Preparation of cryofixed cells for improved 3D ultrastructure with scanning transmission electron tomography
Preparation of cryofixed cells for improved 3D ultrastructure with scanning transmission electron tomography
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DOI：
10.1007/s00418-010-0765-z
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发表时间：
2011-01-01
影响因子：
2.3
通讯作者：
Walther, Paul
中科院分区：
文献类型：
--
作者：
Hoehn, Katharina;Sailer, Michaela;Walther, Paul
与常规的透射式电子显微镜相比，扫描透射式电子断层成像提供了更强的对比度，并且可以分析更厚的样品，最大可达1微米或更大，因为焦深和非弹性散射不受限制。在这项研究中，我们将这种新的成像方法与最先进的样品制备技术相结合，使用新型的轻质透明蓝宝石样品载体进行高压冷冻，并采用冷冻替代方案来提高膜的对比度。这种组合使得成像膜和其他亚细胞结构具有无与伦比的质量。这一点在线粒体中得到了证实，线粒体膜的内膜和外膜以及冠部的膜看起来非常接近，具有最小的膜间隙。这些发现与使用冷冻压裂法的旧观察结果吻合得很好。在880 nm厚的噬血细胞切片上，在断层图像的虚拟切片上可以观察到膜片的三维结构。在一个样本中可以看到微管、肌动蛋白和中间丝。然而，使用表面扫描电子断层扫描技术甚至可以更好地在3D中观察到中间细丝。
Scanning transmission electron tomography offers enhanced contrast compared to regular transmission electron microscopy, and thicker samples, up to 1 mu m or more, can be analyzed, since the depth of focus and inelastic scattering are not limitations. In this study, we combine this novel imaging approach with state of the art specimen preparation by using novel light transparent sapphire specimen carrier for high-pressure freezing and a freeze substitution protocol for better contrast of membranes. This combination allows for imaging membranes and other subcellular structures with unsurpassed quality. This is demonstrated with mitochondria, where the inner and outer mitochondrial membranes as well as the membranes in the cristae appear in very close apposition with a minimal intermembrane space. These findings correspond well with old observations using freeze fracturing. In 880-nm thick sections of hemophagocytes, the three-dimensional structure of membrane sheets could be observed in the virtual sections of the tomogram. Microtubules, actin and intermediate filaments could be visualized within one sample. Intermediate filaments, however, could even be better observed in 3D using surface scanning electron tomography.