UC Berkeley

Conventional fluorescence microscopy, despite its high sensitivity, lacks resolution at a scale comparable to biomacromolecules due to the diffraction of light. Single-molecule-based super-resolution microscopy (SRM), developed in the past 15 years, has enabled researchers to visualize the structural organization of the cell at molecular resolution with high specificity. Nevertheless, the power of single-molecule measurement in resolving inter-molecular heterogeneities has not been fully explored by the SRM field. There is plenty of room for expanding the scope of SRM to access measurement domains beyond biomolecular structures, such as the heterogeneity of physicochemical properties within the cell, a conceptual framework named “functional super-resolution microscopy” (f-SRM). This dissertation describes the efforts by the author and colleagues in developing f-SRM for living cells and applying f-SRM to discover previously unknown physicochemical heterogeneities within the cell. Part I of this dissertation describes the design, development, and application of f-SRM, which has revealed novel nanoscale properties of the cell invisible to conventional SRM. Specifically, I elaborate on the experiments of fluorescence emission spectrum-encoded single-molecule measurements of chemical polarity and molecular diffusivity at nanoscale resolution. Part II concerns a fluorescence excitation-based strategy for functional imaging, which led to the unexpected discovery of a tubular ER-Golgi intermediate compartment (t-ERGIC) in transporting a specific group of soluble proteins. In-depth biochemistry and cell biology analyses elucidated the biogenesis mechanism of the t-ERGIC involving the cargo receptor.