Optimization and dissemination of non-linear Acousto-Optic Lens two-photon microscopy for high speed multiscale 3D imaging

项目总结 为了了解大脑的功能，确定信息在神经元群体中是如何表现的是至关重要的。 活动，以及当它流经微电路时，单个神经元是如何转化的。​双光子(2P) 显微镜是这方面的核心工具，因为它能够以高空间分辨率监测神经元的活动。 在行为动物​的脑组织深处。然而，​的​t​传统的时间分辨率 基于检流计的2P显微镜严重限制了对3D神经元电路中快速信号的测量。 声光透镜(AOL)显微镜，可实现感兴趣区域的快速聚焦和选择性成像 分布在成像体积内，大大提高了3D 2P显微镜的时间分辨率。 但目前依赖于​线性声学驱动波形的美国在线显微镜受到限制，使 它们是有效的​监测结构中的信号投射在Z维。​焦点中的每个变化 需要24​？​S‘死区时间’来重新填充aol光圈，并且连续行扫描仅限于选定的 X-Y焦平面，将3D树状树的成像速率限制在几赫兹，而不是所需的100-1000赫兹 用于监测神经递质报告器和电压指示器。​这个项目的主要目标是优化和 传播​非线性​美国在线3D显微镜，这是我们发明的一种技术，通过以下方式克服这些限制 实现在X、Y和Z方向上的任意方向的超快行扫描(最高可达40 kHz)。 我们已经演示了具有实时校正脑运动的​非线性AOL 2P显微镜的原型 这种技术对清醒行为神经回路的高速多尺度3D成像的性能 动物。我们将在这些结果的基础上，通过优化​非线性人工耳机显微镜来成像整个3D树突 树木和周围的神经元数量以前所未有的速度增长。我们将开发它的变种 树枝状“树状成像”方法，可提供低空间分辨率、超高速3D成像(高达1 KHZ)，结合了​非线性​光学光学的快速扫描和自适应光学特性。我们还将延长 实时的现场可编程门阵列分析应用于我们的闭环系统3D运动校正，以实现“注意力成像” 树状树或回路的活动区域在更高的时空上被快速检测和成像 决议。这些应用程序​将提供监控电压所需的时间分辨率 首次在清醒的动物中发现了锥体细胞的完整3D树状突起。此外，注意力成像将 使神经递质释放能够以高时空分辨率进行映射。低成本传播这一点 强大的新技术将通过提供美国实验室和具有紧凑型​非线性的成像设备来实现 AOL模块将被添加到他们现有的传统2P显微镜上。通过扩展我们的开源 显微镜图形用户界面软件，用NWB2标准化数据格式，精炼自动分析管道， 我们还将提供可靠的用户友好型显微镜控制和半自动数据分析框架 合作者在一系列不同的神经回路上进行实验。

PROJECT SUMMARY To understand brain function, it is essential to identify how information is represented in neuronal population activity and how it is transformed by individual neurons as it flows through microcircuits. ​Two-photon (2P) microscopy is a core tool for this because it enables neuronal activity to be monitored at high spatial resolution deep within brain tissue in behaving animals​. ​However, ​t​he temporal resolution of conventional galvanometer-based 2P microscopy severely limits measurements of fast signaling in 3D neuronal circuits. Acousto-optic lens (AOL) microscopy, which enables fast focussing and selective imaging of regions of interest distributed within the imaging volume, has substantially improved the temporal resolution of 3D 2P microscopy. But current AOL microscopes, which rely on ​linear acoustic drive waveforms, suffer from limitations that make them ine​fficient to monitor signaling in structures that project in the Z dimension. ​Each change in the focus requires a 24 ​µ​s ‘dead time’ to refill the AOL aperture and continuous line scanning is restricted to the selected X-Y focal plane, limiting imaging rates for 3D dendritic trees to a few Hz, rather than the 100-1000 Hz required for monitoring neurotransmitter reporters and voltage indicators. ​The main aim of this project is to optimize and disseminate ​nonlinear ​AOL 3D microscopy, a technology we have invented to overcome these limitations by enabling ultra-fast line scanning (up to 40 kHz) in any arbitrary direction in X, Y and Z. By developing a prototype ​nonlinear AOL 2P microscope with real time correction of brain movement, we have demonstrated the performance of this technology for high-speed multiscale 3D imaging of neural circuits in awake behaving animals. We will build on these results by optimizing ​nonlinear AOL microscopy for imaging entire 3D dendritic trees and the surrounding neuronal population at unprecedented speeds. We will develop variants of this dendritic ‘arboreal imaging’ approach to provide low spatial resolution, ultra-high-speed 3D imaging (up to 1 kHz) by combining the fast scanning and adaptive optics properties of ​nonlinear ​AOLs. We will also extend the real time FPGA analysis used in our closed loop 3D movement correction to enable ‘attentional imaging’ where active regions of a dendritic tree, or circuit, are rapidly detected and imaged at higher spatio-temporal resolution. These applications ​will provide the temporal resolution required for monitoring voltage across the entire 3D dendritic tree of pyramidal cells in awake animals for the first time. Moreover, attentional imaging will enable neurotransmitter release to be mapped at high spatiotemporal resolution. Low cost dissemination of this powerful new technology will be achieved by providing US labs and an imaging facility with compact ​nonlinear AOL modules that will be added to their existing conventional 2P microscopes. By extending our open source microscope GUI software, standardizing data formats with NWB2 and refining automated analysis pipelines, we will also deliver reliable user-friendly microscope control and a semiautomated data analysis framework for the collaborators to carry out experiments on a range of different neural circuits.