利用聚焦离子束技术制备低温电镜冷冻含水切片方法的研究

Cryo-electron tomography is an important research method in the structural biology and cell biology. There are a few cellular specimens can be examined directly by cryo-TEM. However, the majority of the eukaryotic cell volume is impossible, and thus requires additional preparation techniques before imaging. Rapid freezing and high pressure freezing is the common method to prepare the frozen-hydrated specimens. .Conventional methods achieve this by cutting thin sections of frozen-hydrated samples with an ultramicrotome. However, sections of vitrified cells inevitably suffer from distortions and deformations caused by the mechanical cutting process. The most significant artifact is unavoidable sample compression of up to 30–50% occurring in the cutting direction, and the cut samples are poor adhesion ability with the copper grid. These disadvantages limit its application, and also hinder the development of cell biology..A novel alternative to cryo-ultramicrotomy for thinning of frozen-hydrated biological specimens uses focused ion beam (FIB) method. In contrast to mechanical sectioning, thinning of the specimen occurs by sputtering with focused ions, typically gallium. In 2006, M. MAKRO et al., found there was no heat-induced devitrification effect of FIB milling of vitreous water observed by TEM. Researchers show that cryo-FIB milling can indeed be applied to frozen-hydrated material, resulting in specimens that contained eukaryotic cells and native mammalian cells transparent enough for TEM. The FIB method becomes a hotspot in the international research field due to peculiar advantages..The purpose of this project: (1) Development of the freezing sample clamp which are suitable for electron microscopy (SEM and TEM). (2) By using unicellular organisms as test samples, exploring the optimal parameters of preparing the ultrathin sections by FIB and discussing the mechanism of the FIB milling frozen-hydrated material. (3) Electron tomography three-dimensional reconstruction of the fabricate samples..This project is aimed at the international frontier of the biology research of methodology, we hope to do an important contribution in the field.

低温电子断层成像三维重构（cryo-ET）技术是发展结构生物学和细胞生物学研究的重要技术。该技术可以得到更真实和接近天然的细胞内部高分辨率三维结构以及蛋白质大分子定位及相互作用的信息，是蛋白质组学研究的重要的辅助手段。现有的样品制备方法--冷冻切片技术，存在诸多问题,如：样品的压缩、振颤、皱缩、断裂以及载网附着能力差等，严重影响了该研究领域的发展。.本项目用聚焦离子束（FIB）减薄技术，制备细胞冷冻含水超薄切片。研究内容：（1）适用于不同电镜和低温冷冻杆样品夹具和传输装置的研制；（2）以单细胞生物作为测试样品，优化聚焦离子束制备超薄冷冻切片的参数，研究作用机理；（3）对制备出的样品进行电子断层扫描三维重构，并对该技术进行评价。该项目的完成可以促进cryo-ET技术在结构生物学和细胞生物学的应用，进而推动学科的发展。

低温电子断层成像三维重构（cryo-ET）技术是发展原位结构生物学和细胞生物学研究的重要技术 。该技术可以得到更真实和接近天然的细胞内部高分辨率三维结构以及蛋白质大分子定位及相 互作用的信息，是蛋白质组学研究的重要的辅助手段。现有的样品制备方法--冷冻切片技术（CEMOVIS）， 存在诸多问题,如：样品的压缩、振颤、皱缩、断裂以及载网附着能力差等，严重影响了该研 究领域的发展。 本项目用新的样品制备方法---聚焦离子束（FIB）减薄技术，制备细胞冷冻含水超薄切片。研究内容：（1）适用于不同电镜和低温冷冻杆样品夹具和传输装置的研制；（2）研制特殊形状的透射电镜载网方便在双束扫描电镜和透射电镜中转移并确保切片样品的方向一致性；（3）以单细胞生物作为测试样品，优化聚焦离子束制备超薄冷冻切片的参数，研究作用机理；（4）对制备出的样品进行电子断层扫 描三维重构，并对该技术进行评价。该项目的完成可以促进cryo-ET技术在结构生物学和细胞 生物学的应用，进而推动学科的发展。