PDF(Pubmed)
Abstract：
Mapping neuronal networks is a central focus in neuroscience. While volume electron microscopy (vEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide molecular information to identify cell types or functions. We developed an approach that uses fluorescent single-chain variable fragments (scFvs) to perform multiplexed detergent-free immunolabeling and volumetric-correlated-light-and-electron-microscopy on the same sample. We generated eight fluorescent scFvs targeting brain markers. Six fluorescent probes were imaged in the cerebellum of a female mouse, using confocal microscopy with spectral unmixing, followed by vEM of the same sample. The results provide excellent ultrastructure superimposed with multiple fluorescence channels. Using this approach, we documented a poorly described cell type, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.
摘要：
映射神经元网络是神经科学的中心焦点。虽然体积电子显微镜（vEM）可以揭示神经元网络（连接组学）的精细结构，它不提供分子信息来识别细胞类型或功能。我们开发了一种方法，该方法使用荧光单链可变片段（scFvs）对同一样品进行多重无去污剂免疫标记和体积相关的光镜和电子显微镜检查。我们产生了八个靶向脑标记的荧光scFvs。在雌性小鼠的小脑中成像了六个荧光探针，使用共聚焦显微镜与光谱解混合，然后是相同样品的VEM。结果提供了与多个荧光通道叠加的优异的超微结构。使用这种方法，我们记录了一种描述不佳的细胞类型，两种苔藓纤维终端，和一种类型的离子通道的亚细胞定位。因为scFvs可以从现有的单克隆抗体中获得，可以产生数百个这样的探针，以使分子覆盖用于连接组学研究。
