NeuroExM

摘要 该项目描述了NeuroExM™的开发，这是一个高度创新的执行系统 组织中信使RNA和蛋白质种群的综合空间分布分析 进行了扩张显微镜(EXM)处理)。Exm突破性的技术优势，这是 最近由Edward S.Boyden博士开发(Dep.比奥尔。引擎、媒体实验室和部门。大脑认知。麻省理工学院理工学院 马萨诸塞州坎布里奇)和他的同事们，是一种各向同性扩张组织并增加生物大小的能力 结构。这使得可以对太小而不能 由于光的衍射极限，在没有膨胀的情况下分解。在其他好处中，exm允许那些小的 使用更广泛的显微技术对结构进行成像。对Exm的组织进行处理还允许 重复杂交(用于研究mRNA)和/或重复抗体染色(用于研究 蛋白质)，结合重复的显微镜成像轮次。每一轮的收益率都是相邻的， 高倍率、单视野图像堆叠，由至少一个形态参考通道组成 显示神经元亚细胞结构(胞体、轴突、树突、树突棘、突触)以及一个 或显示mRNA和/或蛋白质的几个信息频道。从空间分布的角度进行综合分析 原位神经元中的mRNAs和蛋白质群体需要组装所有执行的图像堆栈 四舍五入为单个、无缝且对齐的三维(3D)EXM图像，该图像是高维的 可以是几个TB大小。然而，这带来了许多关于以下方面的计算挑战 显微图像的配准、分割和分析。NeuroExM中改变游戏规则的创新是 能够执行所有这些任务，而不需要员工中的计算机科学家来运行现有的 为这类复杂分析的每一步开发的基于实验室的单独软件脚本。这是做的 通过在NeuroExM中实施一系列重大技术创新，这是可能的。基于试点工作 在准备本提案期间与博伊登实验室合作进行的，我们相信 NeuroExM将对神经科学研究领域产生重大影响。具体地说， Exm和NeuroExM将使专注于心脏功能改变的研究取得实质性进展 神经元的空间转录组和蛋白质组与神经发育，神经精神， 神经退行性疾病和神经疾病，以及老龄化研究和药物开发。最终， 这将为开发针对广泛的复合体的新的治疗策略提供更好的基础 脑部疾病。在第一阶段，我们将通过开发原型来证明这项新技术的可行性。 软件；第二阶段的工作将专注于为商业发布创造NeuroExM的全部功能。我们 将对NeuroExM进行广泛的可行性研究、产品验证和可用性研究 与博伊登实验室的合作。目前还没有与之竞争的技术。

Abstract This project describes the development of NeuroExM™, a highly innovative system for performing comprehensive spatial distribution analysis of populations of messenger RNAs (mRNAs) and proteins in tissue processed for expansion microscopy (ExM)). The groundbreaking technological advantage of ExM, which was recently developed by Dr. Edward S. Boyden (Dept. Biol. Engin., Media Lab and Dept. Brain Cognit. Sci., MIT, Cambridge, MA) and colleagues, is the ability to isotropically expand tissue and increase the size of the biological structures. This allows nanoscale-resolution, light-microscopic imaging of small objects that are too small to be resolved without expansion due to the diffraction limit of light. Among other benefits, ExM allows those small structures to be imaged with a wider range of microscopy techniques. Processing tissue for ExM also allows repeated hybridization (for investigations of mRNAs) and/or repeated antibody staining (for investigations of proteins) of the same tissue, combined with repeated microscopic imaging rounds. Each round yields adjacent, high-magnification, single field-of-view image stacks, consisting of at least one morphology reference channel showing neuronal sub-cellular structures (somas, axons, dendrites, dendritic spines, synapses) as well as one or several info channels showing mRNAs and/or proteins. Comprehensive analysis of the spatial distribution of populations of mRNAs and proteins in neurons in situ requires assembling the image stacks of all performed rounds into a single, seamless and aligned, three-dimensional (3D) ExM image, which is high-dimensional and can be several terabytes in size. However, this presents a number of computational challenges with respect to microscopy image registration, segmentation and analysis. The game-changing innovation in NeuroExM is the ability to perform all of these tasks without the need to have a computer scientist on staff to run the existing, individual lab-based software scripts developed for each step of this kind of complex analysis. This is made possible by implementing a number of significant technical innovations into NeuroExM. Based on pilot work performed in collaboration with the Boyden lab during preparation of this proposal, we are convinced that NeuroExM will make a significant impact on the field of neuroscience research. Specifically, the combination of ExM and NeuroExM will enable substantial advancements in research studies focusing on alterations in the spatial transcriptome and proteome of neurons associated with neurodevelopmental, neuropsychiatric, neurodegenerative and neurological disorders as well as in aging research and drug development. Ultimately, this will result in an improved basis for developing novel treatment strategies for a wide spectrum of complex brain diseases. In Phase I we will demonstrate feasibility of this novel technology by developing prototype software; work in Phase II will focus on creating the full functionality of NeuroExM for commercial release. We will perform extensive feasibility studies, product validation and usability studies of NeuroExM in close collaboration with the Boyden lab. A competing technology is not available.