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 trafficking
bottlenecks, while illustrating the potential of super-resolution 2PE microscopy techniques to advance
knowledge discovery in biomedicine.