UC Berkeley

This dissertation focuses on the design, synthesis and application of fluorogenic enzymatically cleavable fluorophores for biological imaging in genetically targetable cells.Chapter 1 provides an overview of chemigenetic approaches for studying membrane potential in electrically excitable cells. The mechanisms of action, along with the spectral and physical properties of catalytically cleavable and covalently linked chemigenetic indicators, are discussed.Chapter 2 examines improved esterase-cleavable chemigenetic voltage-sensitive fluorophores (VFs) for targeted imaging in cells. Introducing a benzyl linker between the xanthene alcohol of fluorescein and the ester caging group increases in vivo enzyme activity substantially. In cellular systems, switching from mono-sulfonated to di-sulfonated caged VFs had a greater effect on enzyme activity than the benzyl linker.Chapter 3 explores additional enzymatically cleavable chemigenetic indicators for targeted voltage imaging. VFs for β-galactosidase, cellulase, and protease were successfully synthesized. Their physical and spectral properties were characterized.Appendix I presents additional caged fluorophores for assessing enzyme cleavage in mitochondria.Appendix II provides supplementary information on the loading conditions for di-sulfonated VFs in both HEK293T cells and primary hippocampal neurons. Appendix III provides a protocol for imaging catalytically cleavable chemigenetic VFs in mouse brain slice.