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

用于高速多尺度 3D 成像的非线性声光透镜双光子显微镜的优化和推广

基本信息

批准号：
10005501
负责人：
JESSICA A CARDIN
金额：
$ 46.67万
依托单位：
UNIVERSITY COLLEGE LONDON
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10005501
关键词：
3-Dimensional Acoustics Animals Attention Axon Biological Brain Cells Computer software Data Analyses Dendrites Development Diamond Dimensions Feedback Functional Imaging Funding Generations Hybrids Image Individual Knowledge Lasers Length Maps Measurement Methods Microscope Microscopy Monitor Motion Movement Neurons Neurosciences Neurosciences Research Neurotransmitters Optics Performance Population Property Pyramidal Cells Reporter Resolution Scanning Ships Signal Transduction Speed Spottings Standardization Structure System Technology Testing Three-Dimensional Imaging Time Trees Variant adaptive optics analysis pipeline arbitrary spin automated analysis awake base brain tissue commercialization cost data format data standards data structure experimental study high resolution imaging imaging approach imaging facilities imaging software improved interest lens microscopic imaging nervous system disorder neural circuit neuronal circuitry neurotransmission neurotransmitter release new technology novel open source optical imaging prototype spatiotemporal structured data temporal measurement tool two photon microscopy two-photon ultra high resolution user-friendly voltage

项目摘要

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, the 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 inefficient to monitor signaling in structures that project in the Z dimension. Each change in the focus requires a 24 µs ‘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.

项目概要要了解大脑功能，必须确定信息在神经群体中的表示方式活动及其在流经微电路时如何被单个神经元转化。双光子 (2P) 显微镜是实现这一点的核心工具，因为它能够以高空间分辨率监测神经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检流计的 2P 显微镜严重限制了 3D 神经元回路中快速信号传导的测量。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。但目前的 AOL 显微镜依赖于线性声学驱动波形，存在局限性，使得它们无法有效地监控 Z 维度上投射的结构中的信号。焦点的每次变化。需要 24 µs“死区时间”来重新填充 AOL 孔径，并且连续行扫描仅限于选定的 X-Y 焦平面，将 3D 树突树的成像速率限制为几赫兹，而不是所需的 100-1000 赫兹用于监测神经递质产生和电压指标。该项目的主要目的是优化和传播非线性 AOL 3D 显微镜，这是我们发明的一项技术，旨在克服这些限制能够在 X、Y 和 Z 的任意方向上进行超快线扫描（高达 40 kHz）。我们已经证明了具有实时校正大脑运动功能的非线性 AOL 2P 显微镜原型该技术在清醒状态下对神经回路进行高速多尺度 3D 成像的性能我们将在这些结果的基础上，优化非线性 AOL 显微镜对整个 3D 树突进行成像。我们将以前所未有的速度开发树木和周围的神经群体。树突“树木成像”方法提供低空间分辨率、超高速 3D 成像（最多 1 kHz），通过结合非线性 AOL 的快速扫描和自适应光学特性，我们还将扩展我们的闭环 3D 运动校正中使用实时 FPGA 分析，以实现“注意力成像” 树突树或电路的活动区域可以在更高的时空条件下快速检测和成像这些应用将提供监测整个电压所需的时间分辨率。首次在清醒动物中获得完整的锥体细胞 3D 树突状树，此外，注意力成像将首次实现。能够以高时空分辨率绘制神经递质释放图，并以低成本传播。通过为美国实验室和成像设施提供紧凑的非线性技术，将实现强大的新技术通过扩展我们的开源，AOL 模块将添加到其现有的传统 2P 显微镜中。显微镜 GUI 软件，使用 NWB2 标准化数据格式并完善自动化分析流程，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对一系列不同的神经回路进行实验。