Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes
Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes
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DOI：
10.1073/pnas.1121558109
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发表时间：
2012-04-17
影响因子：
11.1
通讯作者：
Hess, Harald F.
中科院分区：
文献类型：
--
作者：
Kopek, Benjamin G.;Shtengel, Gleb;Hess, Harald F.
特定蛋白质在其细胞环境中的显微图像产生对生物过程和细胞结构的重要见解。超分辨率光学显微镜技术的出现提供了增强EM与纳米分辨率荧光显微镜的可能性，以获得蛋白质在细胞超微结构中的精确位置。不幸的是，努力结合联合收割机超分辨荧光和EM已受阻的分歧和不兼容的样品制备协议的两种方法。在这里，我们描述了一个协议，既保留了微妙的光激活荧光蛋白标签必不可少的超分辨率显微镜和精细的超微结构的EM背景。这种制备方法可以在500- 750-nm的切片中直接进行3D成像，采用干涉光活化定位显微镜，然后扫描聚焦离子束烧蚀产生的EM图像。我们使用这个过程来“着色”的详细EM图像的标记蛋白质的位置的电子探针。这里提出的方法提供了一个新的水平的定义在体内的性质组织的线粒体类核，我们希望这种简单的方法适用于许多其他生物学问题，可以回答直接成像。
Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to "colorize" detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging.