Bioluminescence imaging (BLI) is a powerful tool for in vivo detection of biological events or processes. BLI relies on light production from a chemical reaction, the luciferase-catalyzed oxidation of a small molecule substrate (luciferin). While powerful, BLI has been historically limited in scope. One challenge is imaging more than one population or cellular feature. Selective luciferase-luciferin pairs have been developed to address this limitation. However, the field has largely been stuck at imaging only two populations. Diverse luciferin architectures are required for higher order multiplexing. A second major limitation is that applying bioluminescent pairs in tandem requires long imaging times, so BLI often cannot be used to monitor dynamic multicellular interactions. Finally, multiplexing at the microscale is especially challenging, as bioluminescent tools are often too dim to detect on standard microscopes.
To address these limitations, I have developed new probes and platforms for rapid, multiplexed bioluminescence imaging from the macroscale to the microscale. First, I synthesized a panel of luciferin analogs comprising a naphthalene core and amine substituents. These analogs were robust substrates for the native luciferase in vitro and in cellulo. They could easily be applied in tandem with other structurally distinct luciferins. Next, I developed a platform for rapid imaging of bioluminescent tools. Complex mixture of reporters could be resolved and quantified on the minutes-to-hours time scale, a substantial improvement over conventional approaches. Finally, I worked with collaborators to establish a method for multiplexed bioluminescence imaging at the microscale. This work merged BLI with phasor analysis, a method commonly used to distinguish spectrally similar fluorophores. With the bioluminescent phasor method, we were able to perform multiplexed, continuous imaging of subcellular features and tumor spheroids. Overall, the bioluminescence imaging tools that I have developed will dramatically expand the number of targets that we can image in tandem, and broaden our understanding of biological processes and interactions.