Freezing resistant plant organs are capable to manage ice formation, ice propagation, and ice accommodation down to variable temperature limits without damage. Insights in ice management strategies are essential for the fundamental understanding of plant freezing and frost survival. However, knowledge about ice management is scarce. Ice crystal localisation inside plant tissues is challenging and is mainly based on optical appearance of ice in terms of colour and shape, investigated by microscopic methods. Notwithstanding, there are major uncertainties regarding the reliability and accuracy of ice identification and localisation. Surface light reflections, which can originate from water or resin, even at non-freezing temperatures, can have a similar appearance as ice. We applied the principle of birefringence, which is a property of ice but not of liquid water, in reflected-light microscopy to localise ice crystals in frozen plant tissues in an unambiguous manner. In reflected-light microscopy, water was clearly visible, while ice was more difficult to identify. With the presented polarised cryo-microscopic system, water, including surface light reflections, became invisible, whereas ice crystals showed a bright and shiny appearance. Based on this, we were able to detect loci where ice crystals are accommodated in frozen and viable plant tissues. In Buxus sempervirens leaves, large ice needles occupied and expanded the space between the adaxial and abaxial leaf tissues. In Galanthus nivalis leaves, air-filled cavities became filled up with ice. Buds of Picea abies managed ice in a cavity at the bud basis and between bud scales. By observing the shape and attachment point of the ice crystals, it was possible to identify tissue fractions that segregate intracellular water towards the aggregating ice crystals. Cryo-microscopy in reflected-polarised-light allowed a robust identification of ice crystals in frozen plant tissue. It distinguishes itself, compared with other methods, by its ease of ice identification, time and cost efficiency and the possibility for high throughput. Profound knowledge about ice management strategies, within the whole range of freezing resistance capacities in the plant kingdom, might be the link to applied science for creating arrangements to avoid future frost damage to crops.

中文翻译：

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在反射偏振光下通过冷冻显微镜确定植物组织中的冰适应。

抗冻植物器官能够控制冰的形成、冰的传播和冰适应，降低到可变的温度限制，而不会损坏。了解冰管理策略对于从根本上了解植物冻结和霜冻生存至关重要。然而，关于冰管理的知识很少。植物组织内的冰晶定位具有挑战性，主要基于冰在颜色和形状方面的光学外观，通过显微镜方法进行研究。尽管如此，冰识别和定位的可靠性和准确性仍存在很大的不确定性。来自水或树脂的表面光反射，即使在非冰点温度下，也可能具有与冰相似的外观。我们应用了双折射原理，这是冰的特性，但不是液态水的特性，在反射光显微镜下以明确的方式定位冷冻植物组织中的冰晶。在反射光显微镜下，水清晰可见，而冰更难以识别。使用所呈现的偏振低温显微镜系统，包括表面光反射在内的水变得不可见，而冰晶则呈现出明亮而有光泽的外观。基于此，我们能够检测到冰晶容纳在冷冻和有活力的植物组织中的位点。在长生黄杨的叶子中，大的冰针占据并扩大了正面和背面叶组织之间的空间。在雪花莲的叶子中，充满空气的空腔被冰填满。Picea abies 的芽在芽基和芽鳞之间的空腔中管理冰。通过观察冰晶的形状和附着点，可以识别将细胞内水与聚集的冰晶分离的组织部分。反射偏振光中的冷冻显微镜可以可靠地识别冷冻植物组织中的冰晶。与其他方法相比，它的独特之处在于其易于识别冰块、时间和成本效率以及高通量的可能性。在植物界的整个抗冻能力范围内，对冰管理策略的深入了解可能是与应用科学的联系，以制定避免未来对作物的霜冻损害的安排。反射偏振光中的冷冻显微镜可以可靠地识别冷冻植物组织中的冰晶。与其他方法相比，它的独特之处在于其易于识别冰块、时间和成本效率以及高通量的可能性。在植物界的整个抗冻能力范围内，对冰管理策略的深入了解可能是与应用科学的联系，以制定避免未来对作物的霜冻损害的安排。反射偏振光中的冷冻显微镜可以可靠地识别冷冻植物组织中的冰晶。与其他方法相比，它的独特之处在于其易于识别冰块、时间和成本效率以及高通量的可能性。在植物界的整个抗冻能力范围内，对冰管理策略的深入了解可能是与应用科学的联系，以制定避免未来对作物的霜冻损害的安排。