Problem: High Content Analysis instruments that use CCD image capture offer high optical resolution and provide high information cellular data for secondary screening. However, these systems are limited for primary screening for certain assays because microscope objectives are unable to fully visualize each well. They typically capture only a 1 mm x 1 mm area per image using a 10x objective (2.5% of well area for a 96- well plate). CCD imagers are impractical for applications requiring multiple image capture such as rare event detection, tissue scanning, or colony counting, where they generate very large quantities of data (up to terabytes) and offer limited throughput.
Solution: TTP LabTech’s Acumen range of microplate cytometers, launched in 2001, use laser scanning to excite fluorescence which is then detected by a series of photo multiplier tubes and displayed as 3- dimensional object profiles. Acumen microplate cytometers do not generate image files which require analysis by complex algorithms but instead apply cytometry principles to offer a rapid and user friendly approach to high content analysis. Importantly, they also give a greatly increased scan area of 20 mm x 20 mm (or 4 wells of a 96 well plate). This wide field of view allows the Acumen eX3 to report data for all cells in each well at throughputs of ten minutes a plate.
Whole-well scanning has many advantages over restricted reporting from a small well area:
- It gives statistically robust data from a truly representative cell population — essential for rare event detection assays.
- It can overcome problems of variable stimulation and random cell distribution often observed in screening plates.
- It enables normalization of biological responses to total cell number, offering a simple toxicity or proliferation readout with every test.
- It allows easy analysis of large objects such as C. elegans, tissue sections, or cell colonies, without the need for image stitching which is required by microscope based systems.
To exemplify the benefits of whole well scanning, cell colony formation assays traditionally involve laborious and subjective analysis by hand using a microscope. The Acumen eX3 offers a new approach through the application of a volume algorithm. This assay readout is more representative of cell colony formation in vivo since it correlates well with the growth of higher stage and higher grade tumours, not supported by simply counting the number of colonies above a certain size.
In some cases a cytometric approach to analysis is inappropriate, for example cell segmentation within monolayers. Here, the Acumen eX3 offers the flexibility of exporting whole well TIFF images for subsequent batch analysis by third party image analysis software, increasing the instrument’s utility in certain biological areas.
Whole well scanning can be used to generate reproducible and robust screening assays, with small data files and speed advantages over imagebased technologies, ensuring the Acumen eX3 is entirely suited to a screening environment.