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Cells engineered to express bioluminescence generate photons through a biochemical reaction catalyzed by a luciferase enzyme and a luciferin substrate. Conventional bioluminescent imaging approaches require that the luciferin substrate be added to the cell externally since it cannot be generated intracellularly. Thus, these cells remain “dark,” only emitting their bioluminescent signal in concert with the extracellular addition of the chemical substrate. Autobioluminescence represents an alternative signaling strategy whereby a synthetic bacterial luciferase, optimized for expression in eukaryotic cells, is used to sustain continuous photon emission without the necessary external addition of a substrate to drive reaction kinetics. This substrate-independent generation of light thereby allows autobioluminescent cells to be imaged at any longitudinal time scale desired to generate real-time metabolic data under …