A versatile bioluminescent probe with tunable color

Bioluminescence is a powerful method for imaging in vivo, but applications at the microscale are far from routine. This is due, in part, to a lack of versatile tools for visualizing dynamic events. To address this void, we developed a new platform-Bioluminescence Resonance Energy mAKe over with a Fluorescence-Activating absorption-Shifting Tag (BREAKFAST). BREAKFAST features a bright luciferase combined with a chemogenetic tag (pFAST) for rapid color switching. In the presence of luciferin and a discrete fluorogenic ligand, signal is observed via resonance energy transfer. We evaluated spectral outputs with various fluorogens and established the utility of BREAKFAST for combined fluorescence and bioluminescence imaging. Dynamic, four-color visualization was achieved with sequential ligand addition and spectral phasor analysis. We further showed selective signal quenching with a dark fluorogen. Collectively, this work establishes a new method for bioluminescence imaging at the cellular scale and sets the stage for continued probe development.