3D reconstruction of biological structures: automated procedures for alignment and reconstruction of multiple tilt series in electron tomography.
3D reconstruction of biological structures: automated procedures for alignment and reconstruction of multiple tilt series in electron tomography.
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DOI：
10.1186/s40679-016-0021-2
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发表时间：
2017
影响因子：
--
通讯作者：
Ellisman MH
中科院分区：
文献类型：
--
作者：
Phan S;Boassa D;Nguyen P;Wan X;Lanman J;Lawrence A;Ellisman MH
透射电子显微镜允许收集标本的多个视图及其计算机三维重建和电子断层扫描分析。在这里，我们描述了自动化多倾斜数据采集、倾斜序列处理和校准方法的发展，这些方法允许从更多倾斜序列中组装电子层析数据，从而提高数据质量并增加与弱染色结构相关的对比度。该方案有助于在所有维度上可视化从生物标本的塑料嵌入样品中提取的纳米尺度精细结构细节。由于重金属对比塑料包埋样品对总剂量比电子剂量率更不敏感，因此可以通过在更大范围的样品取向上积累相同区域的较低剂量电子显微照片来实现重建空间的最佳重采样。计算机化的多倾斜系列收集方案与自动化的高级程序一起实施，使收集，图像对齐和处理多倾斜断层扫描数据成为一个无缝的过程。我们从描述良好的生物结构样品中展示了高质量的重建。这些包括巨大的Mimivirus和网格蛋白包被的囊泡，在它们正常的细胞内环境中原位成像。在环氧树脂包埋前，通过高压冷冻和冷冻取代以及化学固定制备的培养细胞样品提供了实例。
Transmission electron microscopy allows the collection of multiple views of specimens and their computerized three-dimensional reconstruction and analysis with electron tomography. Here we describe development of methods for automated multi-tilt data acquisition, tilt-series processing, and alignment which allow assembly of electron tomographic data from a greater number of tilt series, yielding enhanced data quality and increasing contrast associated with weakly stained structures. This scheme facilitates visualization of nanometer scale details of fine structure in volumes taken from plastic-embedded samples of biological specimens in all dimensions. As heavy metal-contrasted plastic-embedded samples are less sensitive to the overall dose rather than the electron dose rate, an optimal resampling of the reconstruction space can be achieved by accumulating lower dose electron micrographs of the same area over a wider range of specimen orientations. The computerized multiple tilt series collection scheme is implemented together with automated advanced procedures making collection, image alignment, and processing of multi-tilt tomography data a seamless process. We demonstrate high-quality reconstructions from samples of well-described biological structures. These include the giant Mimivirus and clathrin-coated vesicles, imaged in situ in their normal intracellular contexts. Examples are provided from samples of cultured cells prepared by high-pressure freezing and freeze-substitution as well as by chemical fixation before epoxy resin embedding.