Methods for serially multiplexed labeling in EM reconstructions of brain tissue

脑组织电镜重建中连续多重标记的方法

• 批准号: 9892040
• 负责人: LINNAEA E OSTROFF
• 金额: $ 24.15万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-13 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9892040
• 关键词: Affect; Antibodies; Antigens; Array tomography; Brain; Cell physiology; Cells; Chemicals; Chemistry; Classification; Classification Scheme; Crosslinker; Data; Disease; Electron Microscopy; Elements; Fixatives; Gene Expression Profile; Gene Transfer Techniques; Genetic; Glutaral; Goals; Image; Impairment; Label; Light; Manuals; Maps; Measures; Membrane; Mental disorders; Methods; Molecular; Molecular Biology; Morphology; Neuronal Plasticity; Neurons; Neuropeptides; Neurosciences; Osmium; Pattern; Plant Resins; Preparation; Procedures; Proteins; Protocols documentation; RNA Probes; Reporter; Research; Sampling; Speed; Synapses; Taxonomy; Testing; Time; Tissue Preservation; Tissue Sample; Tissues; Transgenic Organisms; Variant; brain cell; brain tissue; brain volume; cell type; computerized data processing; connectome; crosslink; design; epon; fly; genetic manipulation; immunoreactivity; innovation; molecular marker; nervous system disorder; novel; novel strategies; preservation; receptor binding; reconstruction; sample fixation; small molecule; structured data; tissue preparation; tool

Project Summary/Abstract Tremendous technological progress in the use of serial electron microscopy (EM) for brain circuit mapping has been made over the last decade, and it is possible to directly visualize synaptic connectivity in larger volumes of brain tissue than ever before. Meanwhile, advances in molecular biology have shed new light on the diversity among neurons, particularly with respect to their patterns of gene expression. Currently, there are no methods available to efficiently integrate molecular labels into serial EM reconstructions. The ability to distinguish many molecular cell type markers in serial EM volumes would greatly enhance our ability to study circuit function, neuronal diversity, and neuroplasticity, and to determine how these are affected in disease states. The major barrier to visualizing molecules in serial EM is that the methods used to preserve morphology and generate contrast for serial EM are incompatible with most labels. Several methods have been developed to accommodate this limitation, mainly by using genetic tools to introduce labels before tissue samples are prepared for EM. These approaches are restrictive in that only a few labels can be used in a single sample, genetic manipulation is required, and endogenous molecules cannot be localized. This project will develop approaches that allow many different molecular labels to be differentiated within a single serial EM tissue volume. An innovative strategy will be used: instead of working around the standard serial EM protocol by designing labels that are compatible with it, the focus will be on replacing the incompatible elements of the standard protocol. Circuit reconstruction by serial EM requires a high degree of morphological preservation, which is typically accomplished with harsh chemicals that damage and denature molecules. However, all that is fundamentally required to preserve morphology is to retain as many molecules in the tissue as possible, which is also necessary for molecular labeling. Therefore, tissue preservation protocols will be developed to minimize extraction of molecules without damage or irreversible denaturation. A combination of strategies will be employed, including novel combinations of chemical crosslinkers and embedding resins. These new approaches will offer a means of revealing valuable information about circuit organization and neuronal diversity that is presently inaccessible.

项目摘要/摘要 脑电路连续电子显微镜（EM）的使用方面的巨大技术进步 在过去的十年中进行了映射，可以直接可视化突触连接 比以往任何时候都大的脑组织。同时，分子生物学的进步已经摆脱了新的 关于神经元之间的多样性，特别是在其基因表达模式方面。现在， 没有可有效地将分子标签整合到串行EM重建中的方法。这 在串行EM体积中区分许多分子细胞类型标记的能力将大大提高我们的能力 研究电路功能，神经元多样性和神经塑性，并确定它们在 疾病状态。在串行EM中可视化分子的主要障碍是用于保存的方法 串行EM的形态和对比度与大多数标签不兼容。有几种方法 已开发以适应这一限制，主要是使用遗传工具在组织前引入标签 为EM准备样品。这些方法是限制性的，因为只能在 需要单个样本，遗传操作，并且不能定位内源分子。这个项目 将开发出允许许多不同分子标签在单个串行EM中区分的方法 组织体积。将使用创新策略：而不是围绕标准串行EM协议进行工作 通过设计与之兼容的标签，重点将是更换不兼容的元素 标准协议。串行EM的电路重建需要高度的形态保存， 通常，这是用苛刻的化学物质来损害和变性分子来完成的。但是，所有这些 从根本上需要保留形态，就是保留组织中尽可能多的分子， 这对于分子标记也是必需的。因此，将制定组织保护方案 最小化分子的提取而没有损害或不可逆变性。策略的结合将 被采用，包括化学交联和嵌入树脂的新型组合。这些新 方法将提供一种揭示有关电路组织和神经元的有价值信息的方法 目前无法访问的多样性。