UC Berkeley

True molecular understanding of cellular and chemical biology requires the characterization of cellular structures with high spatial resolution in living cells. Over the past two decades, super-resolution microscopy (also called ‘nanoscopy’) has emerged as a powerful tool for the visualization of cellular structures with nanoscale resolution. The efficiency of nanoscopy; however, is limited by the fuel—the chemical tools that support the vast lists of requirements for stimulated emission depletion (STED) and single molecule localization microcopy (SMLM). This dissertation describes worked pursued during my graduate degree towards the development of new and unique chemical tools to expand the capabilities of live cell nanoscopy and accordingly advance cell and chemical biology. Chapter 1 provides an introduction to and in-depth discussion of relevant concepts (e.g., nanoscopy, spontaneously blinking fluorophores), ultimately concluding with an overview and analysis of a class of dual component chemical tools developed by our lab termed high density environmentally sensitive (HIDE) probes. Chapter 2 describes the application of HIDE probes to multicolor imaging via simultaneous development of a mutually orthogonal bioorthogonal labeling platform and development of a novel stimulated emission depletion (STED)-compatible rhodamine fluorophore. Chapter 3 describes the development of novel bright and near-IR emitting spontaneously blinking fluorophores for two-color live cell single molecule localization microscopy (SMLM) without chemical additives and with only a single 642 nm laser. Chapter 4 briefly summarized a series of projects that were designed, but not executed during my time in the lab.