Compartmental Analysis utilizes the first fluorescence channel to define a primary object, which can be a particular cellular region or organelle labeled with a fluorescent marker. The primary object is a major constituent of the cell (e.g. nucleus, cytoplasm, or large organelles), and is used to first identify individual cells and then to define the different sub-cellular regions for each cell. In each of the target channels (Channels 2-6), this primary object is used to define four different regions of the cell (called Circ, Ring, Ring Spots and Circ Spots - see schematic in Figure 2) in which measurements are made. Each of these four regions in the cell is independently and separately configurable in each of the five dependent channels for maximum flexibility.

The features measured by Compartmental Analysis include intensities in these regions as well as related features such as intensity ratios, differences, spot numbers,and spot areas. Morphological and intensity properties of the primary object are also reported. The colocalization of targets in different compartments are quantitatively determined by intensity ratios. In addition, status information on whether individual cells are within a defined subpopulation is also reported for certain measured features and is used to automatically characterize and classify cells based on their response compared to a control population (defined manually or by Reference Wells). This feature is illustrated in the example shown in Figure 3, where a time-course of exposure of cells to fluorescently labeled EGF was performed. The flexibility of this BioApplication allows many different types of measurements to be made in each cell. Furthermore, the BioApplication reports events as a percentage of the population responding to treatment, allowing a different view of the cellular events in addition to well-level mean responses.

Detailed cell-level analysis is also possible with the Compartmental Analysis BioApplication. In this example, Swiss 3T3 cells were stained for two transcription factors, NFkB and CREB. The population distributions of cells treated with various compounds is shown in Figure 4. Images were collected in three channels by the ArrayScan HCS Reader, and activation and translocation of these proteins was measured as the ratio of intensities between the nucleus and cytoplasm. Compartmental Analysis can measure translocation of up to five such targets at the single-cell level. Cell-level data were exported through an interface in vHCS™:View to Spotfire® Decision Site® for multi-dimensional visualization. Treatment of cells with TNF-a causes strong activation of the NFkB pathway (Y-axis) and slight activation of the CREB pathway (X-axis). Forskolin, however, causes activation of the CREB pathway but no activation of NFkB. Furthermore, there is a slight narrowing of the distribution of the NFkB activation, revealing subtle effects of the drug that would otherwise be obscured by looking at the well-level data only. Thus, the Compartmental Analysis BioApplication enables drug discovery researchers to understand at a deeper level their compounds’ effects on cells.

Features

• Measures and reports features in up to six different channels (which can represent different fluorophores or exposure conditions)
  
• Reports the intensity in four regions of the cell in each channel for each cell, including sub-cellular regions defined by masks and discrete punctate intracellular objects
  
• Reports ratios and differences in intensity between particular subcellular regions within a channel, as well as ratios in intensity for subcellular regions across channels
  
• Automatically characterizes and classifies the cell population according to individual cell responses
  
• Provides reference wells and gating to allow analysis of subpopulations
  

Benefits

• Maximum flexibility to allow scientists to design and optimize their own quantitative multiparameter assays
  
• Enables analysis of multiple processes that take place within or between different sub-cellular compartments
  
• May be applied to many different biological situations
  
• Allows scientists to do exploratory assay feasibility and development
  

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