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No abstract

Blotting has been the standard technique for preparing aqueous samples for single-particle electron cryomicroscopy (cryo-EM) for over three decades. This technique removes excess solution from a TEM grid by pressing absorbent filter paper against the specimen prior to 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 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 topological 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.

No abstract