New strategies for molecular cell-type labeling in volume electron microscopy

体积电子显微镜中分子细胞类型标记的新策略

• 批准号: 10413454
• 负责人: LINNAEA E OSTROFF
• 金额: $ 105.97万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10413454
• 关键词: Address; Aftercare; Antibodies; Biochemical; Brain; Brain Mapping; Brain imaging; Cells; Complex; Data; Data Analyses; Data Set; Detection; Electron Beam; Electron Microscopy; Engineering; Exposure to; Fixatives; Fluorescence; Fluorescence Microscopy; Goals; Gold; Histology; Image; Imaging technology; Immunohistochemistry; In Situ Hybridization; Label; Light; Literature; Maps; Methodological Studies; Methodology; Methods; Modality; Molecular; Molecular Structure; Molecular Target; Morphology; Neurons; Paraffin; Paraffin Embedding; Pattern; Plant Resins; Preparation; Problem Solving; Proteins; Protocols documentation; RNA; Reagent; Reporter; Resolution; Sampling; Series; Specimen; Structure; Synapses; Techniques; Tissue Embedding; Tissues; Transcript; Transgenic Organisms; base; brain morphology; brain tissue; brain volume; cell type; flexibility; fluorescence imaging; imaging modality; improved; innovation; light microscopy; microscopic imaging; molecular imaging; molecular marker; molecular phenotype; neural circuit; novel strategies; preservation; reconstruction; theories

Project Summary Recent years have seen major breakthroughs in methodology for studying two complex yet fundamental aspects of brain structure: synaptic connectivity patterns and the heterogeneous distribution of molecules. Due to an ongoing technical barrier that has endured for decades, advances in circuit imaging and molecular imaging have progressed almost entirely in parallel, and there are still no routine methods for integrating molecular information into synaptic circuit maps. Imaging brain structure with enough resolution to visualize synapses requires electron microscopy (EM), and EM is not compatible with the standard methods used to identify molecules by light microscopy. It is clear from biochemical data that highly multiplexed labeling of proteins and RNA transcripts will be necessary to generate comprehensive maps of the brain’s molecular structure. To address this need, a number of approaches aiming to extend the spatial resolution and limits of multiplexed labeling of fluorescence microscopy have been developed. The resolution of EM is still orders of magnitude higher than any light-level technique, however, and EM remains the only modality that reveals structural details. EM also presents a unique opportunity for molecular labeling. EM image volumes are reconstructed from serial ultrathin sections, and by applying a different probe to each section a large number of molecules can be localized in a single structure – hundreds or more in the case of a neuron. In contrast to tissue specimens used in light microscopy, ultrathin EM sections are not readily amenable to simple immunohistochemistry (IHC) or in situ hybridization (ISH) protocols. A major reason for this is incompatibility between sample preparation practices: the strong fixatives and dense embedding resins used in EM damage or occlude molecular targets, while the harsh treatments used to facilitate molecular detection degrade fine tissue structure. The problem can be circumvented by the use of specially engineered transgenic reporters, but these do not solve the problem of detecting endogenous molecules in large numbers. In this project, we will draw on long-established methods from the EM and histology fields to develop an unconventional approach to labeling EM sections, and apply this approach to identify molecular cell and synapse types using three different workflows. Our strategy employs removable embedding media, which are standard in light microscopy and which, contrary to traditional assumptions, we have found to be perfectly compatible with EM imaging. To maximize efficiency and flexibility in imaging workflows, we will develop labeling protocols that prioritize resolution, sensitivity, and throughput to different degrees. If successful, this project will produce methods uniquely capable of combining EM-level structural imaging with multiplexed labeling of endogenous molecules, and will dramatically increase the depth of information obtained from EM volume reconstructions.

项目概要 近年来，研究两种复杂的方法在方法上取得了重大突破 大脑结构的基本方面：突触连接模式和异质分布 由于几十年来持续存在的技术障碍，电路成像和分子方面取得了进步。 分子成像几乎完全并行进展，并且仍然没有常规方法 分子将信息整合到突触回路图中，以足够的分辨率对大脑结构进行成像。 可视化突触需要电子显微镜 (EM)，而 EM 与标准方法不兼容 用于通过光学显微镜识别分子 从生化数据可以清楚地看出，高度多重标记。 蛋白质和 RNA 转录本的分析对于生成大脑分子的全面图谱是必要的。 为了满足这种需求，许多方法旨在扩展空间分辨率和限制。 荧光显微镜的多重标记已经发展起来，电镜的分辨率仍然是几个数量级。 然而，其幅度高于任何光级技术，并且 EM 仍然是揭示 结构细节也为分子标记提供了独特的机会。 由连续的超薄切片重建，并通过对每个切片应用不同的探针，获得大量 与神经元相比，分子可以定位在单个结构中——数百个或更多。 光学显微镜中使用的组织标本，超薄电镜切片不容易进行简单的分析 免疫组织化学 (IHC) 或原位杂交 (ISH) 方案的一个主要原因是不兼容。 样品制备实践之间的区别：电磁损伤中使用的强固定剂和致密包埋树脂 或遮挡分子目标，而用于促进分子检测的严厉处理会降低精细度 这个问题可以通过使用专门设计的转基因生产者来解决，但是 这些并不能解决大量检测内源分子的问题，在这个项目中，我们将。 借鉴 EM 和组织学领域长期确立的方法，开发一种非常规方法 标记 EM 切片，并应用这种方法使用三种不同的方法来识别分子细胞和突触类型 我们的策略采用可移动的包埋介质，这是光学显微镜和 与传统假设相反，我们发现它与电磁成像完美兼容。 为了最大限度地提高成像工作流程的效率和灵活性，我们将开发优先考虑的标签协议 如果成功，该项目将产生不同程度的分辨率、灵敏度和通量。 独特地能够将 EM 水平结构成像与内源性分子的多重标记相结合， 并将显着增加从电磁体重建中获得的信息深度。