Cryo-electron microscopy is one of the most powerful imaging tools in a scientist’s toolbox. The technique, referred to as cryo-EM, is used to visualize proteins, pathogens, and various components of biological cells down to near the level of individual atoms. Cryo-EM has been used to visualize the structure of the spike protein, which coronavirus particles use to attach to human cells. A new technique makes sample preparation for cryo-EM safer and simpler using liquid nitrogen.
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What is Cryo-EM?
In 2017, the Nobel Prize in Chemistry was awarded to a team of researchers who developed “cryo electron microscopy for the high resolution structure determination of biomolecules in solution”. The achievement of Nobel Laureates Jacques Dubochet, Joachim Frank, and Richard Henderson was built on ground-breaking innovations in cryo-EM, which was named “Method of the Year” by Nature Methods in 2016.
Cryo-EM is an electron microscopy technique that cools samples to cryogenic temperatures (colder than 120 K, or -143 °C) and embeds them in a vitreous water environment. Technologists apply an aqueous sample solution to a grid mesh, then plunge freeze the sample in either liquid ethane or a mixture of liquid ethane and propane.
The technique was first developed in the 1970s, but recent advances in detector technology and computer algorithms enabled scientists to determine biomolecular structures at near-atomic resolution.
Sample Preparation Remains a Challenge for Cryo-EM
Modern cryo-EM has gained significant attention as an alternative to X-ray crystallography or NMR spectroscopy for applications in macromolecular structure analysis, as it can perform these tasks without needing to crystallize the samples.
Preparing samples for cryo-EM is a complicated process. It relies on ethane, which is a powerful coolant in its liquid state. Ethane is also a flammable gas at room temperature and researchers must handle it with great care to avoid explosions.
Replacing Ethane with Liquid Nitrogen
The new study, published in the International Union of Crystallography journal, prepares samples for cryo-EM with liquid nitrogen instead of ethane, as it is safer and less expensive.
The samples prepared with liquid nitrogen also yielded sharper image results than comparable samples prepared with ethane.
The findings of this landmark study contradict accepted wisdom that dates back to the 1980s and could be the basis for a much safer and higher quality cryo-EM industry in the future.
The study’s senior author is Robert Thorne, a professor of physics in the College of Arts and Sciences at Cornell University and a Weiss Presidential Fellow. He was motivated to replace ethane in cryo-EM as it is a non-standard laboratory chemical, which adds hazards as well as other complications to cryo-EM processes. For Thorne, liquid nitrogen should be the coolant of choice for modern cryo-EM.
How Does Cryo-EM Work?
After flash freezing samples in a glassy sheet of water, cryo-EM operators need to fire electrons through the material’s molecules. This enables electron microscopes to capture multiple, yet blurred, images of the molecules in the frozen sample. Sophisticated algorithms are employed to average the blurred images into a crisp 3D image, although they are not always consistent.
The image fuzziness sometimes comes from the sample itself. Water encasing the molecules can form ice crystals if it is cooled too slowly, degrading the image. Scientists usually get around this by cooling water with ethane rapidly, making sure it snaps into a glassy sheet without crystallizing.
However, this rapid freezing can also add stress to the sheet. Because of this, molecules can move under the intense beam of electrons applied for microscopy, creating more blurring in the final image. This phenomenon is known as beam-induced motion.
Why Does Liquid Nitrogen Improve Cryo-EM?
Cryo-EM is faced with two opposing challenges. Firstly, the technique requires fast cooling of samples to prevent the formation of ice crystals and capture molecules’ biological structures. Secondly, the technique requires controlled cooling of samples – as slowly as possible – to minimize the beam-induced motion effect.
Liquid nitrogen is already used in the technique to convert ethane gas into a liquid, and then to store samples after they have been frozen. The new technique removes dangerous ethane from the equation.
Unprocessed liquid nitrogen can freeze samples around 50 times slower than ethane, but that is too slow to form glassy ice without crystallization. Researchers discovered previously that cold gas hovering above the liquid’s surface was the main factor slowing down nitrogen cooling.
An automated cooling instrument developed by Cornell researchers and an offshoot company, MiTeGen, was previously used for X-ray crystallography. The instrument was adapted in the new research for use with cryo-EM samples.
Using this technique, liquid nitrogen can cool samples at the perfect speed for cryo-EM. The technique uses only liquid nitrogen, replacing dangerous ethane as well as reducing the effects of beam-induced motion.
Now, researchers claim that all liquid nitrogen cooling can be introduced to any cryo-EM workflows to remove the extra steps demanded by methane and simplify the automated cooling instruments needed to keep laboratories safe.
References and Further Reading
Cheng Y., N. Grigorieff N, P.A. Penczek, and T. Walz (2015) A primer to single-particle cryo-electron microscopy. Cell. https://doi.org/10.1016/j.cell.2015.03.050.
Engstrom, T. et al. (2021) High-resolution single-particle cryo-EM of samples vitrified in boiling nitrogen. IUCrJ. https://doi.org/10.1107/S2052252521008095.
Krisch, J. (2021) New technique boosts cryo-electron microscopy clarity, safety. Cornell. Available at: https://news.cornell.edu/stories/2021/09/new-technique-boosts-cryo-electron-microscopy-clarity-safety.