NeuroExM

摘要 该项目描述了NeuroExM™的开发，这是一种高度创新的系统，用于执行 组织中信使RNA（mRNA）和蛋白质群体的综合空间分布分析 处理用于扩增显微术（ExM））。ExM的突破性技术优势， 最近由Edward S. Boyden（部门）生物工程，媒体实验室和部门大脑认知科学，麻省理工学院， 剑桥，MA）及其同事所提出的一种新的方法，是能够各向同性地扩张组织并增加生物组织的尺寸。 结构.这使得纳米级分辨率，光显微镜成像的小物体太小， 由于光的衍射极限，在没有膨胀的情况下解决。除其他好处外，ExM允许那些小型 结构成像范围更广的显微镜技术。用于ExM的处理组织还允许 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。每一轮都产生相邻的， 高放大率、单视场图像堆栈，由至少一个形态学参考通道组成 显示了神经元亚细胞结构（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通道。空间分布的综合分析 原位神经元中的mRNA和蛋白质群体需要组装所有执行的图像堆栈， 四舍五入成一个单一的，无缝和对齐，三维（3D）的ExM图像，这是高维， 可以是数TB的大小。然而，这就提出了许多计算挑战， 显微图像配准、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，而不需要有一个计算机科学家的工作人员来运行现有的， 为这种复杂分析的每一步开发的单独的实验室软件脚本。规定了这一点 通过在NeuroExM中实施一些重要的技术创新，这是可能的。根据试点工作 在准备本提案期间与Boyden实验室合作进行的，我们确信， NeuroExM将对神经科学研究领域产生重大影响。具体来说，结合 ExM和NeuroExM将使专注于改变的研究取得实质性进展， 与神经发育、神经精神 神经退行性疾病和神经系统疾病以及衰老研究和药物开发。最后， 这将为开发针对广泛的复杂性疾病的新型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;第二阶段的工作将侧重于创建NeuroExM的完整功能，以供商业发布。我们 将进行广泛的可行性研究，产品验证和NeuroExM的可用性研究， 与博伊登实验室合作。没有竞争技术。

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.