Serial Blockface SEM Labels for Assessing Nervous System Plasticity

描述（由申请人提供）：连续块面扫描电子显微镜（SBFSEM）是最近发展起来的一种电子显微镜连续重建技术，它通过将切片机放入扫描电子显微镜（SEM）的真空室中，自动对组织块进行切片和成像。sfsem提供的分辨率足以追踪最薄的轴突并识别突触。它还允许用户自动获得几百个切片（50-70纳米厚），以完全重建神经元电路的连接，同时潜在地为免疫金标记提供纳米级分辨率。SBFSEM将与预包埋免疫金标记相结合，开发一种在非常大的标本中进行分子水平靶标三维定位的方法。与神经元映射并行进行，这将允许在神经元互连的高分辨率映射背景下对感兴趣的特定分子的定位进行关联。将开发试剂用于使用不同大小的金颗粒进行多重标记，以优化渗透，最小背景，高溶解度和与样品特征的最大兼容性。我们的目标是使免疫金- sbrsem成为一种常规方法，可用于在空间大的标本中绘制靶标。在0.8 ~ 5nm范围内，我们将鉴定出三种粒径大小的颗粒，这些颗粒可以在多个标记研究中使用SBFSEM进行可视化、识别和区分，并用于演示SBFSEM同时对含有混合突触的磷酸化和非磷酸化连接蛋白35 （Cx35）的神经元束跟踪和预嵌入标记。(a)单一和(b)双重免疫标记的神经元和突触靶点将在西蚊鱼，Gambusia affinis中进行评估，使用新的探针，并为标记和显微镜建立最佳方案。公共卫生相关性：这项研究将为细胞和结构生物学家提供新的工具，使他们能够在细胞和大分子水平上对生物系统，特别是神经系统，以及细胞生物学中的许多其他应用中的大型标本的结构和过程进行观察。这将使人们对这些过程如何工作，以及分子和宏观结构和功能在正常和疾病过程中的关系有新的认识。除了提供改进的免疫金探针和促进纳米生物技术的发展外，这些知识还将导致改进的诊断和治疗方法。

DESCRIPTION (provided by applicant): Serial blockface scanning electron microscopy (SBFSEM) is a recently developed electron microscopy serial- reconstruction technique which automates the process of sectioning and imaging blocks of tissue by incorporating a microtome into the vacuum chamber of a scanning electron microscope (SEM). SBFSEM provides resolution that is sufficient to trace even the thinnest axons and to identify synapses. It also allows the user to automatically obtain several hundred sections (50-70 nm thick) needed to completely reconstruct the connectivity of neuronal circuits, while potentially providing nanometer-scale resolution for immunogold labeling. SBFSEM will be combined with pre-embedding immunogold labeling to develop a method for the three-dimensional mapping of targets at the molecular level in very large specimens. Conducted in parallel with neuronal mapping, this will allow correlation of localization of specific molecules of interest in the context of high-resolution mapping of neuronal interconnectivity. Reagents will be developed for multiple labeling using different sized gold particles optimized for penetration, minimal background, high solubility, and maximum compatibility with specimen characteristics. Our goal is to make immunogold-SBRSEM a routine method that can be used to map targets within spatially large specimens. Three particle sizes in the range 0.8 to 5 nm will be identified that can be visualized, identified and differentiated using SBFSEM in multiple labeling studies, and used to demonstrate concomitant SBFSEM neuronal tract tracing and pre-embedding labeling of phos- phorylated and non- phosphorylated connexin 35 (Cx35) containing mixed synapses: (a) single and (b) double immunolabeling of neuronal and synaptic targets will be evaluated in the Western Mosquitofish, Gambusia affinis, using the new probes, and best protocols established for labeling and microscopy. PUBLIC HEALTH RELEVANCE: This research will provide new tools with which cell and structural biologists can correlate the observation of structures and processes in large specimens from biological systems, particularly the nervous system but also many other applications in cell biology, at the cellular and macromolecular level. These will enable new insights into how such processes work, and how molecular and macroscopic structure and function are related in normal and disease processes. This knowledge will lead to improved diagnostics and therapeutics in addition to providing improved immunogold probes and contributing to the development of nanobiotechnology.