Nagayama Kuniaki, Danev Radostin
Okazaki Institute for Integrative Bioscience and National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki 444-8787, Japan.
Philos Trans R Soc Lond B Biol Sci. 2008 Jun 27;363(1500):2153-62. doi: 10.1098/rstb.2008.2268.
Phase contrast transmission electron microscopy (TEM) based on thin-film phase plates has been developed and applied to biological systems. Currently, development is focused on two techniques that employ two different types of phase plates. The first technique uses a Zernike phase plate, which is made of a uniform amorphous carbon film that completely covers the aperture of an objective lens and can retard the phase of electron waves by pi/2, except at the centre where a tiny hole is drilled. The other technique uses a Hilbert phase plate, which is made of an amorphous carbon film that is twice as thick as the Zernike phase plate, covers only half of the aperture and retards the electron wave phase by pi. By combining the power of efficient phase contrast detection with the accurate preservation achieved by a cryotechnique such as vitrification, macromolecular complexes and supermolecular structures inside intact bacterial or eukaryotic cells may be visualized without staining. Phase contrast cryo-TEM has the potential to bridge the gap between cellular and molecular biology in terms of high-resolution visualization. Examples using proteins, viruses, cyanobacteria and somatic cells are provided.
基于薄膜相板的相差透射电子显微镜（TEM）已被开发并应用于生物系统。目前，研究重点集中在两种采用不同类型相板的技术上。第一种技术使用泽尼克相板，它由均匀的非晶碳膜制成，完全覆盖物镜的孔径，除了在中心钻有一个小孔的地方外，能使电子波的相位延迟π/2。另一种技术使用希尔伯特相板，它由厚度是泽尼克相板两倍的非晶碳膜制成，仅覆盖孔径的一半，并使电子波相位延迟π。通过将高效相差检测的能力与诸如玻璃化等冷冻技术所实现的精确保存相结合，完整细菌或真核细胞内的大分子复合物和超分子结构无需染色即可可视化。相差冷冻TEM在高分辨率可视化方面有潜力弥合细胞生物学和分子生物学之间的差距。文中提供了使用蛋白质、病毒、蓝细菌和体细胞的示例。
