Cryogenic electron microscopy method aids development of COVID-19 vaccine

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What you will learn:

  • How has cryogenic electron microscopy (cryo-EM) helped researchers during the COVID-19 pandemic?
  • Camera Link HS (CLHS) applications outside the factory.

Advances in imaging technology continue to benefit society in disparate ways. For example, cryogenic electron microscopy (cryo-EM) has played a fundamental role in the development of the COVID-19 vaccine. Combining cameras and detectors, sample handling technology, automation and software, cryo-EM is an easy-to-use method for collecting high-quality data. It ultimately helped researchers identify the spike protein of SARS-CoV-2, the virus that causes COVID-19.

A long and automated process

Cryo-EM (Fig.1) involves rapidly freezing a sample to -180°C, leaving the protein particles suspended in ice. The sample sets are placed in a holder containing a grid usually made of gold or carbon. Inside the grid is a sheet with small holes or pockets. The material imaged by the system is instantly frozen inside the fluid contained in these holes.

To start the process, researchers use Thermo Fisher Scientific’s EPU 2 software to set acquisition and optics parameters, as well as verify proper microscope alignment for high-resolution data acquisition. The software then acquires an overview of the grid to determine if the grids are of sufficient quality for an automated recording session. Grids are sorted in batches, with the software automatically grouping grid squares into categories of similar quality.

Finally, researchers select grid squares and leaf holes for acquisition and define the acquisition pattern for each leaf hole. Square grid and leaf hole selection can be done manually or automatically by applying user-defined filter settings. The system uses a beam of electrons to illuminate individual holes, allowing the camera to capture an image. From there, the cryo-EM system moves the beam to multiple locations in the hole or to multiple holes, and then the stage moves, allowing the beam to move to the next location. The beam effectively travels through multiple locations at each sample hole.

Since the process uses low doses of electrons to avoid sample damage and to allow detection of individual electron impacts, images come out noisy and have low contrast. The system must acquire thousands of images, which are aligned and summed to produce an appropriate image. The reconstruction software also removes noise.

“Inspecting individual images would not allow a system operator to distinguish anything in the image due to the low level of detail, so you would only see noise,” said engineer Hans Roeven. system design personnel at Thermo Fisher Scientific.

“The system can combine one or two thousand individual images to produce an image with sufficient contrast that can be fed into the reconstruction process,” he continues. “Creating a full 3D reconstruction requires tens of thousands of such images, which is why the process takes so long. Increasing camera speed (frame rate) plays a significant role in reducing overall data collection time, thereby facilitating workflow and maximizing time to results for our customers.

Cryo-EM and COVID-19

Single-particle cryo-EM involves picking particles extracted from thousands of acquired images to construct 3D representations of viruses or proteins (Fig.2). This process produced the first 3D structures of the isolated SARS-CoV-2 spike protein. The spike protein is a key point of engagement with other cells. It binds to receptors on human host cells before starting the process of virus replication.

Disabling the binding ability of the spike protein is crucial according to Eric Chen, director of market development for Asia and the Pacific at Thermo Fisher Scientific. Prior to its identification, the spike protein was central to vaccine development. To understand its structure and produce an epitope map of the binding sites, the researchers sought to image it. This would allow them to determine which antibodies or inhibitors would prevent viral docking, Chen said.

To learn more about the spike protein, a team from the University of California, Berkeley recently used Thermo Fisher Scientific’s Krios G4 cryo-EM instrument equipped with a cold field emission gun (CFEG) operated at 300kV. A Falcon 4 detector was used for image capture, which features a custom-designed 4096×4096 CMOS image sensor and an internal frame rate of 250 fps (Fig.3). It has been thinned down to 30 µm to ensure electron hits do not generate too large an electron cloud, which would appear as a large clump in an image. Electronic impact and captured images were read with an FPGA through a Camera Link HS (CLHS) interface; its data acquisition system consisted of custom FPGA boards.

Leveraging cryo-EM technology, UC Berkeley researchers discovered that the spike protein has nothing to do with other known ion channels: it consists of a half-channel, not piercing the membrane only half cell. The SARS-CoV-2 ORF3a protein is a putative viral ion channel involved in autophagy inhibition, inflammasome activation, and apoptosis. Protein 3a and anti-3a antibodies are found in infected tissues and plasma, according to the UC Berkeley team’s study, published in Nature.

Researchers found that deletion of 3a in SARS-CoV-2 and deletion of a related 3a gene in the original SARS virus, SARS-CoV-1, reduce disease severity in models animals. The UC Berkeley researchers noted that targeting this protein in the vaccine helped reduce the severity of human COVID-19 infections.

CLHS meets data challenges

Although most commonly associated with machine vision applications, the Association for Advancing Automation’s (A3) CLHS standard offers benefits that extend far beyond the factory floor. Thermo Fisher Scientific leverages the CLHS standard in its transmission electron microscopy (TEM) cameras and detectors, including the system used to identify the spike protein.

TEM applications involve high-resolution, high-speed cameras that generate huge amounts of data (images). While Thermo Fisher Scientific has a long history of using cameras and detectors, reducing overall data collection time is an important factor in new product development, according to Roeven.

“Workflow automation and speed of image acquisition are important factors, and new camera developments involve ever-increasing data rates. Robust, high-throughput interfaces are therefore critical to success,” did he declare. “We not only wanted to standardize the interface, but we also needed low latency triggering to synchronize processes in the microscope, which CLHS provides.”

Integrating resources into the FPGA presented some challenges during development, as the company combines up to eight channels in a single FPGA. Specifically, the interface needed to fit into a design that uses multiple resources for other functions. This required careful partitioning of clock resources to add an eight-lane 10 Gb/s interface.

In turn, the system can efficiently share resources for multiple instances of the CLHS X-Protocol IP core – 64b/66b line encoding with forward error correction designed for 10 Gb/s and higher bit rates. “In addition to the crucial timing task, the low latency, jitter-free link trigger offered by CLHS gave us more freedom in partitioning functions and allowed the system to perform to its full potential,” Roeven said. .

The use of fiber optic cables in the system provides galvanic isolation to prevent ground faults in the system, while providing cost effectiveness, as FPGAs with QSFP+ interfaces are generally commercially available. In addition, according to Roeven, the fiber cables help the system avoid image degradation by noise.

“Using fiber also allows us to place the image processing system at an acceptable distance from the instrument, which is important because computer servers can make a lot of noise and acoustic vibrations can be a source of image degradation when resolving images up to such levels”. levels,” Roeven said.

Future innovations

While cryo-EM methods have attracted a lot of attention for their use in developing COVID-19 vaccines, the technology has already been popular for several years. Its creators won the Nobel Prize in Chemistry in 2017 for “the development of cryo-electron microscopy for high-resolution structure determination of biomolecules in solution”. The process was named Method of the Year 2015 by Nature.

Cryo-EM systems are also used in drug discovery, plant biology research, pathology research, cancer research, materials science, and semiconductor inspection. Going forward, the technology is something to watch as it could have significant impacts in the life sciences and beyond.