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，研究两个复杂的方法论都取得了重大突破 大脑结构的基本方面：突触连通性模式和异质分布 分子。由于持续的技术障碍已经持续了数十年，因此电路成像的进步和 分子成像几乎完全并联进展，并且仍然没有常规方法 将分子信息整合到突触电路图中。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），并且EM与标准方法不兼容 用于通过光学显微镜鉴定分子。从生化数据可以清楚地看出高度多重标记 为了生成大脑分子的综合图，必须需要蛋白质和RNA转录本 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 已经开发了荧光显微镜的多重标记。 EM的分辨率仍然是 但是 结构细节。 EM还为分子标记提供了独特的机会。 em图像量是 从串行超薄部分重建，并通过对每个部分应用不同的探针 分子可以定位在单个结构中 - 在神经元的情况下数百个或更多。与 在光学显微镜中使用的组织标本，超薄EM部分不容易适应简单 免疫组织化学（IHC）或原位杂交（ISH）方案。造成这种情况的主要原因是不兼容 在样品制备实践之间：固定的固定和密集的嵌入树脂用于EM损伤 或遮挡分子靶标，而用于促进分子检测的HARMSH治疗 组织结构。可以通过使用专门设计的转基因记者来规避问题，但是 这些不能解决大量检测内源分子的问题。在这个项目中，我们将 利用来自EM和组织学领域的悠久方法，以开发一种非常规的方法 标记EM部分，并采用这种方法使用三种不同的分子细胞和突触类型 工作流程。我们的战略员工可移动嵌入媒体，这是光学显微镜和 与传统假设形成鲜明对比的是，我们发现与EM成像完全兼容。到 最大化成像工作流程的效率和灵活性，我们将开发优先级的标签协议 分辨率，灵敏度和吞吐量在不同程度上。如果成功，该项目将产生方法 独特能够将EM级结构成像与内源分子的多重标记相结合， 并将大大增加从EM体积重建获得的信息深度。