Many methodological approaches have focused so far on physiological and molecular responses of plant tissues to freezing but only little knowledge is available on the consequences of extracellular ice-formation on cellular ultrastructure that underlies physiological reactions. In this context, the preservation of a defined frozen state during the entire fixation procedure is an essential prerequisite. However, current techniques are not able to fix frozen plant tissues for transmission electron microscopy (TEM) without interrupting the cold chain. Chemical fixation by glutaraldehyde and osmium tetroxide is not possible at sub-zero temperatures. Cryo-fixation methods, such as high pressure freeze fixation (HPF) representing the state-of-the-art technique for best structural preservation, are not equipped for freezing frozen samples. In order to overcome this obstacle, a novel technical approach for maintaining the cold chain of already frozen plant samples prior and during HPF is presented. Different algae (Micrasterias denticulata, Klebsormidium crenulatum) and higher plant tissues (Lemna sp., Ranunculus glacialis, Pinus mugo) were successfully frozen and prepared for HPF at freezing temperatures (− 2 °C, − 5 °C, − 6 °C) within a newly developed automatic freezing unit (AFU), that we manufactured from a standard laboratory freezer. Preceding tests on photosynthetic electron transport and ability to plasmolyse show that the temperatures applied did not impair electron transport in PSII nor cell vitality. The transfer of the frozen specimen from the AFU into the HPF-device and subsequently cryo-fixation were performed without intermediate thawing. After cryo-substitution and further processing, the resulting TEM-micrographs showed excellent ultrastructure preservation of the different organisms when compared to specimens fixed at ambient temperature. The method presented allows preserving the ultrastructure of plant cells in the frozen state during cryo-fixation. The resulting high quality TEM-images represent an important step towards a better understanding of the consequences of extracellular ice formation on cellular ultrastructure. It has the potential to provide new insights into changes of organelle structure, identification of intracellular injuries during ice formation and may help to understand freezing and thawing processes in plant tissues. It may be combined with analytical TEM such as electron energy loss spectroscopy (EELS), X-ray analyses (EDX) and various other electron microscopic techniques.

中文翻译：

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一种制备电子显微镜冷冻生物样品的新技术方法

迄今为止，许多方法学方法都集中在植物组织对冷冻的生理和分子反应上，但关于细胞外冰形成对作为生理反应基础的细胞超微结构的影响的知识很少。在这种情况下，在整个固定过程中保持定义的冷冻状态是必不可少的先决条件。然而，目前的技术无法在不中断冷链的情况下固定冷冻植物组织用于透射电子显微镜 (TEM)。在零下温度下，戊二醛和四氧化锇的化学固定是不可能的。冷冻固定方法，例如代表最佳结构保存的最先进技术的高压冷冻固定 (HPF)，不适合冷冻冷冻样品。为了克服这一障碍，提出了一种在 HPF 之前和期间维持已冷冻植物样品冷链的新技术方法。不同的藻类（Micrasterias denticulata、Klebsormidium crenulatum）和高等植物组织（Lemna sp.、Ranunculus glacialis、Pinus mugo）在冷冻温度（− 2 °C、− 5 °C、− 6 °C）下成功冷冻并准备用于 HPF在我们用标准实验室冷冻机制造的新开发的自动冷冻装置 (AFU) 内。先前对光合电子传递和质壁分解能力的测试表明，施加的温度不会损害 PSII 中的电子传递和细胞活力。将冷冻标本从 AFU 转移到 HPF 装置中，随后进行冷冻固定，无需中间解冻。经过低温替代和进一步处理后，与在环境温度下固定的标本相比，所得的 TEM 显微照片显示不同生物体的超微结构保存良好。所提出的方法允许在冷冻固定过程中将植物细胞的超微结构保持在冷冻状态。由此产生的高质量 TEM 图像代表了朝着更好地了解细胞外冰形成对细胞超微结构的影响迈出的重要一步。它有可能为细胞器结构的变化、冰形成过程中细胞内损伤的识别提供新的见解，并可能有助于了解植物组织中的冷冻和解冻过程。它可以与分析 TEM 结合使用，例如电子能量损失光谱 (EELS)，