Development of a sample preparation protocol for brain ultrastructural analysis, immunolabeling, and neuronal tracing by light microscopy

开发用于脑超微结构分析、免疫标记和光学显微镜神经元追踪的样品制备方案

• 批准号: 10483978
• 负责人: Ons M'Saad
• 金额: $ 24.85万
• 依托单位: PANLUMINATE INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-16 至 2023-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10483978
• 关键词: 3-Dimensional; Academia; Anatomy; Antibodies; Architecture; Biological; Brain; Brain Diseases; Brain imaging; Cells; Characteristics; Color; Complement; Complex; Confocal Microscopy; Crista ampullaris; Data; Data Set; Development; Electron Microscopy; Fluorescence Microscopy; Future; Goals; Hand; Heavy Metals; Human body; Hydrogels; Image; Imaging technology; Individual; Industry; Label; Lipids; Location; Maps; Measurement; Membrane; Membrane Lipids; Microscope; Microscopic; Microscopy; Mitochondria; Modality; Molecular; Mus; Nature; Neurobiology; Neurons; Optical Methods; Optics; Organ; Outcome; Pattern; Phase; Preparation; Proteins; Proteome; Protocols documentation; Research; Research Personnel; Resolution; Sample Size; Sampling; Services; Small Business Innovation Research Grant; Specificity; Sphingosine; Stains; Structure; Synapses; Techniques; Technology; Three-Dimensional Imaging; Time; Tissues; Validation; Work; brain research; brain tissue; cell type; commercialization; cost; density; fluorescence microscope; imaging capabilities; instrumentation; interest; light microscopy; nanoscale; nervous system disorder; neural circuit; novel; optical imaging; preservation; protein distribution; relating to nervous system

Abstract The brain is the most complex organ in the human body. Understanding its neural connections and molecular anatomy requires imaging technology that is capable of mapping the 3D nanoscale distribution of specific proteins in the context of brain ultrastructure. The currently best available option is correlative light and electron microscopy (CLEM), which combines the resolving power and global contrast of EM with the high molecular specificity of fluorescence microscopy. While very powerful, CLEM is laborious, low throughput, expensive and often inadequate in spatial correlation resolution. To date, there is no imaging technology that can resolve specific proteins within the ultrastructure of a synapse. We recently developed an all-optical method called pan-Expansion Microscopy (pan-ExM) that has the potential to allow standard confocal microscopy users to do just that: by combining ~20-fold linear expansion of biological samples with novel bulk (pan) staining of total proteins, hallmark ultrastructural features such as post- and pre-synaptic densities and mitochondria cristae can now be resolved by their characteristic pan-staining pattern, analogous to heavy-metal stains in EM. Panluminate Inc. is a new company built around this core technology. Our proposed Phase I project is to validate pan-ExM protocols that will establish pan-ExM as a brain imaging technology. Our overall goal is to enable every neuroscientist to locate specific protein labels within their 3D contextual compartments as well as perform EM-like neuronal tracing in brain tissue. We specifically propose to (1) validate and optimize antibody-labeling compatible protocols for synapse identification, and (2) optimize and validate a novel lipid pan-stain we developed to delineate cellular boundaries to complement our proteome pan-stain. Ultimately, the developments proposed here will enable Panluminate to streamline and deploy pan-ExM sample preparation kits to every neuroscientist interested in simultaneous 3D ultrastructural analysis, antibody labeling, and neuronal tracing in brain tissue and to offer pan-ExM sample preparation and imaging as a service.

抽象的 大脑是人体中最复杂的器官。了解其神经连接和分子 解剖学需要能够映射特定的3D纳米级分布的成像技术 蛋白质在大脑超微结构的背景下。当前最好的选项是相关光和 电子显微镜（CLEM），它结合了EM的分辨能力和全局对比度 荧光显微镜的分子特异性。虽然非常强大，但Clem却是费力，低吞吐量， 昂贵且通常不足以解决空间相关性分辨率。迄今为止，没有成像技术 可以在突触的超微结构中解析特定的蛋白质。我们最近开发了 称为Pan-expansion显微镜（Pan-Exm）的全光学方法，具有允许标准的潜力 共聚焦显微镜使用者可以做到这一点：通过将生物样品的约20倍线性扩展与 总蛋白质的新型体积（PAN）染色，标志性超微结构特征，例如后和突触前的特征 现在可以通过其特征性的泛染色，类似的方式来解决密度和线粒体cristae 到EM中的重金属污渍。 Panluminate Inc.是一家围绕此核心技术建立的新公司。我们的 提议的第一阶段项目是验证将建立pan-exm作为大脑成像的PAN-EXM协议 技术。我们的总体目标是使每个神经科学家在其3D中找到特定的蛋白质标签 上下文隔室以及在脑组织中执行类似神经元的跟踪。我们特别提出 （1）验证和优化抗体标记的兼容协议以进行突触识别，（2）优化 并验证我们开发的一种新型的脂质泛污物，以描绘细胞边界以补充我们的蛋白质组 泛污。最终，此处提出的发展将使泛滥成灾能够简化和部署 pan-exm样品制备套件，每个对同时3D超微结构分析感兴趣的神经科学家， 抗体标记和脑组织中的神经元跟踪，并提供Pan-Exm样品制备和成像为 服务。