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.

中文翻译：

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生物结构的3D重建：用于电子断层扫描中多个倾斜序列的对齐和重建的自动化程序。

透射电子显微镜可以收集标本的多个视图，并通过电子断层扫描对其进行计算机化的三维重建和分析。在这里，我们描述了自动多倾斜数据采集，倾斜系列处理和对齐方法的开发，这些方法允许从大量倾斜系列中组装电子断层扫描数据，从而提高数据质量并增强与弱染色结构相关的对比度。该方案有利于可视化从各种尺寸的生物样本塑料包埋样品中获取的精细结构的纳米尺度细节。由于带有重金属的塑料包埋样品对总剂量而不是电子剂量率较不敏感，通过在更大范围的样本方向上累积相同区域的低剂量电子显微照片，可以实现对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高级程序一起实施，从而使收集，图像对齐以及多倾斜层析成像数据的处理成为无缝过程。我们证明了从描述良好的生物结构的样品的高质量重建。这些包括巨型Mimivirus和网格蛋白包被的囊泡，它们在正常的细胞内环境中就地成像。实例提供了通过高压冷冻和冷冻替代以及在环氧树脂包埋之前通过化学固定制备的培养细胞样品。