Factors that Influence the Formation and Stability of Thin, Cryo-EM Specimens

Abstract

Poor consistency of the ice thickness from one area of a cryo-electron microscope (cryo-EM) specimen grid to another, from one grid to the next, and from one type of specimen to another, motivates a reconsideration of how to best prepare suitably thin specimens. Here we first review the three related topics of wetting, thinning, and stability against dewetting of aqueous films spread over a hydrophilic substrate. We then suggest that the importance of there being a surfactant monolayer at the air-water interface of thin, cryo-EM specimens has been largely underappreciated. In fact, a surfactant layer (of uncontrolled composition and surface pressure) can hardly be avoided during standard cryo-EM specimen preparation. We thus suggest that better control over the composition and properties of the surfactant layer may result in more reliable production of cryo-EM specimens with the desired thickness.

Figure 1

Spherical cap on a hydrophilic surface. As is illustrated by the side view that is shown in this cartoon, the phrase “spherical cap” refers to the topmost portion of a sphere. For completeness, the radius of the sphere, and its center, are shown below the plane of the hydrophilic substrate. In the top view (not shown), the perimeter of the cap would be a circle of radius a. The thickness of the spherical cap (at the center of the plano-convex lens), h, is given by Eq. 3.

Figure 2

Marangoni flow on freshly cleaved mica. Individual frames extracted from real-time movies, included in the Supporting Material, in which we recorded how Marangoni flow, rather than blotting with filter paper, might be used to thin an aqueous specimen before vitrification. Frames (A) and (B) are extracted from Movie S2, while frames (C) and (D) are from Movie S1. (A) A thin puddle, ∼1 cm in diameter, forms when 10 μL of water is applied to the surface of freshly cleaved mica. Imperfections in the reflectance of light near the periphery of the mica are due to air gaps created by inadvertent cleavage of planes of the mica during cutting the one-inch square from stock. (B) Interference fringes, centered around the point of closest approach to the aqueous puddle, are formed when a syringe needle, made of Teflon (DuPont, Wilmington, DE) and loaded with chloroform, is brought close to the center of the puddle. As Movie S1 shows, this pattern of local thinning is largely reversible when the tip of the syringe is removed. On the other hand, a permanently dry area forms when liquid chloroform touches the aqueous puddle. As is shown in Movie S2, this dry area is again wetted relatively well when a new droplet of water is applied to it, but wetting is no longer as perfect as is the case for freshly cleaved mica. (C) A permanently dry area again forms when a solution of 1 mg/mL of phospholipid in chloroform touches an aqueous puddle. Note a small amount of residue is left at the point where the transferred chloroform sat as it evaporated. (D) In this case, the dry area is largely hydrophobic, as indicated by the fact that an added droplet of water does not spread. Exceptions occur when the applied droplet contacts surrounding areas of the puddle. To see this figure in color, go online.