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Bioluminescence has long been used to image biological processes in vivo. This technology features luciferase enzymes and luciferin small molecules that produce visible light. Bioluminescent photons can be detected in tissues and live organisms, enabling sensitive and noninvasive readouts on physiological function. Traditional applications have focused on tracking cells and gene expression patterns, but new probes are pushing the frontiers of what can be visualized. The past few years have also seen the merger of bioluminescence with optogenetic platforms. Luciferase-luciferin reactions can drive light-activatable proteins, ultimately triggering signal transduction and other downstream events. This review highlights these and other recent advances in bioluminescence technology, with an emphasis on tool development. We showcase how new luciferins and engineered luciferases are expanding the scope of optical imaging. We also highlight how bioluminescent systems are being leveraged not just for sensing-but also controlling-biological processes.

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]