The Maastricht Multimodal Molecular Imaging Institute (M4i), Division of Nanoscopy, Maastricht University, Maastricht, Netherlands.
CryoSol-World, Maastricht, Netherlands.
Nat Commun. 2020 May 22;11(1):2563. doi: 10.1038/s41467-020-16392-5.
The increasing demand for cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Here, we present several technical developments that provide efficient sample preparation for cryo-EM studies. Pin printing substantially reduces sample waste by depositing only a sub-nanoliter volume of sample on the carrier surface. Sample evaporation is mitigated by dewpoint control feedback loops. The deposited sample is vitrified by jets of cryogen followed by submersion into a cryogen bath. Because the cryogen jets cool the sample from the center, premounted autogrids can be used and loaded directly into automated cryo-EMs. We integrated these steps into a single device, named VitroJet. The device's performance was validated by resolving four standard proteins (apoferritin, GroEL, worm hemoglobin, beta-galactosidase) to ~3 Å resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies.
越来越多的人对 cryo-electron microscopy (cryo-EM) 的需求揭示了当前样品制备方案的一些缺陷，例如样品浪费和缺乏重现性。在这里，我们提出了一些技术发展，为 cryo-EM 研究提供了高效的样品制备方法。Pin 打印通过仅将亚纳升级别的样品体积沉积在载体表面上，大大减少了样品浪费。通过露点控制反馈回路减轻了样品蒸发。通过低温液体射流将沉积的样品玻璃化，然后将其浸入低温浴中。由于低温液体射流从中心冷却样品，可以使用预先安装的自动网格，并直接加载到自动化 cryo-EM 中。我们将这些步骤集成到一个名为 VitroJet 的单一设备中。该设备的性能通过使用 200kV 电子显微镜将四个标准蛋白质（脱铁蛋白、GroEL、虫血红蛋白、β-半乳糖苷酶）解析至~3Å分辨率得到了验证。VitroJet 为 cryo-EM 研究中改进的自动化样品制备提供了一个有前途的解决方案。
