Blotting has been the standard technique for preparing aqueous samples for single-particle electron cryo-microscopy for over three decades. This technique removes the excess solution from a transmission electron microscope grid by pressing absorbent filter paper against the specimen before vitrification. However, this standard technique produces vitreous ice with inconsistent thickness from specimen to specimen and from region to region within the same specimen, the reasons for which are not understood. Here, high-speed interference contrast microscopy is used to demonstrate that the irregular pattern of fibers in the filter paper imposes tortuous, highly variable boundaries during the removal of excess liquid from a flat, hydrophilic surface. As a result, aqueous films of nonuniform thickness are formed while the filter paper is pressed against the substrate. This pattern of nonuniform liquid thickness changes again after the filter paper is pulled away, but the thickness still does not become completely uniform. We suggest that similar topographical features of the liquid film are produced during the standard technique used to blot EM grids and that these manifest in nonuniform ice after vitrification. These observations suggest that alternative thinning techniques, which do not rely on direct contact between the filter paper and the grid, may result in more repeatable and uniform sample thicknesses.

中文翻译：

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低温 EM 样品印迹过程中的微观流体行为

三十多年来，印迹一直是为单粒子电子冷冻显微镜制备水样的标准技术。该技术通过在玻璃化之前将吸水滤纸压在样品上，从透射电子显微镜网格中去除多余的溶液。然而，这种标准技术产生的玻璃冰厚度在不同样品之间以及同一样品中不同区域之间的厚度不一致，其原因尚不清楚。在这里，高速干涉对比显微镜用于证明滤纸中不规则的纤维图案在从平坦的亲水表面去除多余液体的过程中施加了曲折的、高度可变的边界。因此，当滤纸压在基材上时，会形成厚度不均匀的水膜。这种不均匀的液体厚度图案在拉开滤纸后再次发生变化，但厚度仍然没有变得完全均匀。我们建议在用于印迹 EM 网格的标准技术中产生液体膜的类似地形特征，并且这些在玻璃化后在不均匀的冰中表现出来。这些观察结果表明，不依赖于滤纸和网格之间直接接触的替代变薄技术可能会导致更可重复和均匀的样品厚度。我们建议在用于印迹 EM 网格的标准技术中产生液体膜的类似地形特征，并且这些在玻璃化后在不均匀的冰中表现出来。这些观察结果表明，不依赖于滤纸和网格之间直接接触的替代变薄技术可能会导致更可重复和均匀的样品厚度。我们建议在用于印迹 EM 网格的标准技术中产生液体膜的类似地形特征，并且这些在玻璃化后在不均匀的冰中表现出来。这些观察结果表明，不依赖于滤纸和网格之间直接接触的替代变薄技术可能会导致更可重复和均匀的样品厚度。