Optimization, Application, and Dissemination of Imaging Modules for High-speed Mesoscopic Volumetric Recording of Neuroactivity in Scattering Brains

项目摘要/摘要 最近的一些观察表明，哺乳动物大脑中的复杂大脑功能来自高度并行的计算，在这种计算中，有关感觉输入、内部状态和行为参数的信息被映射到高度分布的全脑神经元群体。这就需要神经技术，允许在生理时间尺度上大规模记录组织深度和大脑区域的神经活动，以及清醒和行为动物的细胞分辨率。基于双光子扫描荧光显微镜(2PM)和基因编码钙指示剂(GECI)作为神经活动记录器的光学工具开发的最新进展旨在通过开发更快、更大规模和体积的钙(Ca2)成像技术来满足这些需求，但在这方面一个根本未解决的挑战是以有原则和可扩展的方式在速度、分辨率和记录体积大小之间进行内在的权衡。我们的实验室最近为这种最佳记录方案建立了标准，这导致了一种新的高速体积钙成像方法的实现，称为光珠显微镜(LBM)。通过LBM，我们已经展示了在单细胞分辨率下对清醒、行为正常的小鼠两个皮质半球内高达100万个神经元的神经活动的荧光寿命有限体积记录。在这个项目中，我们将采取多管齐下的战略，在扩大我们的LBM技术性能的同时，实现其优化、生物应用和有效传播。这将产生一个更健壮、更简单、更用户友好的LBM技术版本。为了使其能够广泛和有效地传播，在项目的第二部分，我们将利用我们的α测试员的反馈来设计、构建和传播我们系统的β原型，这些原型将分发给几个最终用户实验室，这些实验室将在他们的生物学问题的背景下测试和应用我们的LBM技术。这一β原型还将构成我们技术的商业传播的基础，以及为其开放源码传播的平行努力。

Project Summary / Abstract A number of recent observations suggest that complex brain functions in the mammalian brain emerge from highly parallel computation in which information about sensory inputs, internal states, and behavioral parameters are mapped onto highly distributed brain-wide neuronal populations. This calls for neurotechnologies that allow for large-scale recording of neuro-activity across tissue depths and brain regions at physiological timescales and cellular resolution in awake and behaving animals. While recent advancements in optical tool development based on the combination of two-photon scanning fluorescence microscopy (2p M) and genetically-encoded calcium indicators (GECIs) as reporters of neuro-activity have been aimed at addressing these needs by developing faster, larger-scale, and volumetric calcium (Ca2+) imaging technologies, a fundamental unsolved challenge in this context is navigating the inherent tradeoffs between speed, resolution, and the size of the recording volume in a principled and scalable manner. Our lab has recently established criteria for such optimal recording schemes which has led to the realization of a new high-speed volumetric Ca2+ imaging approach termed Light Beads Microscopy (LBM). Through LBM, we have demonstrated fluorescence lifetime limited volumetric recording of neuro-activity at a single-cell resolution of up to 1 million neurons within both cortical hemispheres of awake, behaving mice. In this project, we will pursue a multipronged strategy towards the optimization, biological applications, and effective dissemination of our LBM technology while extending its performance. This will result in a more robust, less complex, and more user-friendly version of our LBM technology. To enable its broad and effective dissemination, in the second part of the project, we will utilize feedback from our α-testers to design, build, and disseminate β-prototypes of our system that will be distributed to several end-user laboratories who will be testing and applying our LBM technology in the context of their biological questions. This β-prototype will also form the basis for commercial dissemination of our technology as well as a parallel effort for its open-source dissemination.