High-content screening

High-content screening (HCS), also known as high-content analysis (HCA) or cellomics, is a method that is used in biological research and drug discovery to identify substances such as small molecules, peptides, or RNAi that alter the phenotype of a cell in a desired manner. Hence high content screening is a type of phenotypic screen conducted in cells involving the analysis of whole cells or components of cells with simultaneous readout of several parameters. HCS is related to high-throughput screening (HTS), in which thousands of compounds are tested in parallel for their activity in one or more biological assays, but involves assays of more complex cellular phenotypes as outputs. Phenotypic changes may include increases or decreases in the production of cellular products such as proteins and/or changes in the morphology (visual appearance) of the cell. Hence HCA typically involves automated microscopy and image analysis. Unlike high-content analysis, high-content screening implies a level of throughput which is why the term 'screening' differentiates HCS from HCA, which may be high in content but low in throughput. High-content screening (HCS), also known as high-content analysis (HCA) or cellomics, is a method that is used in biological research and drug discovery to identify substances such as small molecules, peptides, or RNAi that alter the phenotype of a cell in a desired manner. Hence high content screening is a type of phenotypic screen conducted in cells involving the analysis of whole cells or components of cells with simultaneous readout of several parameters. HCS is related to high-throughput screening (HTS), in which thousands of compounds are tested in parallel for their activity in one or more biological assays, but involves assays of more complex cellular phenotypes as outputs. Phenotypic changes may include increases or decreases in the production of cellular products such as proteins and/or changes in the morphology (visual appearance) of the cell. Hence HCA typically involves automated microscopy and image analysis. Unlike high-content analysis, high-content screening implies a level of throughput which is why the term 'screening' differentiates HCS from HCA, which may be high in content but low in throughput. In high content screening, cells are first incubated with the substance and after a period of time, structures and molecular components of the cells are analyzed. The most common analysis involves labeling proteins with fluorescent tags, and finally changes in cell phenotype are measured using automated image analysis. Through the use of fluorescent tags with different absorption and emission maxima, it is possible to measure several different cell components in parallel. Furthermore, the imaging is able to detect changes at a subcellular level (e.g., cytoplasm vs. nucleus vs. other organelles). Therefore a large number of data points can be collected per cell. In addition to fluorescent labeling, various label free assays have been used in high content screening. High-content screening (HCS) in cell-based systems uses living cells as tools in biological research to elucidate the workings of normal and diseased cells. HCS is also used to discover and optimize new drug candidates. High content screening is a combination of modern cell biology, with all its molecular tools, with automated high resolution microscopy and robotic handling. Cells are first exposed to chemicals or RNAi reagents. Changes in cell morphology are then detected using image analysis. Changes in the amounts of proteins synthesized by cells are measured using a variety of techniques such as the green fluorescent proteins fused to endogenous proteins, or by fluorescent antibodies. The technology may be used to determine whether a potential drug is disease modifying. For example, in humans G-protein coupled receptors (GPCRs) are a large family of around 880 cell surface proteins that transduce extra-cellular changes in the environment into a cell response, like triggering an increase in blood pressure because of the release of a regulatory hormone into the blood stream. Activation of these GPCRs can involve their entry into cells and when this can be visualised it can be the basis of a systematic analysis of receptor function through chemical genetics, systematic genome wide screening or physiological manipulation. At a cellular level, parallel acquisition of data on different cell properties, for example activity of signal transduction cascades and cytoskeleton integrity is the main advantage of this method in comparison to the faster but less detailed high throughput screening. While HCS is slower, the wealth of acquired data allows a more profound understanding of drug effects. Automated image based screening permits the identification of small compounds altering cellular phenotypes and is of interest for the discovery of new pharmaceuticals and new cell biological tools for modifying cell function. The selection of molecules based on a cellular phenotype does not require a prior knowledge of the biochemical targets that are affected by compounds. However the identification of the biological target will make subsequent preclinical optimization and clinical development of the compound hit significantly easier. Given the increase in the use of phenotypic/visual screening as a cell biological tool, methods are required that permit systematic biochemical target identification if these molecules are to be of broad use. Target identification has been defined as the rate limiting step in chemical genetics/high-content screening. High-content screening technology is mainly based on automated digital microscopy and flow cytometry, in combination with IT-systems for the analysis and storage of the data.“High-content” or visual biology technology has two purposes, first to acquire spatially or temporally resolved information on an event and second to automatically quantify it. Spatially resolved instruments are typically automated microscopes, and temporal resolution still requires some form of fluorescence measurement in most cases.This means that a lot of HCS instruments are (fluorescence) microscopes that are connected to some form of image analysis package. These take care of all the steps in taking fluorescent images of cells and provide rapid, automated and unbiased assessment of experiments. HCS instruments on the market today can be separated based on an array of specifications that significantly influence the instruments versatility and overall cost. These include speed, a live cell chamber that includes temperature and CO2 control (some also have humidity control for longer term live cell imaging), a built in pipettor or injector for fast kinetic assays, and additional imaging modes such as confocal, bright field, phase contrast and FRET. One of the most incisive difference is whether the instruments are optical confocal or not. Confocal microscopy summarizes as imaging/resolving a thin slice through an object and rejecting out of focus light that comes from outside this slice. Confocal imaging enables higher image signal to noise and higher resolution than the more commonly applied epi-fluorescence microscopy. Depending on the instrument confocality is achieved via laser scanning, a single spinning disk with pinholes or slits, a dual spinning disk, or a virtual slit. There are trade offs of sensitivity, resolution, speed, photo-toxicity, photo-bleaching, instrument complexity, and price between these various confocal techniques.