Rapid, multiplexed bioluminescence imaging at all length scales
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). No excitation light is required, so bioluminescence is a bio-friendly and sensitive method for imaging in tissues. While powerful, BLI has been historically limited in scope. One challenge is imaging multiple populations or cellular features in a single sample. 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 typically 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 and exhibit broad, overlapping spectra. Collectively, these limitations prevent efforts to visualize complex networks at all length scales, such as interactions between tumors and immune cells.
To address these limitations, I have developed new probes and platforms for rapid, multiplexed bioluminescence imaging from the macroscale to the microscale. In Chapter 1, I review recent advances in small molecules that have been developed for multi-component BLI. In Chapter 2, I describe my efforts to synthesize 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 further be used in tandem with other structurally distinct luciferins. In Chapter 3, I report on a method to classify a collection of luciferins using a machine learning method, hierarchical clustering. Sources of luciferase selectivity were identified for each compound class; these data provided starting points for new orthogonal enzyme designs. In Chapter 4, I detail a platform for rapid imaging of bioluminescent tools. Complex mixtures of reporters could be resolved and quantified on the minutes-to-hours time scale, a substantial improvement over conventional approaches. In Chapter 5, I describe a collaborative effort 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. Finally, in Chapter 6, I report on an expanded toolkit of reporters that can be applied to bioluminescent phasor analysis. These probes were applied to imaging in complex environments, including whole organisms. 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.