ABSTRACT
Blotting has been the standard technique for preparing aqueous samples for single-particle electron cryo-microscopy (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.
STATEMENT OF SIGNIFICANCE Multiple imaging techniques are used to observe dynamic, micro-scale events as excess water is removed from a substrate by blotting with filter paper. As a result, new insight is gained about why the thickness values of remaining sample material are so variable across a single EM grid, as well as from one grid to the next. In addition, quantitative estimates are made of the shear forces to which macromolecular complexes can be exposed during blotting. The fact that sample thicknesses and flow rates are seen to be inherently under poor control during blotting suggests that other methods of removing excess water may be better suited for consistently achieving large sample areas that are suitable for use in electron cryo-microscopy.
