Correlative light- and electron microscopy with chemical tags.
Correlative light- and electron microscopy with chemical tags.
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DOI：
10.1016/j.jsb.2014.03.018
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发表时间：
2014-05
影响因子：
3
通讯作者：
Frangakis AS
中科院分区：
文献类型：
--
作者：
Perkovic M;Kunz M;Endesfelder U;Bunse S;Wigge C;Yu Z;Hodirnau VV;Scheffer MP;Seybert A;Malkusch S;Schuman EM;Heilemann M;Frangakis AS
相关显微镜将荧光蛋白标记的特异性融入高分辨率电子显微照片中。相关显微镜有多种方法，其中大多数使用绿色荧光蛋白（GFP）作为光学显微镜的标记。在这里，我们使用化学标记和合成荧光团来代替，以实现蛋白质特异性标记，并进行多色成像。我们表明，合成荧光团在乙酸双氧铀存在下保留其嵌入后荧光。嵌入后荧光的质量如此之高，以至于可以使用扫描电子显微镜 (SEM) 和透射电子显微镜 (TEM) 的相同方案来制备样本；当检查单一或其他困难的样品时，这一点特别有价值。我们证明，合成荧光团在超分辨率光学显微镜中可提供明亮、分辨率良好的信号，使我们能够在电子显微照片上的 x-y 平面上叠加精度高达 25 nm 的光学显微图像。为了例证我们新方法的保存质量，我们可视化小鼠上皮细胞粘附连接中钙粘蛋白的分子排列。
Correlative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25 nm in the x–y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells.