Use Of Phase Contrast Imaging To Track Morphological Cellular Changes Due To Apoptotic Activity

By Brad Larson and Peter Banks, Applications Department, BioTek Instruments, Inc.

Phenotypic screening, or the determination of the effects (phenotypes) that a molecule has on a cell, tissue, or whole organism, dates back to earliest drug discovery efforts. Due to advances in molecular biology and biochemistry in the 1990s, this approach was de-emphasized in favor of a more “reductionist” target-based approach1. Through mounting evidence, however, the shift appears not only to have failed in accelerating discovery of new first-in-class medicines, but has also led to higher attrition rates of new lead molecules2. Therefore, a more balanced “holistic” approach, which incorporates both discovery methods is now being implemented.

One of the most important and widely studied phenotypic responses is apoptosis; particularly in oncology research. Understanding apoptosis as it relates to a particular disease helps in understanding its pathogenesis, as well as how it can be treated. Here we demonstrate a method to incorporate automated, digital widefield phase contrast microscopy to monitor potential apoptotic effects of lead molecules. The ability to control conditions within the imaging chamber at 37 ºC/5% CO2 enabled kinetic images to be captured on an hourly basis throughout the entire incubation period. Gen5™ cellular analysis also allowed calculation of rounded apoptotic cells. Determination of apoptotic activity was also performed with a fluorescent live cell assay. Results confirmed the validity of the image-based method to provide accurate analysis of apoptotic induction.