Jewel Ashbrook Qualifier

A central challenge in neuroscience is the inability to chronically observe neuronal structure and activity in deep brain regions with sufficient spatial resolution to resolve subcellular features. Dendritic spines are small neuronal protrusions that mediate over 90% of excitatory synaptic transmission, but their small size (typically <1 μm) makes them technically difficult to image. Electron microscopy has revealed much about spine structure and function but requires fixed tissue, precluding observation of dynamic changes. Two-photon fluorescence microscopy enables dynamic imaging of spines but its diffraction-limited resolution (~400 nm) makes it difficult to quantify subtle changes in spine morphology.

We propose a new super-resolution optical microscopy technique for imaging dendritic spines in intact brain tissue, aiming to reach tens-of-nanometer resolution several hundred microns deep. The approach uses non-degenerate two-photon mixed-mode excitation: subtracting images acquired from different mode combinations narrows the effective point spread function, with a predicted fivefold improvement in resolution over standard two-photon microscopy. This project aims to establish a new approach for studying dendritic spine structure and function in deep brain tissue, enabling longitudinal studies of synaptic plasticity and disease progression not currently feasible.