UC Irvine

Cellular and molecular interactions drive almost all aspects of human biology. Visualizing these interactions can provide critical insight into disease progression, immune function, and cognitive behavior. However, most imaging technologies are not well poised to monitor intricate biological processes, especially in deeper tissues. Existing methods have historically utilized fluorescent proteins and small molecule fluorophores. These imaging agents are typically difficult to apply in thick tissue due to the need for an invasive, external excitation source. Furthermore, due to their large size, they can often perturb native biological processes, rendering them ill-suited for monitoring small molecular species. As a result, we need new minimally invasive and non-perturbing imaging modalities to effectively probe native cellular and molecular processes in relevant environments. In Part I of my thesis work, I leverage bioluminescence as a non-invasive imaging modality. Bioluminescence is a naturally occurring processes in which light is produced via an enzymatic (luciferase) catalyzed oxidation of a small molecule substrate (a luciferin). Bioluminescence imaging (BLI) has long been used for monitoring biological processes in living organisms, but suffers from a lack of sufficiently bright and red-shifted probes for applications in deep tissue. This section of my dissertation aims to address this limitation by developing and optimizing novel, red-shifted luciferase-luciferin pairs for in vivo imaging applications. In Chapter 1, I summarize recent advances in luciferin design for improved BLI, with a focus on whole-organism imaging. In Chapter 2, I report the synthesis and characterization of a minimally-modified benzoxadiazole luciferin. This analog displayed environmentally-sensitive and red-shifted emission with very minimal enzyme engineering, presenting an attractive design element than can be harnessed for future luciferin development. In Chapter 3, I describe a collaborative effort to engineer a luciferase enzyme to yield bright and NIR emission with a coumarin-luciferin. I also detail my work to optimize the formulation of this probe for improved in vivo performance. Lastly, in Chapter 4, I highlight my efforts to expand the palette of bright and red coumarin-luciferins using enzyme evolution. This work results in a new BLI probe that can be applied for multicomponent imaging in deep tissues. Overall, this work will help to facilitate new discoveries relevant to cancer biology, immune function, and a multitude of other processes in complex in vivo environments. In Part 2 of my thesis work, I harness vibrational spectroscopy as a minimally perturbing imaging modality. This technique relies on molecular bond vibrations, either from naturally occurring bonds, or small bioorthogonal vibrational tags. Vibrational tags have been widely used for monitoring intricate biochemical processes and tracking small molecules, but they suffer from reduced sensitivity compared to conventional imaging techniques, such as fluorescence. This section of my dissertation aims to address this disparity by engineering a palette of new and resolvable vibrational tags with increased sensitivity for biological imaging. In Chapter 5, I summarize recent advances in bioorthogonal vibrational tags and their imaging applications in chemical biology. In Chapter 6, I report the design and characterization of a series of sensitive alkyne-based vibrational reporters for multicomponent Raman and IR imaging. This collaborative work leverages key molecular design elements including vibrational insulation and enhancement. We show that several probes were robust metabolic reporters in cellular environments. Overall, this work will provide a platform for high-throughput, molecular interrogation of biological processes ranging from disease mechanisms to cell signaling pathways.