Kumar Anil, Sengupta Nayanika, Dutta Somnath
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
Nanomaterials (Basel). 2021 Mar 5;11(3):643. doi: 10.3390/nano11030643.
In this manuscript, we report the application of graphene oxide (GO) in the preparation of cryo-electron microscopy (cryo-EM) and transmission electron microscopy (TEM) grids. We treated GO with water and organic solvents, such as, methanol, ethanol and isopropanol separately to isolate significantly large GO monolayer flake to fabricate the grids for cryo-EM and TEM study. We implemented a simplified approach to isolate flakes of GO monolayer for constructing the TEM grids, independent of expensive heavy equipment (Langmuir-Blodgett trough, glow-discharge system, carbon-evaporator or plasma-cleaner or peristaltic pumps). We employed confocal microscopy, SEM and TEM to characterize the flake size, stability and transparency of the GO monolayer and atomic force microscopy (AFM) to probe the depth of GO coated grids. Additionally, GO grids are visualized at cryogenic condition for suitability of GO monolayer for cryo-EM study. In addition, GO-Met-HO grids reduce the effect of preferred orientation of biological macromolecules within the amorphous ice. The power-spectrum and contrast-transfer-function unequivocally suggest that GO-Met-HO fabricated holey grids have excellent potential for application in high-resolution structural characterization of biomolecules. Furthermore, only 200 movies and ~8000 70S ribosome particles are selected on GO-coated grids for cryo-EM reconstruction to achieve high-resolution structure.
在本论文中，我们报道了氧化石墨烯（GO）在冷冻电子显微镜（cryo-EM）和透射电子显微镜（TEM）载网制备中的应用。我们分别用水和有机溶剂（如甲醇、乙醇和异丙醇）处理GO，以分离出显著大尺寸的GO单层薄片，用于制备cryo-EM和TEM研究的载网。我们采用了一种简化方法来分离GO单层薄片以构建TEM载网，无需昂贵的重型设备（Langmuir-Blodgett槽、辉光放电系统、碳蒸发器或等离子清洗器或蠕动泵）。我们使用共聚焦显微镜、扫描电子显微镜和透射电子显微镜来表征GO单层的薄片尺寸、稳定性和透明度，并使用原子力显微镜（AFM）来探测GO涂层载网的深度。此外，在低温条件下观察GO载网，以评估GO单层用于cryo-EM研究的适用性。另外，GO-Met-HO载网可降低生物大分子在非晶冰中择优取向的影响。功率谱和对比度传递函数明确表明，GO-Met-HO制备的多孔载网在生物分子高分辨率结构表征方面具有优异的应用潜力。此外，在GO涂层载网上仅选择200个电影和约8000个70S核糖体颗粒用于cryo-EM重建，以获得高分辨率结构。
