高压冷冻及冷冻替代样品制备方法在体扫描电镜中的应用  

Application of the preparation technique of high⁃pressure freezing and freeze substitution sample in volume scanning electron microscopy

作　　者：顾嘉敏 许淑嫚 郭帅 巩志伟 倪兵[3] 刘铮 GU Jia-min;XU Shu-man;GUO Shuai;GONG Zhi-wei;NI Bing;LIU Zheng(Cryo-Electern Microscopy Center,Southern University of Science and Technology,Shenzhen Guangdong 518055;School of Physics and Electronic Science,East China Normal University,Shanghai 200241;School of Life Science,East China Normal University,Shanghai 200241,China)

机构地区：[1]南方科技大学冷冻电镜中心,深圳广东518055 [2]华东师范大学物理与电子科学学院,上海200241 [3]华东师范大学生命科学学院,上海200241

出　　处：《电子显微学报》2023年第4期471-480,共10页Journal of Chinese Electron Microscopy Society

基　　金：国家自然科学基金专项资助项目(No.32241028);国家自然科学基金面上资助项目(No.82070329).

摘　　要：高压冷冻及冷冻替代技术是一种利用高压和低温对含水组织样品进行瞬间冷冻固定和低温脱水的电镜样品制备技术,它因可以保存更加真实的细胞超微结构而常应用于透射电镜观察和分析。本文利用模式动物小鼠为材料,将这种技术应用于体扫描电镜的样品制备,并与常规化学固定的样品制备进行比较。结果显示在小鼠心肌组织中,高压冷冻及冷冻替代技术可以有效避免常规制备样品中的细胞器膜结构皱缩、细胞质分布不均匀、细胞核膜收缩形成锯齿状、甚至局部断裂等现象,使细胞超微结构更接近其生活时的状态。High⁃pressure freezing and freeze substitution is a sample preparation technique of electron microscopy that uses high⁃pressure cryogenics for quick frozen fixation and low⁃temperature dehydration of hydrous tissue samples.This technique can preserve celluar ultrastructures that are close to natural states.Thus,it is frequently used for sample preparations in transmission electron microscopy.This work used one of the most commonly used mouse animal models as material,and promoted this technique to sample preparation for volume scanning electron microscopy.This technique was also compared with a conventional chemical fixation for sample preparation.Our result show that high⁃pressure freezing and freeze substitution techniques can effectively avoid the organelle membrane wrinkling,uneven cytoplasmic distribution,contraction of the cell nuclear membrane to form a jagged shape or even local fracture that are frequently observed in conventionally prepared samples.This technique can make the cellular ultrastructure closer to its in situ state.

关 键 词：高压冷冻-冷冻替代 心肌组织 超微结构 体扫描电镜成像 三维重构 

分 类 号：Q2-3[生物学—细胞生物学] Q24Q6-3Q6-3Q952