High-content screening (HCS), also known as High-Content Imaging (HCI), is a well-established approach for the multi-parametric visualization and analysis of cellular events. Since its first introduction more than a decade ago, high-content screening systems have continually evolved with many improvements enabled to meet user demands of greater flexibility and the growing requirements of 2D and 3D cell-based fluorescence assays involving complex cellular disease models and studying pharmacokinetics.
Today, HCS/HCI systems play an important analytical role in cell-based assays and screening labs and a wide selection of high content imaging systems are available. Recognizing the importance of such a system and its contribution to laboratory’s ongoing research, the question arises – how do you choose an HCI system for your lab or project? What are the most important things to consider with an automated fluorescence microscope to ensure you make the right choice?
In this article, we will address five key points that constitute guidelines researchers need to consider when choosing a new HCS/HCI system.
At its foundation, a high content imaging system is first and foremost a fluorescence microscope and as such, the image quality it produces is the most important characteristic to consider. When scanning a very large number of samples at high speeds, it is important to note that the speed does not come at the expense of quality. Proper focus to give good image sharpness and quality are important qualities, not only for achieving “publication grade images” but also for extracting reliable and correct information from the images. A sharp image enables accurate and reliable segmentation and minimizes the number of statistical errors in automated image analysis accompanying HCS/HCI.
Image sharpness and quality is substantially affected by the quality of autofocus technology and optical path of the system.
Laser-based autofocus is a highly reliable focusing method for High content imaging systems. It is important to check that the focus algorithm is automatically adapted to the magnification (objective) used, since autofocus with high magnifications of 40X is challenging. Strict testing during equipment demonstrations should ensure the high-content screening system does not fail at high magnification.
In addition, it is important to have flexibility in the definition of autofocus procedure, which can also be optimized for different magnifications. In some unique systems the user can choose a process that maps the focal plane for a particular well in a multiwell plate using four separate autofocus points. Such a ‘fast’ method saves autofocusing steps when imaging at multiple fields of view within a well and hence can greatly reduce scanning times. This multipoint option makes scan time slightly longer compared with a ‘very fast’ process taking only a single autofocus point at the center of a well, but is still substantially shorter than ‘slow’ processes performing autofocus for each individual field of view within a well. Autofocus options are more common at intermediate magnification of around 10x, where the depth of field is still relatively large, compared to higher magnification where identifying the focal plane requires greater precision.
High content imaging involves rapid scanning of multi-well plates at multiple time points. When following dynamic processes and monitoring individual cells over time, a key essential factor is the accuracy of the scanner, i.e. motion repeatability. A typical Eukaryote cell varies in size between tens to hundreds of microns across their widest point. When working with high Numerical Aperture (NA) and high magnification objectives of 40X and above it is important to make sure that the same cells are imaged and monitored in each imaging cycle. Such a requirement means that the positioning accuracy of the scanner holding the objective should be as high as possible, preferably on the scale of a few hundred nanometers. Such high precision in scanner motion is challenging and requires sophisticated engineering, but is necessary to ensure the objective revisits the same exact spot repeatedly.
The term “time is money” takes on a whole new meaning when it comes to compound screens of tens of thousands of different chemicals and/or their combination. Such massive screens often involve a comprehensive scan of the entire plate, followed by an automated image analysis of all acquired images. Higher scan speeds allow single labs to become more high-throughput and process more samples at any given time. This issue of throughput has an acute significance not only in large pharma companies that run large-scale drug screening campaigns, but also in academic laboratories or core facilities that serve multiple laboratories simultaneously. A higher scan speed means more users can access the system on any given day and they more rapidly obtain results for publication or for the design of further experiments.
High scanning speed, which does not require compromise on image quality, necessitates high-performance hardware components, such as linear encoders to minimize motional friction, as well as moving small elements, such as the microscope objective, rather than the hefty microscope stage holding the sample plate.
HCS/HCI systems designed with these considerations in mind can readily scan 96 locations (i.e. wells) in four fluorescent channels in less than 1 minute and 40 seconds, using 50ms camera exposure time per channel.
Ease of use
HCS systems are based on physics and precision engineering, but must be usable by biologists. The wide range of tools and capabilities high content imaging systems offer to biologists need not reflect the complex hardware and software that enable them through a complicated user interface. Rather, biologists should be empowered to perform more experiments faster, so training for use of a new HCS/HCI system needs to be as fast as possible.
Therefore, when choosing a new system, it is important to pay attention to the user interface, evaluate how intuitive it is to operate the software both for acquisition and image analysis to get a sense of its general ease of use. The more friendly systems are designed with an interface that requires pressing only a few buttons on a touch screen and quick navigation when manually visualizing your sample. Some of the more user-friendly options include an analog joystick for a familiar and smooth visual scanning experience prior to initiating automated imaging. Training for imaging with such a system does not take longer than half a day for any level of user, from the junior laboratory worker to the senior professor or the most experienced technician. Ideally, only one additional half day should be needed to learn how to use accompanying automated image analysis software.
Value for money
When trying to assess the cost-effectiveness of an HCS system, we need to take into account both the cost of the system and its productivity. The best Return on Investment (ROI) is obtained from a system whose price is reasonable and offers very high scanning speeds that double the throughput of parallel systems. Also, quick user training on systems with an easy and intuitive user interface also effectively increases the ROI because it saves on the training time and facilitates passing of knowledge and know-how between colleagues – each user should become productive with the system in no time.
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