National Institute of Advanced Industrial Science and Technology, Biomedicinal Information Research Center, 2-3-26, Aomi, Koto-ku, Tokyo 135-0064, Japan.
Micron. 2011 Feb;42(2):100-6. doi: 10.1016/j.micron.2010.08.006. Epub 2010 Sep 8.
To obtain high-resolution images of biological macromolecules, a cryo-electron microscope (cryo-EM) is useful to preserve the hydrated state of the molecules. In addition, the cryo-conditions reduce some of the electron radiation damage, although averaging is necessary to obtain the molecular resolution from biological macromolecules. For the averaging, the three reconstruction methods of electron crystallography, helical reconstruction, and single particle analysis facilitate the determination of the atomic models. Here we describe suitable techniques for high-resolution imaging in these three different methods, based on our experiences using cryo-EM with a liquid-helium cooled stage. Irradiation damage by the electron beam is the most serious problem in the structural analysis of a biological specimen, and we clearly show that further reduction of the specimen temperature, from liquid nitrogen to liquid helium temperature, improves the cryo-protection factor by at least two-fold for both glucose-embedded and frozen-hydrated specimens. We review the image processing, and then discuss the future prospects of cryo-EM.
为了获得生物大分子的高分辨率图像，使用 cryo-electron microscope（cryo-EM）有助于保持分子的水合状态。此外，cryo 条件减少了一些电子辐射损伤，尽管需要平均化才能从生物大分子中获得分子分辨率。对于平均化，电子晶体学、螺旋重建和单颗粒分析这三种重构方法有助于确定原子模型。在这里，我们根据使用带有液氦冷却台的 cryo-EM 的经验，描述了这三种不同方法中进行高分辨率成像的合适技术。电子束的辐照损伤是生物样本结构分析中最严重的问题，我们清楚地表明，进一步降低样本温度，从液氮降至液氦温度，对于葡萄糖嵌入和冷冻水合样本， cryo 保护因子至少提高两倍。我们回顾了图像处理，然后讨论了 cryo-EM 的未来前景。
