Bioluminescence is a sensitive technique for imaging biological features over time. While versatile, the modality has been challenging to employ for simultaneous, multi-component tracking due to a lack of resolvable luciferase-luciferin pairs. An expanded bioluminescent toolkit is necessary for examining complex cellular interactions. Toward this end, we and others have been developing orthogonal bioluminescent probes for multiplexed imaging. These tools comprise engineered luciferase enzymes that preferentially process chemically modified luciferins. While successful, the approach is quite time intensive. Many of the luciferin analogs are not readily available and require lengthy syntheses. Extensive enzyme engineering is also typically required to generate complementary luciferases. In this work, we report an alternative method for multiplexed bioluminescence imaging that leverages already-orthogonal caged luciferins and established enzymes. The masked luciferins do not undergo a light-emitting reaction until uncaged by complementary enzymes. Three caged luciferin/uncaging enzyme pairs were shown to be orthogonal to one another in vitro. The resulting bioluminescent signals were readily unmixed using a custom linear unmixing algorithm. The caged luciferins were then applied to multiplexed cellular imaging. Cells were engineered to express unique uncaging enzymes along with firefly luciferase. Each population could be readily differentiated upon sequential addition of the corresponding caged luciferins. Three-component imaging was possible on the minutes time scale. Collectively, this work establishes a user-friendly platform for rapid, multiplexed bioluminescence imaging.

Caged luciferins enable rapid multi-component bioluminescence imaging

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.