IN Cell Investigator high-content analysis software v1.3

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1 GE Healthcare Data file AA IN Cell Investigator IN Cell Investigator high-content analysis software v1.3 Flexible software for automated analysis of high-content cellular images IN Cell Investigator software v1.3 offers a flexible and comprehensive solution for automated high-content analysis of live and fixed cell assays. The software combines Spotfire DecisionSite, an interactive visual data analysis tool, with the latest versions of the award winning IN Cell Developer Toolbox and the IN Cell Analysis Modules. Automated cell imaging devices like the IN Cell Analyzer 1000 and IN Cell Analyzer 3000 generate high volumes of data, especially where complex subcellular assays are used in drug discovery and cellular biology research. Decisions and understanding depend on efficient analysis and data interpretation. This is why IN Cell Investigator software was designed by biologists for biologists, offering a choice of tools to address the breadth of analysis activities necessary for subcellular research and screening. Simply make your analysis selection and start work. IN Cell Investigator software consists of three major components. IN Cell Investigator Analysis Modules v3.5 provide a comprehensive range of preconfigured and fully validated image analysis routines Together, these components allow the user to: Rely on validated analysis protocols with modules for more than 50 applications Develop protocols for unique assays without specialist scripting knowledge Measure cellular processes such as cell cycle, receptor internalization, and micronuclei formation Track cell movements and monitor parameter changes in individual cells over time Choose segmentation methods for stained and unstained samples Sort, filter, and visualize result data using an intuitive and interactive interface Make decisions from complex multiparametric data using Spotfire DecisionSite guided visual analysis tools In addition, IN Cell Investigator can be used to analyze data files from other imagers with the optional IN Cell Translator software. IN Cell Investigator Developer Toolbox v1.7 enables users to construct their own image analysis protocols for specialized applications Spotfire DecisionSite Basic v8.2 allows for interactive visual analysis of results Fig 1. Screenshots showing the transfer of results from IN Cell Investigator Developer Toolbox to Spotfire DecisionSite for visualization.

2 IN Cell Investigator Analysis Modules v3.5 The current range of ten IN Cell Investigator Analysis Modules is fully validated for over 50 applications and enables the user to obtain reliable results without the need to create and validate protocols. This version includes a dedicated analysis module for the micronuclei genotoxicity assay and flexible classification tools for subpopulation analysis. MTA provides true high-content data by analyzing highly multiplexed assays with up to seven fluorescent markers and up to seven different object types, reporting more than 10 intensity and morphology parameters for each type. Nuclear Trafficking: Calculates a nuclear to cytoplasmic fluorescence intensity ratio for many assays Granularity: Quantitates images with respect to granule count, area, and intensity in relation to size scales Morphology: Measures the morphology and the fluorescence intensity of user-defined nuclear and cytoplasmic compartments Cell Cycle Trafficking: Assigns cells into one of three phase categories: G2, M, or G1/S Object Analysis: Quantitates changes in intensity of fluorescent labels. It applies to a wide range of cellular assays in both end-point and kinetic formats Dual Area Object Analysis: Allows simultaneous quantitation of subcellular inclusions such as grains, vesicles, and fibers (e.g., for apoptosis analysis) Neurite Outgrowth: Performs proprietary segmentation of images to quantitate neurite and cell morphological features Membrane Trafficking: Allows quantitation of subcellular inclusions and can be tailored to a variety of different applications (e.g., translocation of proteins to and from the plasma membrane) Micronuclei Formation: Automated analysis of micronucleus assays allows significantly faster analysis and consistently objective scoring of this important component of toxicology evaluation of new drug candidates Fig 2. Decision tree classification of a G1S cell cycle assay. At each node of the decision tree a different filter divides the input population into two subpopulations. By connecting an output population to another filter node it can be further classified, resulting here in four subpopulations S, G2, G1, and M. The plate map shows the percentage of G1 cells in each well. Cells can be classified into subpopulations by applying one or more filters, each of which is based on one or two userselectable fluorescence or morphology measures. Thresholds and discrimination functions are set using interactive graphs, where each data point is linked to the corresponding cell image. Complex classification schemes can be designed by combining multiple filters to build a decision tree. Using morphology and fluorescence parameters, decision trees can report multiple subpopulations or phenotypes with their accompanying measures, thereby allowing subpopulation analysis. Multi Target Analysis: The most versatile of the modules allowing analysis of up to seven object types and flexible classification The Multi Target Analysis (MTA) module is designed for the analysis of cell-based assays where multiparametric classification of cells into subpopulations is required including cell viability, cell cycle monitoring, apoptosis, and cell signaling. Additionally, it can be applied to almost any assay that examines cellular processes. Fig 3. Classification of live and dead cells in a viability assay. The integrated nuclear intensity of calcein-am (green channel; x-axis) and propidium iodide (red channel; y-axis) were visualized for individual objects using a 2-D scatter plot to assign live (green), dead (red) and unclassified (blue) subpopulations. Clicking on a data point (arrow) highlights the cell in the corresponding image. 2 06/ AA

3 IN Cell Investigator Developer Toolbox v1.7 IN Cell Investigator Developer Toolbox is designed for specialized high-content analysis applications where predeveloped image analysis modules are not suitable. Designed for biologists, the controlled, fully supported environment helps users build tailored custom routines, enabling them to quickly analyze and interpret results of complex and unique assays. A selection of advanced segmentation, preprocessing, and post-processing tools provides full control over each step of the analysis sequence. The Developer software can be used in the following ways: Construction of simple, user-defined analysis protocols Incorporation of more functionality through the addition of a macro into a protocol As an imaging tool without analysis The software provides access to a vast array of user-definable parameters, calculations, measures covering all aspects of cellular assays, and a large choice of tools for image pre- and postprocessing, segmentation, and classification of cells. The cell tracking feature allows tracking of cell movements over time. IN Cell Developer Toolbox highlights include: Proximity Cell Tracking: To analyze live cells over time Classifiers: To enable the same flexible classification schemes as in the Multi Target Analysis module Complex Wavelet Transformation Denoising: An efficient noise removal that preserves object edges Fig 4. Screenshot of Developer Toolbox showing a Context Module for myotube formation analysis. Left pane: editable protocol, middle pane: HTML interface, right pane: analyzed image. Cell tracking Tracking the movement and behavior of living cells is an indispensable technique for a wide variety of functional studies. The new cell tracking feature complements the IN Cell Analyzer 1000 Environmental Control Module, enabling automated quantitation of cell position, speed, and direction as a function of time. In addition, daughter cells resulting from cell divisions can be identified and tracked. Changes in individual cell parameters such as size, shape, and intensity can be monitored to provide a deeper understanding of the time-dependent behavior of every cell in a population. Results can then be visualized and analyzed using a dedicated Spotfire DecisionSite cell tracking guide. Pseudofluoresce: A preprocess to enhance brightfield images for segmentation with standard methods Context Modules: HTML-wrapped protocols with a selfexplanatory interface for specific applications. They can be used as a starting point for customized analysis Together with the existing features of Developer, these image analysis options can generate a wealth of multiparametric phenotypic data to provide deep insight into cellular function on many levels. Fig 5. Cell tracking in IN Cell Investigator 1.3. The interface allows adjusting a range of parameters. The performance of cell tracking will depend on cell motility, cell density, magnification, and sampling rate. 06/ AA 3

4 Spotfire DecisionSite Basic 8.2 Spotfire DecisionSite is a powerful data analysis package that enables: Rapid interactive visual data analysis Filtering and sorting of high-content data Easy access to the most informative visualizations through guided analysis This industry-standard application allows you to explore indepth changes to cellular phenotypes in response to cellular stimuli and perturbation. Plate result data from the IN Cell Investigator Analysis Modules and IN Cell Investigator Developer Toolbox are transferred to Spotfire DecisionSite software via a dedicated interface. This interface provides a direct link between cellular data points in Spotfire DecisionSite and the corresponding image objects. Dedicated Spotfire guides for IN Cell Investigator enable you to analyze results with just a few mouse clicks. Guided analysis is available for standard views like plate heat-maps and more complex tasks like EC 50 /IC 50 calculation or temporal analysis of cell movements. The guides provide instructions and hyperlinks to the most relevant data views to enable fast decision making. Fig 7. Spotfire DecisionSite screenshot with IN Cell guide pane showing a plate heat-map, IC 50 plot, DNA content histograms and a scatter plot with live and dead cell populations (clockwise from lower left). Supported data input formats IN Cell Analyzer 1000 (*.dce, *.xdce) IN Cell Analyzer 3000 (*.run) Generic stacks of TIFF images Cellomics (dib) (1) BD (MAC/TIFF) (1) Molecular Devices (STK) (1) MIAS (TXT/TIFF) (1) (1) Requires IN Cell Translator software. A B Supported data export formats Data can be exported from IN Cell Investigator software through a number of options, defined when you construct the analysis protocol. Data export options available include: Fig 6. (A) Mouse L929 cells stained with the nuclear dye Hoechst were imaged on IN Cell Analyzer 1000 every 15 min over 22 h. (B) Individual cell tracks for 30 consecutive time-points are graphically presented in X-Y coordinates in Spotfire DecisionSite using the cell tracking guide. Spotfire DecisionSite software enables: Faster insight: Easy to use, interactive, visual analytics speed time to insights Confident decisions: Interactive analysis results enable confident, collaborative decision-making IN Cell Investigator software complements the IN Cell Analyzer 1000 and IN Cell Analyzer 3000 by providing the key components of an efficient high-content analysis workflow. Microsoft Excel (*.xls) Formatted text (space delimited ) (*.prn) Text (tab delimited ) (*.txt) CSV ( *.CSV) Wks (1-2-3) (*.wks) XML Installation requirements Windows XP Professional SP 2 or higher PC-compatible computer with a Pentium IV microprocessor (or higher, Xenon multi-core processor is recommended) High-resolution graphics display (at least 1024 x 768 x 24 bits); 2 monitors recommended Minimum RAM size of 1 GB for analysis (2 GB for single core, 3 GB for dual core, and 4 GB for 4 cores is recommended) The paging file should match RAM size Minimum of 10 GB free disk space on the installation HD drive Microsoft Internet Explorer 6.0 Internet-connectivity preferred (required for license activation of Spotfire) 4 06/ AA

5 New features in version 1.3 With IN Cell Investigator software v1.3, users benefit from: New cell tracking functionality in the Developer Toolbox and Multi Target Analysis Module allow measuring of cell movements and monitoring parameter changes in individual cells over time New interactive graphs and decision trees in Developer Toolbox and the Analysis Modules make it easy to classify cells according to phenotype for cell cycle analysis and other multiplexed assays New multiprocessor support to further increase computing speed on multi-core and multi-cpu computers Improved access to the most informative Spotfire visualizations through dedicated Spotfire guides for IN Cell Investigator New Cell Viability Context Module provides an expandable protocol for multiplexed cell viability assays New Spotfire DecisionSite Guides give easy access to the most relevant visual data analysis tools New Segmentation Parameters have been added to Intensity, Vesicle, Nuclear, and Cytoplasm segmentation. They allow users to optimize speed and performance of their protocols Improved ability to analyze transmitted light image stacks as Pseudo Fluorescence preprocessing parameters can now be adjusted Improved Memory Management ensures robust performance with very large stacks Dev 1.6 Dev 1.7 0% 20% 40% 60% 80% 100% 120% Relative processing time Fig 8. Performance gains on multi-core CPU. This graph shows the time required to analyze an image-stack with the Developer Toolbox on a PC Workstation with two Intel Xeon DualCore processors. The new version of Developer Toolbox requires 60% less time to process an image stack with 96 dual-channel images because it can process multiple images in parallel. A single-user license enables the use of up to four processing cores. Table 1. Validated applications using IN Cell Investigator Cellular processes Signaling pathways Cell cycle Organelle and trafficking Neuronal function Cell morphology and health Genotoxicity Receptor activation GPCR internalization CypHer 5E Transfluor Labeled ligand internalization Calcium flux Receptor-mediated PLC translocation Cell membrane receptor binding Nuclear hormone receptors Toxicity and apoptosis Chromatin condensation Cell proliferation Nuclear count Cytochrome C release and mitochondrial integrity Annexin V binding Viability/cell count Cell rounding GFP translocation assays Transcription factor activation and deactivation Phospholipid sensors Plasma membrane sensors Endosomal sensors Cell survival signaling Cell migration signaling Reporter gene expression Protein expression Stress response signaling Cell cycle status reporting DNA replication studies Cell Proliferation Multiplexed cell cycle analysis Cell cycle based on DNA content Nucleo-cytoplasmic translocation Endosomal trafficking Plasma membrane translocation Membrane ruffling Golgi integrity Mitochondrial translocation Neurite extension analysis Cell rounding Measurement of discrete structures at cell surface Transient transfections Subpopulation analysis Cytoskeletal reorganization Cell viability Micronuclei formation 06/ AA 5

6 Ordering information Product Configuration Code number IN Cell Investigator software single seat license IN Cell Investigator software 1 additional seat license IN Cell Investigator software 5 seat licenses One machine license for the Investigator Suite One additional machine license (requires valid Investigator license) concurrent licenses for Investigator Analysis Modules and Developer and 5 machine licenses for Spotfire DecisionSite IN Cell Investigator SMC Software maintenance contract per license and year IN Cell Translator software Enables the use of third party files GE, imagination at work, and GE monogram are trademarks of General Electric Company. CypHer is a trademark of GE Healthcare companies. The IN Cell Analyzer 1000 is the subject of US patent application number 10/514925, together with other pending family members, in the name of GE Healthcare Niagara, Inc. The IN Cell Analyzer 1000 and associated analysis modules are sold under license from Cellomics Inc. under US patent numbers US , , , , , , , , , , , , , , , ; Canadian patent numbers CA , , , ; Australian patent number AU ; European patent numbers EP , , , , , ; Japanese patent numbers JP , and other pending and foreign patent applications. Any use of IN Cell Investigator software is subject to GE Healthcare Standard Software End-User License Agreement for Life Sciences Software Products. A copy of this Standard Software End-User License Agreement is available on request. All third party trademarks are the property of their respective owners General Electric Company All rights reserved. First published June 2007 GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden GE Healthcare UK Ltd Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Europe GmbH Munzinger Strasse 5, D Freiburg Germany GE Healthcare Bio-Sciences KK Sanken Bldg , Hyakunincho Shinjuku-ku, Tokyo Japan All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. For contact information for your local office, please visit: GE Healthcare Bio-sciences Corp 800 Centennial Avenue P.O. Box 1327 Piscataway, NJ USA AA