Achieving Superior Cryo-EM Results

Cryo-electron microscopy (cryo-EM) is a type of transmission electron microscopy (TEM) which is based on the principle of cryogenically freezing biological samples so that they can be studied under a TEM at high resolution.

Since being developed in 1990, this method has given huge insight into the molecular structure of biological samples. The development of Cryo-EM is such a huge breakthrough that the pioneers of Cryo-EM, Richard Henderson, Jacques Dubochet, and Joachim Frank were honored with the Nobel Prize for Chemistry in 2017.

This technique was created due to organic and biological samples usually exhibiting poor contrast in the TEM and will decompose when they are exposed to high-energy electrons. New techniques needed to be created in order to image these materials, as traditional organic sample preparation for TEM often severely damaged the biological structures.

Cryo-EM also permits researchers to analyze complex, large, and flexible structures, create high-resolution images, and produce accurate, 3D reconstructions of the isolated molecule which is being examined.

Cryo-EM does come with its own challenges, even though it is incredibly useful for groundbreaking biological research. Some are outlined below:

Common Cryo-EM Research Challenges

The hardest challenge with cryo-EM is sample preparation, this is because it is often prone to error and very time-consuming; before it is ready for viewing one sample can take up to a month of preparation. This is largely due the iterative process of deciding the best protocol for selecting, isolating, staining, dispensing, blotting, and freezing the sample. All of these factors influence the final image quality and can obliterate biological samples in the process.

The freezing process is one of the most challenging stages in preparing samples. Also called “plunging”, the sample is quickly quenched to temperatures below -160 ºC, which causes the water to freeze so suddenly that it takes on an amorphous state, called “vitrified” ice which is required for imaging. Often however, the plunge freezing method can cause samples to rupture, meaning the procedure has to be refined until the sample survives the process.

Finding an appropriate carbon support grid for placing cryogenically frozen samples on is another challenge. Grids with carbon films that are flatter normally produce better ice uniformity and image clarity, since for all samples imaged the thickness of the vitrified ice should be consistent. Furthermore, the thickness of the carbon film must be thin enough to maximize image quality, but thick enough to support heavy samples. It is recommended that the carbon thickness and mesh size of the holey carbon grids be optimized for every sample examined in the lab.

Image of several viruses in contact with cell (A), with 3-D reconstruction of individual virus particles (B) with 3-D reconstruction of virus particles contacting the cell (C). Image courtesy Christiane Riedel and Kay Grünewald, University of Oxford

Image of several viruses in contact with cell (A), with 3-D reconstruction of individual virus particles (B) with 3-D reconstruction of virus particles contacting the cell (C). Image courtesy Christiane Riedel and Kay Grünewald, University of Oxford

The Requirement of Holey Carbon Grids for Cryo-EM

Holey carbon grids are the sample support utilized for cryo-EM samples. The sample material is distributed onto the grid and rapidly quenched in liquid ethane near liquid nitrogen temperature. There are a number of reasons why employing a holey carbon grid is vital for cryo-EM research, such as:

  • Compatibility: Usually designed to be compatible with automated data collection software, Holey carbon grids result in more high-quality target sites per grid.
  • Variety: Holey carbon grids vary in mesh size, hole diameter, mesh material, and film thicknesses, to aid a variety of research needs.
  • Pristine Flatness: Holey carbon grids are designed to be as flat as possible. The reason for this flatness is even and thin distribution of ice for the best viewing quality. The pristine flatness of a holey carbon grid permits researchers to obtain superior cryo-EM data collection.

Holey Carbon Grid Use Case: Cryo-EM of Retroviral Envelope

In 2016, to support their samples of retroviral murine leukemia cells, researchers from the University of Vienna and the University of Oxford utilized C-Flat holey carbon grids. The researchers were able to collect cryo-EM images for tomograms, or two-dimensional image slices through a three-dimensional object after dispensing their sample, blotting it dry, and plunge freezing. Afterward, the tomograms were used to show the underlying chemical structures of the murine leukemia cells.

The tomograms displayed “massive structural rearrangements” of the murine leukemia virus envelope protein whilst the virus was attacking host cells. Researchers compared virally-attacked cells with virus-free cells and noticed that the viruses didn’t have a clearly discernible bi-layer at the surface of the plasma membrane. This notable bi-layer structure difference was evidence that mechanical stress or lipid rearrangement might be influential factors in the leukemia virus attack.

Conclusion

Cryo-EM research techniques are becoming increasingly popular, and will no doubt become more common due to its recent attention for being awarded the 2017 Nobel Prize in Chemistry. Cryo-EM pioneers Henderson, Dubochet, and Frank’s work over the past thirty years has filled the technology void in biochemical imaging.

Researchers can now routinely produce 3D structures of biomolecules at near-atomic resolution as a result of their cryo-EM innovations, providing new insight into the molecular structures underpinning biological reactions.

This information has been sourced, reviewed and adapted from materials provided by Protochips.

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