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）是最近开发的电子显微镜连续重建技术，其通过将切片机结合到扫描电子显微镜（SEM）的真空室中来自动化组织块的切片和成像过程。SBFSEM提供的分辨率足以追踪甚至最薄的轴突并识别突触。它还允许用户自动获得完全重建神经元回路连接所需的数百个切片（50-70 nm厚），同时可能为免疫金标记提供纳米级分辨率。SBFSEM将与包埋前免疫金标记相结合，以开发一种在分子水平上在非常大的标本中进行三维定位的方法。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的背景下相关感兴趣的特定分子的定位。将开发用于多种标记的试剂，使用不同尺寸的金颗粒，优化渗透性、最小背景、高溶解度和与标本特征的最大兼容性。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内映射目标。将鉴定在0.8至5 nm范围内的三种颗粒尺寸，其可以在多个标记研究中使用SBFSEM可视化、鉴定和区分，并用于证明含有磷酸化和非磷酸化连接蛋白35（Cx 35）的混合突触的伴随SBFSEM神经束示踪和包埋前标记：将使用新的探针和为标记和显微镜建立的最佳方案，在西方食蚊鱼Gambusia affinis中评价神经元和突触靶的（a）单和（B）双免疫标记。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，从生物系统，特别是神经系统，以及细胞生物学中的许多其他应用中，对大型标本中的结构和过程进行观察。这些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如何相关有新的见解。这些知识将导致改进的诊断和治疗，除了提供改进的免疫金探针和促进纳米生物技术的发展。

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.