Advanced Nonlinear Imaging

Core 3. Abstract Nonlinear Imaging Two-photon laser scanning microscopy (TPLSM) has enabled in vivo studies on the function of neuronal circuitry. However, the penetrating depth of traditional TPLSM is limited to around 400 µm in brain tissue due to scattering and aberration, which limits its application in deep cortical layers and highly scattering white matter. The goal of the parent U19 proposal is to understand medullary circuits and how they can be autonomous but subject to high-level activation and temporal organization from motor cortex and superior colliculus. This requires using our Direct Sensing Adaptive Optics TPLSM (AO-TPLSM) to perform diffraction-limited resolution imaging of scattering and/or deep structures. This technology is fundamental to the goals of Project 1 to image through the scattering tissue of brainstem and through the upper layers of superior colliculus to reach the deep motor layers of superior colliculus. This Core will assist with the transfer of adaptive optics two-photon microscope technology from UCSD to MIT and to Johns Hopkins. It holds the promise to enhance discovery through Project 3, where AO-TPLSM is essential to image scattering tissue such as brainstem (MIT), and Project 5, where AO-TPLSM is essential to image cellular and synaptic functions in deep regions of cortex (Johns Hopkins). The Core will maintain travel funds for Liu. The Core will arrange for the purchase of components for this custom microscope, from which subassemblies will be constructed at UCSD. The MIT and Hopkins performance sites are responsible for upgrading and maintaining their own appropriate fiber 100-fs laser. Our approach will achieve uniformity and consistency in technology across all three performance sites using AO-TPLSM. The use of identical and, as much as possible, standard commercial components will maximize reliability, optimize debugging, and minimize retraining of junior investigators that collaborate across sites.