Feng Xiangsong, Fu Ziao, Kaledhonkar Sandip, Jia Yuan, Shah Binita, Jin Amy, Liu Zheng, Sun Ming, Chen Bo, Grassucci Robert A, Ren Yukun, Jiang Hongyuan, Frank Joachim, Lin Qiao
Department of Mechanical Engineering, Columbia University, New York, NY 10027, USA; School of Mechatronics Engineering, Harbin Institute of Technology, Harbin 150001, China.
Integrated Program in Cellular, Molecular, and Biophysical Studies, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
Structure. 2017 Apr 4;25(4):663-670.e3. doi: 10.1016/j.str.2017.02.005. Epub 2017 Mar 9.
We describe a spraying-plunging method for preparing cryoelectron microscopy (cryo-EM) grids with vitreous ice of controllable, highly consistent thickness using a microfluidic device. The new polydimethylsiloxane (PDMS)-based sprayer was tested with apoferritin. We demonstrate that the structure can be solved to high resolution with this method of sample preparation. Besides replacing the conventional pipetting-blotting-plunging method, one of many potential applications of the new sprayer is in time-resolved cryo-EM, as part of a PDMS-based microfluidic reaction channel to study short-lived intermediates on the timescale of 10-1,000 ms.
我们描述了一种使用微流控装置制备具有可控、高度一致厚度的玻璃态冰的冷冻电子显微镜（cryo-EM）网格的喷雾-浸入法。使用基于聚二甲基硅氧烷（PDMS）的新型喷雾器对脱铁铁蛋白进行了测试。我们证明，通过这种样品制备方法可以将结构解析到高分辨率。除了取代传统的移液-印迹-浸入法外，这种新型喷雾器的众多潜在应用之一是在时间分辨冷冻电子显微镜中，作为基于PDMS的微流控反应通道的一部分，用于研究10-1000毫秒时间尺度上的短寿命中间体。
