Time-resolved cryo electron microscopy (TRCEM) has emerged as a powerful technique for transient structural characterization of isolated biomacromolecular complexes in their native state within the time scale of seconds to milliseconds. For TRCEM sample preparation, microfluidic device [9] has been demonstrated to be a promising approach to facilitate TRCEM biological sample preparation. It is capable of achieving rapidly aqueous sample mixing, controlled reaction incubation, and sample deposition on electron microscopy (EM) grids for rapid freezing. One of the critical challenges is to transfer samples to cryo-EM grids from the microfluidic device. By using microspraying method, the generated droplet size needs to be controlled to facilitate the thin ice film formation on the grid surface for efficient data collection, while not too thin to be dried out before freezing, i.e., optimized mean droplet size needs to be achieved. In this work, we developed a novel monolithic three dimensional (3D) annular gas-assisted microfluidic sprayer using 3D MEMS (MicroElectroMechanical System) fabrication techniques. The microsprayer demonstrated dense and consistent microsprays with average droplet size between 6-9 μm, which fulfilled the above droplet size requirement for TRCEM sample preparation. With droplet density of around 12-18 per grid window (window size is 58×58 μm), and the data collectible thin ice region of >50% total wetted area, we collected ~800-1000 high quality CCD micrographs in a 6-8 hour period of continuous effort. This level of output is comparable to what were routinely achieved using cryo-grids prepared by conventional blotting and manual data collection. In this case, weeks of data collection process with the previous device [9] has shortened to a day or two. And hundreds of microliter of valuable sample consumption can be reduced to only a small fraction.

中文翻译：

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用于时间分辨冷冻电子显微镜样品制备的气体辅助环形微喷雾器

时间分辨低温电子显微镜 (TRCEM) 已成为一种强大的技术，可在几秒到几毫秒的时间尺度内对天然状态下分离的生物大分子复合物进行瞬态结构表征。对于 TRCEM 样品制备，微流体装置 [9] 已被证明是促进 TRCEM 生物样品制备的有前途的方法。它能够在电子显微镜 (EM) 网格上实现快速混合水样、受控反应孵化和样品沉积以实现快速冷冻。关键挑战之一是将样品从微流体装置转移到冷冻电镜网格。通过使用微喷方法，需要控制产生的液滴尺寸，以促进网格表面形成薄冰膜，从而有效地收集数据，虽然在冷冻前不会太薄而无法干燥，即需要实现优化的平均液滴尺寸。在这项工作中，我们使用 3D MEMS（微机电系统）制造技术开发了一种新型整体式三维 (3D) 环形气体辅助微流体喷雾器。微喷雾器表现出密集且一致的微喷雾，平均液滴尺寸在 6-9 μm 之间，满足上述 TRCEM 样品制备的液滴尺寸要求。每个网格窗口的液滴密度约为 12-18 个（窗口大小为 58×58 μm），并且可收集的薄冰区域 >50% 的总润湿面积，我们在 6- 8小时的持续努力。这种输出水平与使用通过常规印迹和手动数据收集制备的冷冻网格常规实现的水平相当。在这种情况下，使用先前设备 [9] 的数周数据收集过程已缩短至一两天。数百微升的宝贵样品消耗量可以减少到很小的一部分。