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

项目摘要 为了了解大脑功能，识别信息在神经元群体中的表达是至关重要的 以及当它流过微电路时，它是如何被单个神经元转化的。 双光子（2 P） 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活动 在行为动物的脑组织深处。 然而，传统的时间分辨率 基于电流计的2 P显微镜严重限制了3D神经元回路中快速信号的测量。 声光透镜（AOL）显微镜，可对感兴趣区域进行快速聚焦和选择性成像 分布在成像体积内，大大提高了3D 2 P显微镜的时间分辨率。 但是，目前的AOL显微镜，它依赖于线性声学驱动波形，遭受的限制，使 它们不能有效地监测在Z维度上投射的结构中的信号。 焦点的每一次变化 需要24 µ s的“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树突状树的成像速率限制在几Hz，而不是所需的100-1000 Hz 用于监测神经递质报告器和电压指示器。 该项目的主要目的是优化和 传播非线性AOL 3D显微镜，我们发明了一种技术，以克服这些限制， 能够在X、Y和Z的任意方向上进行超快速线扫描（高达40 kHz）。通过开发一个 原型非线性AOL 2 P显微镜与脑运动的真实的时间校正，我们已经证明 该技术用于清醒状态下神经回路的高速多尺度3D成像的性能 动物我们将建立在这些结果的基础上，通过优化非线性AOL显微镜成像整个3D树突 树木和周围的神经元群体以前所未有的速度。我们将开发出它的变体 树枝状“树状成像”方法，提供低空间分辨率、超高速3D成像（高达1 kHz）的快速扫描和非线性AOL的自适应光学特性相结合。我们还将延长 在我们的闭环3D运动校正中使用了真实的FPGA分析，以实现“注意力成像”， 树突树或电路的活动区域在更高的时空分辨率下被快速检测和成像， 分辨率这些应用将提供监测跨 第一次在清醒的动物中完整的3D树突状细胞树。此外，注意力成像将 使神经递质释放能够以高时空分辨率被映射。低成本的传播， 强大的新技术将通过为美国实验室和成像设备提供紧凑的非线性 AOL模块将被添加到他们现有的传统2 P显微镜。通过扩展我们的开源 显微镜GUI软件，使用NWB 2标准化数据格式并完善自动化分析管道， 我们还将提供可靠的用户友好的显微镜控制和半自动数据分析框架， 合作者对一系列不同的神经回路进行实验。

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.