Serial Blockface SEM Labels for Assessing Nervous System Plasticity

用于评估神经系统可塑性的串行 Blockface SEM 标签

• 批准号: 7746768
• 负责人: RICHARD DENIS POWELL
• 金额: $ 33.84万
• 依托单位: NANOPROBES, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-18 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7746768
• 关键词: Accounting; Axon; Cells; Cellular Structures; Cellular biology; Characteristics; Chemistry; Connexins; Detection; Development; Diagnostic; Disease; Electron Microscope; Electron Microscopy; Electrons; Evaluation; Goals; Gold; Hand; Human; Image; Imagery; Interneurons; Knowledge; Label; Laser Scanning Confocal Microscopy; Lead; Left; Light; Maps; Methods; Microscopic; Microscopy; Microtome - medical device; Molecular; Motor Neurons; Nervous system structure; Neurons; Particle Size; Penetration; Plastics; Preparation; Process; Protocols documentation; Puerto Rico; Reagent; Research; Resolution; Rosa; Sampling; Scanning; Scanning Electron Microscopy; Signal Transduction; Solubility; Specimen; Structural Biologist; Structure; Surface; Surface Properties; Synapses; Synaptic Potentials; Techniques; Therapeutic; Thick; Tissues; Transmission Electron Microscopy; Universities; Vacuum; Work; axonal sprouting; base; biological systems; biomaterial compatibility; improved; insight; interest; nanoGold; nanobiotechnology; nanoparticle; nanoprobe; nanoscale; particle; public health relevance; reconstruction; tool

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.

描述（由申请人提供）：串行区块扫描电子显微镜（SBFSEM）是一种最近开发的电子显微镜串行 - 重建技术，它通过将微型群掺入扫描电子显微镜（SEM）的真空室中（SEM）来自动化组织的切片和成像组织的过程。 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几百个部分（50-70 nm厚），以完全重建神经元电路的连通性，同时潜在地提供了用于免疫标记的纳米尺度分辨率。 SBFSEM将与预先装饰的免疫金标签结合使用，以开发一种在非常大的标本中分子水平上靶标的三维映射的方法。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，特定感兴趣的特定分子的定位相关。将使用针对穿透性，最小背景，高溶解度以及与样品特性的最大兼容性优化的不同大小的金颗粒开发用于多个标记的试剂。我们的目标是使Immunogold-SBRSEM成为常规方法，可用于在空间大型标本中绘制目标。 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使用新的探针和建立用于标记和显微镜的最佳方案，将评估神经元和突触靶标的免疫标记的神经元和突触靶标。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通过这些工具将来自生物系统的大型标本（尤其是神经系统，以及许多其他在细胞生物学中，细胞和大分子分子水平）中的大型标本中的结构和过程相关联。这些将使新的见解对此类过程的工作原理以及分子和宏观结构和功能如何在正常和疾病过程中相关。这些知识除了提供改进的免疫探针并为纳米局技术的发展做出贡献外，还将导致改善诊断和治疗剂。