Scientists and bioengineers are warming up to cryogenic electron microscopy (cryo-EM), an ultra-low-temperature technique for visualizing the atomic-level inner workings of human cells and other applications.
Life scientists and bioengineers are rapidly warming up to cryogenic electron microscopy (cryo-EM), an ultra-low-temperature technique for visualizing the atomic-level inner workings of human cells. More researchers will undoubtedly grow hotter on the technology following a government-funded initiative to increase the use of these costly and important tools in a variety of applications.
Cryo-EM is the best tool yet for understanding the nanoscale relationships between a biomolecule’s natural structure and its function in health and disease. The technique parts the curtain on the inner workings of life, the individual steps of disease progression, and potential targets for new drugs and vaccines.
Based on decades-old electron microscopy methods, cryo-EM remains far superior in biology because it can image proteins, nucleic acids, viruses, and other fragile biomolecules without the use of structure-altering dyes or fixatives. This capability positions cryo-EM to open new doors in basic research and drug development. But cryo-EM also has potential to reveal previously unseen structural details of materials of growing importance in disciplines like renewable energy, medical devices, and nanoengineering.
Cryo-EM’s three primary developers shared the 2017 Nobel Prize in chemistry for their seminal contributions to a technique that has experienced substantial performance gains over the past five years. Although it has been in development for decades, cryo-EM as we know it today is still in its infancy. Consequently, only a handful of research centers have invested in the technology to date. The hulking-yet-delicate instruments can cost more than $4 million and they require custom-constructed laboratory facilities with expensive MEP mitigations for vibration, humidity, and electromagnetic interference. For the U.S. scientific community to tap into the promise of cryo-EM, more research centers need access to the equipment and a larger workforce of trained cryo-EM operators.
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That’s one reason why the National Institutes of Health recently launched its $130 million Transformative High-Resolution Cryo-Electron Microscopy initiative. Over the next six years, NIH funds will support three national cryo-EM centers at Stanford University, Oregon Health & Science University, and the New York Structural Biology Center. While working to advance the technique’s capabilities and ease of use, each center will provide other U.S. institutions with access to cryo-EM instruments and training courses without bearing the high cost of equipment, staff and facilities.
Life science researchers are embracing cryo-EM because it combines the best traits of two common characterization tools: optical (or light) microscopy and X-ray crystallography. Optical microscopes are nondestructive and can produce detailed images of living cells, but their resolving power is limited to objects larger than a few hundred nanometers. In other words, they can discern bacteria but not viruses.
X-ray crystallography, the current gold standard for molecular structure characterization, can reveal features on the Angstrom scale, but it requires the sample to be in crystalline form prior to inspection. Some materials can’t be crystallized, meaning there has previously been no way to examine their fine structures. Cryo-EM provides the resolution of crystallography with optical microscopy’s ease of use and nondestructive nature, and it can work with virtually any type of biological sample.
Electron microscopes are fixtures in semiconductor research and other applications involving hard, stable samples that can withstand irradiation by high-intensity electron beams in a vacuum chamber. They have had limited use in biology because those operating conditions are too harsh to use on proteins and other delicate biomolecules. The cryogenic sample preparation step in cryo-EM overcomes that barrier by flash-freezing a biological sample in a thin layer of vitrified ice. Not only does the method preserve the molecule in its natural state while in mid-motion, it also suspends it in a stable, transparent layer that can tolerate the experimental conditions.
To prepare a sample for a cryo-EM procedure, as many copies as possible of a biomolecule or molecular complex are removed from a cell, purified, and rapidly cooled to -180 C on a cryo-EM grid. During the experiment, thousands of raw images from multiple perspectives are captured and then converted into sharp 3D images and movies. Processing and storing cryo-EM data consumes massive amounts of computing power. A single 3D visualization can include several thousand individual movies, each requiring up to eight gigabytes of storage. Only research centers with access to adequate supercomputing capabilities can handle the terabyte-level loads generated by a busy cryo-EM lab – another factor limiting access to the technique.
Cryo Beyond Bio
Most of the excitement around cryo-EM focuses on its potential in basic biomedical research and drug discovery. However, the technique is also creating new possibilities for engineers in disciplines like renewable energy, medical devices, and nanomaterials.
Beyond biology, materials scientists and engineers are using cryo-EM to visualize structures and processes that don’t lend themselves to other EM techniques. For example, a team at Stanford recently used cryo-EM to observe sensitive lithium-ion battery components and interfaces at the atomic level. Typical lithium-ion batteries contain certain lithium-containing electrode materials, organic electrolytes and solid electrolyte interphases that are structurally unstable under standard transmission EM analysis. As it does with biological samples, cryo-EM preserves the native shapes of these chemically reactive, beam-sensitive materials, thereby enabling researchers to resolve individual lithium metal atoms. These observations could help researchers better understand failure mechanisms such as the formation of dendrites that reduce performance lifetimes in high-energy batteries.
“With cryo-EM you can look at a material that’s fragile, chemically unstable, and you can preserve its pristine state – what it looks like in a real battery – and look at it under high resolution,” says Yi Cui, a Stanford materials engineering professor who led the study. “This is tremendously exciting. It could explode the whole field of materials design.”
That’s the same idea behind a hybrid microscope-spectrometer system developed at Penn State University. The instrument can help keep pace with changes in materials science.
“Fifty years ago, materials science was all hard materials, metals and ceramics,” says Bernd Kabius, a senior scientist at Penn State’s Materials Research Institute. “Now the emphasis is on soft materials like polymers and combinations of semiconductors with complex molecules for technological applications.”
Conventional methods don’t allow researchers to study the dynamic processes of catalytic reactions at surfaces in liquid environments. However, cryo-EM and spectroscopic methods developed for life science applications can be adapted to bring valuable spatial and chemical data into the materials lab, he says.
At Cornell University, Lena Kourkoutis is using cryo-EM to study the dynamic processes of battery degradation and cooled-state superconductors. The professor of applied and engineering physics is also heading up a Cornell team developing a novel hybrid instrument combining the best traits of cryo-EM with the enhanced resolution possible with scanning transmission cryo-EM. As the first of its kind, the cryo-STEM instrument would bring new levels of resolution to the study of solid-liquid interfaces and non-biological materials that feature soft-hard interfaces.
“Biologists have figured out how to do cryogenic electron microscopy, and materials scientists and physicists have figured out how to get to the atomic scale,” Kourkoutis says. “We are trying to combine the capabilities of both in order to open up new possibilities.”
Michael MacRae is an independent technical writer based in Oregon.
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