Abstract: Bioluminescence is a sensitive technique for imaging biological features over time. 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. In this talk, I will first describe my efforts to streamline multicomponent bioluminescence imaging using caged luciferins and established uncaging enzymes. These caged probes generate unique bioluminescent signals that can be distinguished via a linear unmixing algorithm to differentiate unique cell populations. In the second half of my talk, I will describe an alternative multiplexing strategy for monitoring macrophage polarization. I used a palette of luminescent enzymes and spectral phasor analysis to illuminate signature reporters of cell activation. Changes in reporter expression were ultimately analyzed upon macrophage stimulation with various cytokines, enabling single-cell imaging of macrophage polarization over time. Collectively, my work advances bioluminescence imaging strategies and broadens the scope of tools for visualizing cellular processes.