Cryo-electron microscopy (cryo-EM) allows scientists to visualize biological molecules at near-atomic resolution. Cryo-EM grids are small, specialized supports that hold delicate biological samples for imaging.
Understanding Cryo-EM Grids
Cryo-EM grids are small, circular meshes, about 3 millimeters in diameter, serving as the foundation for biological sample preparation. They are covered with an ultrathin support film that provides a surface for the sample. The primary function of a cryo-EM grid is to enable vitrification, the rapid freezing of biological material.
Vitrification involves plunging the grid and sample into a cryogen, like liquid ethane cooled by liquid nitrogen, at temperatures around -180°C. This rapid cooling prevents water from forming ice crystals, which would damage delicate biological structures. Instead, water freezes into an amorphous, glass-like state, preserving biological molecules in their native conformation.
Materials That Make Up Grids
Cryo-EM grids consist of two main parts: the mesh base and the ultrathin support membrane. The mesh base provides mechanical support and is commonly made from metals like copper, gold, or nickel. Copper grids are widely used for their stability and electrical conductivity. Gold grids offer enhanced purity and biocompatibility, beneficial for samples sensitive to copper ions. Nickel-titanium alloy grids also provide good conductivity and reduce sample drift during imaging.
The support membrane, placed on the mesh base, is an ultrathin film, often made from carbon or graphene. Carbon support films are widely used for their ability to provide a lower background signal for high-resolution data. Graphene-based supports are also used due to their exceptional electron transparency and mechanical strength.
Common Grid Designs and Their Purpose
The design of the support film on cryo-EM grids is crucial for optimal sample preparation. Holey carbon grids are a common design, featuring evenly spaced holes in the carbon film. Examples include Quantifoil and C-flat grids, which create thin, uniform ice layers over these holes where biological particles reside. Hole size and spacing are important considerations; for instance, a 1.2 µm hole size with 1.3 µm carbon in between is a common choice.
These designs manage ice thickness and particle distribution. The goal is to achieve a vitreous ice layer thin enough to minimize electron scattering and maximize image contrast. While larger holes offer a greater imageable area, they can also increase the risk of beam-induced motion during imaging. Some grids may also feature a continuous ultrathin carbon film on top of the holey layer, which helps stabilize proteins by providing a more consistent interface.
How Grid Selection Impacts Imaging
The choice of cryo-EM grid significantly influences high-resolution imaging quality. Grid material, support film type, and hole design affect the resulting cryo-EM data. Selecting the right grid helps achieve optimal ice thickness, crucial for high-resolution images. If ice is too thick, it leads to increased background noise and reduced image contrast.
Grid properties also promote uniform particle distribution, preventing aggregation or sticking. Gold grids, for example, can reduce sample drift caused by electron beam irradiation, maintaining image quality. Careful grid selection minimizes issues like sample denaturation, charging effects, and beam-induced motion, which are important for capturing clear structural information.