FRET-based imaging assays are widely utilized to study protein-protein interactions or signal transduction processes in living cells.

In the study presented here, we take advantage of the 4 sCMOS cameras in the PerkinElmer Opera Phenix™ High Content Screening System. With its proprietary Synchrony™ Optics, the system enables simultaneous acquisition of the donor and the FRET image with the option to acquire two additional markers in parallel. Optimal excitation of the Cerulean donor with a 425 nm laser further increased the sensitivity in FRET measurements. Image analysis using the Harmony® High Content Analysis Software allowed an easy-to-use workflow to quantify the FRET efficiency on a pixel-by-pixel basis.