Facile hermetic TEM grid preparation for molecular imaging of hydrated biological samples at room temperature

Although structures of vitrified supramolecular complexes have been determined at near-atomic resolution, elucidating in situ molecular structure in living cells remains a major challenge. Here, we employ a novel yet simple liquid-cell technique, previously developed for real-time visualization of dynamics at a liquid-gas interface, to image wet biological samples. Due to the scattering effects from the liquid phase, the transmission electron micrographs exhibit amplitude contrast comparable to that observed in negatively stained samples. We successfully resolve subunits within the protein complex GroEL, which has been imaged in a buffer solution at room temperature. Additionally, we capture various stages of virus cell entry, which have limited structural information available due to their transient nature. To further analyze the morphological details, we employ the technique of individual particle electron tomography (IPET). These results demonstrate the potential of this approach as a valuable and cost-effective technique that complements other microscopy techniques in addressing important biological questions at the molecular level.尽管玻璃化超分子复合物的结构已实现近原子分辨率解析，但解析活细胞内的原位分子结构仍是一项重大挑战。本研究采用一种此前专为气液界面动力学实时可视化开发的新型简便液池技术，对湿润生物样品进行成像。由于液相的散射效应，该透射电子显微图像呈现出与负染色样品相当的振幅衬度。我们成功解析了蛋白质复合物GroEL的亚基结构，该样品已在室温缓冲液中完成成像。此外，我们还捕捉到了病毒细胞入侵的多个不同阶段——由于该过程具有瞬时性，此前其相关结构信息十分有限。为进一步分析形态细节，我们采用了单颗粒电子断层扫描（individual particle electron tomography, IPET）技术。上述结果证明，该方法作为一种兼具实用价值与成本效益的技术，可作为其他显微技术的补充，用于解决分子层面的重要生物学问题。