期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 112, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/54066
关键词
Plant Biology; Issue 112; High-pressure freezing; freeze-fracture; double-layer coating; cryo-scanning electron microscopy; resurrection plants; chloroplast; thylakoid membrane; supramolecular organization; exoplasmic fracture face; protoplasmic fracture face; photosystem
资金
- United States-Israel Binational Agricultural Research and Development Fund [US-4334-10]
- Israel Science Foundation [1034/12]
- Human Frontier Science Program [RGP0005/2013]
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.
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