• Applied Microscopy
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• v.39 no.1
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• pp.65-72
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• 2009

Cryo-SEM which enables specimens to be observed in frozen form has been used to study liquid specimens in their native states. Cryo-methods, sample preparation for cryo-SEM, are quite complex and involve several discrete but vitally interconnected steps which are rapid cooling, fracturing, sectioning, etching and coating. It is important to select practical techniques and to optimize conditions of each steps considering analytic purpose and specimen characters, viz., sample dimension, water contents. In this study, etching methods and sample preparation before freezing had been studied for observation of cyanobacteria, Synechocystis sp. PCC 6803 using cryo-SEM and their cryo-SEM images were compared to Conventional SEM (CSEM) images treated by chemical fixation. We could observe the improved morphological images of the pili of the surface and membranes of Synechocystis sp. PCC 6803 and the three-dimensional architectures of their biofilm, which were difficult to observe using chemical fixation and CSEM. These results suggest that cryo-methods/cryo-SEM are useful techniques for morphological study of biological specimen.

• Applied Microscopy
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• v.47 no.4
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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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• v.47 no.4
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• pp.215-217
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• 2017

Dr. Jacques Dubochet, Dr. Joachim Frank, and Dr. Richard Henderson received the 2017 Nobel Prize for Chemistry for their efforts to develop effective ways to obtain high-resolution three-dimensional images of biomolecules using cryo-electron microscopy. Congratulations to the Nobel Prize in the field of electron microscopy, I will explain the scientific contributions of the three winners and introduce the role of cryo-electron microscopy (including cryo technology) in biology.

• Applied Microscopy
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• v.48 no.1
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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.

• Progress in Superconductivity and Cryogenics
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• v.15 no.4
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• pp.53-58
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• 2013

KSTAR in-vessel cryo-pump has been installed in the vacuum vessel top and bottom side with up-down symmetry for the better plasma density control in the D-shape H-mode. The cryogenic helium lines of the in-vessel cryo-pump are located at the vertical positions from the vacuum vessel torus center 2,000 mm. The inductive electrical potential has been optimized to reduce risk of electrical breakdown during plasma disruption. In-vessel cryo-pump consists of three parts of coaxial circular shape components; cryo-panel, thermal shield and particle shield. The cryo-panel is cooled down to below 4.5 K. The cryo-panel and thermal shields were made by Inconel 625 tube for higher mechanical strength. The thermal shields and their cooling tubes were annealed in air environment to improve the thermal radiation emissivity on the surface. Surface of cryo-panel was electro-polished to minimize the thermal radiation heat load. The in-vessel cryo-pump was pre-assembled on a test bed in 180 degree segment base. The leak test was carried out after the thermal shock between room temperature to $LN_2$ one before installing them into vacuum vessel. Two segments were welded together in the vacuum vessel and final leak test was performed after the thermal shock. Commissioning of the in-vessel cryo-pump was carried out using a temporary liquid helium supply system.

• Applied Microscopy
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• v.42 no.2
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• pp.111-114
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• 2012

The scanning electron microscopy is an ideal technique for examining plant surface at high resolution. Most hydrate samples, however, must be fix and dehydrate for observation in the scanning electron microscope. Because the microscopes operate under high vacuum, most specimens, especially biological samples, cannot withstand water removal by the vacuum system without morphological distortion. Cryo-techniques can observe in their original morphology and structure without various artifacts from conventional sample preparation. Rapid cooling is the method of choice for preparing plant samples for scanning electron microscopy in a defined physiological state. As one of cryo-technique, high-pressure freezing allows for fixation of native non-pretreated samples up to $200{\mu}M$ thick and 2 mm wide with minimal or no ice crystal damage for the freezing procedure. In this study, we could design to optimize structural preservation and imaging by comparing cryo-SEM and convention SEM preparation, and observe a fine, well preserved Arabidopsis stem's inner ultrastructure using HPF and cryo-SEM. These results would suggest a useful method of cryo-preparation and cryo-SEM for plant tissues, especially intratubule and vacuole rich structure.

• Applied Microscopy
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• v.46 no.1
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• pp.1-5
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• 2016

Recent cryo-electron microscopy (EM) studies reported the structure of various types of proteins at high resolution which is sufficient to visualize the intermolecular interaction at near atomic level. There are two main factors that cause the advances in cryo-EM; the development of image processing techniques, such as single particle analysis, and the improved electron detection devices. Although the atomic structures of small and asymmetric proteins are not yet to be determined by cryo-EM, this striking improvement implies the bright prospect of the application in biomedical studies. This study reviews the recently published studies reported high resolution structures using improved imaging analysis techniques and electron detectors. Furthermore, we will discuss about the future aspects of cryo-EM application.

• Molecules and Cells
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• v.43 no.3
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• pp.298-303
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• 2020

Cryo-electron microscopy (cryo-EM) is now the first choice to determine the high-resolution structures of huge protein complexes. Grids with two-dimensional arrays of holes covered with a carbon film are typically used in cryo-EM. Although semi-automatic plungers are available, notable trial-and-error is still required to obtain a suitable grid specimen. Herein, we introduce a new method to obtain thin ice specimens using real-time measurement of the liquid amounts in cryo-EM grids. The grids for cryo-EM strongly diffracted laser light, and the diffraction intensity of each spot was measurable in real-time. The measured diffraction patterns represented the states of the liquid in the holes due to the curvature of the liquid around them. Using the diffraction patterns, the optimal time point for freezing the grids for cryo-EM was obtained in real-time. This development will help researchers rapidly determine high-resolution protein structures using the limited resource of cryo-EM instrument access.

• International Journal of Advanced Culture Technology
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• v.3 no.1
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• pp.46-51
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• 2015

The mechanical and electrical properties of aluminium alloy 6061 are reported in this present work. Aluminium alloys were homogenized at $550^{\circ}C$, for 5 hours and cooled in the furnace. The different thickness reductions of 60-90% on homogenized aluminium alloy plates were achieved by cryo-rolling. Later, the as rolled samples were aged by solution treatment at the temperatures of $520^{\circ}C$ for 1 hour, water quenched; subsequently aged at $160^{\circ}C$ for 8-24 hours and partial aged (not solution treatment) at $160^{\circ}C$ for 8- 24 hours. Mechanical and electrical properties of samples were investigated. The experimental result showed that the microhardness of cryo-rolled samples were increase with increasing the percentage of the thickness reduction. Moreover, the microhardness of cryo-rolled, aged by solution treatment samples were higher than those of the cryo-rolled and cryo-rolled, partial aged samples. The cryo-rolled alloys subjected to full aged at $160^{\circ}C$ for 24 hours exhibited the hardness of 125 HV and electrical conductivity values was 45.76 %IACS and the cryo-rolled alloys subjected to partial aged at $160^{\circ}C$ for 20 hours exhibited the hardness of 67 HV and electrical conductivity values was 49.67 %IACS.

• Applied Microscopy
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• v.48 no.1
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• pp.1-5
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• 2018

Cryo-transmission electron microscopy (cryo-TEM) allows us to perform structural analysis of a analyses of large protein complexes, which are difficult to analyze using X-ray crystallography or nuclear magnetic resonance. The most common examples of proteins used are ribosomes and proteasomes. In this paper, we briefly describe the advantage of cryo-TEM and the process of two-dimensional classification by considering a human proteasome as an example.