Optimizing environmental conditions and image processing as a means for simplifying BRET imaging

Abstract

Protein interactions are fundamental to our understanding of biological processes. Bioluminescence Resonance Energy Transfer (BRET) is a method for monitoring protein‐protein interactions (PPIs) and protein conformational changes in living cells in real time. It is based on the well‐established principle of Förster Resonance Energy Transfer (FRET). Unlike the commonly used fluorescence‐based FRET, BRET requires no light input, making it more suitable for monitoring PPIs in light sensitive tissues. Because no phototoxicity occurs, BRET may also lend itself to long‐term time‐resolved measurements. However, producing images with BRET is challenging, as it requires long exposure times, specialized imaging equipment, and extensive image processing. Here, we report BRET imaging results at subcellular resolution that were obtained using an electron‐multiplying camera system. We evaluate various image processing techniques. We also report on two sample preparation techniques that appear to boost luminescence output and thus simplify and shorten the BRET imaging procedures.