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.

项目摘要 近年来，在研究两个复杂的但 脑结构的基本方面：突触连接模式和神经元的异质性分布 分子。由于持续了几十年的技术障碍，电路成像和 分子成像几乎完全平行发展，仍然没有常规方法 将分子信息整合到突触电路图中。用足够的分辨率成像大脑结构， 突触的可视化需要电子显微镜（EM），而EM与标准方法不兼容 用于通过光学显微镜识别分子。从生物化学数据可以清楚地看出，高度多重标记 蛋白质和RNA转录本的合成将是必要的，以产生大脑分子的全面地图。 结构为了满足这一需求，许多旨在扩展空间分辨率和限制的方法被提出。 已经开发了荧光显微术的多重标记。EM的分辨率仍然是 然而，比任何光级技术都高的幅度，EM仍然是揭示 结构细节。EM也为分子标记提供了独特的机会。EM图像体积是 从连续的切片中重建，并通过对每个切片应用不同的探针， 分子可以局限在一个单一的结构中--在神经元的情况下有数百个或更多。相比 在光学显微镜中使用的组织标本中，ECOEM切片不容易进行简单的检查。 免疫组织化学（IHC）或原位杂交（ISH）方案。一个主要原因是不兼容 样品制备实践之间：EM损伤中使用的强固定剂和致密包埋树脂 或封闭分子靶点，而用于促进分子检测的苛刻处理会使精细 组织结构这个问题可以通过使用特别设计的转基因报告基因来解决， 但这些并没有解决检测大量内源性分子的问题。在这个项目中，我们将 利用EM和组织学领域的长期建立的方法，开发一种非传统的方法， 标记EM切片，并应用这种方法来识别分子细胞和突触类型，使用三种不同的 工作流程。我们的策略采用可移动的包埋介质，这是光学显微镜的标准， 与传统的假设相反，我们发现它与EM成像完全兼容。到 最大限度地提高成像工作流程的效率和灵活性，我们将制定标签协议， 分辨率、灵敏度和吞吐量。如果成功，这个项目将产生方法， 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， 并且将显著增加从EM体积重建获得的信息的深度。