利用聚焦离子束技术制备低温电镜组织样品冷冻含水切片的方法研究及应用

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 imaging directly, 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. A novel alternative to cryo-ultramicrotomy for thinning of frozen-hydrated biological specimen uses focused ion beam (FIB) method. In contrast to mechanical sectioning, thinning of the specimen occurs by sputtering with focused ions, typically gallium. The FIB method becomes a hotspot in the international research field due to peculiar advantages. This project is based the previous fabrication of the single cell lamella by FIB method, and we want to develop the more complicated and difficult tissue lamella by FIB method, but this method can solve the closer to the physiological state biological problems. The purpose of this project: (1) Development of the freezing sample clamp which are suitable for electron microscopy (SEM and TEM). (2) Electron tomography three-dimensional reconstruction of the fabricated samples. (3) By using desmosomes 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. The project can be solved the cryo-ET technology bottleneck problems in situ macromolecular structure analysis in structural biology, and we hope to do an important contribution in the field.

低温电子断层成像（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、天然的细胞内部三维结构以及蛋白质大分子的原位结构信息，是蛋白质组学研究的重要辅助手段，是结构生物学的重要的研究手段。现有的冷冻切片制备技术，存在诸多问题（样品的压缩、断裂等），严重影响了该研究领域的发展。本项目基于已开发的聚焦离子束法制备单细胞样品冷冻含水切片技术的工作基础上，拟进一步研发适用范围更广、制备难度更大、更有科学意义的组织样品高质量冷冻含水切片技术。研究内容：（1）建立一套冷冻含水组织样品制备实验方法以及相关配套硬件装置；（2）对制备出的样品进行电子断层成像，并对其进行评价；（3）利用建立的技术研究皮肤组织细胞桥粒的钙黏着蛋白排列的三维空间结构。该项目的完成可以解决cryo-ET技术在结构生物学和细胞生物学在原位结构解析中的瓶颈问题，进而推动学科的发展。

低温电子断层成像（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、天然的细胞内部三维结构以及蛋白质大分子的原位结构信息，是蛋白质组学研究的重要辅助手段，而且是结构生物学的必然趋势。该方法的样品制备技术，存在诸多问题（样品的压缩、断裂等），严重影响了该研究领域的发展。基于现有的开发单细胞样品冷冻含水切片技术的工作基础上，本项目拟进一步研发适用范围更广、制备难度更大的组织样品高质量冷冻含水切片技术。研究内容：（1）建立一套基于聚焦离子束技术制备冷冻含水组织样品制备实验方法以及相关配套硬件装置；（2）对制备出的样品进行电子断层成像，并对其进行评价；（3）利用建立的技术研究皮肤组织细胞桥粒的钙黏着蛋白排列的三维空间结构和小鼠肝脏核糖体原位的三维结构。该项目的完成可以解决cryo-ET技术在结构生物学和细胞生物学在原位结构解析中的瓶颈问题，进而推动整个学科的发展。