Rapid Multicomponent Bioluminescence Imaging via Substrate Unmixing

Abstract

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..

Coumarin luciferins and mutant luciferases for robust multi-component bioluminescence imaging

“… 29 Substrate unmixing and image processing algorithms can rapidly detect unique enzyme-substrate pairings within complex mixtures. 31,70 Multiple classes of orthogonal probes are available for multicomponent imaging via these methods, but only a few exhibit the necessary optical properties (NIR bioluminescence) for sensitive imaging.…”

“…Pecan was particularly attractive for orthogonal probe development as it has been previously used in vivo. 29,31 When CouLuc-1-NMe 2 was incubated with puried Pecan, more intense light emission (77-fold) was observed compared to Fluc (Fig. S12 †).…”

“…[26][27][28] A handful of such orthogonal probes have been co-opted for dual imaging in vivo, but applications in deep tissue remain challenging. 11,[29][30][31] This is due to a shortage of luciferins with both sufficient red emission and distinct molecular architectures. Candidate luciferins must also be sufficiently bright, bioavailable, and easy to synthesizecriteria that few existing probes meet.…”

Multicomponent Bioluminescence Imaging with Naphthylamino Luciferins

“…Selectivity between orthogonal luciferase‐luciferin pairs has been historically achieved via mutual exclusion: “unmatched” luciferins and luciferases do not interact. We have recently found, though, that perfect selectivity among the probes is not required for multi‐component imaging [11,12] . Rather, unique patterns of light emission are sufficient.…”

“…Rather, unique patterns of light emission are sufficient. If the dimmer substrate is imaged first, the change in brightness when the second substrate is added allows for two unique luciferases to be resolved (Figure 1B) [11] . Multi‐component imaging via this approach still relies on diverse substrates, though, as distinct emission profiles are required.…”