Abstract: In 2012, Promega introduced NanoLuc, an ATP-independent marine luciferase mutant exhibiting a high photon production rate in the presence of a synthetic luciferin analog, furimazine. However, NanoLuc has several unfavorable features, including low tissue penetration of its blue emission, and limited substrate solubility and stability. Recent studies (including our efforts) have partially addressed these issues, but there are remaining key hurdles that prevent a broader adoption of NanoLuc-derived bioluminescence systems for in vivo animal imaging. The overall objective of this 4-year R01 project is to develop next-generation ATP-independent luciferases, luciferins, and bioluminescent calcium indicators to greatly facilitate imaging experiments in small animals. Leveraging our strong preliminary results and sustained research interests and expertise in the relevant areas, we will pursue the following three Specific Aims: 1. Develop shelf-stable and water-soluble luciferins. 2. Develop further red-shifted luciferase-luciferin pairs. 3. Engineer next-generation bioluminescent calcium indicators with enhanced physiological responsiveness. The outcomes of this project will be bioluminescent tools to facilitate imaging studies in animal models. These fundamental research tools will further catalyze an extensive array of biological and biomedical studies.