• Applied Microscopy
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• 제47권3호
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• pp.160-164
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• 2017

Electron crystallography has been used as the one of powerful tool for studying the structure of biological macromolecules at high resolution which is sufficient to provide details of intramolecular and intermolecular interactions at near-atomic level. Previously it commonly uses two-dimensional crystals that are periodic arrangement of biological molecules, however recent studies reported a novel technical approach to electron crystallography of three-dimensional crystals, called micro electron-diffraction (MicroED) which involves placing the irregular and small sized protein crystals in a transmission electron microscope to determine the atomic structure. In here, we review the advances in electron crystallography techniques with several recent studies. Furthermore, we discuss the future direction of this structural approach.

• Applied Microscopy
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• 제47권4호
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• pp.223-225
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• 2017

Cryo-electron microscopy (cryo-EM) is arguably the most powerful tool used in structural biology. It is an important analytical technique that is used for gaining insight into the functional and molecular mechanisms of biomolecules involved in several physiological processes. Cryo-EM can be separated into the following three groups according to the analytical purposes and the features of the biological samples: cryo-electron tomography (cryo-ET), cryo-single-particle reconstruction, and cryo-electron crystallography. Cryo-tomography is a unique EM technique that is used to study intact biomolecular complexes within their original environments; it can provide mechanistic insights that are challenging for other EM-methods. However, the resolution of reconstructed three-dimensional (3D) models generated by cryo-ET is relatively low, while single-particle reconstruction can reproduce biomolecular structures having near-atomic resolution without the need for crystallization unless the samples are large (>200 kDa) and highly symmetrical. Cryo-electron crystallography is subdivided into the following two categories according to the types of samples: one category that deals with two-dimensional (2D) crystalline arrays and the other category that uses 3D crystals. These two categories of electron-crystallographic techniques use different diffraction data obtained from still diffraction and continuous-rotation diffraction. In this paper, we review crystal-based cryo-EM techniques and focus on the recently developed 3D electron-crystallographic technique called microcrystal-electron diffraction.

• Applied Microscopy
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• 제28권3호
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• pp.299-306
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• 1998

T-butyl alcohol (TBA) freeze-drying method originally designed by Inoue and Osadake (1989) was adopted to dry specimens for scanning electron microscopy and the results were compared with those dried using critical point dryer (CPD). Small pieces $(1\times1\times3mm)$ of liver of Sprague-Dawley rats were cut and fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer after anesthesia, and processed for scanning electron microscopy by several modifications of TBA freeze-drying methods and by the standard preparation method using CPD. The bile canaliculi and sinusoidal endothelial surface were observed. Tissue dehydrated with TBA before TBA freeze-drying preserved the structures best comparable to those prepared with CPD. This result suggests that combination of dehydration with TBA and TBA freeze-drying is a superior method to the original TBA freeze-drying method dehydrated with ethanol.

• Applied Microscopy
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• 제48권1호
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• pp.6-10
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• 2018

Cryo-electron microscopy (cryo-EM) allows the analysis of the near-native structures of samples such as proteins, viruses, and sub-cellular organelles at the sub-nano scale. With the recent development of analytical methods, this technique has achieved remarkable results. The importance of cryo-EM gained wide recognition due to last year's award of the Nobel Prize in Chemistry. To help promote the knowledge of this technique, this paper introduces the basic workflows of cryo-EM and domestic cryo-EM service institutes.

• Applied Microscopy
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• 제25권4호
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• pp.71-82
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• 1995

The ultrastructure and storage reserves of the Capsicum annuum seeds were studied in order to identify structure and to localize storage components in the endosperm using light microscopy, scanning and transmission electron microscopy. The seed coat was composed of one cell layer which contained a large number of lipid bodies, while most of the endosperm cells did not showed many lipid bodies. During seed maturation, the endosperm cells were continuously degenerated by the autophagy. Various types of plastids were also distinguished in the endosperm cells. They contained starch grains surrounded by electron-dense tiny particles, plastoglobuli, and vasicular bodies.

• Applied Microscopy
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• 제48권4호
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• pp.81-86
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• 2018

The respiratory chain complex forms a supercomplex (SC) in the inner mitochondrial membrane. This complex facilitates the process of electron transfer to produce the proton gradient used to synthesize ATP. Understanding the precise structure of the SC is considered an important challenge. However, it has not yet been reported. The development of a Cryo-electron microscopy (EM) technique provides an effective way to obtain high-resolution micrographs to determine the high-resolution three-dimensional structure of biomolecules. In this brief review, the currently reported Cryo-EM structures of the mammalian respirasome have been described in order to establish a direction for further research in the respiratory system.

• Applied Microscopy
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• 제52권
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• pp.11.1-11.9
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• 2022

Zoonotic poxvirus infections pose significant threat to human health as we have witnessed recent spread of monkeypox. Therefore, insights into molecular mechanism behind poxvirus replication cycle are needed for the development of efficient antiviral strategies. Virion assembly is one of the key steps that determine the fate of replicating poxviruses. However, in-depth understanding of poxvirus assembly is challenging due to the complex nature of multi-step morphogenesis and heterogeneous virion structures. Despite these challenges, decades of research have revealed virion morphologies at various maturation stages, critical protein components and interactions with host cell compartments. Transmission electron microscopy has been employed as an indispensable tool for the examination of virion morphology, and more recently for the structure determination of protein complexes. In this review, we describe some of the major findings in poxvirus morphogenesis and the contributions of continuously advancing electron microscopy techniques.

• Applied Microscopy
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• 제47권1호
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• pp.43-49
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• 2017

A new method was introduced to distinguish the ferrite, bainite and martensite in transformation induced plasticity (TRIP) steel by using electron backscatter diffraction (EBSD) and transmission electron microscopy (TEM). EBSD is a very powerful microstructure analysis technique at the length scales ranging from tens of nanometers to millimeters. However, iron BCC phases such as ferrite, bainite and martensite cannot be easily distinguished by EBSD due to their similar surface morphology and crystallographic structure. Among the various EBSD-based methodology, image quality (IQ) values, which present the perfection of a crystal lattice, was used to distinguish the iron BCC phases. IQ values are very useful tools to discern the iron BCC phases because of their different density of crystal defect and lattice distortion. However, there are still remaining problems that make the separation of bainite and martensite difficult. For instance, these phases have very similar IQ values in many cases, especially in deformed region; therefore, even though the IQ value was used, it has been difficult to distinguish the bainite and martensite. For more precise separation of bainite and martensite, IQ threshold values were determined by a correlative TEM analysis. By determining the threshold values, iron BCC phases were successfully separated.

• Applied Microscopy
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• 제45권3호
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• pp.170-176
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• 2015

In this work, various electron microscopy and analysis techniques were used to investigate the microstructural evolution of a 9% Cr tempered martensite ferritic (TMF) steel T91 upon ultrasonic nanocrystalline surface modification (UNSM) treatment. The micro-dimpled surface was analyzed by scanning electron microscopy. The characteristics of plastic deformation and gradient microstructure of the UNSM treated specimens were clearly revealed by crystal orientation mapping of electron backscatter diffraction (EBSD), with flexible use of the inverse pole figure, image quality, and grain boundary misorientation images. Transmission electron microscope (TEM) observation of the specimens at different depths showed the formation of dislocations, dense dislocation walls, subgrains, and grains in the lower, middle, upper, and top layers of the treated specimens. Refinement of the $M_{23}C_6$ precipitates was also observed, the size and the number density of which were found to decrease as depth from the top surface decreased. The complex microstructure and microstructural evolution of the TMF steel samples upon the UNSM treatment were well-characterized by combined use of EBSD and TEM techniques.

• Applied Microscopy
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• 제48권4호
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• pp.122-125
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• 2018

The focused ion beam (FIB) method is widely used to prepare specimens for observation by transmission electron microscopy (TEM), which offers a wide variety of imaging and analytical techniques. TEM has played a significant role in material investigation. However, the FIB method induces amorphization due to bombardment with the high-energy gallium ($Ga^+$) ion beam. To solve this problem, electron beam induced deposition (EBID) is used to form a protective layer to prevent damage to the specimen surface. In this study, we introduce an optimized TEM specimen preparation procedure by comparing the EBID of carbon and tungsten as protective layers in FIB. The selection of appropriate EBID conditions for preparing specimens for TEM analysis is described in detail.