Project Summary One of the most powerful approaches for studying biological function relies on the use of genetically encoded light-emitting proteins to visualize cell physiology. However, existing reporter genes - the prototypical green fluorescent protein (GFP) and luciferase − have two major limitations. First, oxidation by molecular oxygen is central to the mechanism by which GFP, luciferase, and derivative reporters emit light. Second, optical photons are scattered and absorbed by opaque tissue, which effectively blocks light penetration in intact animals. As a result, GFP and luciferase based reporters fail to produce light in complex settings such as hypoxia (oxygen < 1%) or deep inside intact animals. An immediate impact of these shortcomings is on medical research. Hypoxia plays a central role in the pathophysiology of tumors and polymicrobial infections with consequences ranging from drug resistance to inflammation. To understand how hypoxia reprograms cell function in these contexts, there is a need for reporter gene technologies that allow biological activity to be dynamically studied in hypoxic cell cultures. Likewise, to understand processes such as tumor biology in their important in vivo context, there is a need for reporter genes that are compatible with optically opaque animals. The goal of our research program is to address these long-standing challenges in biological imaging. To do so, our research will pursue the development of new classes of reporter genes for noninvasive imaging of biological function in hypoxic cell cultures (in vitro) and in live animals (in vivo). Our proposed approach builds on proteins with special properties – photoreceptors, paramagnetic enzymes, and water channels – and applies molecular engineering to develop new reporters for fluorescence and magnetic resonance imaging (MRI). Our research program proposes five core objectives: 1) engineering bright, multi-colored, oxygen-independent fluorescent proteins for hypoxia, 2) developing sensitive and multiplexable MRI reporters for in vivo imaging, 3) designing bioresponsive sensors based on these proteins to detect cell metabolites and gene expression, 4) applying these sensors to study antibiotic tolerance in hypoxic bacteria, and 5) induction of specialized treatment resistant cancer cells in glioblastoma tumors. Success in these goals will provide a breakthrough technique for studying a broad spectrum of biological processes where hypoxia and in vivo milieu provide important pathophysiological contexts.