Assay development for image-based high-content screening
Image-based high-content screening (HCS) is a high-throughput screening (HTS) technology that combines automated fluorescence microscopy from microtiter plates with digital image analysis. This effectively allows phenotypic screening with sufficient throughput to interrogate large compound libraries…
The microscopic images and the data derived from them provide detailed and quantitative phenotypic information about the cells in each well. Therefore, HCS can quantify a number of biological processes and pathways that are intractable to the more `one-dimensional data´ derived from conventional plate readers, for example.
Examples of HCS assays include assays that measure protein translocation, internalisation, externalisation, degradation of target proteins, cellular organelles and substructures, as well as cellular processes like cell migration, cell proliferation and apoptosis. Importantly, phenotypic changes can be measured in live cells (either continuously or at a predetermined endpoint) or in fixed cells (endpoint assays). In addition, several labels can be measured to multiplex the assay and provide additional layers of information.
Obviously, HCS assays are highly complex and can be challenging to design and develop. Their biological relevance, however, has made them an integral part of the early stages of drug discovery.1 The critical importance of HCS assay development is often overlooked in this whole process. Here, we discuss some aspects of assay development that are critical to its successful implementation.
Image information and principle of analysis
Although one could envisage a target agnostic, unbiased phenotypic screen, in practice an image-based assay is usually designed to measure the activity, abundance or location of pathway targets, proteins, and cellular structures of interest. The aim then is to be able to acquire images that can be processed using suitable image analysis software. The image analysis can be a challenge in itself and this topic is more comprehensively covered in a couple of recent publications.2,3 It’s important at the beginning to understand the biology of the system and to be able to acquire images that allow…