UC Irvine

Optical imaging strategies have revolutionized our ability to study complex biological systems. Being able to observe and track functions across length scales has led to the discovery of critical processes important to human health. However, optical imaging strategies suffer from a limited number of distinguishable probes. Historically, existing methods rely on fluorescent proteins, which can suffer from high signal-to-noise ratios. Additionally, greater multiplexing can make distinguishing many tools a difficult task. To overcome these challenges, we need new imaging probes that are flexible and can be used in tandem to study complex biological processes such as molecular and cellular events. In my thesis work, I harnessed bioluminescence to image cellular and subcellular features with novel methods and probes. Bioluminescence is a naturally occurring processes in which a photon of light is produced via the enzymatic (luciferase) oxidation of a small molecule substrate (a luciferin). Bioluminescence has commonly been used to image single populations of cells at the macroscale, but suffers from the lack of distinguishable tools for studying complex processes. In Chapter 1, I summarize recent advances in bioluminescence-based imaging methods, with a focus on the development of new probes and technologies. In Chapter 2, I describe my work on using a machine learning method to understand the interactions between libraries of luciferins and luciferases. I leveraged this knowledge to identify novel luciferin-luciferase pairs for multicomponent imaging. In Chapter 3, I summarize my work on the development of a novel, red-shifted luciferin comprising a common fluorescent protein chromophore. This novel molecule provided bright, near infrared emission useful for deep tissue imaging. In Chapter 4, I spotlight my work on developing a novel luciferin-luciferase pair using substrate walking paired with directed evolution. I synthesized a new vargulin-inspired luciferin that will become the basis for a directed evolution campaign. In the final Chapter, I describe a tunable bioluminescent tool for imaging dynamic cellular processes. This collaborative work leveraged a fluorogenic protein alongside a luciferase. The resulting construct can change emission spectra in the presence of different small molecule fluorogens. Overall, this work broadens the scope of imaging probes for studying multi-scale biological processes.