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

项目摘要 近年来，研究两个复杂结构的方法论取得了重大突破 大脑结构的基本方面：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 分子成像的进展几乎完全是平行的，仍然没有常规的方法来 将分子信息整合到突触电路图中。以足够的分辨率成像大脑结构 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。从生化数据中可以清楚地看到，高度多元化的标记 蛋白质和RNA转录本将是产生大脑分子的全面图谱所必需的 结构。为了满足这一需求，一些旨在扩展空间分辨率和限制的方法 荧光显微镜的多重标记技术已经发展起来。EM的分辨率仍为数量级 然而，EM的震级高于任何光级技术，并且EM仍然是唯一能够揭示 结构详图。EM也为分子标记提供了一个独特的机会。EM映像卷有 从连续的超薄切片重建，并通过在每个切片上应用不同的探头 分子可以定位在一个单一的结构中--对于神经元来说，分子可以定位在数百个或更多的结构中。与之形成鲜明对比的是 用于光学显微镜的组织标本，超薄的EM切片不容易服从于简单 免疫组织化学(IHC)或原位杂交(ISH)方法。造成这种情况的主要原因是不兼容 样品制备方法之间：用于EM损伤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苛刻处理会使细微的 组织结构。这个问题可以通过使用专门设计的转基因记者来规避，但 这些并不能解决大量检测内源性分子的问题。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来 标记EM切片，并应用此方法识别分子细胞和突触类型，使用三种不同的 工作流程。我们的策略使用了可拆卸的嵌入介质，这是光学显微镜和 与传统的假设相反，我们发现它与电磁成像完全兼容。至 最大限度地提高成像工作流的效率和灵活性，我们将制定优先排序的标注协议 不同程度的分辨率、灵敏度和吞吐量。如果成功，这个项目将产生方法 独一无二地能够将EM级结构成像与内源分子的多重标记结合在一起， 并将极大地增加从EM体积重建获得的信息深度。

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.