Bioluminescence from firefly luciferase is a powerful method used in a wide-range of applications ranging from in vitro drug-binding assays to real-time in vivo bioluminescence imaging. The method is relatively inexpensive and easy to use. Bioluminescence uses chemistry to enzymatically convert a small-molecule to a light emitter, which eliminates the background signal seen with other imaging platforms. Either altering the luciferase enzyme or modification to the small-molecule luciferin substrate can modulate the distribution and intensity of light output in vivo. Despite the ability of bioluminescent reagents o produce measurable photon flux in vivo, some of the light is attenuated by hemoglobin absorption before reaching the detector. This highlights the need for brighter substrates and/or substrates that can better access luciferase within whole animals. Access of luciferin to luciferase is an experimental limitation of bioluminescent imaging and D-luciferin has a known biodistribution pattern. However, modification of luciferin can modulate this distribution and enable better imaging in specific tissues. Indeed, a luciferin analog from our lab has been shown to outperform the standard D-luciferin substrate in live mouse brain, even though the analog was supplied at 20-fold lower concentration. Further, mutation of luciferase enables selectivity for these luciferin analogs. Extending the scope of what a luciferase is, even an enzyme from Drosophila (CG6178) revealed latent luciferase activity with our luciferin analogs. Some of the luciferin analogs from our lab are selective substrates for distinct luciferase mutants and may provide a path to luciferase-luciferin pairs that can be used in the same animal or sample without cross-reactivity. In the case where cross-reactivity is minimized to a point where reporters are able to report on distinct events, the reporters are orthogonal. We hypothesize that 1) the combination of luciferin analogs with optimized mutant luciferases or even latent luciferase enzymes can generate more robust light output within the tissue of interest, providing a greater dynamic range for measuring biological events; 2) select pairs of mutated luciferases and luciferin analogs are capable of orthogonality, improving the utility of bioluminescence in vivo for measuring separate events within one animal; and 3) CG6178's structure will enhance our understanding of bioluminescence and open the possibility to designing luciferin substrates capable of bioluminescence with endogenous enzymes. To test these hypotheses, we will use live mice expressing luciferase, luciferase mutants, and CG6178 to assay the biodistribution, intensity, and selectivity of light output with our luciferin analogs. We will combine distinct luciferase mutants within individual mice in separate tissues and test for orthogonality with separate luciferin substrates. We will also solve crystal structures of CG6178 with substrate bound to understand the molecular basis of light emission with this latent luciferase. The success of any of the aims in this fellowship will improve bioluminescence for use by the biomedical research community.