Release date: 2018-06-06

Core technology to be overcome

On May 29, the protein sample of Zhang Shengsen, a doctoral student at the Life Science Institute of Tsinghua University, entered the cryo-electron microscope at 9 o'clock on time. A few days later, the "10-10" grade protein "HD 3D color photo" will be released. Researchers can "look directly" at the molecular structure of a single protein and solve the mechanism of life.

During this time, the electron gun in the cryo-electron microscope will continue to emit electrons, each time looking at a small unit. In order to explain this "small unit", Zhang Sensen has indicated to the Science and Technology Daily reporter a small piece of gold with the size of the "tweezers". "There are about 200 uniform holes in the gold piece, and 150 holes in each hole." The electron beam only 'sees' one of the small holes at a time." The gold film is similar to the "slide" of protein. Unlike the optical microscope, the glass slide is transparent, and the small gold piece is transparent to electrons, allowing the electron beam to pass through the sample. It is scattered. The scattered signal is captured and recorded, and the molecular structure can be presented after calculation.

The production capacity of transmission electron microscopy is one of the foundations of the ability to manufacture cryo-electron microscopy. "No domestic company produces transmissive electron microscopes." Thermo Scientific's technical support Chen Baoqing said without hesitation. He graduated from Peking University with a major in geophysics. He is very familiar with the industry. He said, "There were several companies that were manufactured before, such as the original. Jiangnan Optical Instrument Factory is not built now."

Filament that can be controlled by single electron beam, only imported

"In theory, as long as a sufficiently strong electric field is applied, the electrons will 'swim' out of the material," Chen Baoqing said. But the state of "swim" is far from the state of the electronic that can be used.

What kind of electronics can take high-definition 3D color photos for protein? Professor Wan Keshu from the School of Materials Science and Engineering at Southeast University described the ideal state: electrons of exactly the same speed, emitted from a point at the “source”.

"These requirements are contradictory." Wan Keshu explained that electrons overflow from the surface of the material, and there are many electrons to be emitted. The area is large, but it is difficult to meet the consistency requirement when the area is large.

If the electron gun is imagined as a gun, it must complete the machine gun's sweep with the accuracy of "sniping". The angle and speed of the "bullet" are exactly the same.

"Electronic energy needs to be highly consistent. Although it does not reach the laser level, it must be a very narrow distribution." Chen Baoqing explained that electronic "bullet" consistency is the prerequisite for improving image resolution.

To this end, the core structure of the electron gun is actually a very fine "gyro needle", which looks like a gyro, but the tip is thinner than the needle. The electrons start from the tip and collide with the macromolecules in a vacuum environment to reflect the molecular conformation.

"The previous technical route was to let the electron gun emit electrons by heating. The emission source (commonly known as "filament") used tungsten wire or lanthanum hexaboride. It needs about 2500 °C, and the high temperature promotes electron emission, but it also makes the electrons extremely active and difficult to control. Therefore, the electron microscope of the thermal emission electron gun has low precision," Wankeshu said.

"The field emission is through the high-voltage electric field, pulling the electrons out of the 'filament', and it can be done at room temperature." Wankeshu said, "The filaments used are not produced domestically, and all rely on imports, each of which is about tens of thousands of dollars."

He mentioned the normal temperature field emission gun (Schottky electron gun) which deposits zirconia on a specific surface of the crystal of single crystal tungsten. FEI Company later made a new breakthrough in the production of electron guns, combining heat and field, and further improving stability. In the introduction of the instrument of the cryo-electron microscope laboratory of Tsinghua University, it can be seen that the latest model of the electron microscope purchased in 2013 and arrived in 2015 is marked with “X-FEG” in the column of the electron gun, and the Chinese translation is an ultra-stable high-brightness electron gun. “The filament used is the same in material as before, and the process can make the brightness stronger.” Chen Baoqing introduced.

The scientific achievements of the stamp were interrupted

Another key part of the field emission is the external electric field that pulls out the electrons, with an electric field voltage of up to 300 kV. "Being at such a high voltage to maintain a stable voltage, can 'pull' out a stable and consistent electron, professionally known as 'monochrome good'." Wan Keshu said.

According to an article entitled "The History of the Development of Transmission Electron Microscopy in China", in 1963, China began the experiment on the stability factors of high-voltage 100kV electron guns. In 1965, the prototype was completed. China independently developed transmission electron microscopes in 1979. At the international advanced level, commemorative stamps have also been issued for domestically produced electron microscopes.

R&D in this area was interrupted for various reasons. “Until a few years ago, China also tried to restart the company in this area. It also decided to complete the independent research and development of field-transmitted transmission electron microscopy.” Chen Baoqing recalled that there were related researchers who turned to find him and asked why FEI had no relevant patent.

"One of the shortcuts they think of is to use the manufacturer's patents for reference, but many of the production processes are secret-level, and will not be rumored at all." Chen Baoqing said.

From "seeing people" to "discriminating freckles", China's research and development did not make it strong.

“Now, there is only one company in China that produces SEM, and TEM is not produced at all.” Chen Baoqing said that Zeiss also stopped the production of TEM. At present, there are only three manufacturers of TEM in the world, namely Japanese electronics. Hitachi, FEI (acquired by Thermo Fisher in 2016 for $4.2 billion.)

No market is why equipment giants have abandoned transmission electron microscopy. "The clarity before transmission electron microscopy makes the cryo-electron microscopy basically have no practical effect in scientific research and development." Chen Baoqing said. It can be understood that before I could only see the personal shadow, I can now recognize the freckles on my face.

In addition to the principle changes of the electron gun, other technologies on the cryo-electron microscope, such as the realization of the three-dimensional reconstruction algorithm and the successful development of the sample-making robot, have made the resolution of the cryo-electron microscope greatly improved, and become a weapon of life science research.

In the development of cryo-electron microscopy from "seeing people" to "discriminating freckles", China has not made a strong effort. In the cryo-electron microscope laboratory, everything from consumables to accessories must be imported. "The sample station is 100,000 yuan, the small gold piece is 50 yuan, and the external support is 150 yuan." Zhang Sensen said that all the tools used to match the cryo-electron microscope need to be original, there is no "cottage version". If the part is broken, no one can repair it. It can only be replaced after the part is mailed to the goods. For Chinese users of cryo-electron microscopes, this experience may last for a short time.

Source: Technology Daily

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