Sample behavior on cryoEM grids

Sample behavior on cryoEM grids

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Lesson summary:

What happens to samples when they are added to grids and frozen? In this lesson, we will explore critical aspects of cryoEM grid preparation, known effects on samples, and common hurdles that must be overcome during sample screening and grid optimization.

Lesson concepts: 

  • How air/water and water/surface interfaces strongly affect sample behavior on grids
  • How grid supports, grid substrates, and ice thickness affect sample behavior

Online educational resources:

Workshops & presentations

Introduction to cryoEM sample preparation

EM grids are ~3mm in diameter and are typically nanofabricated to have regularly spaced patterns - a mesh pattern on which is suspended a thin foil (i.e., carbon or gold) with a regular array of micrometer-sized holes - that not only provides structural rigidity but can also be used for automated data acquisition. EM samples are directly applied to the prepared surface of the grids whereby an excess solution is removed to leave a thin film of solution suspended across the open holes in the foil, typically 0.5 - 2 µm in diameter.  

Ultimately, within the holes are cryoEM specimens for either single particle or cryoET data collection. An example of which is shown here:

To see other examples of what it looks like to visualize cryoEM grids across different orders of magnitude:

Dr. Janet Iwasa's 3D rendered zoom & narration (via cryoEM101.org):

To understand sample behavior, it is useful to consider a 'side-on' view of single particles within a hole. When considered in this direction, we are looking at a slice through a hole to show a cross-section of a cryoEM sample. In general, there are two types of approaches to prepare cryoEM grids: 'open holes' vs. 'support film'. In the open hole approach, the sample is incubated with grids at high concentrations so that the sample 'spills over' into the holes. As an orthogonal approach, support films can be added on top of the holes to allow samples to adhere directly to the support.

 

Grid preparation

Before discussing the details of open-hole vs. support film grids it is important to have a basic understanding of how cryoEM grids are prepared for the sample application. To facilitate automated data collection, cryoEM grids can be purchase from vendors or prepared in-house (e.g. Marr et al. 2014). Once obtained, cryoEM grids are typically hydrophobic, which means that they will resist wetting by your sample + buffer. To ensure sample adherence, grids are made more hydrophilic by exposing them to plasma through glow-discharging or plasma cleaning. While similar, glow-discharging and plasma cleaning utilize distinct approaches.

  • Glow discharging creates a plasma from air to modify grid surfaces
  • Plasma cleaning refers to a different device that specifically makes a plasma for surface modification. Plasma cleaning uses gas mixtures (e.g. Argon:Oxygen) to impart specific effects.

Support film grids are prepared using glow discharging or specific plasma. For amorphous carbon, glow charging or plasma cleaning is appropriate, whereas grids that have graphene require plasma such as hydrogen gas.

 

Grid types

Open holes
Support film
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