Figure 2.

Side-view schematics of a hole of cryoEM grid, highlighting particle behavior and distribution in relation to AWI. A Time-lapsed illustration of AWI adsorption of particles in the buffer. First, particles stay in the bulk volume (Left panel). A1 denotes the surface area of the buffer layer at the AWI, i. e., the size of the grid hole. There are two A1’s, one at the top and one at the bottom. After a short period, particles adsorb to one of the two AWIs, achieving a more favorable energy state (one-side AWI adsorption) (Center panel). A2 denotes the overall sum of the area of particles’ surfaces exposed to the air, upon migrating to the AWI. At the top AWI, the overall A1 decreases as the particles occupy the surface, unlike the unoccupied A1 at the bottom AWI. Eventually, particles adsorb to the remaining AWI as the buffer layer thins, achieving the most favorable energy state (two-side AWI adsorption) (Right panel). At the bottom AWI, the overall A1 also decreases due to the same particles occupying that surface. B Three common problems encountered by the sample particles at AWI, namely, particle deformation, denaturation, and preferred orientation. C Time-lapsed illustration of how surfactants reduce or even eliminate AWI adsorption of particles during sample preparation. First, the sample-surfactant mixture is loaded from the top side, denoted by the bulge (first panel). Surfactants migrate more quickly than the sample particles (see explanation in the result section) to occupy the two AWIs, thereby blocking sample particles from migrating to the surfaces. Upon blotting, some particles and surfactants are removed by the filter paper (second panel), exposing a second surface at the blotted side (third panel), which would also be occupied by the surfactant molecules, preventing the remaining sample particles from migrating to the free surface (last panel)