Development and dissemination of high speed 3D acousto-optic lens two-photon microscopy for in vivo imaging

为了了解健康和疾病中的大脑功能，必须快速监测神经回路中的信号。双光子显微镜是神经科学研究的核心工具，因为它能够以高空间分辨率监测脑组织深处的神经元活动。然而，传统设计的机械扫描和聚焦严重限制了成像的时间分辨率，并且大脑运动使记录复杂化。我们已经开发出一种新型的紧凑型声光透镜（AOL）的3D双光子显微镜，克服了这些限制，允许高速成像的体积跨越数百微米和真实的时间校正的大脑运动，发生在清醒的行为动物。20-40 kHz的敏捷3D随机访问指向和扫描（RAPS）弥补了光学成像和电生理学之间时间分辨率的差距。此外，我们的AOL的紧凑设计功能使其能够以相对较低的成本添加到现有的双光子显微镜。该提案旨在完善和迅速推广这一强大的新技术。技术改进包括扩大可以成像的体积，并将基于AOL的实时运动校正从20扩展到30。与我们实验室的实验人员和分布在世界各地的其他主要领导团体（技术将首先传播给他们）的密切合作将确保AOL的当前和新功能以及开源GUI软件是强大的，并满足神经科学界的要求。这将提供研究神经回路所需的新的、迫切需要的技术开发，并在商业制造商将产品推向市场所需的3年时间之前加速这一世界领先技术的传播。

To understand brain function in health and disease it is essential to rapidly monitor signaling in neural circuits. Two-photon microscopy is a core tool for neuroscience research because it enables neuronal activity to be monitored at high spatial resolution deep within brain tissue. However, the mechanical scanning and focusing of conventional designs severely limits the temporal resolution of 30 imaging and brain movement complicates recordings. We have developed a novel compact acousto-optic lens (AOL) 3D two-photon microscope that overcomes these limitations, allowing high speed imaging over volumes spanning hundreds of micrometres and real time correction of the brain movement that occurs in awake behaving animals. The agile 3D random access pointing and scanning (RAPS) at 20-40 kHz bridges the gap in the temporal resolution between optical imaging and electrophysiology. Moreover, compact design features of our AOL enable it to be added to existing two-photon microscopes at relatively low cost. This proposal aims to refine and rapidly disseminate this powerful new technology. Technology refinements include expanding the volumes that can be imaged and extending AOL-based real-time movement correction from 20 to 30. The close collaboration with experimentalists in our lab and other key leading groups distributed worldwide (to whom the technology will be initially disseminated) will ensure that current and new features of the AOL, as well as the open source GUI software, are robust and meet the requirements of the neuroscience community. This will deliver new, urgently needed technological developments required for investigating neural circuits and accelerate dissemination of this world leading technology well ahead of the 3 year timescale that is required by commercial manufacturers to bring a product to market.