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

项目概要/摘要 了解认知相关的行为功能如何从已识别细胞的活动模式中产生 类型取决于从基因识别和记录大规模系综动态的能力 跨多个大脑区域和层的纵向跟踪神经元群，具有高空间和 行为相关时间尺度上的时间分辨率。双光子扫描显微镜组合 具有基因编码的钙（Ca2）指标是目前体内光学最重要的工具 神经元活动的记录及其在大脑深部区域的应用。然而，目前商业 由于与可获得的成像相关的限制，可用的 2pM 系统的应用受到限制 深度、体积视场 (VFOV) 和时间分辨率，可在这些分辨率下神经元群体动态 有效捕获。我们最近开发并论证了一种新的高速原理 体积 Ca2 成像平台，称为混合多重雕刻光 (HyMS) 显微镜，结合了 2pM，三光子显微镜 (3pM)。 HyMS 允许对单细胞神经活动进行体积记录 在高达 17 Hz 的体积内分辨率高达 ~1 × 1 × 1.22 mm 清醒的老鼠。该工具的影响将取决于成功的优化、神经生物学 神经科学界的应用和传播策略。虽然我们将提供开源 考虑到此类系统的技术复杂性和成本，技术熟练的实验室的访问权限是最有效的 战略是通过与工业界的合作以及系统的商业化。在这里我们提出一个 实现这一目标的路线图。在现有系统的基础上，我们将实施一系列措施 技术改进和优化。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 系统来执行高速多光子体积测量 小鼠背侧海马 (HPC) 整个深度功能电路的 Ca2 成像， 涵盖 HPC 三突触电路的所有主要区域。该应用程序将为我们提供有价值的 进一步优化、完善和开发我们的 HyMS 原型系统的反馈。同时， 我们将与我们的工业合作伙伴一起开发 HyMS 系统的第一个原型 (-HyMS) 原型将再次由Losonczy 实验室使用和测试。从他们那里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，该原型将用于吸引更广泛的本地参与 用户作为 Beta 测试人员。 9 个用户实验室，主要来自纽约地区，具有广泛的生物学问题和 应用程序，将作为 Beta 测试人员参与并向我们提供迭代的用户反馈，最终将 推动并纳入 HyMS 及其开源模型的商业化 获得这项技术。

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.