Expanding the bioluminescent tool box for imaging macroscopic cell networks

Optical imaging strategies have revolutionized our understanding of living systems. Among the most popular techniques for imaging in whole tissues and organisms is bioluminescence. The most widely used bioluminescent system comprises the luciferase enzyme from the firefly (Fluc) and its small molecule substrate, D-luciferin (D-luc). When introduced into cells, these components produce photons that can be captured by sensitive cameras. Since mammalian cells and tissues produce little to no endogenous light, bioluminescence imaging (BLI) is well suited for use in whole organisms. Despite its sensitivity and broad dynamic range, BLI has largely been limited to imaging one cell type at a time. Multicellular networks have been refractory to BLI owing to lack of distinct luciferase-luciferin probes. BLI also lacks adequate spatial resolution to directly visualize cell-to-cell contacts. To address these voids, my thesis work focused on: (1) developing “caged” luciferins to report on cell-cell interactions and (2) creating novel bioluminescent pairs via luciferin analog synthesis and luciferase enzyme engineering. Collectively, the tools developed from my thesis work will provide a better understanding of complex interactions in vivo