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Assay Tutorials : Nov 15, 2012 ( )
Improving HCA with Laser Scanning Imaging
Tool Allows Positional Analysis of Every Cell in a Single Scan!--h2>
High-content screening (HCS) was developed to meet the increasing requirements of research scientists in basic biomedical research and early-phase drug discovery for cell target validation and multiparametric functional studies as well as for the screening of large compound libraries on key cellular targets.
The concept of HCS can be said to have arisen from the combination of single-cell analysis offered by flow cytometry with automated fluorescence microscopy. Whereas flow cytometry, which analyzes single cells by passing a cell suspension through a laser beam, is unable to analyze adherent cell populations and tissue sections in situ, image-based systems are capable of analyzing adherent cell populations, cell colonies, tissues, and even small organisms presented in microplate format.
Image-based systems are commonly employed for the study of changes in cell morphology, colony formation, cell migration, cell health, and apoptosis, as well as for the study of cell-signalling events. HCS allows large numbers of images within microplates to be acquired and processed rapidly thereby increasing the scale and speed of throughput of biological data.
HCS instrument technology has evolved from the employment of a charged couple device (CCD) camera to capture images from automated fluorescence microscopy and integrated application and processing software. Application software enables establishment of focus and microscope resolution, controlled movement and imaging of the microplate, and acquisition of the required number of images to satisfy data point or cell numbers ensuring statistically significant results while logging the exact position of each data point.
The number of images required can also vary according to the number of fluorophore labels employed per study, and analysis of individual images or multicolor image sets is calculated by the application of a series of complex algorithms. The resultant morphological and fluorescence parameters associated with each cell allows definition of sub-populations within the dataset, which is a key enabling benefit of high-content technologies over traditional bulk fluorescence readers.
Imaging studies can be distinguished as either HCS or high-content analysis (HCA)-based, where HCA is defined as the automated extraction and analysis of cellular images obtained by high-resolution automated microscopes. HCS, however, studies the effect of compound libraries and small molecules on cellular activities or parameters as required for the primary and secondary screening stages of drug discovery.
CCD-Based Imaging Systems
There are a range of different CCD-based HCS instruments available on the market today that vary in instrument complexity and capabilities, offering a trade-off between image quality, speed of microplate read, assay capability, and cost. Instruments may consist of basic, automated microscopes coupled with information software for image capture and processing or may be integrated with additional instrumentation such as liquid-handling robots or incubators to enhance the laboratory automation process and workflow.
Instruments that are at the higher end of the market may also provide higher resolution (100x), confocal options, and laser light sources to support a broad range of applications from simple whole-cell biology and phenotyping, to high-resolution subcellular analysis of proteins, organelles or multi-parametric functional studies. Collation of multiple images and datasets routinely produces gigabyte-size files that require powerful computer processing capacities for image analysis and storage.
Moreover, as resolution increases, a greater number of images are required to achieve satisfactory cell numbers for statistical robustness impacting on data collection time and storage capacity. For screening purposes, however, although data is collected from image analysis, the need to store large datasets is not generally required following assay validation, therefore easing data storage issues.
Laser-scanning imaging (LSI) as employed by the acumen® eX3 (TTP Labtech) complements CCD-based HCS instruments, providing rapid “on the fly” analysis of multiplexed assays.
In this system, in place of a microscope, a scanning laser beam is passed through an F theta lens, enabling rapid analysis over a large field of view and depth of field. Emitted fluorescence from labelled cells is detected via a series of photomultiplier tubes (PMT), providing a 2D image similar to that generated by a CCD camera.
Serial PMTs enable simultaneous four-color fluorescence detection for multiplexing studies enabling high-content information to be calculated for every object. High-content data can be exported to proprietary or open-source software, allowing the system to address a range of applications routinely requiring a 20x objective using CCD-based technologies.
Although lacking the higher optical resolutions of the more costly CCD-based imagers, acumen’s widefield objective provides whole well imaging capabilities enabling the capture and positional analysis of every cell in a single scan. This feature eliminates the need for multiple image capture to achieve suitable cell numbers for data normalization and overcomes issues of uneven cell distribution across a well.
In addition, it is possible to filter cell debris or fluorophore crystals based on size, to provide statistically robust data from every well. Whole-well imaging and simultaneous collection of multiparametric data eliminates the need to process multiple images or colors in each well resulting in small files sizes (50KB CSV), which can be easily transported to screening databases. In screening mode, acumen allows validated assays to be routinely and rapidly carried out without the need for detailed image processing and large data file storage.
acumen’s large depth of field also enables high-content image analysis of cells, spheroids and cell colonies, or objects growing on within or a gel for the study of a range of applications such as cell-cycle analysis, cell migration, and live-cell studies, as well as automated analysis of tissues and small organisms. This attribute overcomes the additional imaging times required following refocusing and reimaging using CCD-based imagers or the restricted depth of field imposed by microscopes.
Laser-scanning imaging technology enables rapid plate scanning and analysis independent of plate size (from 96 to 1,536 wells). With an acquisition rate up to 40 images per second, assay miniaturization and minimal cell culture usage, the system saves valuable sample and reagent costs without impacting assay throughput.
With “on the fly” data acquisition and analysis, acumen is an ideal instrument for HCS of compound libraries where rapid data output is required.
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