Direct wavefront sensing and adaptive optics to enable two-photon imaging axons and spines throughout all of cortex

主要研究者（最后一名、第一名、中间名）：克莱菲尔德，大卫 项目摘要 双光子激光扫描显微镜是不可或缺的成像的结构和功能， 哺乳动物大脑的亚细胞分辨率。然而，分辨率和效率随着组织而降低 深度作为散射和光学像差的结果。自适应光学可以通过以下方式改善多光子成像： 将失真合成到激发光束的波前，其补偿在激发光束中的像差。 由组织产生的波前。我们的系统利用自适应光学，使调查人员能够探测 亚细胞动力学在个别突触沿着整个深度的皮层。这是一个重要的进步，特别是 因为第5层和第6层皮层输出神经元位于大脑表面深处。许多当代研究在 神经影像学界受到当前不能记录输出内的突触动力学的限制 皮层区域，例如，层5，与主输入层内相反，即，第四层，中间层 水平，例如，2/3层。我们将消除这一限制，从而开辟一个新的子领域，在体内研究 皮质输出的亚细胞决定因素。 我们提出的工作纳入了“良好的工程实践”，在我们目前的自适应光学设计 双光子显微镜设计。我们将发布准确的计划和施工细节，以使其他 实验室复制这个系统。我们将进一步教育神经影像学界的原则， 自适应光学以及基于自适应光学的双光子显微镜的设计和应用。这种努力包括 加州大学圣地亚哥分校的研讨会。在建议拨款期内，我们会继续推动 自适应光学双光子系统和更新和扩大我们的用户群。拟议的新方向包括 快速聚焦移动和像差校正，用于在间隔为As的平面上的衍射限制聚焦 高达300 µm，并结合共振扫描仪进行快速细胞成像。最后，我们将形成一个 用户之间的团队努力，并结合团队的反馈，将自适应光学扩展到新的领域， 在神经科学中的探索。

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.