Examining the architecture of synapses in brain tissue at nanometer resolution

描述（由申请人提供） 翻译后摘要：在我们的知识的巨大进步往往是创新技术进步的结果。例如，将光学显微镜与高尔基染色相结合导致了现代神经科学的诞生。我的建议旨在为神经科学研究方法提供一个重大飞跃，具有改变未来大脑功能和功能障碍研究的潜力。大脑功能依赖于突触处的微小专门结构，其中蛋白质分子以纳米精度排列。突触机制的微小变化被视为许多神经系统疾病和神经退行性疾病的早期表现，随着人口老龄化，这些疾病越来越普遍。然而，在足够高的分辨率水平上检查突触蛋白组织一直是具有挑战性的，特别是在脑组织中。令人惊讶的是，这种限制并不在于显微镜方法。荧光超分辨率显微镜方法，如光激活定位显微镜（PALM），可以探测10 nm的分辨率已经开发出来。然而，与蛋白质标记、样品制备和数据解释相关的障碍阻碍了它们在脑组织中的应用。在这里，我建议开发创新的方法来标记内源性突触蛋白和制备脑样品PALM。我们还将建立一种新的方法来可视化细胞背景下的超分辨率蛋白质组织，以帮助解释数据。我们将通过结合多个学科的创新发展来实现这一目标，包括分子生物学，生物化学，遗传学，超分辨率荧光显微镜，电子显微镜和计算机编程。如果建立起来，我们的技术将通过提供前所未有的能力来获得突触结构的细节，从而彻底改变神经科学研究中使用的方法。这些方法将适用于其他细胞蛋白质的研究。识别以前无法检测到的细微变化的能力也将使早期诊断和影响突触和其他细胞结构的神经系统疾病的机械理解得到增强。 公共卫生相关性：大脑中蛋白质组织的细微变化是许多神经系统疾病和神经退行性疾病的早期表现，随着人口的不断老龄化，这些疾病越来越普遍。我们的目标是开发可以可视化这些以前无法检测到的变化的方法，以实现早期诊断和增强对影响突触结构的神经系统疾病的机制理解。

DESCRIPTION (Provided by the applicant) Abstract: Great advances in our knowledge are often the result of innovative technological advances. For example, combining light microscopy with Golgi staining led to the birth of modern neuroscience. My proposal aims to provide a major leap in the methodology of neuroscience research that holds the potential of transforming future studies of brain function and dysfunction. Brain function relies on tiny specialized structures at the synapse where protein molecules are arranged with nanometer precision. Small changes in synaptic machinery are viewed as early manifestations of many neurological disorders and neurodegenerative diseases, which are increasingly prevalent as the population ages. However, it has been challenging to examine synaptic protein organization at a sufficiently high level of resolution, especially in brain tissue. Surprisingly, this limitation does not lie in the microscopic methods. Fluorescence super-resolution microscopic methods such as the photoactivated localization microscopy (PALM) that can probe with 10-nm resolution have been developed. However, obstacles associated with protein labeling, sample preparation and data interpretation have prevented their application to brain tissue. Herein, I propose to develop innovative methodologies to label endogenous synaptic proteins and prepare brain samples for PALM. We will also establish a novel approach for visualizing the super-resolution protein organization within a cellular context to aid the interpretation of the data. We will do so by combining innovative developments across multiple disciplines, including molecular biology, biochemistry, genetics, super-resolution fluorescence microscopy, electron microscopy and computer programming. If established, our techniques will revolutionize the methodologies used in neuroscience research by providing unprecedented abilities to obtain fine details of synaptic architecture. These methods will be applicable to the study of other cellular proteins. The ability to identify previously undetectable subtle changes will also enable early diagnoses and an enhanced mechanistic understanding of neurological diseases affecting synaptic and other cellular structures. Public Health Relevance: Subtle changes in protein organization in the brain are an early manifestation of many neurological disorders and neurodegenerative diseases, which are increasingly prevalent as the population continues to age. We aim to develop methods that can visualize these previously-undetectable changes to enable early diagnoses and an enhanced mechanistic understanding of neurological diseases affecting synaptic structures.