Electron tomography of ice-embedded prokaryotic cells
Electron tomography of ice-embedded prokaryotic cells
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DOI：
10.1016/s0006-3495(98)74028-7
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发表时间：
1998-02-01
影响因子：
3.4
通讯作者：
Baumeister, W
中科院分区：
文献类型：
--
作者：
Grimm, R;Singh, H;Baumeister, W
通过骤冷将古细菌的整个细胞嵌入玻璃冰中，并通过 120 kV 的自动能量过滤电子断层扫描进行研究。嵌入的细胞厚度在 300 至 750 nm 之间，其结构通过包含 50-140 个图像的倾斜系列重建至 20-40 nm 的分辨率，剂量保持在可容忍的限度内。 20 nm 的分辨率可以在三个维度上可视化已经历部分裂解的嗜气火杆菌细胞 S 层的各个茎。从理论上研究了不同实验条件下低剂量电子断层扫描可达到的分辨率与样品厚度的关系。要在 120 kV (300 kV) 下获得 2 nm 分辨率，样品厚度不得超过 100 nm (150 nm)。对于 10 nm 的分辨率，最大厚度为 450 nm (700 nm)。 300 kV 的加速电压是有利的，主要对于厚度超过 100 nm 的样品。迄今为止的实验研究得出的分辨率比理论值差 2-5 倍。
Whole cells of archaea were embedded in vitreous ice by plunge freezing and investigated by automated energy-filtered electron tomography at 120 kV. The embedded cells were between 300 and 750 nm thick, and their structures were reconstructed to a resolution of 20-40 nm from tilt series comprising 50-140 images, The dose was kept within tolerable limits. A resolution of 20 nm allowed visualization of the individual stalks of the S-layer of Pyrobaculum aerophilum cells, which had undergone partial lysis, in three dimensions. The attainable resolution for low-dose electron tomography under different experimental conditions was theoretically investigated in terms of the specimen thickness. To obtain 2-nm resolution at 120 kV (300 kV), the specimen must not be thicker than 100 nm (150 nm). For a resolution of 10 nm, the maximum thickness is 450 nm (700 nm). An accelerating voltage of 300 kV is advantageous, mainly for specimens thicker than 100 nm. Experimental investigations so far have resulted in a resolution that is worse by a factor of 2-5 as compared to theory.