ADVANCED INTERFEROMETRIC SUPER-RESOLUTION TECHNIQUES FOR VISUALIZING AND QUANTIFYING SYNAPTIC PLASTICITY IN MOUSE BRAIN MODELS

This dissertation presents the development and application of advanced imaging systems for ultrahigh-resolution visualization of synaptic structures in mouse brain tissues. The work focuses on optimizing the 4Pi single-molecule switching nanoscopy system to achieve sub-15 nm resolution imaging, involving precise instrument assembly, phase delay and dispersion control, and aberration calibration to approach theoretical imaging conditions. Building on this foundation, I developed 4Pi-BRAINSPOT, an integrated imaging platform that combines tissue clearing, light-sheet illumination, and in-situ point spread function retrieval. This integration overcomes challenges posed by tissue opacity and scattering, enabling high-precision single-molecule localization in brain sections up to 50 μm thick. The advancements in imaging depth and clarity significantly enhance the detection of fine molecular details, particularly within neurological studies. 4Pi-BRAINSPOT facilitates the detailed visualization of nanoscale structures, such as dendritic spines and synaptic proteins, while preserving the integrity of neural circuits, thus providing unprecedented insights into the brain's architecture. To demonstrate the capabilities of 4Pi-BRAINSPOT, I investigated synaptic and network alterations in response to experience-dependent neural activity in mouse models. Specifically, I examined changes in dendritic spine plasticity in the visual cortex and correlated it to functional synaptic connectivity, contributing to an improved understanding of the molecular basis of synaptic dysfunction and connectivity impairments associated with neurodevelopmental disorders. Overall, this work provides powerful tools for visualizing and quantifying synaptic architecture in native tissue environments, significantly advancing our knowledge of synaptic function, plasticity, and the molecular mechanisms underlying complex neurological conditions, including neurodevelopmental and neurodegenerative diseases.