Bioluminescence imaging with luciferase-luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for imaging in vivo utilize the same substrate, and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme-substrate “hits” were identified. These tools produced light when complementary enzyme-substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multi-component imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and will expedite the identification of more unique luciferases and luciferins, further expanding the bioluminescence toolkit.

Bioluminescence imaging with luciferase enzymes and luciferin small molecules is a well-established technique for tracking cells and other biological features in rodent models. Despite its popularity, bioluminescence has long been hindered by a lack of distinguishable probes. Here we present a method to rapidly identify new substrate-selective luciferases for multicomponent imaging. Our strategy relies on parallel screening of luciferin analogues with panels of mutant enzymes. The compiled data set is then analyzed in silico to uncover mutually orthogonal sets. Using this approach, we screened 159 mutant enzymes with 12 luciferins. Thousands of orthogonal pairs were revealed with sufficient selectivity for use in biological environments. Over 100 pairs were validated in vitro, and three were applied in cell and animal models. The parallel screening method is both generalizable and scalable and will streamline the search for larger collections of orthogonal probes.

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Bioluminescence with luciferase-luciferin pairs is an attractive method for surveying cells in live tissues and whole organisms. Recent advances in luciferin chemistry and luciferase engineering are further expanding the scope of the technology. It is now possible to spy on cells in a variety of deep tissues and visualize multicellular interactions—feats that are enabling new questions to be asked and new ideas to be explored. This perspective piece highlights recent successes in bioluminescent probe development and their applications to imaging in live cells, tissues, and animals.

We report a set of brominated luciferins for bioluminescence imaging. These regioisomeric scaffolds were accessed using a common synthetic route. All analogs produced light with firefly luciferase, although varying levels of emission were observed. Differences in photon output were analyzed via computation and photophysical measurements. The “brightest” brominated luciferin was further evaluated in cell and animal models. At low doses, the analog outperformed the native substrate in cells. The remaining luciferins, while weak emitters with firefly luciferase, were inherently capable of light production and thus potential substrates for orthogonal mutant enzymes.