ^^ íS ^ c) changes in the ratio of the total luminescence intensity or the average luminescence intensity of the cytoplasm to the total luminescence intensity or the average luminescence intensity on or in the nucleus or 5 d) changes in the difference of the total luminescence intensity or the average luminescence intensity of the cytoplasm and the total luminescence intensity or the average luminescence intensity on or in the nucleus.

5. The method according to claim 1, wherein the transcription factor of interest is luminescently labeled by contacting the cell with a luminescently labeled antibody or antibody fragment.

6. The method according to claim 1, wherein the transcription factor of interest is selected from the group consisting of c-fos and homologs fos and homologues c-jun, NF-KB, NFAT and STATs.

7. A kit for the identification of compounds that modify the activation of the transcription factor comprising: a) a primary antibody that binds specifically to a transcription factor of interest eb) instructions for using the primary antibody to identify compounds that modify the activation of the transcription factor in a cell of interest according to the method of claim 1. ". ^ Aat. _ ,. & ..-. riMuÉ-_í - f - i a ^^ s ^ faith ....._ -. ,

8. The equipment according to claim 7, wherein the primary antibody is luminescently labeled.

9. The kit according to claim 7, further comprises a secondary antibody that can detect the primary antibody.

10. The kit according to claim 9, wherein the secondary antibody is luminescently labeled.

11. The kit according to claim 7, further comprising at least one of the following: a) cells expressing the transcription factor of interest or b) a compound that is known to modify the activation of the transcription factor of interest

12. The kit according to claim 7, wherein the transcription factor is selected from the group of c-fos and fos homologs, c-jun and c-jun homologs, NF-KB, NFAT and STATs.

13. A kit for the identification of compounds that modify the activation of the transcription factor comprising: a) an expression vector comprising a nucleic acid encoding a transcription factor of interest that translocates from the cytoplasm to the nucleus in the activation and -Jaaaa ^ a - J - - - ^ --- - - ~ - - - - - ^ _ - ^ a, -! -, ..-- ^ ». .. . _- »^ _ ^ - ^^ ¿s? T, b) instructions for using the expression vector to identify compounds that modify the activation of the transcription factor in a cell of interest, in accordance with the method of claim 1 .

14. The kit according to claim 13, wherein the expression vector comprises a nucleic acid encoding a luminescent protein, wherein the luminescent protein and the transcription factor of interest are expressed as a fusion protein.

15. The kit according to claim 13, further comprising at least one of the following: a) an antibody or fragment thereof that specifically binds to the transcription factor of interest; b) cells that are transfected with the expression vector or c) at least one compound that is known to modify the activation of the transcription factor of interest.

16. The kit according to claim 13, wherein the transcription factor is selected from the group consisting of c-fos and c-fos homologs, c-jun and c-jun homologs, NF-KB, NFAT and STATs.

17. A kit for identifying compounds that modify the activation of the transcription factor comprising: a) an isolated protein comprising a luminescently labeled transcription factor eb) instructions for using the luminescently labeled transcription factor to identify compounds that modify the activation of the transcription factor transcription in a cell of interest, according to the method of claim 1.

18. The kit according to claim 17, further comprising at least one compound that is known to modify the activation of the transcription factor of interest.

19. The kit according to claim 17, wherein the transcription factor is selected from the group consisting of c-fos and fos homologs, c-jun and c-jun homologs, NF-KB, NFAT and STATs.

20. An automated method for identifying compounds that modify protein kinase activation comprising: a) providing a series of locations containing multiple cells to be treated with a test compound, wherein the cells possess a luminescently labeled protein kinase or protein substrate kinase, wherein the luminescently labeled protein kinase or protein kinase substrate translocates from the cytoplasm to the nucleus in protein kinase activation and wherein the luminescently labeled protein kinase or protein kinase substrate is present in the cells, before, together with or after treatment with the test compound; b) contacting the cells with the test compound; c) scanning the multiple cells in each of the cell-containing locations to obtain luminescent signals of the luminescently labeled protein kinase or the protein kinase substrate in the subcellular compartments of the cells being analyzed, wherein the subcellular compartments comprise the cell nucleus and the cell cytoplasm; d) convert the luminescent signals into digital data and e) use the digital data to make measurements automatically, wherein the measurements are used to calculate the changes automatically in the distribution of the luminescently labeled kinase protein or the protein kinase substrate in or between the cell nucleus and cell cytoplasm of the cells being analyzed and where the change in distribution correlates to the modification of protein kinase activity by the test compound.

21. The method according to claim 20, further comprising scanning multiple cells in each of the locations containing cells in a high throughput mode and scanning selectively from only a subset of locations containing cells in a high-end mode. content to obtain luminescent signals of the luminescently labeled protein kinase or protein kinase substrate in the subcellular compartments of the cells being analyzed.

22. The method according to claim 20, wherein the measurements comprise determining one or more of the following: - ^ F ^ sm ^ -í a) a total or luminescence intensity average of the luminescently labeled protein kinase or the substrate of the kinase protein on or in the cell nucleus; b) a total or luminescence intensity average of the luminescently labeled protein kinase or the protein kinase substrate outside the cell nucleus, representing the cell cytoplasm; c) an area of the cell nucleus or d) an area of the cytoplasm.

23. The method according to claim 22, wherein the calculated changes comprise one or more of the following. a) changes in the total or average luminescence intensity of the luminescently labeled kinase protein or the protein kinase substrate on or in the cell nucleus of the cells being analyzed; b) changes in the total or average luminescence intensity of the luminescently labeled kinase protein or the protein kinase substrate in the cell cytoplasm of the cells being analyzed; c) changes in the ratio of the total luminescence intensity or the average luminescence intensity of the cytoplasm to the total luminescence intensity or the average luminescence intensity on or in the nucleus or d) changes in the difference of the total luminescence intensity or from the average luminescence intensity of the cytoplasm to the total luminescence intensity or the average luminescence intensity on or in the nucleus.

24. The method according to claim 20, wherein the protein kinase or protein kinase substrate of interest is luminescently labeled by contacting the cell with a luminescently labeled antibody or antibody fragment. 25. The method according to claim 20, wherein the protein kinase of interest is selected from the group consisting of kinase proteins regulated by extracellular signals (ERKs), amino-terminal kinase c-Jun (JNKs), regulatory kinase proteins. Fos (FRKs), activated p38 mitogen protein kinase (p38MAPK), protein kinase

10 A (PKA) and protein kinase of activated mitogen cinases (MAPKKs).

26. A kit for identifying compounds that modify the kinase protein activation comprising: a) a primary antibody that specifically binds to a protein kinase or a protein kinase substrate of interest eb) instructions for using the antibody to identify compounds that modify the activation of protein kinase in a cell of interest, according to the method of claim 20.

27. The kit according to claim 26, wherein the primary antibody is luminescently labeled.

28. The kit according to claim 26, further comprising a secondary antibody that can detect the primary antibody. 25 J --fÉf_f¡lr -_-_-_-------- Í ----_

29. The kit according to claim 28, wherein the secondary antibody is luminescently labeled.

30. The kit according to claim 26, further comprising at least one of the following: a) cells expressing the protein kinase or the protein kinase substrate of interest or b) a compound that is known to modify the activation of the protein kinase of interest.

31. The kit according to claim 26, wherein the protein kinase is selected from the group consisting of protein kinases regulated by extracellular signals (ERKs), amino-terminal kinase c-Jun (JNKs), protein kinases of Fos regulation (FRKs) , p38 activated mitogen protein kinase (p38MAPK); protein kinase A (PKA) and protein kinase of activated mitogen kinases (MAPKKs).

32. A kit for the identification of compounds that modify protein kinase activation comprising: a) an expression vector comprising a nucleic acid encoding a protein kinase or a protein kinase substrate of interest that translocates from the cytoplasm to the nucleus in activation eb) instructions for using the expression vector to identify compounds that modify the activation of the protein kinase in a cell of interest, according to the method of claim 20. - * teA ~ - - .. »-. * -_-_-.-

33. The kit according to claim 32, wherein the expression vector further comprises a nucleic acid encoding a luminescent protein, wherein the Luminescent protein and protein kinase or protein kinase substrate of interest are expressed as a fusion protein.

34. The kit according to claim 32, further comprising at least one of the following: a) cells that are transfected with the expression vector; b) an antibody or fragment thereof that specifically binds to the protein kinase or protein kinase substrate of interest or c) a compound that is known to modify the activation of the protein kinase of interest.

35. The kit according to claim 32, wherein the protein kinase is selected from the group consisting of protein kinases regulated by extracellular signals (ERKs), amino-terminal kinase c-Jun (JNKs), protein kinases of Fos regulation (FRKs) , p38 activated mitogen protein kinase (p38MAPK); protein kinase A (PKA) and protein kinase of activated mitogen kinases (MAPKKs).

36. A kit for the identification of compounds that modify the activation of the kinase protein comprising: c) an isolated protein comprising a luminescently labeled protein kinase or the luminescently labeled protein kinase substrate and - ^ ^. _, _ ^ _. ^. _____ _____; _ d) instructions for using the luminescently labeled protein kinase or the luminescently labeled protein kinase substrate, in accordance with the method of claim 20.

37. The kit according to claim 36, further comprising a compound that is known to modify the activation of the protein kinase.

38. The kit according to claim 36, wherein the protein kinase is selected from the group consisting of protein kinases regulated by extracellular signals (ERKs), amino-terminal kinase c-Jun (JNKs), protein phosphorylating kinases (FRKs) , p38 activated mitogen protein kinase (p38MAPK); protein kinase A (PKA) and protein kinase of activated mitogen kinases (MAPKKs).

39. A machine-readable storage medium comprising a program containing a set of instructions for causing a cellular analysis system for executing the method of claim 1, wherein the cellular analysis system comprises an optical system with a state adapted to sustain a plate containing cells, a digital camera, a means for directing the fluorescence or luminescence emitted from the cells to the digital camera and a computational means for receiving and processing the digital data of the digital camera.

40. A machine-readable storage medium comprising a program containing a set of instructions for causing a cellular analysis system for executing the method of claim 20, wherein the cellular analysis system comprises an optical system with a state adapted to sustain a plate containing cells, a digital camera, a means for directing the fluorescence or luminescence emitted from the cells to the digital camera and a computational means for receiving and processing the digital data of the digital camera.

41. An automated method for analyzing compounds that modify cell morphology comprising: a) providing a series of locations containing multiple cells that are contacted with a test compound, wherein the cells possess one or more luminescent reporter molecules and wherein one or more than the luminescent reporter molecules can be expressed by the cells or added to the cells before, together with or after the cells are contacted with the test compound, b) contacting the cells with the test compound; c) scanning multiple cells in each of the cell-containing locations to obtain luminescent signals from the luminescent reporter molecules in the cellular compartments of the cells, wherein the subcellular compartments comprise the cell nucleus, the cell cytoplasm, the cell membrane and the cell membrane; cellular cytoskeleton; d) convert the luminescent signals into digital data and e) use the digital data to make measurements automatically, where the measurements are used to calculate the changes automatically in the distribution of the luminescent reporter molecules in or between the cellular compartments and where the changes calculated correlate with changes in cell morphology induced by the test compound.

42. The method according to claim 41, which comprises scanning the multiple cells in each of the locations containing cells in a high performance mode and selectively scanning only a subset of the locations containing cells in a high content mode to obtain luminescent signs of the 5 reporter molecules luminescently labeled in subcellular compartments of the cells being analyzed.

43. The method according to claim 41, further comprising at least one of the following: 10 a. acquire an image of the cell nucleus; b. acquire an image of the cellular cytoplasm; c. acquire an image of the cell membrane or d. acquire an image of the cellular cytoskeleton.

44. The method according to claim 43, wherein the measurements comprise one or more of the following: a. an aggregate of the entire area of the nucleus; b. a complete area of the average core; c. a total cytoplasmic area, 20 d. a cytoplasmic intensity of the aggregate; and. one cytoplasmic area per cell nucleus; F. a cytoplasmic intensity per cell nucleus; g. an average intensity of the cellular cytoplasm; h. a proportion of the cell nucleus - cellular cytoplasm; 25 i. a measurement of an actin cytoskeleton structure or j. an average cell area. -S * - * - - - ... .. . _ att Efflnfe? »?

45. The method according to claim 44, wherein the calculated changes comprise one or more of the following: a. changes in the entire area of the aggregate core; b. changes in the entire area of the average core; c. changes in the total cytoplasmic area; d. changes in the cytoplasmic intensity of the aggregate; and. changes in the cytoplasmic area of the cell nucleus, f. changes in cytoplasmic intensity per cell nucleus; g. changes in the intensity of the average cell cytoplasm; h. changes in the cell nucleus-cell cytoplasm ratio; i. changes in the measurement of the actin cytoskeleton structure or j. changes in the average cell area.

46. The method according to claim 41, wherein the sub-regions of the series of locations containing multiple cells are sampled multiple times at intervals to provide the kinetic measurement of changes in cell morphology.

47. The method according to claim 41, wherein the cells possess a luminescent reporter molecule selected from the group consisting of cytosketal markers, cytosolic volume markers and cell surface markers.

48. The method according to claim 47, wherein the luminescent reporter molecule selectively detects the actin filaments. ^ j ^^^ á a _. ^ ..-. ^ -..- A. ~ ~ 6a * tB & ~ ....

49. A team to identify the stimulus that modifies the cell morphology, which includes: a. a luminescent compound that specifically marks the cell membrane, the cytoplasm or the cytoskeleton and 5 b. instructions for using the luminescent compound to identify test compounds that modify cell morphology in accordance with the method of claim 41.

50. The equipment according to claim 49, further comprising at least one of the following: a) a luminescent compound that specifically marks the cell nucleus or b) at least one compound that is known to modify cell morphology.

51. A machine-readable storage medium comprising a set of instructions for causing a cellular analysis system for executing the method of claim 41, wherein the cellular analysis system comprises an optical system with a state adopted to sustain a plate containing cells, a digital camera, 20 a means for directing the fluorescence or luminescence emitted from the cells to the digital camera and a computational means for receiving and progressing the digital data of the digital camera.

52. An automated method for identifying compounds that modify the microtubule structure comprising: a) providing a series of locations containing multiple cells to be treated with a test compound, wherein the cells possess a luminiscently labeled microtubule labeling molecule which is expressed by or added to the cells before, together with or after contacting the cells with the test compound; b) contacting the cells with the test compound; c) scanning the multiple cells in each of the cell-containing locations to obtain luminescent signals from the luminiscently labeled microtubule signaling molecule in the cellular cytoplasm or the cellular cytoskeleton; d) convert the luminescent signals into digital data and e) use the digital data to make measurements automatically, where the measurements are used to calculate the changes automatically in the distribution of the luminiscently labeled microtubule labeling molecule in the cellular cytoplasm or the cytoskeleton and where the calculated changes in the distribution of the luminiscently labeled microtubule labeling molecule in the cellular cytoplasm or the cellular cytoskeleton correlates with changes in microtubule structure induced by the test compound.

53. The method according to claim 52, further comprising scanning multiple cells in each of the locations containing cells in a high throughput mode and scanning selectively from only a subset of the locations containing cells in a single mode. high content to obtain luminescent signals of the microtubule signaling molecule luminescently labeled in the subcellular compartments of the cells being analyzed.

54. The method according to claim 52, wherein the luminiscently labeled microtubule labeling molecule is selected from the group consisting of the α and β sofmas of tubulin, MAP4 and antibodies that specifically recognize a protein selected from the group consisting of isoforms a ß tubulin, 5 MAP 4, MAP 2 and tau.

55. The method according to claim 52, wherein the measurements comprise determining at least one of the following: g. edge resistance; 10 h. cellular area; i. cell size; j. cellular form; k. integrated luminescence intensity and I. cell microtubule morphology; 15 m. dot distribution of the cytoskeleton of the cellular mi- crotubule; n. interconnection or branching of the cellular microtubule cytoskeleton; or. length and amplitude of the microtubule; p. microtubule aggregation; q. Microtubule depolymerization or 20 r. microtubule reorganization.

56. The method according to claim 55, wherein the calculated changes comprise the determination of at least one of the following: a. changes in edge strength; 25 b. changes in the cell area; c. changes in cell size; ^ ** g ^ d. changes in cellular form; and. changes in integrated luminescence intensity; F. changes in cell microtubule morphology; g. changes in the distribution by points of the cellular microtubule cytoskeleton; h. changes in the interconnection or branching of the cellular microtubule cytoskeleton; i. changes in the length and amplitude of the microtubule; j. changes in microtubule aggregation; k. changes in microtubule depolymerization or I. changes in microtubule rearrangement.

57. The method according to claim 52, wherein the sub-regions of the series of locations containing multiple cells are sampled multiple times at intervals to provide kinetic measurements of the changes in the structure of the microtubule.

58. A computer for the automated analysis of the microtubule structure comprising: a. an expression vector comprising a nucleic acid encoding a microtubule binding protein e b. instructions for using the expression vector to carry out the method of claim 52.

59. The kit according to claim 58, wherein the expression vector comprises a nucleic acid encoding a luminescent protein, wherein the microtubule binding protein and the luminescent protein thereof are expressed as a fusion protein.

60. The kit according to claim 58, further comprising at least one of the following: a) cells that are transfected with the expression vector or b) a compound that is known to modify the structure of the microtubule.

61. A computer for the automated analysis of the microtubule structure comprising: a. a primary antibody that binds specifically to a microtubule labeling molecule of interest e b. instructions for using the expression vector to carry out the method according to claim 52.

62. The kit according to claim 61, wherein the primary antibody is luminescently labeled.

63. The equipment according to claim 61, further comprising a secondary antibody that can detect the primary antibody.

64. The kit according to claim 63, wherein the secondary antibody is luminescently labeled.

65. The equipment according to claim 61, further comprising at least one of the following: a) cells expressing the microtubule labeling molecule of interest or b) a compound that is known to modify the structure of the microtubule.

66. The kit according to claim 61, wherein the microtubule labeling molecule is selected from the group consisting of the isoforms a and β of tubulin, MAP 4, MAP 2 and tau.

67. A computer for the automated analysis of the microtubule structure comprising: a. an isolated protein comprising a luminescently labeled microtubule molecule e b. instructions for using the luminescently labeled microtubule labeling molecule to carry out the method of claim 52.

68. The kit according to claim 67, wherein the labeling molecule of the luminescently labeled microtubule is selected from the group consisting of the isoforms a and β of tubulin, MAP 4, MAP 2 and tau.

69. A machine readable storage medium comprising a program containing a set of instructions for causing a cellular analysis system for executing the method according to claim 52, wherein the cellular analysis system comprises an optical system with an adapted state to hold a plate containing multiple cells, a digital camera, a means to direct the fluorescence or luminescence emitted from the cells to the digital camera and a computerized means to receive and process the digital data of the digital camera. ^^^ ¿gg ^