Ninagawa Takako, Eguchi Akemi, Kawamura Yukio, Konishi Tadashi, Narumi Akira
Graduate School of Mechanical Engineering, Kanagawa Institute of Technology, 1030 Shimo-ogino, Atsugi, Kanagawa, 243-0292, Japan.
Toyo Engineering Works, LTD., 1634, Shimotsuruma, Yamato, Kanagawa, 242-0001, Japan.
Cryobiology. 2016 Aug;73(1):20-9. doi: 10.1016/j.cryobiol.2016.06.003. Epub 2016 Jun 21.
Intracellular ice crystal formation (IIF) causes several problems to cryopreservation, and it is the key to developing improved cryopreservation techniques that can ensure the long-term preservation of living tissues. Therefore, the ability to capture clear intracellular freezing images is important for understanding both the occurrence and the IIF behavior. The authors developed a new cryomicroscopic system that was equipped with a high-speed camera for this study and successfully used this to capture clearer images of the IIF process in the epidermal tissues of strawberry geranium (Saxifraga stolonifera Curtis) leaves. This system was then used to examine patterns in the location and formation of intracellular ice crystals and to evaluate the degree of cell deformation because of ice crystals inside the cell and the growing rate and grain size of intracellular ice crystals at various cooling rates. The results showed that an increase in cooling rate influenced the formation pattern of intracellular ice crystals but had less of an effect on their location. Moreover, it reduced the degree of supercooling at the onset of intracellular freezing and the degree of cell deformation; the characteristic grain size of intracellular ice crystals was also reduced, but the growing rate of intracellular ice crystals was increased. Thus, the high-speed camera images could expose these changes in IIF behaviors with an increase in the cooling rate, and these are believed to have been caused by an increase in the degree of supercooling.
细胞内冰晶形成（IIF）给冷冻保存带来了几个问题，并且它是开发能够确保活体组织长期保存的改进冷冻保存技术的关键。因此，获取清晰的细胞内冷冻图像的能力对于理解IIF的发生和行为都很重要。作者开发了一种配备高速相机的新型低温显微镜系统用于本研究，并成功地用它获取了草莓天竺葵（Saxifraga stolonifera Curtis）叶片表皮组织中IIF过程的更清晰图像。然后使用该系统检查细胞内冰晶的位置和形成模式，并评估由于细胞内冰晶导致的细胞变形程度以及在不同冷却速率下细胞内冰晶的生长速率和晶粒尺寸。结果表明，冷却速率的增加影响细胞内冰晶的形成模式，但对其位置的影响较小。此外，它降低了细胞内冷冻开始时的过冷度和细胞变形程度；细胞内冰晶的特征晶粒尺寸也减小了，但细胞内冰晶的生长速率增加了。因此，高速相机图像可以揭示随着冷却速率增加IIF行为的这些变化，并且据信这些变化是由过冷度增加引起的。
