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使用内源性组织标志物的无标记三维细胞电子显微镜技术

Label-free 3D-CLEM Using Endogenous Tissue Landmarks.

作者信息

Luckner Manja, Burgold Steffen, Filser Severin, Scheungrab Maximilian, Niyaz Yilmaz, Hummel Eric, Wanner Gerhard, Herms Jochen

机构信息

Department of Biology I, Biocenter Ludwig-Maximilians-University Munich, Planegg-Martinsried 82152, Germany; German Center for Neurodegenerative Diseases (DZNE), Translational Brain Research, Munich 81377, Germany.

German Center for Neurodegenerative Diseases (DZNE), Translational Brain Research, Munich 81377, Germany; Center for Neuropathology, Ludwig-Maximilians-University Munich, Munich 81377, Germany; Carl Zeiss Microscopy, Oberkochen 73447, Germany.

出版信息

iScience. 2018 Aug 31;6:92-101. doi: 10.1016/j.isci.2018.07.012. Epub 2018 Jul 20.

DOI:10.1016/j.isci.2018.07.012
PMID:30240628
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6137285/
Abstract

Emerging 3D correlative light and electron microscopy approaches enable studying neuronal structure-function relations at unprecedented depth and precision. However, established protocols for the correlation of light and electron micrographs rely on the introduction of artificial fiducial markers, such as polymer beads or near-infrared brandings, which might obscure or even damage the structure under investigation. Here, we report a general applicable "flat embedding" preparation, enabling high-precision overlay of light and scanning electron micrographs, using exclusively endogenous landmarks in the brain: blood vessels, nuclei, and myelinated axons. Furthermore, we demonstrate feasibility of the workflow by combining in vivo 2-photon microscopy and focused ion beam scanning electron microscopy to dissect the role of astrocytic coverage in the persistence of dendritic spines.

摘要

新兴的三维相关光电子显微镜方法能够以前所未有的深度和精度研究神经元结构与功能的关系。然而，已有的光镜和电镜图像关联方案依赖于引入人工基准标记，如聚合物微球或近红外标记，这可能会掩盖甚至损害被研究的结构。在此，我们报告一种普遍适用的“平面包埋”制备方法，仅利用大脑中的内源性标志物（血管、细胞核和有髓轴突）就能实现光镜和扫描电镜图像的高精度叠加。此外，我们通过结合体内双光子显微镜和聚焦离子束扫描电子显微镜来剖析星形胶质细胞覆盖在树突棘持久性中的作用，证明了该工作流程的可行性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7efe/6137285/d5b767c9905a/gr5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7efe/6137285/1bb6ba08f51d/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7efe/6137285/09d03f97f942/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7efe/6137285/059c66e66669/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7efe/6137285/9c71a4771540/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7efe/6137285/8b0789e5ceb5/gr4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7efe/6137285/d5b767c9905a/gr5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7efe/6137285/1bb6ba08f51d/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7efe/6137285/09d03f97f942/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7efe/6137285/059c66e66669/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7efe/6137285/9c71a4771540/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7efe/6137285/8b0789e5ceb5/gr4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7efe/6137285/d5b767c9905a/gr5.jpg

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