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

首席调查员(最后、第一、中间)：大卫·克莱菲尔德 项目总结 双光子激光扫描显微镜是对细胞的结构和功能进行成像所必不可少的 具有亚细胞分辨率的哺乳动物大脑。然而，分辨率和效率随着组织的变化而降低 由于散射和光学像差而产生的深度。自适应光学可以通过以下方式改进多光子成像 合成对激发光束的波前的失真，以补偿 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 沿着整个皮质深度的个体突触的亚细胞动力学。这是一个至关重要的进步，尤其是 作为第5层和第6层，皮质输出神经元位于大脑表面深处。许多当代研究中 神经成像界目前无法记录输出中的突触动态，这一点受到了限制 皮质区域，例如，层5，而不是在主要输入层，即，层4和中间层内 水平，例如，层2/3。我们将取消这一限制，从而开辟体内研究的新领域 皮质输出的亚细胞决定因素。 我们建议的工作将“良好的工程实践”融入到我们目前的自适应光学设计中。 双光子显微镜的设计。我们将发布准确的计划和施工细节，以使其他 实验室复制这一系统。我们将进一步教育神经成像社区，让他们了解 自适应光学和基于自适应光学的双光子显微镜的设计和应用。这一努力包括 加州大学圣地亚哥分校的研讨会。在建议拨款的整段期间，我们会继续把 自适应光学双光子系统，并更新和扩大我们的用户基础。建议的新方向包括 As分离平面衍射极限焦距的快速移焦和像差校正 高达300微米，并结合了用于快速细胞成像的共振扫描仪。最后，我们将形成一个 用户之间的团队合作，并纳入团队的反馈，以将自适应光纤扩展到 随着神经科学的兴起而产生的问题。

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.