Speaking of cryo-electron microscopy, Xiao Bian thinks that no matter whether it is a graduate student or a professor of big coffee, people who may have a little connection with scientific research are not strange to this name, because it is too famous! Many scientific research results based on cryo-electron microscopy are published in the top journals of Nature, Science, and Cell (envy face), which is called NSC artifact. The development of cryo technology directly contributing to the life sciences, especially the rapid development of structural biology, and this year is delivered (to bully me big chemical) winning the "explosive" chemistry prize! However, these are not important, after all, he is the biological family of the child next door to the passerby attitude (envy but I do not show it) look like, anyway, it would not open! Unexpectedly, on a clear day, there are still some windy and beautiful, Stanford's Cui Da Shen (Cui Wei) hard to get it into my big material circle, and blockbuster, engaged in a Science! (Ask you Fubu Fu) heard the news, small series can not help but fall into a deep meditation, you are doing the material, how such a big difference it? (Next table Kobayashi: young people are Daniel Daniel, do you, huh ~!!) However, after a brief meditation, small sense of shame and then courage, decided to take a look at this "chaos into" My biological material circle next to the house The child, the boss of Wanda, also made a set-up in the lab one day (big boss: I just don't talk), that little editor is not a big advantage, wit like me! The singularity is not as good as the wit, so below, Xiaobian takes everyone to learn about the dragon knives of this biosphere - cryo-electron microscopy! 1. What is a cryo-electron microscope? Cryo-electron microscopy (Cryo-EM) (I'm also a good friend of my big material to remember this word, I believe that the future will become a popular keyword in the literature of search materials) Refers to the method of imaging the sample by transmission electron microscopy in a low temperature environment after rapid freezing of the biological macromolecule, and then obtaining the three-dimensional structure of the sample through image processing and reconstruction calculation [1]. Figure 1 shows the FEI Titan Krios 300kV cryo-electron microscope from the Institute of Biophysics of the Chinese Academy of Sciences. It is said that a single unit should be more than $6 million. After more than 30 years of development, cryo-electron microscopy has even surpassed X-ray crystallography and nuclear magnetic resonance (NMR) to support the foundation of high-resolution structural biology research. Figure 1 FEI Titan Kiros 300kV cryo-electron micrograph So why do you need cryo-electron microscopy? It is well known that X-ray crystallography is a classical method for analysing structures. However, it requires obtaining single crystals of biological samples, and it is very difficult to grow crystals of biological macromolecules. At the same time, atomic images have been directly observed by electron microscopy in materials science research [ 2] (As a material Wang, the importance of TEM, SEM I don't think there is no need to rumor), so biologists also want to use electron microscopy to take a high-resolution photo of biomacromolecules, analyze its structure, to understand its biochemical reaction mechanism, However, things are not so simple, the application of electron microscopy in the biological field is severely limited: (1) biological samples are rich in water, while TEM works under high vacuum; (2) high-energy electron beams can seriously damage biological samples. (3) Biological samples are mainly light elements such as C, O, N, H, etc. The reflection and scattering of electrons are similar to the background, and the image contrast is very low; (4) the protein molecules will drift, resulting in blurred images. After many scientists' long-term efforts and constantly overcome various difficulties, the cryo-electron microscopy technology has finally developed, and the high-resolution structural analysis of biomolecules in solution has been realized, which has brought biochemistry into a new era, among which three have pioneering contributions. Scientists thus won the 2017 Nobel Prize in Chemistry. They are: Professor Jacques Dubochet of the University of Lausanne, Switzerland, Professor Joachim Frank of Columbia University, and Professor Richard Henderson of the University of Cambridge, UK. The cryo-electron microscopy technique for biomacromolecular HD photography is [1]: sample freezing → low-dose electronic cryo imaging → three-dimensional reconstruction. Figure 2 3 scientists who won the 2017 Nobel Prize in Chemistry (1) Sample freezing Sample freezing is actually a thought that scientists have long thought of, but after freezing, the water molecules in the sample form ice crystals, which not only produce strong electron diffraction to mask the sample signal, but also change the sample structure. Until 1974, Kenneth A. Taylor and Robert M. Glaeser when viewed in the aqueous biological sample -120 ℃ No ice crystal formation, and found frozen sample can withstand a higher dose and longer electron irradiation, before turning to bring the samples were frozen. The Jacques Dubochet father mentioned above went one step further and discovered the glass state of water, which successfully solved the problem of cryo-electron microscopy, as shown in Figure 3 (a) [1]. Figure 3 Schematic diagram of sample preparation (a) and three-dimensional reconstruction (b) of cryo-EM (2) Low dose electronic cryo imaging The material Wang knows that when the TEM and SEM are generally used, the conductivity of the sample is better, and the higher the electron dose, the better the image quality. However, high-dose electrons are devastating to biomacromolecules, so Professor Richard Henderson proposed imaging at the lowest possible electron dose at low temperatures. He and his collaborators reconstructed a rough (7?) and high-resolution (3.5?) bacterial rhodopsin model in 1975 and 1990, as shown in Figure 4, demonstrating cryo-EM Feasibility for high resolution structural analysis of biological macromolecules. However, this 15-year improvement seems to be inferior to Hartmut Michel et al. (1988 Nobel Laureate in Chemistry), which obtained a membrane protein 3.0-resolution atomic model as early as 1984. Although the situation is not optimistic, Professor Henderson continues to theoretically guide the development of cryo-electron microscopy technology and predicts that with the development of electron microscopy technology and sample preparation level, cryo-electron microscopy will become a powerful analytical solution for difficult samples and macromolecular structures. tool. Figure 4 3D structural model of bacterial rhodopsin (3) 3D reconstruction The small partners who have done TEM know that the TEM obtains a two-dimensional projection image. To obtain a three-dimensional structure, a series of modeling and transformation is required. This process is three-dimensional reconstruction. Joachim Frank, the third prize winner mentioned above, and his collaborators established a method for the asymmetric particle from two-dimensional projection to three-dimensional structure (random cone tilt method), which laid the foundation for single-dimensional three-dimensional reconstruction of cryo-electron microscopy. The principle is shown in Figure 3(b) [3, 4]. Subsequently, the SPIDER program was developed for cryo-electron microscopy structural analysis and has been widely used. At present, the widely used 3D reconstruction software in the field of cryo-electron microscopy is Dr. Sjors Scheres from the Richard Henderson Father's Laboratory at the University of Cambridge. It is said that Dr. Sjors Scheres did not have an NSC paper, but Professor Richard Henderson still introduced it to Cambridge. RELLION developed by the MRC Molecular Biology Laboratory. However, even with the help of Ren Duo two veins (the above three key processes), cryo-electron microscopy did not immediately get such a red burst. This is mainly because (1) the signal-to-noise ratio of the cryo-electron microscope is low, and (2) the drift of the image is taken, so that the two-dimensional projection that can be acquired is still in a fuzzy state, so it can only be applied to the structural analysis of a limited single particle of biological macromolecule. , severely limited its application. Until 2013, Professor Cheng Yifan of the University of California, San Francisco (UCSF) used direct electronic detectors (DDD) to record single-particle images of cryo-electron microscopy, greatly improving signal-to-noise ratio and resolution, and achieving near-atom resolution ( The analysis of the membrane protein structure of 3.3) caused a sensation in the industry, as shown in Figure 5. Subsequently, the cryo-electron microscopy technique has no disadvantage in the analysis of the 3D structure of biomacromolecules. At present, the Subramaniam laboratory of NIH in the United States successfully analyzed the structure of glutamate dehydrogenase with a resolution of 1.8 Ã…, creating the world record for the highest resolution. Figure 5 Comparison of resolution before and after direct electron detector application It can be seen that it is the three Nobel Prize scientists who have completed their breakthrough work in their respective fields: Jacques Dubochet broke through the bottleneck of freezing technology, Joachim Frank made an original contribution to the 3D reconstruction algorithm, and Richard Henderson used the low electron dose for the first time. The imaging completes the analysis of the 3D structure of biomacromolecules and has been guiding the development of cryo-electron microscopy technology in theory. Finally, a leap from 0 to 1 has been formed. The cryo-electron mirror, the dragon knives, has been cast, and the new research in structural biology has been initiated. situation. The above three scientists have won the Nobel Medal and can be said to be well deserved! 2. The history of cryo-electron microscopy in structural biology From the issue of NSC and other top publications and the continuous flow of biomacromolecular structures, the huge success of cryo-electron microscopy in the field of structural biology need not be repeated. Taking China as an example, the significant progress made in the field of structural biology based on cryo-electron microscopy is very impressive, as shown in Table 1 [5] (2016). With the great heat of cryo-electron microscopy technology, many universities and research institutes in China have spent a lot of money to purchase cryo-electron microscopy equipment. More than 24 independent laboratories have used cryo-electron microscopy to study the 3D structure of biological macromolecules such as proteins. , as shown in Figure 6 [5]. Table 1 The landmark achievements of China's mainland based on cryo-electron microscopy in 2016 Figure 6 Distribution map of major domestic cryo-electron microscopes 3. Cryo-electron microscopy emerges in materials science Xiao Bian did not find any report on the application of cryo-electron microscopy technology to the field of materials science before Professor Cui Wei, but with or without it, Professor Cui Wei of Stanford published online on October 27, 2017 in Science entitled "Atomic structure". The research paper [of] of sensitive battery materials and interfaces revealed by cryo-electron microscopy[6] is a beginning of cryo-electron microscopy in the study of materials science. Xiaobian thinks it is appropriate. After all, it is the stone of other mountains. A model can be said to open a new world of materials science research. Lithium- ion partners know that lithium dendrites are the biggest safety hazard in lithium batteries. It is not unrelated to the spontaneous combustion accidents that occur in Samsung and Apple products from time to time. Today, the generation, growth and piercing of the dendrites cause internal short circuits in the battery, which is a problem that battery experts have to face, and is also the research direction of “continuous high temperature†in the field of materials. However, it is well known that lithium is very active and extremely sensitive to the environment. How to study the formation and growth of lithium dendrites from the atomic level is extremely challenging. The traditional high-resolution TEM electron beam energy is very high, which will seriously damage the dendrite structure and even meltdown; while the low-resolution TEM, direct imaging, surface probe and other technologies obtain limited information. In this Science paper, Professor Cui Wei and others were inspired by the "frozen electron microscopy to obtain the atomic level structure of fragile biomacromolecules", and creatively introduced cryo-electron microscopy technology into the study of sensitive battery materials and interface fine structures. For the first time, the structural problems of the lithium dendritic atomic resolution level were obtained. The results show that the cryo-electron microscopy technique completely preserves the original morphology and related structure and chemical information of dendrites, and remains intact under electron beam bombardment for 10 min. The high-resolution Cryo-EM photo shows that the lithium dendrite is a long strip of perfect hexagonal crystal, completely different from the irregular shape observed by traditional electron microscopy; and its growth behavior shows obvious <111> preferential orientation, growth. "Bending" may occur during the process, but no crystal defects are formed and the perfect crystal structure is not affected. In addition, the research results also include the composition and structure of the solid electrolyte interface (SEI). Professor Cui Wei said that the results are very exciting, which proves that Cryo-EM can effectively characterize the fragile and unstable battery materials, such as lithium silicon, sulfur, etc., and keep them in real batteries. Original state. Figure 7 Preserving and stabilizing lithium metal with Cryo-EM Greatly accelerates the process of literature, the paper also found another article using Cryo-EM Lithium batteries "New Insights on the Structure of Electrochemically Deposited Lithium me tal and Its Solid Electrolyte Interphases via Cryogenic TEM", by the University of California, San Diego Professor Ying Shirley Meng of the branch school (UCSD) and others published in Nano Lett. On, published on November 1, 2017, online publication is only four days later than Professor Cui's Science Masterpiece. The article [9] also adopted the cryo-electron microscopy technique to stabilize the electrochemically deposited active lithium metal while reducing the damage caused by electron beam, and then studied its nanostructure, chemical composition and solid electrolyte interface. It can be said that Professor Cui Wei is the same, which proves that Cryo-EM is a powerful tool for studying electron beam and heat sensitive battery materials, and can obtain relevant information from the most basic level. Fig. 8 Study on Cryo-EM for electrodeposition of Li metal In addition to the above two papers on the use of Cryo-EM in sensitive battery materials and SEI research in lithium batteries, the limited knowledge of small series may be in organic/inorganic hybrid perovskite materials, certain polymer materials, water. In the characterization of fine structures and intermediate states such as gels and quantum dots, the advantages that Cryo-EM will have are self-evident. It is foreseeable that in the near future, the atomic-level characterization of these electron beam- and heat-sensitive active materials may be the potential of Cryo-EM in the field of materials. 4. to sum up On October 4, 2017, the announcement of the Nobel Prize in Chemistry caused a crazy circle of friends. He believed that cryo-electron microscopy is a chemistry prize given to physicists to reward their outstanding contributions to the biological field. Of course. Now, Professor Cui Wei's Science paper and Professor Meng Ying's Nano Lett. The paper finally made this chemistry prize more chemical. The exciting work done by the children of the next-door biologist's “cross-border†material circle has faintly incited research on materials science. Xiao Bian believes that this technology that leads biochemical research into a new era, stirring my big material rivers and lakes is also a day! references [1] Yang Hui, Li Shentao, Xue Bing. Cryo-electron microscopy: Life from an atomic scale [J]. Journal of Capital Medical University, 2017, 38(5): 770-776. [2] Liu Zheng, Zhang Jingqiang. A major breakthrough in structural biology research methods - the application of electronic direct detection cameras in cryo-electron microscopy [J]. Journal of Biophysics, 2014, 30(6): 405-415. [3] Yin Changcheng. If you want to do something good, you must first sharpen your weapon! —— 2017 Nobel Prize in Chemistry Evaluation [J]. Chinese Journal of Biochemistry and Molecular Biology, 2017, 33(10): 979-984. [4] Milne JLS, Borgnia MJ, Bartesaghi A, et al. Cryo-electron microscopy-a primer for the non-microscopist [J]. FEBS Journal, 2013, 280: 28-45. [5] Wang H, Lei J, Shi Y. Biological cryo-electron microscopy in China [J]. Protein Science, 2017, 26: 16-31. [6] Li Y, Li Y, Pei A, et al. Atomic structure of sensitive battery materials and interfaces revealed by cryo-electron microscopy [J]. Science, 2017, 358: 506-510. [7] Material Cow: Cui Wei's latest science: Fun-frozen electron microscopy - revealing battery materials and interface atomic structures, http://www. cailiaoniu. Com/108826. Html. [8] X-MOL: The Chemical Nobel Prize-free cryo-electron microscope re-emerges, and the Cui Wei team brings a heavy science, http://www. X-mol. Com:8081/news/9696. [9] Wang X, Zhang M, Alvarado J, et al. New Insights on the Structure of Electrochemically Deposited Lithium me tal and Its Solid Electrolyte Interphases via Cryogenic TEM [J]. Nano Letter, 2017, 17: 7606-7612. Ftth Drop Cable Assembly,Ftth Drop Cable Assembly Assembly,Ftth Drop Cable Assembly Adapter,Ftth Drop Cable Assembly Access Huizhou Fibercan Industrial Co.Ltd , https://www.fibercan-network.com