by Caitlin Smith
High-content analysis (HCA) is the automated extraction and analysis of cellular images taken during high-resolution light microscopy. Add to HCA today’s more advanced optical probes and complex bioinformatics, and you have a powerful tool for the bioresearcher asking complex questions. HCA can yield information about cell morphology, subcellular localization, intracellular signaling and more. It can be performed on cultured cells, tissue sections or even 3D cultures. It is being used in a wide range of applications, such as drug screening, multiparametric functional studies, chemical genomics, target validation and toxicity testing. “The power of HCA as an enabling technology for basic research and drug discovery stems from its ability to automatically perform and analyze large numbers of complex experiments, and to replace human bias in image interpretation by numerical representation of cellular behavior,” says Jacob Tesdorpf, director of high-content instruments and applications at PerkinElmer. “This enables academic researchers to survey genome-wide interaction networks by RNAi knockdown, to screen for rare events or to effectively tease out even subtle changes between cell populations.”
Because HCA is performed on whole cells, and researchers can use multiple probes in the same assay, they can gain exceptional understanding of complex biological mechanisms in a surprisingly short time—for example, in minutes instead of hours or days. This also can accelerate research by helping researchers reach decision points faster—for example, if HCA tells you sooner that you should do X instead of Y, then you can begin preparing for the change in workflow earlier. “In drug discovery, productivity advantages make HCA a valuable technology for target identification, for example through the use of RNAi screens, whilst the detailed information helps give greater confidence in both hit selection and safety and efficacy performance, prior to lead candidate selection,” says Leighton Howells, program manager for cellular imaging at GE Healthcare Life Sciences. “In research, the insights and cellular context of HCA [are] proving highly valuable in . . . complex disease research in areas such as cancer, neurology, immunology and infectious diseases.”
Several considerations bear mentioning, though. In HCA, each captured image may contain hundreds of cells (each of which may yield numerous data points); in one day, a researcher could obtain tens of thousands of images. Storing the images and data analysis is a burgeoning issue for many HCA researchers. Thus, before purchasing an HCA instrument, it is important to consider your ability to manage large amounts of data, the data analysis and management tools included in the instrument’s software and whether those tools are compatible with other software tools in your lab (if instrument interfacing is relevant for you). Another point to consider is whether you need a system designed especially for speed, a system geared more toward high-quality images (at the cost of some speed)—or a system in between. There is no best choice, only what is right for your research environment and your type of experiments.
Going for speed
If speed is paramount in your research, and you need quick answers with high-quality imaging,
Molecular Devices recently released the ImageXpress® Micro XL Wide Field High Content Screening System, which is designed to increase throughput. It uses state-of-the-art detector and illumination technologies to capture cellular resolution images in a single field encompassing one 384-well plate. “This maximizes content up to three-fold of what is acquired with standard HCS [high-content screening] systems,” says Grischa Chandy, bioimaging product manager at Molecular Devices. “Assay window and reliability are enhanced with 3-log dynamic range and <5% CV for intensities across the plate.” The new accompanying software, MetaXpress® 4, increases acquisition and image analysis to more than 10 million cells per hour. “Additionally, the MetaXpress 4 Software has a digital confocal option to enhance image contrast during acquisition, enabling researchers to decrease exposure time more than two-fold, thus increasing the acquisition speed,” says Chandy.
Chandy believes one of the most important aspects of choosing a system for HCA is the speed of the entire workflow. “The industry as a whole needs to increase the speed at which data can be acquired and processed into meaningful results,” says Chandy. “No other technology is as information-rich for biological research as HCS.”
Balancing speed and image quality
Pushing the HCA instrument to operate at higher and higher speeds will lead eventually to lower-quality images, so a balance must be struck between the two. GE Healthcare Life Sciences’ latest HCA release is the IN Cell Analyzer 6000 system, a laser-based confocal imaging platform designed to optimize both speed and image quality. It lets you adjust the imaging along the wide-field to confocal spectrum for each assay channel, creating better control of the speed and image quality. “The IN Cell Analyzer 6000 is designed for challenging applications and high-resolution imaging, extending the reach of HCA into applications more typically associated with low-throughput confocal microscopy, such as 3D imaging, co-localization studies and assays with low signals,” says Howells. “The system is designed to enable increased throughput for a wide variety of assay and sample types, without compromising image quality.”
David Andrews, professor of biochemistry and biomedical sciences at McMaster University, uses HCA in his research and recommends sacrificing speed and throughput for higher image quality, when appropriate. “More and more HCA is being used to do smaller, data-rich screens,” says Andrews. “Ninety percent of our users scan less than 10 plates.” He notes that relatively high operating costs can be a potential drawback of HCA systems. “Make sure that you are able to support what you buy—you need an income that covers all your costs plus 15% of the purchase price [per] year,” he says. “Make sure you have a team of people. People that do image analysis, bioinformatics and cell biology are all different phenotypes.” Andrews hopes the upcoming second generation of HCA instruments will have “lower operating costs, label-free detection with automated data analysis, non-toxic dyes that allow segmentation and determination of cell physiology, and FLIM [fluorescence lifetime imaging microscopy] and spectral imaging.”
HCA tools for challenging new applications
If you aren’t sure what experiments are on your horizon because you are stymied by how to extract all the information you know is packed into your experimental models, consider a new system or software package that can examine your data in new ways. “The analysis software should make it straightforward for the biologist to extract even complex multiparametric data from the images,” says Tesdorpf.
An example is the new Thermo Scientific ArrayScan Infinity HCS Reader. This platform features variable pinhole confocal technology, solid-state LED illumination and live-cell and label-free capabilities. “We designed the new system in response to the fact that scientists are increasingly applying HCA to stem cell characterization, multi-dimensional cell models, primary cultures and tissues,” says Mark Collins, director of global marketing for the Cellomics business within Thermo Scientific. “These types of samples pose challenges, and developing the applications (for example, analyzing a thick stem cell colony) demands new types of imaging techniques.”
The ArrayScan Infinity HCS Reader also includes Thermo Scientific’s image analysis and bioinformatics software featuring Intelligent Acquisition (iQ), which allows the instrument to complete image analysis measurements in real time during data acquisition. “This not only speeds up the whole ‘time-to-decision’ (we can scan a 96-well plate and deliver data to the researcher in under four minutes), but also allows ‘rules’ to be built that give feedback to the instrument to modify the acquisition,” says Collins. The iQ technology lets “the instrument acquire only the images it needs for statistically robust and biologically relevant results, no more and no less. Other systems that collect images and analyze offline have to wait until the analysis is complete to find out if enough of the right kinds of images were acquired, which hampers productivity.”
Collins believes that not enough researchers are currently reaping the benefits of HCA. Thermo aims to make HCA more accessible by rendering it less complex and better understood. “We recently released a new compact, more affordable high-content reader, capable of doing about 70 [to] 80% of the key high-content biologies,” says Collins. “The Thermo Scientific CellInsight Personal Image Cytometer offers easier accessibility to high content, with easy-to-use software and a budget-friendly price tag within the reach of an individual researcher.”
Regardless of your imaging platform, you can benefit from the new version of PerkinElmer’s Columbus™ Image Data Management and Analysis System, a platform-agnostic analysis and storage system for HCA research. The technology is especially advantageous for labs with more than one type of HCS instrument. The latest version, to be released in January 2012, is the Columbus 2.3, an HCA workflow system that includes secondary data analysis capabilities. “This enables scientists to run high-content data quality control across whole screens and to identify statistically significant hits from multiparametric data,” says Tesdorpf. “Columbus 2.3 also allows results to be compared to chemical concentration, generating dose response curves. It also features a further improved version of PerkinElmer’s unique machine-learning technology, PhenoLOGIC™, which makes cell classification as easy as point and click.” PerkinElmer additionally offers its own HCA platforms, the Opera® High Content Screening System and Operetta® High Content Imaging System. “Our latest release for the Opera system includes new hardware and software options, such as brightfield illumination with digital phase contrast, increased objective range and new analysis algorithms to enhance its live-cell imaging capabilities,” says Tesdorpf.
Even as greater numbers of researchers turn to more accessible HCA platforms, data management remains daunting. “As HCA evolves, the technologies will simplify, become more robust and more accessible to a wider range of scientists on a global level,” says Howells. “Today, HCA systems provide scientists with the tools to generate more data than ever before. Interrogating, integrating and managing these data remains one of the biggest challenges for high-content scientists.”
The image at the top of this page is from GE Healthcare Life Sciences IN Cell Analyzer 6000.