Serial Blockface SEM Labels for Assessing Nervous System Plasticity

描述(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，它通过将切割器合并到扫描电子显微镜(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电路连接所需的数百个切片(50-70 nm厚)，同时潜在地为免疫金标记提供纳米级的分辨率。SBFSEM将与包埋前的免疫金标记法相结合，开发一种在非常大的样本中在分子水平上绘制靶标的三维图谱的方法。在进行神经元映射的同时，这将允许在神经元互连的高分辨率映射的背景下关联感兴趣的特定分子的定位。将开发用于多重标记的试剂，使用不同大小的金粒子进行优化，以穿透、最小背景、高溶解度和与样品特征的最大兼容性进行优化。我们的目标是使免疫金-SBRSEM成为一种常规方法，可以用来在空间大样本中绘制靶标。将确定三种尺寸在0.8至5 nm的颗粒，可以使用SBFSEM在多个标记研究中进行可视化、鉴定和区分，并用于演示伴随的SBFSEM神经束跟踪和包含混合突触的非磷酸化连接蛋白35(Cx35)的包埋前标记：(A)单和(B)双免疫标记神经元和突触靶标将使用新的探针在西方蚊虫中进行评估，并建立标记和显微镜的最佳方案。公共卫生相关性：这项研究将提供新的工具，细胞和结构生物学家可以利用这些工具对来自生物系统的大样本的结构和过程的观察进行关联，特别是神经系统，以及细胞生物学中的许多其他应用，在细胞和大分子水平上。这些将使人们能够对这些过程是如何工作的，以及在正常和疾病过程中分子和宏观结构和功能是如何相关的有新的见解。这一知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，还将导致改进的诊断和治疗。

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.