High-content imaging (HCI), often referred to as high-content screening (HCS), is an automated variant of multiplexed fluorescence microscopy. It makes use of multi-parameter techniques for objective computer analysis of images, known as high-content analysis (HCA).
Ever since its origin, high-content imaging has proven to be a crucial method for quantifying cellular event-related changes and exploring their underlying processes through multi-parametric visualization.
The number of high-content screening applications has greatly increased due to the ability to obtain high-content data at the single-cell or subcellular level with exceptionally high throughput. Platforms for high-content imaging were subsequently developed to accommodate the constantly shifting requirements of various users.
Greater adaptability is beneficial across the board, and methods like cell painting have been crucial for drug development and cytological characterization. Additional specifications include the necessity for pharmacokinetic research, complicated cellular disease models, and 2D and 3D analysis based on fluorescence tests.
High-content imaging now plays an important analytical role in live-cell imaging and in screening labs. For this expanding scope of applications, a variety of HCI systems are readily available.
Considering how important they are, choosing the best system is a crucial step. There are five crucial parameters that users should consider when choosing a high-content imaging platform, and these are outlined below.
Image Credit: IDEA Bio-Medical Ltd.
1. Image quality
The main component of a high-content imaging system is a fluorescent microscope. The most crucial factor to take into account is thus the image quality.
The objective of high-content imaging is fast throughput, however, speed should never come at the price of quality. The quality of the focusing mechanism and the optical path of the system have a significant impact on the sharpness and clarity of the images.
For high-content image systems, laser-based autofocus is a very trustworthy focusing technique. When scanning several samples, it guarantees appropriate focus, which is essential for getting better image sharpness and quality.
These are essential characteristics for producing photos of a publication grade from which accurate information may be extracted. Precise and dependable object identification is made possible by a crisp image, which also helps to reduce mistakes in automated image analysis.
Since autofocus at high magnifications of 40× and higher is difficult, it is crucial to check that the focusing algorithm is automatically adjusted to the magnification (objective) utilized when choosing a system.
The system should be rigorously tested during equipment demos to ensure that it will not malfunction at high magnification, especially when using high numerical aperture (NA) objectives.
A flexible description of the focusing process is also necessary so that it can be tuned for various magnifications. In certain special systems, the user can select a method that uses four different focusing points to map the focal plane for a specific well in a multi-well plate.
When photographing at various fields of view within a well, such a “rapid” approach eliminates the need for autofocusing stages. This can significantly shorten scanning times.
This multipoint option slows down the operation, especially when compared to a “very fast” method that uses a single focusing point near the well’s center. Even so, it still works more quickly than a “slow” method that executes focusing for each field of vision in the well separately.
As opposed to higher magnification, determining the focal plane needs more accuracy; autofocus alternatives are more prevalent at an intermediate magnification of around 10×, where the depth of field is still reasonably big.
Image quality is the most important characteristic to consider when choosing a High Content Imaging system. Image Credit: IDEA Bio-Medical Ltd.
2. Motion accuracy
Multi-well plates must be rapidly scanned, frequently at various times, in high-content imaging. The precision of the scanner, or motion repeatability, is a crucial element when tracking dynamic processes and keeping track of individual cells over time.
The size of a normal eukaryotic cell ranges from tens to hundreds of micrometers at its broadest point. It is crucial to make sure that the same cells are imaged and observed in each imaging cycle when dealing with high NA and high magnification objectives, often 40× and higher.
Due to this need, the scanner carrying the objective must have positioning precision that is as high as possible, preferably on the order of a few hundred nanometers. Scanner motion with such great accuracy is difficult and calls for complex engineering. However, the demand is required to make sure the target continually returns to the same location.
3. Acquisition speed
When it comes to compound screens that include several distinct compounds and/or their combinations, the term “time is money” takes on a whole new meaning. A thorough scan of the entire plate is frequently used for such large displays, followed by automatic image analysis of all captured images.
Higher scan speeds enable single labs to become more high-throughput and process more samples at any given time. Large pharmaceutical corporations that conduct extensive drug screening campaigns, as well as university laboratories or core facilities that support several projects or laboratories simultaneously, are only two places where the problem of throughput is of utmost importance.
More people can access the system each day with a faster scan speed. Results that are meant for publishing or study design can be attained more quickly.
High-content imaging platforms created with these factors in mind can quickly scan 96 locations (i.e., wells) in four fluorescence channels utilizing a 50-millisecond camera exposure time per channel in less than 1 minute and 40 seconds. As a result, if a system’s maker lists anticipated scan durations for particular situations, systems can be standardized and reviewed early on.
4. Ease of use
Although high-content imaging systems are a masterpiece of precise engineering, biologists are its main clientelle. The interface of any HCI platform should not reflect the underlying design complexity. Instead, it should enable biologists to use the vast array of tools and capabilities of the platform as quickly and effectively as possible.
Thus, it is crucial to consider the user interface while choosing a new system. To gain a feel for the software’s overall usability, potential buyers should always assess how easy it is to use the software for both image processing and data collecting. Equipment demonstrations indicate the intuitiveness of a system with how quickly users become comfortable independently using the system.
This feature will facilitate the onboarding of new users and guarantee that biologists can complete more experiments more quickly.
The best user-friendly systems are made with a straightforward user interface that just requires a few pushes of a button on the touchscreen and provides easy navigation and manual sample representations. For a comfortable and seamless visual scanning experience before beginning automated imaging, there are additional alternatives like an analog joystick.
Users from any level, from the entry-level lab assistant to the senior professor or the most seasoned technician, can learn imaging with such a system in less than a half-day. The related automatic image analysis program should preferably just require one more half-day of training.
5. Value for money
Users must take into account both the system’s price and productivity when determining if a high-content imaging system is cost-effective. The best return on investment (ROI) is provided by a platform that is reasonably priced and offers very high scanning speeds that double the throughput of parallel systems.
Additionally, rapid user training on systems with an easy-to-use user interface significantly enhances ROI since it cuts down on training time and allows the sharing of information and know-how among colleagues. Each user can become productive with the system within a short period.
About IDEA Bio-Medical Ltd.
IDEA Bio-Medical is founded in 2007 through a partnership between YEDA (the Weizmann Institute’s commercialization arm) and IDEA Machine Development (an innovation hub).
We specialize in automated imaging systems and image analysis software, offering a broad range of biological applications based on the company’s unique algorithms library. The company is developing novel image-based screening platforms for the pharmaceutical industry and medical centers, dedicated to broadening the scope of personalized medicine.
Our WiScan Hermes system incorporates the most advanced technologies currently available in the machine vision field, integrated with engineering methodologies of high reliability and quality at the level of semi-conductors and digital printing industries, which are the specialty of our mother company, IDEA Machine Development Design and Production Ltd.
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