Principal Investigator (Last, first, middle):KLEINFELD, DAVID Project summary Two-photon laser scanning microscopy is indispensable for imaging the structure and function of the mammalian brain with subcellular resolution. However, the resolution and efficiency decreases with tissue depth as a result of scattering and optical aberrations. Adaptive optics can improve multi-photon imaging by synthesizing a distortion to the wavefront of the excitatory beam that compensates for aberrations in the wavefront that are created by the tissue. Our system utilized adaptive optics to enable investigators to probe subcellular dynamics in individual synapses along the full depth of cortex. This is a crucial advance, particularly as layer 5 and 6 cortical output neurons lie deep to the surface of the brain. Many contemporary studies within the neuroimaging community are limited by the current inability to record synaptic dynamics within output regions of cortex, e.g., layer 5, as opposed to within the dominant input layer, i.e., layer 4, and the intermediate levels, e.g., layers 2/3. We will remove this limit and thus open up a new subfield of in vivo studies on subcellular determinates of cortical output. Our proposed work incorporates "good engineering practice" in the design of our current adaptive optics two-photon microscope design. We will disseminate accurate plans and construction details to enable other laboratories to duplicate this system. We will further educate the neuroimaging community on the principles of adaptive optics and the design and utility of adaptive optics-based two-photon microscopes. This effort includes workshops at UC San Diego. Throughout the period of the proposed grant, we will continue to advance the adaptive optics two-photon system and update and expand our user base. Proposed new directions include a rapid shift in focus together with aberration correction for diffraction limited focus over planes separated by as much as 300 µm and the incorporation of a resonant scanner for fast cell-based imaging. Lastly, we will form a team effort among users and incorporate feedback from the team to extend adaptive optics into new areas of inquiry in neuroscience as they arise.