Analysis of the dynamics of drug candidates’ action on cellular processes yields important mechanistic information.  This data can be efficiently obtained using sensitive and specific fluorescence applications combined with real time (confocal) microscopy.  Single living (or fixed) cells are interrogated with batteries of assays developed to detect changes in the intracellular concentration and distribution of second messengers and other cellular regulatory molecules over time and space. High Content Analysis (HCA) and High Content Screening (HCS) are terms that describe such single cell imaging approach as it applies to current drug discovery.

In the Center, automated fluorescence microscopy for HCA and HCS is performed on a BD™ Pathway Bioimager (BD Bioscience, Rockville, MD).   This system is equipped with two mercury lamps as light sources, 16 excitation and 5 emission filters, high sensitivity/high resolution Hamamatsu Orca ER CCD camera, spinning disk-based confocal module, temperature and CO2 control, LED for transmitted light observation, and an on-stage fluidic head for automated compound addition using disposable tips compatible with 96 well plates. The system can perform a wide variety of end-point, time lapse, and real-time kinetic single cell imaging (wide-field or confocal) assays on cells grown on coverslips, chamber slides, 96, and 384 well plates. In addition, fluorescent arrays of cells or tissues on standard microscope slides can be imaged. The instrument’s highly reproducible x-y-z mechanical resolution (100 nm in x-y and 50 nm in z) allows the objective to accurately reposition on previously imaged fields for interleaving time lapse experiments. Furthermore, because of the ultra-precise x-y-z movement, large montages of slides, cell layers, and small organisms can be created. The software (AttoVision®) directs the instrument to perform automated autofocus routines, cell segmentation (Regions of Interest, ROIs), image capture, drug-addition, intensity and ratio calculations, and real-time data display. Numbered ROIs in the images and corresponding traces are color-coded and interactive; thus, “clicking” on a trace highlights the cell of origin and vice versa, allowing the identification of individual cell responses within a heterogeneous population. A hierarchical Data Classification structure is used to bin experimental data into user-defined classes to generate useful summaries, which can be further imported in BD™ Image Data Explorer (IDE), a Microsoft® Excel®- based program that performs single cell-based data analysis and generates reports and charts (e.g., dose-response curves; EC50, Z’ and S/N calculations; histograms; scatterplots; etc.). 

High Content Single Cell Screening Assays