No abstract

Bioluminescent probes are powerful tools for visualizing biology in live tissues and whole animals. Recent years have seen a surge in the number of new luciferases, luciferins, and related tools available for bioluminescence imaging. Many were crafted using classic methods of optical probe design and engineering. Here we highlight recent advances in bioluminescent tool discovery and development, along with applications of the probes in cells, tissues, and organisms. Collectively, these tools are improving in vivo imaging capabilities and bolstering new research directions.

Bioluminescence imaging with luciferase-luciferin pairs is commonly used for monitoring biological processes in cells and whole organisms. Traditional bioluminescent probes are limited in scope, though, as they cannot be easily distinguished in biological environments, precluding efforts to visualize multicellular processes. Additionally, many luciferase-luciferin pairs emit light that is poorly tissue penetrant, hindering efforts to visualize targets in deep tissues. To address these issues, we synthesized a set of π-extended luciferins that were predicted to be red-shifted luminophores. The scaffolds were designed to be rotationally labile such that they produced light only when paired with luciferases capable of enforcing planarity. A luciferin comprising an intramolecular “lock” was identified as a viable light-emitting probe. Native luciferases were unable to efficiently process the analog, but a complementary luciferase was identified via Rosetta-guided enzyme design. The unique enzyme–substrate pair is red-shifted compared to well-known bioluminescent tools. The probe set is also orthogonal to other luciferase-luciferin probes and can be used for multicomponent imaging. Four substrate-resolved luciferases were imaged in a single session. Collectively, this work provides the first example of Rosetta-guided design in engineering bioluminescent tools and expands the scope of orthogonal imaging probes.

Near-infrared probes were developed from coumarin-modified luciferins and engineered luciferases, enabling facile multiplexed bioluminescence imaging.

Engineered luciferases and luciferins have dramatically expanded the scope of bioluminescence imaging in recent years. Multicomponent tracking remains challenging, though, due to a lack of streamlined methods to visualize combinations of bioluminescent reporters. Here we report a strategy for rapid, multiplexed imaging with a wide range of luciferases and luciferins. Sequential addition of orthogonal luciferins, followed by substrate unmixing, enabled facile detection of multiple luciferases in vitro and in vivo. Multicomponent imaging in mice was also achieved on the minutes-to-hours time scale, a vast improvement over conventional protocols..