通过低温扫描电子显微镜研究光合膜内冻结，割断叶片组织的超分子组织
Summary
Here we describe a procedure for studying freeze-fractured plant tissues. High-pressure frozen leaf samples are freeze-fractured and double-layer coated, yielding well preserved frozen-hydrated samples that are imaged using the cryo-scanning electron microscope at high magnifications with minimal beam damage.
Abstract
冷冻断裂样品的冷冻扫描电子显微镜（SEM）允许在接近天然条件的生物结构的调查。在这里，我们描述了叶片样品中学习光合（类囊体）膜的超分子组织的技术。这是由叶组织的高压冷冻，冷冻压裂，双层涂层和最后低温SEM成像实现的。双层涂覆方法的使用允许获得高倍率（> 100000）与到冷冻水合样品最小光束损害以及最小充电效果的图像。使用所描述的过程，我们在采取复苏植物Craterostigma细叶脱水过程中进行，现场光系统和捕光天线蛋白复合物超分子分布调查改变。
Introduction
氧光合作用，原产于古代的蓝藻，是由内共生事件，导致叶绿体细胞器的开发继承了藻类和陆生植物。在所有现代的产氧光养，光合作用电子传输和质子动力的产生，并减少功率被称为“类囊体”膜扁平囊样小泡内进行。这些膜容纳该进行光合作用的光驱动反应和提供能量转换介质中的蛋白复合物。植物和（部分）藻类的类囊体膜分化成两个不同的形态结构域：紧紧贴伏膜的区域称为“基粒”和互连基粒未叠加膜，称为“基质片层1。植物和藻类类囊体膜的各种冷冻断裂研究已经进行，在70年代初开始。当冷冻断裂，膜沿着它们的疏水芯2分割，生成exoplasmic面（EF）和原生质面（PF），这取决于蜂窝隔室其中半膜的边界，如最初由Branton 等人提出的。 。在1975年3植物和藻类类囊体有四种不同的裂缝面：EF文件，EFU，PFS和PFU，用's'和'U'表示'堆积'和'拆散'膜的区域，分别为。膜蛋白复合物，这是不分裂或破裂，必须保持与E或膜的P一侧的倾向。最初的意见，即类囊体的不同裂缝面包含不同大小的颗粒和密度4，以及随之而来的大量调查，导致了开展光反应所观察到的颗粒和膜蛋白复合物的鉴定和相关5-13 （见还回顾14,15）。
FRE类囊体膜的结-断裂试验通常进行于叶绿体或分离的类囊体膜的制剂（但见16,17），在任何改变的在结构和/或超分子组织的分离过程期间可能发生的危险。继骨折，复制是铂/炭（PT / C）的蒸发法制备，再由碳（C）厚厚的一层，最后是生物材料18的消化。复制是透射电子显微镜（TEM）观察。传统的冷冻断裂-复制技术继续作为用于研究光合膜的超分子组织和它们适应于不同的一个重要的工具， 例如 ，光，条件19-23。
在我们最近的homoiochlorophyllous复苏植物Craterostigma细叶 24的研究中，我们的目的是探讨在超分子组织为O的变化˚F类囊体膜，以及在整体蜂窝组织，脱水和复水过程中。该homoiochlorophyllous复活的物种的独特性在于它们能够生存在他们的营养组织（叶子）干燥的条件下，同时保持其光合机构。一旦水可用时，这些植物恢复和恢复小时内光合活性几天25。在这项研究中，冻结，割断叶样品的冷冻扫描EM（SEM）成像用高压冷冻样品冷冻固定组合。这些程序提供了在接近其原生状态26的状态可视化冷冻水合生物样品的方法。一个主要的好处是，样品冷冻断裂和涂层，没有连续的步骤后直接检查。这是在不同的相对含水量（RWC）植物的调查尤为重要，因为他们的水合状态，制备过程中保持不变。怎么样以往，人们关键缺点是，当在高放大倍率，光合复合物的大小的精确测量需要扫描冷冻水合样品可从光束损坏成像期间遭受，更是如此。为了克服这一点，一种方法被称为“双层涂层'（DLC）27,28与分别利用特定冷冻SEM成像条件相结合。这导致那些显著光束敏感较少样本和允许的有价值的信息，对光合蛋白质超分子组织和复苏植物C.其他细胞成分的阐明细叶 在原地高放大倍率。
Protocol
Representative Results
Discussion
本文中所描述的技术允许保存完好的高压冷冻的植物组织的由低温扫描电子显微镜的范围内冻结，割断膜调查。使用这些程序的主要优点是样品制备是纯物理;涉及化学品或脱水任何步骤是必要的。因此，它可以在一个近乎原生状态26,32研究生物结构。用叶组织的好处是，可以得到在整体蜂窝组织的信息，以及作为研究特定的膜，如类囊体膜，其天然生理环境， 即内，叶绿体内。另?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢安德烈Kaech（苏黎世大学）为他的扫描电子显微成像有用的建议。这项工作是由美国 – 以色列两国农业研究和发展基金（批准号：US-4334-10，ZR），以色列科学基金会（批准号：1034年至1012年，ZR），和人类前沿科学计划支持（RGP0005 / 2013，ZR）。透射电镜研究在欧文和Cherna莫斯科维茨中心纳米和生物纳米成像在魏茨曼科学研究所进行。
Materials
| ethanol abs | Bio-Lab | 052505 | |
| isopropanol | Bio-Lab | 162605 | |
| 1-hexadecene | Sigma-Aldrich | H7009 | |
| 0.1/0.2 platelets | Engineering Office M. Wohlwend GmbH, Switzerland | 241 | Platelets are of 3-mm diameter and 0.5-mm-thick (Type A) with 0.1/0.2-mm-deep cavities (of diamater 2 mm). Similar platelets can be obtained from Leica Microsystems. |
| high-precision-grade tweezers | Electron Microscopy Sciences | 72706-01 | Dumont (Switzerland) Durostar style #5 tweezers; Can be substituted with other high-precision tweezers. |
| high-pressure freezing machine | Bal-Tec | HPM 010 | High-pressure freezing alternatives: 1. HPF Compact 02, Wohlwend GmbH; 2. HPM 010, RMC Boeckeler; 3. EM PACT2, Leica Microsystems; 4. EM HPM 100, Leica Microsystems; 5. EM ICE, Leica Microsystems. |
| freeze-fracture system | Leica Microsystems | EM BAF 060 | |
| cryo preparation loading stage | Leica Microsystems | 16770228 | |
| specimen holder for univeral freeze fracturing | Leica Microsystems | 16LZ04746VN | Clamp holder for specimen carriers of diameter 3 mm |
| vacuum cryo-transfer shuttle | Leica Microsystems | EM VCT 100 | |
| scanning electron microscope | Zeiss | Ultra 055 | |
| cryo SEM stage | Leica Microsystems | 16770299905 | |
| image acquisiton software | SmartSEM, Carl Zeiss Microscopy GmbH | ||
| image analysis software | Fiji/Image J, National Institute of Health | http://fiji.sc/Fiji |
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