Genetically Encoded Activity Sensors for Photoacoustic Imaging of the Brain

项目摘要 用于大规模记录活的和行为的大脑中的神经元活动的新工具对于 更好地了解大脑功能，有效分析和治疗神经元疾病。时间 分辨率体积光声成像为大规模脑记录提供了巨大的潜力 因为它精致地渗透到活的组织中。仪器仪表的最新发展 光声神经成像正在以不断提高的分辨率、灵敏度、 视场和帧速率。然而，这些发展需要与之相匹配的工程。 合适的造影剂和活动记者。现有的基因编码的钙指示剂 在原理证明光声研究中显示出了希望，但他们在 光谱的蓝绿范围极大地受到血红蛋白强吸收的干扰 哺乳动物的大脑。相反，细菌光敏色素的吸光度理想范围在 红外线。该项目将把基于细菌光敏色素的荧光蛋白改造成强大的 用于光声学的钙传感器。我们将把小的钙结合结构域插入细菌 通过使用现有的结构信息或通过使用随机插入策略来 将它们转化为高效的基于吸光度的钙报告程序。原型记者将会更进一步 采用多样化和大规模变种筛选设计，使用独特的新自动化 申请者自行开发的机器人筛选站。最后，高性能的传感器变种将是 在活体小鼠视皮层中得到验证。因此，该提案在以下方面结合了互补的专门知识 发展光声仪器和大规模生物传感器工程以实现FAST 完整散布的哺乳动物脑的体积活动成像。

Project Summary New tools for large-scale recording of neuronal activity in a living and behaving brain are essential for a better understanding of brain function, efficient analysis and treatment of neuronal disorders. Time resolved volumetric photo-acoustic imaging offers tremendous potential for large-scale brain recording due to its exquisite penetration into living tissues. Most recent developments in instrumentation for photo-acoustic neuroimaging are rapidly advancing the field with ever increasing resolution, sensitivity, field of view and frame rates. Yet, these developments need to be matched by concomitant engineering of suitable contrast agents and activity reporters. The existing genetically encoded calcium indicators have shown promise in proof-of-principle photoacoustic studies but their absorbance maxima in the blue-green ranges of the spectrum is vastly interfered by the strong absorption of hemoglobin in mammalian brains. On the contrary, absorbance of bacterial phytochrome ideally ranges into the near infrared. This project will engineer fluorescent proteins based on bacterial phytochrome into powerful calcium sensors for photo-acoustics. We will insert small calcium binding domains into bacterial phytochromes either by using existing structural information or by using random insertion strategies to turn them into efficient absorbance-based calcium reporters. Prototypical reporters will then be further engineered using diversification and large scale screening of variants using a unique new automated robotic screening station developed by the applicants. Finally, high performing sensor variants will be validated in mouse visual cortex in vivo. Thus, the proposal combines complementary expertise in development of photo-acoustic instrumentation and large scale biosensor engineering to enable fast volumetric activity imaging of the intact scattering mammalian brain.