1. Background
The field of “cellomics” emerged from the early success of genomics and pro-teomics and the term has been used interchangeably with “functional genomics” and more recently with “functional proteomics”. Our definition of cellomics, as a field, is broader: “the study of the temporal and spatial activity of cells and cellular constituents that are responsible for cell functions”.
High Content Screening (HCS) is a set of methods and tools that were originally created, developed, commercialized, and defined by Cellomics, Inc. in 1997 (Giuliano et al., 1997; Giuliano et al., 2003) as a platform technology for the emerging field of cellomics. In today’s language, this would be called a “systems cell biology” platform. The original definition of HCS was “an automated method for analyzing arrays of cells that contain one or more fluorescent reporter molecules, where the fluorescent signals are converted into digital data and then the digital data are used to automatically make measurements of intensity and/or distribution of the fluorescent signals on or in the cells, where changes indicate a change in distribution, environment, or activity of the fluorescent reporter molecules”. Variations in the original definition of HCS have evolved over the last few years as more scientists have used the technology in drug discovery and basic biomedical research. The most general definition offered to date has been “the use of biological probes in combination with imaging technologies to probe within individual cells and to screen for activity on a target at a subcellular level (Screening Review, 2004)”.
HCS was conceived as a high-throughput, cell biology platform aimed at “industrializing” cell biology. The concept was analogous to the creation of automated DNA sequencing. The developers of the automated DNA sequencers that eventually allowed the human genome project to be successful in a reasonable amount of time and at a reasonable cost, did not develop all the technologies from scratch. They integrated previously developed methods of fluorescent dye tagging of nucleotides, running gels containing DNA fragments, reading the “ladders” in the gels and incorporating the sequence data into searchable databases. The major step from a technical and an intellectual property perspective was the integration and automation of the whole set of processes into one turnkey method. The output of the process of automated DNA sequencing was not a “ladder” read on the gel, but a DNA sequence that could be fed into a DNA search engine to find new genes.
The development of HCS followed the same path as automated DNA sequencing. Cellomics, Inc. successfully integrated and automated multiple, previously single technologies including fluorescence microscopy; imaging science; fluorescence-based reagents (Taylor, 1992; Taylor et al., 2001); cell assays; and searchable databases. The result, HCS, formed a platform technology that was positioned to industrialize the field of cellomics.
2. Present applications
To date, a wide range of cellular processes have been explored with HCS. The first applications were measuring the activation of transcription factors (e.g., NF-kB) by quantifying the translocation from the cytoplasm into the nucleus (Ding et al., 1998) and quantifying the internalization of receptors into vesicles (Conway et al., 1999). Subsequently, screens for a wide range of transcription factors and receptors have been performed. An increasing number of cellular processes ranging from apoptosis, cell migration, cell cycle, G-protein coupled receptor activation (Walker et al., 1999), microtubule stability (Giuliano, 2003), and cytotoxicity (Abraham et al., 2004) have been implemented. It is believed that any cellular constituent including “biosensor” proteins and organelles can be specifically labeled and quantitative measurements can form the basis of an HCS screen (Giuliano and Taylor, 1998; Meyer and Teruel, 2003).
An ability to take more than one measurement from a cell has allowed the so-called multiplexing. For example, while the function (e.g., translocation) of the target protein in question can be monitored using one fluorescence channel (e.g., using green fluorescent protein tags (see Article 50, Using photoactivatable GFPs to study protein dynamics and function, Volume 5), or staining with antibodies coupled to fluoresein (see Article 58, Immunofluorescent labeling and fluorescent dyes, Volume 5)), other parameters of the cell can be simultaneously measured in another fluorescence channel, such as nuclear volume by staining with Hoechst dyes. This increases the information content of the assay.
3. Future developments
HCS has been accepted as an important approach to drug discovery by the pharmaceutical industry and it is now being incorporated into large-scale screens in academic research. The first generation platforms will now evolve as the market demands more from the platform. There will be advances in the individual component technologies that make up HCS, as well as in the integrated solutions. The optical and mechanical systems will become more sophisticated with the inclusion of modules for: reading microarrays of cells on chips; spectral deconvolution in order to analyze greater than four fluorescent reporters in the same assay; large area detection to increase the throughput; and additional fluorescence spectroscopic measurements to broaden the molecular information gained. Imaging science will be harnessed to perform faster and more powerful pattern recognition analyses that will increase the robustness of the measurements. Training sets of data will allow the end user to better define the positive and negative attributes that the systems will be programmed to detect and to measure. In addition, the increased speed will permit the measurement of all possible morphometric and fluorescence parameters that will help define the optimal parameters for making decisions.
More specific, as well as multiplexed fluorescence-based reagents will be created; especially for live-cell screens. Multiplexing reagents with distinct spectral distributions, fluorescence lifetimes, and fluorescence anisotropy will be used in combination with reagents that can “modulate” cell constituent functions such as RNA-based reagents that can “knock down” coding and even noncoding genes (see Article 60, siRNA approaches in cell biology, Volume 5), as well as “chemical switches” that can regulate specific gene expression. Individual, as well as multiple cellular pathways will be interrogated with these advanced reagents opening up more complicated assays involving multiplexed measurements in four dimensions (x,y ,z space and time). The creation of even more data per assay/screen (many terabytes) with these new tools will drive the development and implementation of more advanced informatics and cellular bioinformatics. The process of rapidly flowing through the production of data, the extraction of information and finally, the creation of knowledge will become faster and more powerful.
HCS promises to have the same impact on the field of cellomics, as automated DNA sequencing had on the field of genomics.
