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.

摘要 大脑是人体中最复杂的器官。了解它的神经连接和分子 解剖学需要成像技术，该成像技术能够映射特定组织的3D纳米级分布。 蛋白质在大脑超微结构中的作用。目前最好的选择是相关光， 电子显微镜（CLEM），它结合了分辨率和全球对比度的EM与高 荧光显微镜的分子特异性。虽然非常强大，但CLEM费力，吞吐量低， 昂贵且空间相关分辨率常常不足。到目前为止，还没有成像技术 它可以分辨突触超微结构中的特定蛋白质。我们最近开发了一种 全光学方法称为泛扩展显微镜（pan-ExM），有可能允许标准 共聚焦显微镜用户做到这一点：通过结合生物样品的~20倍线性扩展， 总蛋白质的新的整体（PAN）染色，标志性的超微结构特征，如突触后和突触前 密度和线粒体嵴现在可以通过其特征性的泛染色模式来分辨，类似于 到EM中的重金属染色泛光公司是一家围绕这一核心技术建立起来的新公司。我们 拟议的第一阶段项目是验证泛ExM协议，将建立泛ExM作为脑成像 技术.我们的总体目标是让每一位神经科学家都能在他们的3D模型中定位特定的蛋白质标签。 背景区室以及执行EM样神经元追踪脑组织。我们特别建议 （1）验证和优化用于突触识别的抗体标记兼容方案，以及（2）优化 并验证我们开发的一种新的脂质泛染色剂，以描绘细胞边界，补充我们的蛋白质组 泛染色。最终，这里提出的发展将使Panluminate能够简化和部署 pan-ExM样品制备套件提供给每一位对同步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示踪，并提供泛ExM样品制备和成像， 一种服务。