Optimization, application and dissemination of high-speed hybrid multiphoton volumetric imaging technologies

项目摘要/摘要 理解认知相关的行为功能是如何从已识别的细胞的活动模式中出现的- 类型是基于记录大规模合奏动态的能力，从遗传识别和 纵向跟踪跨多个脑区域和层的神经元群体，具有高空间和 在行为相关时间尺度上的时间分辨率。双光子扫描显微镜的组合 具有遗传编码的钙(钙)指示器是目前体内光学最基本的工具 神经元活动的记录及其在脑深部区域的应用。然而，目前商业上 由于与可获得的成像相关的限制，现有的2 PM系统在其应用方面受到限制 深度、体积视野(VFOV)和时间分辨率，在该分辨率下神经元种群动态 有效地被俘虏了。我们最近开发并演示了一种新的高速电机的原理证明 体积钙成像平台称为混合多路雕刻光(HYMS)显微镜 下午2点，三光子显微镜检查(下午3点)。HYMS允许对单细胞的神经活动进行体积记录 在最高17赫兹的大脑皮层和皮质下区域，分辨率高达~1×1×1.22毫米 行为正常的老鼠醒过来。这个工具的影响将取决于成功的优化，神经生物学 在神经科学界的应用和传播战略。虽然我们将提供开放源代码 对于技术熟练的实验室来说，考虑到这种系统的技术复杂性和成本，最有效的 战略是通过与业界的伙伴关系和系统的商业化来实现的。在这里，我们提出一种 实现这一目标的路线图。在现有系统的基础上，我们将实施多项 技术上的改进和优化。利用与Losonczy实验室的持续合作 哥伦比亚大学，我们将使用我们优化的HYMS系统进行高速多光子体积测量 小鼠背侧海马区(HPC)整个深度的功能回路的CA2成像， 包括HPC三突触回路的所有主要区域。这个应用程序将为我们提供有价值的 为我们的HYMS原型系统的进一步优化、改进和开发提供反馈。同时， 我们将与我们的工业合作伙伴共同开发首个HYMS系统原型(-HYMS)。 样机将再次由Losonczy实验室使用和测试。从以下方面获得的见解和用户反馈 应用程序将推动测试原型(-HYMS)的开发，该原型将用于与更广泛的本地 用户作为测试版测试者。9个用户实验室，主要来自纽约地区，有广泛的生物学问题和 应用程序，将作为测试者参与，并为我们提供迭代的用户反馈，最终将 推动并纳入HYMS的商业化以及其开源模型 使用这项技术。

PROJECT SUMMARY / ABSTRACT Understanding how cognitively-relevant behavioral functions emerge from activity patterns of identified cell- types is predicated on the ability to record large-scale ensemble dynamics from genetically-identified and longitudinally-tracked neuronal populations across multiple brain regions and layers with high spatial and temporal resolution over behaviorally-relevant time-scales. Two-photon scanning microscopy in combination with genetically-encoded calcium (Ca2+) indicators is currently the most essential tool for in vivo optical recording of neuronal activity, its application to deep brain regions. However, currently the commercially available 2pM systems are limited in their applications due to constraints related to the obtainable imaging depth, volumetric field-of-view (VFOV), and temporal resolution at which neuronal population dynamics can be effectively captured. We have recently developed and demonstrated the proof of principle of a new high-speed volumetric Ca2+-imaging platform termed Hybrid Multiplexed Sculpted Light (HyMS) Microscopy that combines 2pM with three-photon microscopy (3pM). HyMS allows for volumetric recording of neuroactivity at single-cell resolution within volumes up to ~1 × 1 × 1.22 mm at up to 17 Hz in cortical as well as sub-cortical regions of awake behaving mice. The impact of this tool will depend on a successful optimization, neurobiological application and dissemination strategy within the neuroscience community. While we will provide open source access for technically skilled labs, given the technical complexity and costs of such a system, the most effective strategy is through partnership with industry and through commercialization of the system. Here we propose a roadmap towards this objective. Building on our current existing system, we will implement a number of technical refinements and optimizations. Leveraging the ongoing collaboration with the Losonczy Lab at the Columbia University, we will use our optimized HyMS system to perform high-speed multiphoton volumetric Ca2+ imaging of functional circuitry across the entire depth of the mouse dorsal hippocampus (HPC), encompassing all major regions of the HPC trisynaptic circuitry. This application will provide us valuable feedback for further optimization and refinement and development of our HyMS prototype system. In parallel, we will develop together with our industrial partner a first prototype of the HyMS system (-HyMS) This prototype will be again used and tested by the Losonczy Lab. The obtained insights and user feedback from their application will drive the development of a beta prototype (-HyMS) which will be used to engage broader local users as beta testers. 9 user labs, mainly from the NYC area, with a broad range of biological questions and applications, will participate as beta testers and provide us with iterative user feedback which will ultimately drive and be incorporated both into the into the commercialization of HyMS as well its open source model of the access to this technology.