High-content screening

High Content screening is an automated cell biology method drawing on optics, chemistry, biology and image analysis to permit rapid, highly parallel biological research and drug discovery.

High Content screening&mdash;a definition
High-content screening is a powerful new drug discovery method that uses living cells as the test tube for molecule discovery. "High-content screening" describes the use of spatially or temporally resolved methods to discover more in an individual experiment than one single experimental value. It is the combination of modern cell biology, with all its molecular tools, with automated high resolution microscopy and robotic handling. It differs from most life science work because experiment evaluation is entirely automated.

High Content screening is also known as chemical biology, cell based screening, chemical genetics, phenotypic screening, visual screening, and it has a major place in pharmaceutical company drug discovery. All these terms refer to the systematic search for new drugs, small molecule inhibitors or chemical entities that could have use in biology or medicine. While it combines technologies that have been used individually, the scale, speed and automation of HCS make it more than the sum of it parts.

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 priori knowledge of the biochemical targets that are affected by compounds and while this may be a benefit for compound discovery, the biochemical target itself must be subsequently identified. 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 (Burdine and Kodadek, 2004). Target identification has been defined as the rate limiting step in chemical genetics/high-content screening (Eggert and Mitchison, 2006).



High-content screening- seeing into the future of drug discovery
In high-content screening, automated microscopes take images of cellular models where the proteins of interest are detected because they bear a fluorescent tag, such as the green fluorescent protein, or where they can be identified by fluorescent antibodies. Image analysis is then used to measure changes in properties of the cells caused by external treatment such as chemical inhibitors or RNA interference- for example if the cells division is slowed or entry of a protein into the cell is arrested.

High-content screening is an extension of the more classical High-throughput screening that has been used in the pharmaceutical industry since the early 1990's. Microscope images- almost exclusively from fluorescent cells- are rich in spatial and quantitative information, that is extracted by computer aided image analysis. This data is used to determine whether a potential drug affects aspects of cell function involved in or that describe a disease. For example, 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.

The impact of high-content screening
High-content screening technology allows for the evaluation of multiple biochemical and morphological parameters in cellular systems. Through combining the imaging of cells in microtiter plates with powerful image analysis algorithms, pharmaceutical and biotech companies can acquire deeper knowledge on multiple biochemical or morphological pathways at the single-cell level at an early stage in the development new drugs.

The utility of automated cell biology requires an examination of how automation and objective measurement can improve the experimentation and the understanding of disease. First, it removes the influence of the investigator in most, but not all, aspects of cell biology research and second it makes entirely new approaches to possible.

In review, classical 20th century cell biology used cell lines grown in culture where the experiments were measured using very similar to that described here, but there the investigator made the choice on what was measured and how. In the early 1990’s, the development of CCD cameras (charge coupled device cameras) for research created the opportunity to measure features in pictures of cells- such as how much protein is in the nucleus, how much is outside. Sophisticated measurements soon followed using new fluorescent molecules, like FURA-2 to measure calcium, or elegant fluorescent molecules for measuring the pH of internal cell compartments. The wide use of the green fluorescent protein, a natural fluorescent protein molecule from jellyfish, then accelerated the trend toward cell imaging as a mainstream technology in cell biology. Despite these advances, the choice of which cell to image and which data to present and how to analyse it was still selected by the investigator.

By analogy, if one imagines a football field and dinner plates laid across it, instead of looking at all of them, the investigator would choose a handful near the score line and had to leave the rest. In this analogy the field is a tissue culture dish, the plates the cells growing on it. While this was a reasonable and pragmatic approach automation of the whole process and the analysis makes possible the analysis of the whole population of living cells, so the whole football field can be measured.

From one to many&mdash;what this offers for research
This technology allows a (very) large number of experiments to be performed, dramatically more than were possible. What is this good for? As described, its current use is in chemical genetics where large, diverse small molecule collections are systematically tested for their effect on cellular model systems. Novel drugs can be found using screens of tens of thousands of molecules, and these have promise for the future of drug development. Beyond drug discovery, chemical genetics is aimed at functionalizing the genome by identifying small molecules that acts on most of the 21’000 gene products in a cell. High-content technology will be part of this effort which could provide useful tools for learning where and when proteins act by knocking them out chemically. This would be most useful for gene where knock out mice (missing one or several genes) can not be made because the protein is required for development, growth or otherwise lethal when it is not there. Chemical knock out could address how and where these genes work. The large datasets produced by automated cell biology contain spatially resolved, quantitative data which can be used for building for systems level models and simulations of how cells and organisms function. Systems biology models of cell function would permit prediction of why, where and how the cell responds to external changes, growth and disease.

High-content screening technology
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.

Technology providers
The instruments on the market can be divided on the basis of price, footprint and the ethereal design qualities of the box they come in - but the most incisive difference is whether the instruments are optical confocal or not. Confocal imaging summarizes as imaging/resolving a thin slice through an object and rejecting out of focus light that comes from outside this slide. This gives higher image signal to noise and higher resolution than the more commonly applied epi-fluorescence microscopy. For many biological assays, confocal imaging is not ideal (e.g. phototoxicity issues or the need for a larger focal depth etc). What all instruments share is the ability to take, store and interpret images automatically and most integrate into large robotic cell/medium handling platforms.

Alternatively more generic microscope instrumentation such as the Cell Observer by Carl Zeiss can be used for high content screening. While such instruments are less specialized they can be more appealing to academic settings, where tasks and experiments change more rapidly than in industrial research.
 * The current confocal platforms are the point scanning 4 color ImageXpress ULTRA (Molecular Devices, Union City, USA), the spinning disk (nipkow disk) Pathway 855 and 435 from BD Biosciences (formerly Atto Biosciences, Rockville, Maryland), OPERA (Evotec Technologies, Hamburg, Germany) and the slit scanning Incell 3000 (GE/Amersham Biosciences, Cardiff, UK).
 * The current widefield platforms are: Arrayscan (Cellomics), Incell 1000 (GE/Amersham Biosciences, Cardiff, UK), Scanalyzer (Scanalyzer LemnaTec, Aachen Germany) and "ImageXpress MICRO" (Molecular Devices, Union City, USA). Several instruments are ready for live cell temperature controlled imaging - such as the "Pathway HT" (Becton Dickinson Biosciences) and the "ImageXpress MICRO" (Molecular Devices, Union City, USA), "Scan^R" (Olympus Soft Imaging Solutions, Germany), which may be of use to biologists interested in following events over relatively long time courses in vivo.

Dedicated software for image analysis is available from these vendors or from specialised firms such as DCILabs (Belgium) and Definiens (Germany), as well as from some groups which provide open-source software for image analysis, such as BioImageXD, CellProfiler, and ImageJ.

Kits for high-content screening of various target proteins (e.g. p53, c-jun and NFkB) have recently become available from commercial suppliers.

Where we are
The technology can be defined as being at the same development point as the first automated DNA sequencers in the early 1990’s. Automated DNA sequencing was a disruptive technology when it became practical and -even if early devices had shortcomings- it enabled genome scale sequencing projects and created the field of bioinformatics. The impact of a similarly disruptive and powerful technology on molecular cell biology and translational research is hard to predict but what is clear is that it will cause a profound change in the way cell biologists research and medicines are discovered.

Timeline of the evolution of the science and technology

 * (5/96) Introduction of the BDI High Content Screening System ArrayScan system introduces high throughput cell-based screening
 * (10/96) Founding of Cellomics
 * (1999) Introduction of the Cellomics ArrayScan II
 * (date) Introduction of the Universal Imaging Discovery-1
 * (date) Introduction of the Incell 3000
 * (date) Introduction of the Evotec Opera
 * (date) Introduction of the InCell 1000
 * (date) Introduction of Transfluor assay
 * (date) Introduction of the Atto Pathway HT
 * (date) Inroduction of Axon Instruments ImageXpress 5000
 * (1999) Introduction of Scanalyzer Platform Scanalyzer
 * (5/05) Introduction of the Molecular Devices ImageXpress Micro
 * (1/06) Introduction of the Molecular Devices ImageXpress Ultra
 * (3/06) Introduction of the Olympus scan^R
 * 1) 48h life cell imaging on cell arrays - Life cell imaging with 1536 experiments in parallel.
 * 2) Cell detecting autofocus system with memory function

Please contribute with dates and key assay and image analysis technologies...