Microscale Fluid Behavior during Cryo-EM Sample Blotting
Microscale Fluid Behavior during Cryo-EM Sample Blotting
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DOI：
10.1016/j.bpj.2019.12.017
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发表时间：
2020-02-04
影响因子：
3.4
通讯作者：
Glaeser, Robert M.
中科院分区：
文献类型：
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作者：
Armstrong, Maxim;Han, Bong-Gyoon;Glaeser, Robert M.
在过去的三十年里，印迹法一直是制备单粒子电子冷冻显微镜水溶液样品的标准技术。该技术通过在玻璃化前将吸收性滤纸压在样品上来从透射电子显微镜网格中去除过量的溶液。然而，这种标准技术产生的玻璃状冰在不同样品之间以及同一样品内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被用来证明，不规则图案的纤维在滤纸施加曲折的，高度可变的边界期间，从一个平坦的，亲水性的表面去除多余的液体。结果，当滤纸压在基底上时，形成厚度不均匀的水性膜。这种液体厚度不均匀的模式在滤纸被拉开后再次改变，但厚度仍然没有变得完全均匀。我们认为，类似的地形特征的液体膜中产生的标准技术用于吸EM网格，这些表现在玻璃化后的不均匀的冰。这些观察结果表明，替代的薄化技术，不依赖于滤纸和网格之间的直接接触，可能会导致更可重复和均匀的样品厚度。
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.