### Abstract

? DESCRIPTION (provided by applicant): Despite the growing availability of optical markers of neuronal activity, as well as genetic tools for optical manipulation, current optical microscopy techniques for imaging the intact brain at cellular resolution have approached their limits, particularly in terms of 3D volumetric imaging speeds. The brain and nervous system is inherently 3D, with cortical layers playing specific roles in information processing. Small organisms such as Drosophila melanogaster (fruit fly), Danio rerio (zebrafish) and Caenorhabditis elegans, have become valuable platforms for neuroscience research and genetic manipulation, and offer the chance to capture the entire nervous system of a complete, behaving organism. However, for both rodent brain and small organism microscopy, current techniques are limited to slow volumetric imaging rates, or single-plane acquisition. We recently developed a transformative new approach to high speed 3D microscopy called Swept, Confocally-Aligned Planar Excitation (SCAPE) microscopy. SCAPE was conceived as a way to dramatically improve volumetric imaging speeds, while maintaining a simple optical layout and image acquisition geometry. SCAPE is a hybrid between light-sheet microscopy and laser scanning confocal which overcomes the major speed barriers of both techniques. Recently published in Nature Photonics, SCAPE can image at volume rates 10-100 x faster than laser scanning microscopy or fast light-sheet imaging. We have demonstrated imaging of cellular-level structure and function in both the awake, behaving rodent brain and freely moving Drosophila melanogaster larvae at 10-20 volumes per second (VPS) over large fields of view. A further feature of SCAPE is its simple, single, stationary objective, permitting 3D imaging with no motion at the sample, making it well suited for integration with pattered optogenetic manipulation of cells during high-speed 3D imaging. Having achieved proof of concept' we now wish to develop SCAPE into a tool for routine use by neuroscientists working in both small organisms, for in-toto imaging of cellular activity and behavior, and in awake, behaving mouse brain. The former will be achieved through development and translation of an improved beta prototypes 1P-SCAPE' system, with development of user friendly acquisition software, data handling and analysis platforms, and ultimately its deployment and support for use in studies of somatosensory integration in adult and larval Drosophila. For mouse brain imaging, we propose to test the limits of SCAPE by exploring two- photon implementation (2P-SCAPE), which will afford deeper penetration imaging into scattering tissues such as the rodent brain.