PROJECT SUMMARY / ABSTRACT The development of optical systems that are able to record calcium dynamics from large networks of somas, axons, and dendrites in freely behaving animals is critical to understanding their functional roles in behavior. While multi-photon miniature microscopes have been developed and presented in the literature, they are either unable to effectively resolve cellular projections or are prohibitively limited in other ways, making their direct application to neuroscience questions difficult. In order to overcome these challenges, I propose the development of a novel miniature multiphoton microscope which is able to resolve submicron cellular features across large, 700um fields of view in freely behaving animals. Doing so necessitates the design and fabrication of custom objective lenses, an innovative optical geometry, highly-tuned scanner control signals, and custom relay lenses. Once assembled, the proposed system will offer new capabilities through technical advances, resulting in an ideally suited system for neuroscience. In Aim 1, the components of the microscope will be individually developed and tested before coming together as a complete system. The custom objective lenses, scanner hardware / control algorithms, and relay lenses will be realized, and individually tested before the microscope is assembled. Aim 2 is centered on validation and use of the technique both in controlled and experimental conditions. First, sub-diffraction fluorescent beads will be used to measure system performance and PSF, before thick, fluorescently labeled tissue-slices are imaged. Once validated, head-fixed imaging experiments will be conducted in transgenic animals expressing fluorescent calcium indicators, and the dynamics will be measured and analyzed. Lastly, animals will be imaged during free behavior in a social interaction task, to investigate thalamic projection dynamics in the anterior cingulate cortex. Aim 3 is focused on the optimization of hardware, and the creation of new subsystems to adapt existing 2P microscopes in labs to conduct miniature 2P microscopy in an efficient and cost-effective manner. Aim 3 also details extensive dissemination of all key information central to the creation and use of the developed micro- scope, following the UCLA miniscope project’s approach. Mechanical design files, analysis / control software, PCB manufacture files, along with instructional videos on alignment and use of the system during experimenta- tion, will be made open-access such that the research community can access and adopt the designed technology for conducting critical experiments central to neuroscience.