Project Summary/ Abstract Nervous system disease may yield devastating impact on cognition, emotion, or sensorimotor function. Gene delivery using adeno-associated virus (AAV) vectors has demonstrated immense potential for treatment of congenital and acquired diseases impacting the central and peripheral nervous systems. Advancing mechanistic understanding of vector uptake and trafficking within nervous system cells would inform viral vector capsid design. Heretofore, visualizing viral vector cellular transduction in vivo has been hampered by a lack of optimal means for resolving nanoscale particles in thick tissues. Imaging viral particles whose dimensions are below the ~250 nm diffraction limit resolution of light microscopy is typically achieved using electron microscopy, a resource-intensive technique incompatible with life. There is a critical need to develop intravital imaging techniques that enable high-speed and deep nanoscale imaging of living systems. Two- photon excitation (2PE) microscopy is a powerful technique for intravital imaging of the nervous system that employs ultrafast near-infrared laser light capable of penetrating deep into tissues. Though the technique enables intravital imaging of thick tissues, the achievable resolution and image quality of 2PE microscopy is inadequate for study of nanoscale processes. 2PE microscopy may be paired with stimulated emission depletion (STED) techniques to enable resolution of nanoscale fluorescent-tagged targets. To enhance imaging depth and signal-to-background, 2PE may be achieved via spatiotemporal overlap of two ultrafast lasers of different wavelengths in a process termed non-degenerate 2PE. Heretofore, wide dissemination of 2PE-STED and non-degenerate 2PE microscopy techniques have been hampered by the cost and complexity associated with synchronization and alignment of two ultrafast laser sources. Herein, we propose to develop efficient super-resolved multiphoton microscopy approaches to enhance spatiotemporal resolution and imaging depth within living tissues by employing a single dual-output commercial ultrafast laser. Once developed, we will employ these novel imaging platforms to study intracellular trafficking of single AAV particles within cells of the murine nervous system. In Aim 1, the dual-output ultrafast laser will be utilized to achieve 2PE-STED microscopy via pulsed depletion and employed to image AAV trafficking in cultured Schwann cells and primary sensory neurons of murine dorsal root ganglia. In Aim 2, the dual-output ultrafast laser will be utilized to achieve non-degenerate 2PE and paired with a continuous wave depletion beam for deep super-resolution imaging of AAV trafficking in corneal Schwann cells and sensory neurons in live anesthetized mice. A liquid lens will be employed to enhance volumetric imaging speed. If successful, this work carries potential to advance understanding of viral vector transduction of nervous system cells by identifying intracellular...