High-content screening (HCS) is a well-established approach for the multiparametric analysis of cellular events. Since its first introduction more than a decade ago, HCS imaging systems have continually evolved with many improvements enabled to meet user demands of greater flexibility and the growing requirements of assays involving complex cellular disease models.
Today, HCS systems are increasingly being deployed for the analysis of 3D spheroids/microtissues and phenotypic assays, and are expected to impact gene editing studies based on CRISPR-Cas9 in the future.
What is more, such analyses are not limited to one type of HCS imaging system.There appears to be roles for high content widefield and confocal systems; for whole-well scanning systems based on F-theta lens; and even multi-mode plate readers with a whole-well imaging capability.
As such we can expect these imaging systems to play an important analytical role in cell-based assay and screening labs in the future.
High-content screening (HCS) (sometimes called high-content analysis (HCA) or high content imaging (HCI)) is now a well-established approach for the multi-parametric analysis of cellular events. HCS describes a set of analytical methods based on automated microscopy, image processing and visualisation tools to extract quantitative data from cell populations. HCS typically involves fluorescence imaging of samples in a high-throughput format and reports quantitatively on multiple parameters such as the spatial distribution of targets and individual cell and organelle morphology. HCS has gained substantial momentum in recent years due to its ability to study many features simultaneously in complex biology systems and the adoption of HCS technology has now spread into many areas of the drug discovery process.
Since the first introduction of HCS systems, now more than a decade ago, instruments have continually evolved with some systems now into their third or fourth generations. This evolution includes both improvements to the automated microscope hardware (eg sCMOS cameras, confocal optics, oil immersion objective lens, throughput capabilities etc) as well as the significant enhancements to the software, not only in the image acquisition, but also in the analysis, evaluation and subsequent data storage. Many of these improvements have been enabled to facilitate the growing HCS requirements for higher-throughput, phenotypic screening and assays involving complex disease models, such as live cells, primary cells, stem cells and 3D spheroids/microtissues.
In January 2016, HTStec undertook a market survey on HCS systems to document current end user opinions, practices and preferences in the use of HCS instruments and software, and to understand future HCS workflow requirements1. Some of the findings of this market survey are now reported together with vendor updates describing the latest developments in their HCS systems.
Main use of HCS assays
The main use of HCS reported by survey respondents was small molecule drug discovery (87% using). This was followed by studying cell behaviour or differentiation (59% using) and then mechanistic studies (57% using). Least used was safety/toxicology (35% using) (Figure 1).
How HCS assays are conducted today
The cell type most used today (2016) by survey respondents for their HCS assays was tumour cell lines (29% of assays). This was followed by primary cells (22% of assays); transformed or recombinant cell lines (17% of assays); native immortalised cells (13% of assays); stems cells or iPS-derived phenotypes (10% of assays); and then transiently transfected cells (9% of assays) (Figure 2). Survey respondents reported a median of three dyes (stains) multiplexed per HCS assay and a median of 6 to 10 different parameters evaluated per HCS assay (Figures 3 and 4). Fluorescent dyes were rated as the main labelling technique/source used in HCS assays. This was closely followed by commercial antibodies; and then fluorescent proteins (eg GFP); and home brew assays. Rated least was quantum dots (Figure 5). The median use of 3D cell cultures (eg spheroids, microtissues, matrices, scaffolds etc) in HCS assays and analysis today was minimal (<25% assays) (Figure 6). In contrast, the median use of HCS assays and analysis to monitorreagents) (Figure 12). phenotypic-based screens (versus target-based screens) today was moderate (25-75% assays) (Figure 7). The type of imaging most used for HCS assays was widefield imaging (72% using). This was followed by confocal imaging (64% using); and then whole-well scanning (44% using); whole-well imaging (multi-mode plate reader-based) (26% using); and digital phase contrast (20% using) (Figure 8). The majority (69%) of survey respondents stated that whole-well imaging technology as offered on several multimode plate readers does not feature in their HCS assays or workflow. The remaining 31% of respondents think whole-well imaging has a role in their HCS assays or workflow and in the full report gave feedback on where it fits in (Figure 9). 3D cell culture was rated as the HCS task that most requires confocal imaging. This was followed by imaging of a tissue slice; and then counting the number of small pits inside the cells; and imaging of small organelles, eg mitochondria. Least requiring confocal imaging was live cell imaging; and assays that are sensitive (easily disrupted) during the washing process (Figure 10).
Use of vendor’s HCS imaging systems
The vendor’s high content imaging instrument that survey respondents have most access to and make most use of today was PerkinElmer (50% accessing). This was followed by ThermoScientific (Cellomics) and Molecular Devices (both 42% accessing) and then GE Healthcare (21% accessing). No respondents reported they had access to Vala Sciences and IDEA Bio-Medical HCS instruments (Figure 11).
Features that influence purchasing of HCS systems
Sensitivity/resolution was ranked as the most important feature when purchasing an HCS system. This was followed by image analysis software and then throughput. Ranked least important was portfolio of related products (eg informatics, software, microplates, assays and reagents) (Figure 12).
What will most impact future of HCS assays
Phenotypic assays and 3D spheroids/cell cultures were rated as the area where new developments are likely to impact and grow the market (increase use and adoption) for HCS assays. This was followed by high content informatics; 3D imaging/reconstruction capability; and then RNAi/gene editing CRISPR-Cas9. Rated least impact was accessory reagents. (Figure 13)
Latest developments in HCS systems:
The following vendor snapshots describe some of the latest developments in HCS systems. BioTek’s (www.biotek.com) Cytation Cell Imaging Multi-Mode Reader is designed for the most common applications in HCS: cell counts, signal expression changes, morphology changes and signal movement (such as translocation). For these applications, Cytation’s accompanying Gen5™ software allows multiple cellular measurement masks to be defined, making it easy to measure cellular compartments such as the nucleus and cytoplasm, or the entire cell. The resulting single cell analysis can quickly measure cell populations or subpopulations of cells. The magnification range, up to 60x and more than 15 colour cubes along with critical temperature and CO2/O2 control, makes it a good alternative for handling many live and fixed cell assays typically performed in HCS labs. In contrast to very complex systems’ software, Gen5’s image analysis offers a short learning curve for analyses ranging from basic cell counting to measuring nuances between cellular populations. The system is designed for users at all levels of image analysis experience who want an easy to use, yet powerful platform for common HCS applications. Cytation Cell Imaging Multi-Mode Reader can also be equipped with multi-mode microplate reading optics to cover a broad range of applications. The combined imaging and multi-mode detection capability adds to its versatility with features such as hit-picking key wells for imaging, which saves time and minimises data storage needs. Cytation is available at a fraction of the cost of standard HCS systems, providing great value over a broad imaging and multi-mode detection application range (Figure 14).
GE Healthcare Life Sciences’ (www.gelifesciences. com) IN Cell Analyzer 6000 is a unique laser-based, line scanning confocal high content imaging system that provides high sensitivity imaging with a rapidly adjustable aperture inspired by the iris mechanism of the eye. The novel variable aperture technology maximises flexibility while the efficient optical design yields exceptional image quality. In addition to precision engineered hardware, the IN Cell acquisition software is constantly advancing, with regular updates to meet increasingly challenging experimental needs. Notable improvements from 2015 include: a 9x increase in achievable frame rate at full frame, a 20% increase in throughput, a novel and robust autofocus algorithm, enhanced tools for quickly reviewing scan data, and numerous improvements in overall system flexibility. With the recent speed improvements allowing acquisition up to 125 frames per second (fps), new applications such as analysis of calcium flux in beating cardiomyocytes are enabled. Cardiotoxicity is a common cause of drug candidate failures and improved analysis tools to evaluate compounds are needed. Calcium flux regulation is essential to cardiac function and is an attractive target for cardiac pharmaceuticals. Recently, the IN Cell 6000 was used to capture rapid changes in calcium flux in cardiomyocytes. Imaging at 42fps for this assay allowed the IN Cell Analyzer to capture a smooth waveform and accurately characterise drug effects on cardiac beat rate. In the near future, GE expects to see additional applications made possible by the recent speed improvements on the IN Cell 6000 (Figure 15).
Temporal analysis studies of rare biological events in live experiments (eg cell divisions; detection of circulating tumour cells) are crucial for understanding complex processes. Such studies however, are often challenging since they are commonly based on individual static ‘snapshots’. In most cases, cell populations are not entirely uniform, and individual cells within such populations are not synchronised. Thus visualising rare events requires systematic, continuous tracking of multidimensional aspects of the system, capturing dynamic processes and identifying transient, sometimes unexpected features. Such tracking must include automatic and highly accurate detection of specific events of interest and the ability to rapidly capture and document those events at high resolution. IDEA bio-Medical (www.idea-bio.com) offers a new feature for spatial detection and characterisation of temporal kinetics of rare events using its advanced HCS system, WiScan® Hermes. This new function offers sophisticated HCS tools for studying rare events in living cells. Based on its ultrafast image acquisition properties, the system first scans the entire plate and acquires images using low magnifications (2x-10x) in pre-defined time intervals (Figure 16A, 16B). This initial scan automatically spots objects of interest, based on multi-parametric morphological features or fluorescence properties (Figure 16C). This ultrafast scanning capacity is achieved thanks to unique capabilities which include stationary plate (the moving component is the objective) and multichannel simultaneous imaging. A rapid, automatic change of objectives is activated between scans, followed by a second scan which acquires images and records movies in high magnifications (20x- 60x) only for the selected objects of interest (Figure 16D), which are accurately spotted and captured thanks to rapid focusing and scanner high positioning repeatability (Figure 16).
Leica HCS A from Leica Microsystems (www. leicamicrosystems. com) can speed up the process of discovery through high content screening. Combining the flexibility of a point scanning confocal with the high speed of a camera-based widefield system in the same assay saves time. Compensation for specimen drift, single object tracking and immersion fluid are taken care of for high quality raw data. The system allows making strong inferences based on robust statistics by screening a large number of samples or conditions. Leica HCS A supports users with all the flexibility they need to incorporate any standard sample dish or multi-well plate. Tissues or organs typically do not fit into one field of view. Leica HCS A offers a powerful stitching solution called Mosaic to combine these large images with time series, multi-well formats or custom positions. With Computer Aided Microscopy (CAM), rare events can be continuously monitored in parallel with image acquisition. Streaming to external storage devices using the OME-TIFF format allows studying all data by any tool reading TIFF. In conjunction with CAM, Leica HCS A can respond to feedback from image analysis software about events detected during acquisition. This powerful approach has proven to simplify large collaborative screening campaigns. The integration of Leica HCS A into the confocal and widefield systems of Leica Microsystems allows users to standardise biological applications for rapid and reproducible results (Figure 17).
There is an increasing interest in using more complex assays and three-dimensional cell models in screening for better modelling of tissue biology and cell interactions. The ability to use 3D models in a high-throughput, high-content screening is a significant step in facilitating identification of chemotherapeutic drug candidates. Molecular Devices’ (www.moleculardevices.com) Image- Xpress® Micro high-content widefield or confocal imaging systems allow users to overcome the common challenges in performing high-throughput 3D imaging. New 3D analysis features have been added to the MetaXpress® High-Content Image Acquisition and Analysis Software allowing for imaging and analysis of spheroids, microtissues, cells in 3D matrix and small organisms. To illustrate, Molecular Devices developed and optimised methods for an in vitro toxicity assessment using 3D liver spheroids derived from human iPS cells (Figure 18). The 3D analysis of the images was done using the newly-integrated features which simplify quantification of 3D structures with volume, intensity and distance measurements in multi-well workflows. This enables users to monitor and quantify complex drug-induced phenotypic changes, including changes of spheroid shape and complexity, cell division, apoptosis and mitochondria integrity in a 3D environment. It has demonstrated that 3D image analysis delivers a number of informative phenotypic readouts that enable screening for deleterious effects of test compounds on cell morphology and viability from a simple assay protocol.
At the 2016 AACR Annual Meeting in New Orleans, Nexcelom Bioscience (www.nexcelom. com) launched a new model of the Celigo image cytometer Celigo-5C. This new model features five imaging channels including one bright field and four fluorescent colours (Ex/Em: 377nm/470nm, 488nm/536nm, 531nm/629nm, 628nm/688nm). By utilising the F-theta lens and galvanometric mirror technology, the Celigo rapidly captures uniform images of entire wells. The new Celigo-5C system is designed to satisfy the needs of high content screening using the increasing numbers of phenotypical models of co-cultured multiple cell types in both 2D and 3D formats, 3D tumour spheroid, cancer associated fibroblasts (CAF), and immune cells. Advances in 3D culture systems have made it possible to expand patient-derived organoids (PDO) from normal or diseased tissues. To perform drug screening, it is necessary to automate the quantification of PDO both in size and in number. The Celigo image cytometer was used to image PDO grown in 24, 48, 96 and 384 well plates. The whole-well image at 1um/pixel provides high quality image for software to segment, count and measure the size of PDO in each well (Figure 19).
Olympus (www.olympus-sis.com) scanR is a modular microscope-based screening platform designed for fully-automated image acquisition and data analysis of biological samples. The system was developed as with an extremely broad application range complemented by sophisticated image and data-analysis software, which is based on a powerful, interactive, cytometry-oriented approach for easy straightforward assay setup, handling and analysis of huge numbers of multidimensional data sets. Handling fixed and live cells with equal ease, Olympus scanR is the perfect screening platform for a wide cross-section of research and specifically targets the need for quantitative imaging and image analysis in modern cell biology, systems biology and medical research. Olympus scanR perfectly combines the modularity and flexibility of a microscope-based setup with the automation, speed, throughput, reliability and reproducibility demands of screening applications. The system can handle many different formats, from any multiwell plates and slides to custom-built arrays. The unmatched flexibility and open design make it adept at both routine and advanced applications. With a powerful analysis module for biological functional assays, it is an ideal tool for assay development and high-content screening. Olympus ScanR provides complex image analysis and advanced data evaluation abilities, enabling it to address a whole range of standard and bespoke assays. The Olympus scanR software features an intuitive graphical user interface based on a strict, workflow-oriented approach. This ensures simple handling in daily operation, easy image acquisition and straightforward system configuration. Extensive expansion capabilities are available to perfectly match the specifications of any application. This includes time-lapse cytometry, 3D deconvolution, combination with Olympus microscopy imaging software cellSens, integration of a plate-loading robot, IR laser hardware autofocus based on IX83 ZDC or an Olympus incubation system (Figure 20).
PerkinElmer (www.perkinelmer.com) launched the new Operetta® CLS™ high-content analysis system in early 2016. It is the first high-content system to combine automated water immersion objectives with confocal optics, stable LED illumination, and a sCMOS camera. The highly efficient lightpath provides scientists performing basic research, assay development or screening with high sensitivity and flexibility to run everyday cellular assays or to retrieve rich content from primary cells or 3D models. The Operetta CLS can be automated by systems such as PerkinElmer’s plate::handler™ for automating simple overnight runs and cell::explorer™ for automating entire high-content screening workflows. Like the Opera® Phenix™ high-content screening system which provides unrivalled performance for speed and sensitivity, the Operetta CLS system is powered by the Harmony® software. With Harmony version 4.5, scientists benefit from PreciScan intelligent acquisition, enabling efficient screening for rare events or 3D microtissues and MultiScale analysis for analysing phenotypes in a larger context. PerkinElmer also released new versions of its Columbus™ software for high volume image storage and analysis and High Content Profiler™ software for multi-parametric hit selection. These latest versions offer improved integration to enable browsing, searching, downloading, and aggregation of content in Columbus directly within High Content Profiler. This eliminates the need to export and import data, and manually merge with metadata – saving precious time and avoiding mistakes during data transfer. In addition, PerkinElmer has broadened its range of HCS microplates to include the CellCarrier™ Spheroid ULA plate, GravityPLUS™ ULA plate and GravityPLUS™ Hanging Drop system for growing 3D microtissues from InSphero AG (Figure 21).
There has been a rapid growth in interest in high content assays, as the industry gradually recognises the need to understand the underlying functional biology associated with new therapeutic targets. As a result, imaging of cell function has become increasingly important, and this in turn has led to demand for the integration of high content imaging within fully automated screening platforms. Tecan (www.tecan.com) is a market leader in laboratory automation for cell biology applications, and is renowned for offering easy integration of third party devices on to its core liquid handing platforms. The ability to incorporate high content imaging systems – such as SynenTec’s Cellavista™, Thermo Scientific’s ArrayScan™ and GE Healthcare’s IN Cell Analyzer – into the same systems that are already being used for cell-based high throughput screening helps to ensure the best return on investment and futureproof labs against changing assay types. Tecan’s recently launched Fluent™ Laboratory Automation Solution for cellbased assays is specifically designed to enhance throughput, streamline workflows and deliver more precise and reliable results. It helps to optimise the performance of cell-based assays by simplifying the handling of precious samples and seamlessly integrating with the various cell biology devices needed for HCS, such as HEPA hoods and wash stations. Critically, it also allows completely automated monitoring of cell cultures – cell counting, viability determination and cell confluence assessment – using the Spark™ series of multimode readers. By providing a walkaway solution for this error-prone and tedious QC work, Tecan can offer truly automated cell-based assay workflows (Figure 22).
The ThermoFisher Scientific (www.thermoscientific. com/HCS) CellInsight™ CX7 High Content Screening Platform incorporates a near-infra red (NIR) channel for use in wide-field and confocal imaging. This extra spectral range is invaluable for researchers using red fluorescent proteins such as mKate or mCherry that occupy a large band within the red spectrum. The NIR channel is well separated from deep red, and enables an optimal combination of green and red fluorescent proteins together with a broad range of immunocytochemistry probes. Taking advantage of the NIR spectrum, AlexaFluor 750 secondary antibody conjugates offer spectral resolution for multiplexing. A 5-plex experiment using the CellInsight CX7 platform in confocal imaging mode to explore the impact on cell health when autophagy is blocked using chloroquine is shown in Figure 23. The CellInsight CX7 platform is a productive tool for multiplex analysis. Rapid results are achieved using laser-based autofocus with simultaneous data collection and analysis (ie results are analysed while the plate is being scanned). With more than 30 pre-established assays in HCS Studio 3.0 Cell Analysis Software, this platform offers the ideal starting point for high content applications.
In order to maximise the benefits of phenotypic screening and minimise the chance of missing a hit, it is beneficial to screen against a full compound library rather than library subsets, using high-content assays. Despite the benefits, such an approach is challenging using an automated high-content microscope as HTS requires: very rapid throughputs, miniaturisation to minimise cell and reagent costs, a simple approach to hit identification and manageable data output. TTP Labtech’s (www.ttplabtech.com) acumen Cellista reduces these challenges by providing the value of a highcontent approach for hit identification in a format that is HTS-friendly. With throughputs of more than two million data points a week, cellular imaging at high-throughput is achieved by laser scanning excitation through an F-theta lens and photomultiplier tube (PMT) signal detection. This design enables rapid whole-well imaging for multiplexed assays. The needs of efficient screening laboratories are met by easy-to-use Cellista software and small data output files that facilitate rapid workflow integration. Full library phenotypic screening can now be easily achieved in high-throughput using a high-content approach. Recently, an acumen Cellista cell viability assay has been developed to deliver improved data quality and cost compared with established ATP-luminescence technologies. Cell viability and proliferation assays are fundamental to the drug discovery process, however, the ATP-luminescence approach is known to underestimate potency and overestimate toxicity for certain compound classes. Through understanding more about compound mechanism of action, TTP Labtech’s acumen Cellista offers an alternative approach that eliminates false positive reporting leading to confident decision making early in drug discovery (Figure 24).
A recent trend in high content screening is 3D live cell analysis which facilitates the acquisition of more relevant biological information, closer to that found in real human organs. Yokogawa Electric Corporation (www.yokogawa.com/scanner), which has a long history of spinning disk confocal scanning units, has developed a new function for its Confocal Quantitative Image Cytometer CQ1. It is high-speed time-lapse image acquisition mode which can get the images at 20 frames/second. CQ1 calculates the feature data of each cell so the researcher can analyse fast phenomenon. An example of toxicity testing with human iPSC-derived cardiomyocytes is shown. Multiple colonies of cardiomyocytes (Cellular Dynamics International, Inc) were cultured in an Elplasia microplate (Kuraray Co, Ltd). The cardiomyocytes stained with calcium indicator changed the fluorescence intensity according to the pulsation of the cells. The oscillation of the fluorescence intensity of each colony were measured (Figure 25). The analysis of the heart rate waveform with CQ1 can offer the results of toxicity test for each colony, which have conventionally been averaged as well data only. This new function can be expected to be applicable to the study of fast phenomenon such as neural firing. CQ1 can also conduct 3D analysis for the confocal images. For example it can digitise the condition and behaviour of each cell in cell sheets and spheroids. Yokogawa also offers a high throughput, high content screening system CV7000S. This has a liquid handling option, so it can analyse the differences of behaviour of cells and spheroids immediately after dispensing a reagent or drug.
Vendor updates on HCS systems can be roughly grouped into the following areas of new development:
Faster image acquisition: Recent speed improvements allowing faster image acquisition permit new applications such as the analysis of calcium flux in beating cardiomyocytes to be enabled, capturing smoother waveforms and more accurately characterising drug effects on cardiac beat rates across the well and on individual cardiomyocyte colonies (GE Healthcare Life Sciences, Yokogawa Electric Corp).
Temporal kinetics of rare events: Computer-aided detection of rare events (based on feedback from image analysis software), continuous monitoring, temporal characterisation and the ability to document at high resolution are seen as increasingly important (IDEA bio-Medical, Leica Microsystems, PerkinElmer).
New spectral channels: Incorporation of an extra spectral range (eg near-infra red (NIR) channel) for use in wide-field and confocal imaging facilitates the combination of green and red fluorescent proteins together with a broad range of immunocytochemistry probes enabling broader multiplexed analysis (ThermoFisher Scientific).
3D analysis: New 3D analysis features have been added to many HCS systems software allowing for the analysis of spheroids, organoids, microtissues, cells in 3D matrix and small organisms. These are applicable to both widefield and confocal images and whole-well scanning systems based on F-theta lens. Users can now easily monitor and quantify complex drug-induced phenotypic changes, including changes of spheroid number, shape, size, complexity, cell division, apoptosis, mitochondria integrity, cell morphology and viability all in a 3D environment (Molecular Devices, Nexcelom Bioscience, PerkinElmer; Yokogawa Electric Corp).
Rapid whole-well imaging: For HCS systems to usefully deployed in HTS they must offer rapid throughput, miniaturisation to minimise cell and reagent costs, a simple approach to hit selection and manageable data output. Instruments using laser scanning excitation through an F-theta lens offer an alternative approach to rapid whole-well imaging ideally suited to multiplexed assays and meeting the needs of phenotypic screening (TTP Labtech, Nexcelom).
New imaging options: These include high-content systems that combine automated water immersion objectives with confocal optics, stable LED illumination, and a sCMOS camera providing the highest sensitivity and flexibility to run every day cellular assays or to retrieve rich content from primary cells or 3D models (PerkinElmer); ins truments available at a fraction of the cost of standard HCS systems, providing great value over a broad imaging and multi-mode detection application range (BioTek); and microscope-based setups with the modularity and flexibility to address the automation, speed, throughput, reliability and reproducibility demands of screening applications (Olympus).
Data analysis and storage: HCS software is constantly a dvancing, with regular updates to meet increasingly challenging experimental needs and faster image acquisition. Improvements include many new features (especially in the area of 3D and phenotypic assays); shorter learning curves; higher volume image storage and profiling software for multi-parametric hit selection (all vendors).
Automation: New solutions for cell-based assays specifically designed to enhance throughput, streamline workflows and deliver more precise and reliable results from HCS imaging systems integrated with automated liquid handing platforms (Tecan, PerkinElmer).
In conclusion it is clear HCS systems are increasingly being deployed for analysis of 3D cell models and phenotypic assays, and are expected to impact gene editing studies based on CRISPR-Cas9 in the future. What is more such analyses are not limited to just one type of HCS imaging system. There appears to be roles for high content widefield and confocal systems; for whole-well scanning systems based on F-theta lens; and even multi-mode plate readers with a whole-well imaging capability. As such we can expect these imaging systems to play an important analytical role in cell-based assay and screening labs in the future.
Dr John Comley is Managing Director of HTStec Limited, an independent market research consultancy whose focus is on assisting clients delivering novel enabling platform technologies (liquid handling, laboratory automation, detection instrumentation; assay methodologies and reagent offerings) to drug discovery and the life sciences. Since its formation 14 years ago, HTStec has published 126 market reports on enabling technologies and Dr Comley has authored 57 review articles in Drug Discovery World. Please contact email@example.com for more information about HTStec reports.
1 High Content Screening Trends 2016. Published by HTStec Limited, Godalming, UK, February 2016.
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