基于囊泡的生命大分子液相电镜技术开发与应用

生命大分子在体内外液体环境中的实时动态成像是超高分辨率显微技术领域的重要发展方向。其中，制备能维持液体状态的电镜样品是生命大分子液相电镜技术研究的关键。本研究拟以液相电镜样品制备为切入点，以解决传统氮化硅液体池厚度大、强电子散射导致图像衬度低，以及石墨烯液体池尺寸难以控制、制备重复率低等问题为目的，开发基于囊泡的液相电镜液体池。首先从囊泡的结构稳定性、支撑囊泡的薄膜固化成型等角度，筛选囊泡和薄膜材料组成，优化制备流程。同时考察囊泡内液体在高真空下的稳定性及制备流程对内部样品的影响，分析薄膜厚度、成分等参数与图像分辨率的相互关系，实现可获取高分辨动态图像的液体池精准制备。在此基础上选择DNA、蛋白质等生命大分子以及细菌等活细胞样品，研究生命大分子在体内外的运动和动态结构变化。本研究可丰富和发展液相电镜技术，为进一步对生命大分子体内外时空动态变化成像的深入研究提供理论和实验基础。

Real-time imaging of the dynamic processes of biomacromolecules in liquid environment is of great importance for ultra-high resolution microscopy. The key aspect of liquid-phase TEM is the preparation of specimen which can keep the biomacromolecules in liquid state. Herein, we propose a new type of liquid cell based on vesicle. The vesicle based-liquid cell is able to overcome the drawbacks in traditional liquid cells, such as large specimen thickness, strong electron scattering in the silicon nitrite liquid cell which lowers image contrast, and difficulties in controlling the sizes, low efficiency in the graphene liquid cell. To prepare the vesicle based-liquid cell, we will firstly screen the vesicle and thin film materials, and optimize the protocols by assessing the vesicle structural stability and solidification process of the thin film, etc. Meanwhile, we will evaluate the liquid stability in vesicles in ultra-high vacuum environments and the influence of the preparation process on the internal specimens. The relationship between the thin film parameters such as thickness, chemical components and the image quality will be analyzed. On the basis of obtaining vesicle based-liquid cell that can capture high resolution time-lapsed images, the dynamic processes of biomacromolecules such as DNA, protein molecules and cell specimens such as living bacteria will be investigated. This study will expand the frontier of liquid-phase TEM technique and provide a theoretical and experimental basis for deeper investigations on the real-time imaging of the dynamic processes of biomacromolecules in liquid.