Charuvi Dana, Nevo Reinat, Kaplan-Ashiri Ifat, Shimoni Eyal, Reich Ziv
Department of Biological Chemistry, Weizmann Institute of Science; Institute of Plant Sciences, Agricultural Research Organization, Volcani Center;
Department of Biological Chemistry, Weizmann Institute of Science.
J Vis Exp. 2016 Jun 23(112):54066. doi: 10.3791/54066.
Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we describe a technique for studying the supramolecular organization of photosynthetic (thylakoid) membranes within leaf samples. This is achieved by high-pressure freezing of leaf tissues, freeze-fracturing, double-layer coating and finally cryo-SEM imaging. Use of the double-layer coating method allows acquiring high magnification (>100,000X) images with minimal beam damage to the frozen-hydrated samples as well as minimal charging effects. Using the described procedures we investigated the alterations in supramolecular distribution of photosystem and light-harvesting antenna protein complexes that take place during dehydration of the resurrection plant Craterostigma pumilum, in situ.
对冷冻断裂样品进行低温扫描电子显微镜（SEM）观察，能够在接近自然状态的条件下研究生物结构。在此，我们描述一种用于研究叶片样品中光合（类囊体）膜超分子组织的技术。这是通过对叶片组织进行高压冷冻、冷冻断裂、双层镀膜，最后进行低温SEM成像来实现的。使用双层镀膜方法能够获取高放大倍数（>100,000倍）的图像，同时对冷冻水合样品的束损伤最小，充电效应也最小。利用所描述的程序，我们原位研究了复苏植物矮小景天脱水过程中光系统和捕光天线蛋白复合体超分子分布的变化。
