UC Irvine

Bioluminescence is a sensitive technique for imaging biological features over time. This technique relies on the oxidation of a small molecule luciferin by a luciferase enzyme to produce light. Historically, though, the modality has been challenging to employ for multiplexed tracking due to a lack of resolvable bioluminescence tools. This is largely due to a limited number of tools that can be used in tandem for simultaneous detection. Additionally, many of the multiplexing tools that have been generated have required extensive enzyme engineering, chemical synthesis, and screening to identify orthogonal luciferin–luciferase pairs. In order to visualize complex multicellular processes in vivo, more accessible multiplexing tools need to be developed. A multiplexed readout is needed for visualizing complex biological processes involving multiple cell types, such as immune cell function. Further understanding of these cellular networks could provide a system for evaluating the effects of potential therapeutics. To address these limitations, I applied chemical tools for visualizing cellular processes in a multiplexed readout. In Chapter 1, I introduce bioluminescence imaging probes and how they have been engineered to provide a variety of tools for multiplexed imaging. In Chapter 2, I describe my efforts in applying caged probes for a multicomponent imaging strategy. The caged luciferins enabled a readout on individual and mixed cell populations. Additionally, this imaging strategy was amenable for reading out on endogenous enzymatic activity, enabling future work on detecting endogenous cell populations in complex biological environments. In Chapter 3, I report on gene expression probes for imaging immune cell processes. I applied our bioluminescence spectral phasor analysis for multiplexing macrophage polarization reporters for a dynamic readout. In Chapter 4, I detail further application into model systems for visualizing biological processes, such as endogenous macrophage activation and circadian rhythm in cells. Specialized cell lines were generated for translatable models for future in vivo experiments. Overall, the imaging strategies I have developed and applied towards tracking multicellular interactions will enable a deeper understanding of biological processes in complex environments.