UC Berkeley

Single-molecule and super-resolution microscopy have transformed biological imaging by enabling nanoscale visualization of structures that are inaccessible to conventional diffraction-limited microscopy. However, many biological processes are not defined by structure alone. They evolve over extended time scales, depend on heterogeneous chemical microenvironments, and are regulated by dynamic molecular motion. In this dissertation, I develop and apply functional single- molecule and super-resolution microscopy approaches to investigate biological systems across these temporal, chemical, and dynamic dimensions. First, I introduce a simple oil-sealing strategy for long-term single-molecule and super- resolution microscopy. STORM imaging is fundamentally limited by fluorophore photobleaching and oxygen-dependent degradation of imaging buffer performance. By forming a physical barrier at the air–liquid interface, mineral oil sealing suppresses oxygen diffusion into the imaging buffer, prolongs the effectiveness of enzymatic oxygen scavenging systems, and extends STORM imaging from less than 6 h to more than 24 h. This method enables sustained two-color super- resolution imaging with minimal structural degradation and allows slow biological processes, including Aβ42 aggregation and insulin-degrading enzyme-mediated degradation, to be observed at the nanoscale. Second, I apply spectrally resolved STORM to map stage-specific chemical changes during Aβ42 aggregation, a central process in Alzheimer’s disease pathology. By combining Nile Red, an environment-sensitive hydrophobicity probe, with CRANAD-2, a β-sheet-selective amyloid probe, I resolve the chemical microenvironments of Aβ42 assemblies with single-aggregate resolution. Nile Red spectral shifts reveal stage-dependent changes in hydrophobicity and polarity from monomers to oligomers and fibrils, while CRANAD-2 identifies ordered fibrillar aggregates.1 Two-color and sequential labeling experiments demonstrate that this dual-probe strategy distinguishes aggregate morphology from local chemical environment and reveals nanoscale heterogeneity within individual aggregates. These findings provide a stage-resolved chemical portrait of Aβ42 aggregation and suggest that exposed hydrophobicity in oligomeric species may contribute to disease-relevant toxicity. Third, I combine multicolor STORM with live-cell single-molecule displacement mapping to investigate the nanoscale architecture and dynamics of the septin cytoskeleton. In COS-7 cells, septin 7 forms stress-fiber subtype-specific structures, including double-layered arrays along ventral actin stress fibers and peripheral localization along transverse arcs. Septin filaments are excluded from focal adhesions and associate with acetylated microtubules primarily in actin-poor regions, revealing spatial partitioning between cytoskeletal networks. Disruption of actin polymerization with Latrunculin A reorganizes linear septin filaments into ring-like assemblies and increases septin molecular mobility, whereas washout restores linear architecture and reduces mobility. These results show that septin organization and dynamics are reversibly regulated by actin network integrity. Together, these studies demonstrate how functional super-resolution microscopy can connect nanoscale structure, chemical environment, molecular motion, and biological function. By extending imaging duration, enabling chemical mapping of protein aggregation, and linking cytoskeletal architecture to molecular dynamics, this dissertation establishes a framework for studying complex biological systems beyond static structural imaging.