Genetically Encoded Activity Sensors for Photoacoustic Imaging of the Brain

项目摘要 大规模记录活体和行为大脑中神经元活动的新工具对于研究神经元活动至关重要。 更好地了解大脑功能，有效地分析和治疗神经元疾病。时间 分辨体积光声成像为大规模脑记录提供了巨大的潜力 因为它能很好地渗透到活组织中仪器仪表的最新发展 光声神经成像正以不断增加的分辨率，灵敏度， 视野和帧速率。然而，这些发展需要配套工程 合适的造影剂和活性报告物。现有的基因编码钙指标 已经在原理证明光声研究中显示出前景，但是它们在光声中的吸收最大值在 光谱的蓝绿色范围被血红蛋白的强烈吸收极大地干扰， 哺乳动物的大脑相反，细菌光敏色素的吸光度理想地在接近100 nm的范围内。 红外线该项目将基于细菌光敏色素的荧光蛋白工程化， 光声钙传感器我们将小的钙结合结构域插入到细菌中， 通过使用现有的结构信息或通过使用随机插入策略， 将其转化为高效的基于吸收的钙报告基因。典型的记者将进一步 使用独特的新自动化技术，通过多样化和大规模筛选变体进行工程设计， 申请人开发的机器人筛查站。最后，高性能传感器变体将 在小鼠体内视觉皮层中得到验证。因此，该提案结合了以下方面的互补专长： 开发光声仪器和大规模生物传感器工程， 完整的散射哺乳动物大脑的体积活性成像。

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.