Caged luciferins enable rapid multicomponent bioluminescence imaging.

Bioluminescence is a sensitive technique for imaging biological features over time. Historically, though, the modality has been challenging to employ for multiplexed tracking due to a lack of resolvable luciferase–luciferin pairs. Recent years have seen the development of numerous orthogonal probes for multi‐parameter imaging. While successful, generating such tools often requires complex syntheses and lengthy enzyme evolution campaigns. This work showcases an alternative strategy for multiplexed bioluminescence that takes advantage of already‐orthogonal caged luciferins and established uncaging enzymes. These probes generate unique bioluminescent signals that can be distinguished via a linear unmixing algorithm. Caged luciferins enabled two‐ and three‐component imaging on the minutes time scale. We further showed that the tools can be used in conjunction with endogenous enzymes for multiplexed studies. Collectively, this approach lowers the barrier to multicomponent bioluminescence imaging and can be readily adopted by the broader community. [ABSTRACT FROM AUTHOR]