Micro-scale fluid behavior during cryo-EM sample blotting

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.

Current limitations to high-resolution structure determination by single-particle cryoEM

“…The new preparation techniques avoid blotting the grid with filter paper (Fig. 10 a ), which not only causes mechanical stress (Armstrong et al ., 2019), but – depending on chemical composition – may release small amounts of Mg 2+ and Ca 2+ ions that can affect the structure of biological macromolecules (Walker et al ., 1994). The use of ashless filter paper or glass fibre filters circumvents this problem.…”

“…With large complexes, damage during isolation or cryoEM grid preparation is a more likely reason. Most of this damage is probably caused by surface forces, although shear forces during blotting can also play a role (Armstrong et al ., 2019).…”

Next generation vitrification robot

“…All three cryo-EM methods have in common that the specimen is flash-frozen in their native, hydrated state by a vitrification step as described by Nobel-laureate Jacques Dubochet [4]. In this step, the sample is applied on a supporting grid carrier, followed by thinning of sample -liquid film to sub-100 nm thickness [5] and subsequent rapid cooling by plunging in liquid ethane. We have developed a new and highly flexible multi-purpose vitrification robot.…”