Murata Kazuyoshi, Esaki Masatoshi, Ogura Teru, Arai Shigeo, Yamamoto Yuta, Tanaka Nobuo
National Institute for Physiological Sciences, Okazaki, Aichi 444-8585, Japan.
Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan.
Ultramicroscopy. 2014 Nov;146:39-45. doi: 10.1016/j.ultramic.2014.05.008. Epub 2014 Jun 2.
Electron tomography using a high-voltage electron microscope (HVEM) provides three-dimensional information about cellular components in sections thicker than 1 μm, although in bright-field mode image degradation caused by multiple inelastic scattering of transmitted electrons limit the attainable resolution. Scanning transmission electron microscopy (STEM) is believed to give enhanced contrast and resolution compared to conventional transmission electron microscopy (CTEM). Samples up to 1 μm in thickness have been analyzed with an intermediate-voltage electron microscope because inelastic scattering is not a critical limitation, and probe broadening can be minimized. Here, we employed STEM at 1 MeV high-voltage to extend the useful specimen thickness for electron tomography, which we demonstrate by a seamless tomographic reconstruction of a whole, budding Saccharomyces cerevisiae yeast cell, which is ~3 μm in thickness. High-voltage STEM tomography, especially in the bright-field mode, demonstrated sufficiently enhanced contrast and intensity, compared to CTEM tomography, to permit segmentation of major organelles in the whole cell. STEM imaging also reduced specimen shrinkage during tilt-series acquisition. The fidelity of structural preservation was limited by cytoplasmic extraction, and the spatial resolution was limited by the relatively large convergence angle of the scanning probe. However, the new technique has potential to solve longstanding problems of image blurring in biological specimens beyond 1 μm in thickness, and may facilitate new research in cellular structural biology.
使用高压电子显微镜（HVEM）进行电子断层扫描可提供有关厚度超过1μm切片中细胞成分的三维信息，尽管在明场模式下，透射电子的多次非弹性散射导致的图像退化限制了可达到的分辨率。与传统透射电子显微镜（CTEM）相比，扫描透射电子显微镜（STEM）被认为具有更高的对比度和分辨率。由于非弹性散射不是关键限制因素，并且可以将探针展宽最小化，因此已使用中压电子显微镜对厚度达1μm的样品进行了分析。在这里，我们采用1 MeV高压的STEM来扩展用于电子断层扫描的有用样品厚度，我们通过对整个厚度约为3μm的出芽酿酒酵母细胞进行无缝断层重建来证明这一点。与CTEM断层扫描相比，高压STEM断层扫描，特别是在明场模式下，显示出足够增强的对比度和强度，以允许对整个细胞中的主要细胞器进行分割。STEM成像还减少了倾斜系列采集过程中的样品收缩。结构保存的保真度受到细胞质提取的限制，空间分辨率受到扫描探针相对较大的会聚角的限制。然而，这项新技术有潜力解决厚度超过1μm的生物标本中长期存在的图像模糊问题，并可能促进细胞结构生物学的新研究。
