Atmospheric Scanning Electron Microscopy Research Articles

大気圧電子顕微鏡ASEMによる水中観察法の開発

大気圧電子顕微鏡ASEMによる水中観察法の開発

大気圧電子顕微鏡ASEMによる水中観察法の開発

大気圧電子顕微鏡ASEMによる水中観察法の開発

Observation of Tissues in Solution by Atmospheric Scanning Electron Microscope (ASEM)

1. Graduate School of Comprehensive Human Sciences, University of Tsukuba. 1-1-1 Tennodai, Tsukuba, Ibaraki Prefecture 305-0006, Japan. 2. Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Umezono 1-1-4, Tsukuba, Ibaraki 305-8568, Japan. 3. Traditional Uighur Medicine Institute of Xinjiang Medical University, 393 Xinyi Rd, Urumqi, Xinjiang Uighur Autonomous Region, 830011 China.

The Atmospheric Scanning Electron Microscope (ASEM) Observes Axonal Segmentation and Synaptic Induction in Solution

The Atmospheric Scanning Electron Microscope (ASEM) Observes Axonal Segmentation and Synaptic Induction in Solution

Atmospheric scanning electron microscope system with an open sample chamber: configuration and applications.

An atmospheric scanning electron microscope (ASEM) with an open sample chamber and optical microscope (OM) is described and recent developments are reported. In this ClairScope system, the base of the open sample dish is sealed to the top of the inverted SEM column, allowing the liquid-immersed sample to be observed by OM from above and by SEM from below. The optical axes of the two microscopes are aligned, ensuring that the same sample areas are imaged to realize quasi-simultaneous correlative microscopy in solution. For example, the cathodoluminescence of ZnO particles was directly demonstrated. The improved system has (i) a fully motorized sample stage, (ii) a column protection system in the case of accidental window breakage, and (iii) an OM/SEM operation system controlled by a graphical user interface. The open sample chamber allows the external administration of reagents during sample observation. We monitored the influence of added NaCl on the random motion of silica particles in liquid. Further, using fluorescence as a transfection marker, the effect of small interfering RNA-mediated knockdown of endogenous Varp on Tyrp1 trafficking in melanocytes was examined. A temperature-regulated titanium ASEM dish allowed the dynamic observation of colloidal silver nanoparticles as they were heated to 240°C and sintered.

Immuno-Electron Microscopy of Primary Cell Cultures from Genetically Modified Animals in Liquid by Atmospheric Scanning Electron Microscopy

High-throughput immuno-electron microscopy is required to capture the protein-protein interactions realizing physiological functions. Atmospheric scanning electron microscopy (ASEM) allows in situ correlative light and electron microscopy of samples in liquid in an open atmospheric environment. Cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required. Here, cells were imaged by optical or fluorescence microscopy, and at high resolution by gold-labeled immuno-ASEM, sometimes with additional metal staining. Axonal partitioning of neurons was correlated with specific cytoskeletal structures, including microtubules, using primary-culture neurons from wild type Drosophila, and the involvement of ankyrin in the formation of the intra-axonal segmentation boundary was studied using neurons from an ankyrin-deficient mutant. Rubella virus replication producing anti-double-stranded RNA was captured at the host cell's plasma membrane. Fas receptosome formation was associated with clathrin internalization near the surface of primitive endoderm cells. Positively charged Nanogold clearly revealed the cell outlines of primitive endoderm cells, and the cell division of lactic acid bacteria. Based on these experiments, ASEM promises to allow the study of protein interactions in various complexes in a natural environment of aqueous liquid in the near future.

Copper oxide nanorods assembly and their whisker transformation at liquid–liquid interface and on solid surfaces

We report on real time fluctuations and chain-like assemblies of copper oxide (CuO) nanorods at the liquid–liquid interface. The rods were found to transform into whiskers and 3D structures upon spin-casting on silicon substrates. The results of atmospheric scanning electron microscope (ASEM) and atomic force microscopy (AFM) provide compelling evidence that both the transformation and the assembly are driven by interaction between the π electrons of toluene and CuO nanorods, and to a smaller extent by breath figure dynamics rather than the solution-liquid–solid mechanism that was previously believed to account for whisker formation. These results open up a way for studying and understanding the dynamics of nanoparticles at liquid–liquid interfaces.

Direct Observation of Swelling and Delamination Behaviors on Layered Double Hydroxides in Solution by Atmospheric Scanning Electron Microscope (ASEM)

Direct Observation of Swelling and Delamination Behaviors on Layered Double Hydroxides in Solution by Atmospheric Scanning Electron Microscope (ASEM)

Atmospheric Scanning Electron Microscope System with an Open Sample Chamber

Atmospheric Scanning Electron Microscope System with an Open Sample Chamber

Nanometer-scale Real-space Observation and Material Processing for Polymer Materials under Atmospheric Pressure: Application of Atmospheric Scanning Electron Microscopy

Nanometer-scale Real-space Observation and Material Processing for Polymer Materials under Atmospheric Pressure: Application of Atmospheric Scanning Electron Microscopy

Positively charged nanogold label allows the observation of fine cell filopodia and flagella in solution by atmospheric scanning electron microscopy

Optical microscopy is generally the first choice to observe microbes and cells. However, its resolution is not always sufficient to reveal specific target structures, such as flagella and pili, which are only nanometers wide. ASEM is an attractive higher resolution alternative, as the sample is observed in aqueous solution at atmospheric pressure. Sample pretreatment for ASEM only comprises simple tasks including fixation, gold labeling, and reagent exchange, taking less than 1 h in total. The lengthy sample pretreatments often required for more classical electron microscopies, such as embedding and dehydration, are unnecessary, and native morphology is preserved. In this study, positively charged nanogold particles were used to label the surfaces of bacteria and cultured animal cells, exploiting their net negative surface charge. After gold enhancement to increase the size of the nanogold particles, ASEM imaging of the bacteria in aqueous solution revealed pili and delicate spiral flagella. This natural shape contrasts starkly with images of dried flagella recorded by standard SEM. Positively charged nanogold labeled the plasma membrane of cultured COS7 cells, and after enhancement allowed filopodia as thin as 100 nm in diameter to be clearly visualized. Based on these studies, ASEM combined with positively charged nanogold labeling promises to become an important tool for the study of cell morphology and dynamics in the near future.

Ultrastructural analysis of nanogold-labeled cell surface microvilli in liquid by atmospheric scanning electron microscopy and their relevance in cell adhesion.

The adhesion of leukocytes circulating in the blood to vascular endothelium is critical for their trafficking in the vasculature, and CD44 is an important cell surface receptor for rolling adhesion. In this study, we demonstrate the correlative observation of CD44 distribution at the lymphocyte cell surface in liquid by fluorescence optical microscopy and immuno-electron microscopy using an atmospheric scanning electron microscope (ASEM). The ultrastructure of the cell surface was clearly imaged by ASEM using positively charged Nanogold particles. ASEM analysis demonstrated microvilli projections around the cell surface and the localization of CD44 on the microvilli. Treatment of cells with cytochalasin D resulted in a loss of the microvilli projections and concomitantly abrogated CD44-mediated adhesion to its ligand hyaluronan. These results suggest the functional relevance of microvilli in CD44-mediated rolling adhesion under shear flow.

The Atmospheric Scanning Electron Microscope (ASEM) Observes the Critical Moment of Platelet Generation from Megakaryocytes in Solution

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

In situ electron microscopy studies of calcium carbonate precipitation from aqueous solution with and without organic additives

For the understanding of mineral formation processes from solution it is important to obtain a deeper insight into the dynamics of crystal growth. In this study we applied for this purpose a novel atmospheric scanning electron microscope that allows the investigation of CaCO3 particle formation in solution under atmospheric conditions with a resolution of approximately 10nm. Furthermore it permits the in situ observation of the dynamics of crystal evolution. With this tool the precipitation of CaCO3 was studied in the absence and presence of additives, namely poly(acrylic acid) and poly(styrene sulfonate-co-maleic acid) which are known to influence the crystal growth rate and morphology. We determined particle growth rates and investigated the formation and dissolution dynamics of an observed transient phase, believed to be amorphous calcium carbonate. This technique also enabled us to study the depletion zones, areas of lower intensity due to reduced ion concentrations. Ion flux rates were obtained from the depletion zone width, which amounted to several μm assuming the formation and dissolution dynamics of amorphous calcium carbonate being the rate determining process. This assumption was confirmed since the obtained fluxes were found to be in good agreement with fluxes derived from the experimentally observed crystal growth rates.

Detection of CD133 (prominin‐1) in a human hepatoblastoma cell line (HuH‐6 clone 5)

We examined CD133 distribution in a human hepatoblastoma cell line (HuH-6 clone 5). We directly observed the cultured cells on a pressure-resistant thin film (silicon nitride thin film) in a buffer solution by using the newly developed atmospheric scanning electron microscope (ASEM), which features an open sample dish with a silicon nitride thin film window at its base, through which the scanning electron microscope beam scans samples in solution, from below. The ASEM enabled observation of the ventral cell surface, which could not be observed using standard SEM. However, observation of the dorsal cell surface was difficult with the ASEM. Therefore, we developed a new method to observe the dorsal side of cells by using Aclar® plastic film. In this method, cells are cultured on Aclar plastic film and the dorsal side of cells is in contact with the thin silicon nitride film of the ASEM dish. A preliminary study using the ASEM showed that CD133 was mainly localized in membrane ruffles in the peripheral regions of the cell. Standard transmission electron microscopy and scanning electron microscopy revealed that CD133 was preferentially concentrated in a complex structure comprising filopodia and the leading edge of lamellipodia. We also observed co-localization of CD133 with F-actin. An antibody against CD133 decreased cell migration. Methyl-β-cyclodextrin treatment decreased cell adhesion as well as lamellipodium and filopodium formation. A decrease in the cholesterol level may perturb CD133 membrane localization. The results suggest that CD133 membrane localization plays a role in tumor cell adhesion and migration.

Visualization and characterization of engineered nanoparticles in complex environmental and food matrices using atmospheric scanning electron microscopy

Imaging and characterization of engineered nanoparticles (ENPs) in water, soils, sediment and food matrices is very important for research into the risks of ENPs to consumers and the environment. However, these analyses pose a significant challenge as most existing techniques require some form of sample manipulation prior to imaging and characterization, which can result in changes in the ENPs in a sample and in the introduction of analytical artefacts. This study therefore explored the application of a newly designed instrument, the atmospheric scanning electron microscope (ASEM), which allows the direct characterization of ENPs in liquid matrices and which therefore overcomes some of the limitations associated with existing imaging methods. ASEM was used to characterize the size distribution of a range of ENPs in a selection of environmental and food matrices, including supernatant of natural sediment, test medium used in ecotoxicology studies, bovine serum albumin and tomato soup under atmospheric conditions. The obtained imaging results were compared to results obtained using conventional imaging by transmission electron microscope (TEM) and SEM as well as to size distribution data derived from nanoparticle tracking analysis (NTA). ASEM analysis was found to be a complementary technique to existing methods that is able to visualize ENPs in complex liquid matrices and to provide ENP size information without extensive sample preparation. ASEM images can detect ENPs in liquids down to 30 nm and to a level of 1 mg L(-1) (9×10(8) particles mL(-1) , 50 nm Au ENPs). The results indicate ASEM is a highly complementary method to existing approaches for analyzing ENPs in complex media and that its use will allow those studying to study ENP behavior in situ, something that is currently extremely challenging to do.

TiO<sub>2</sub> Nanoparticles Induced Genotoxicity in Cultured Cells Using Atmospheric Scanning Electron Microscopy (ASEM)

Nano-sized titanium oxide nanoparticles (TiO2 NPs) are widely used as a dye in food and cosmetics. TiO2 NPs are known to induce DNA damage when incorporated into cells. However, no bioassay is currently available to easily determine the cell incorporation of TiO2 NPs or related DNA damage, and to date, few studies have examined the different degrees of incorporation into cells according to the size of the TiO2 NPs particles and the presence or absence of cell specificity regarding DNA damage. This present study was therefore designed to examine COS7 cells that had incorporated TiO2 NPs using atmospheric scanning electron microscopy (ASEM). The results indicated that absorption of TiO2 NPs into cells and nuclear abnormalities had occurred. ASEM is a rapid and simple technique that enables the observation of samples immediately after fixation with glutaraldehyde and staining with phosphotungstic acid, and this method was suggested to be useful in screening for DNA damage.

Immuno EM–OM correlative microscopy in solution by atmospheric scanning electron microscopy (ASEM)

In the atmospheric scanning electron microscope (ASEM), an inverted SEM observes the wet sample from beneath an open dish while an optical microscope (OM) observes it from above. The disposable dish with a silicon nitride (SiN) film window can hold a few milliliters of culture medium, and allows various types of cells to be cultured in a stable environment. The use of this system for in situ correlative OM/SEM immuno-microscopy is explored, the efficiency of the required dual-tagged labeling assessed and the imaging capabilities of the ASEM documented. We have visualized the cytoskeletons formed by actin and tubulin, the chaperone PDI that catalyses native disulfide bond formation of proteins in the endoplasmic reticulum (ER) and the calcium sensor STIM1 that is integrated in ER membranes, using established cell lines. In particular, a dynamic string-like gathering of STIM1 was observed on the ER in Jurkat T cells in response to Ca(2+) store depletion. We have also visualized filamentous actin (F-actin) and tubulin in the growth cones of primary-culture neurons as well as in synapses. Further, radially running actin fibers were shown to partly colocalize with concentric bands of the Ca(2+) signaling component Homer1c in the lamellipodia of neuron primary culture growth cones. After synapse formation, neurite configurations were drastically rearranged; a button structure with a fine F-actin frame faces a spine with a different F-actin framework. Based on this work, ASEM correlative microscopy promises to allow the dynamics of various protein complexes to be investigated in the near future.

Direct Observation of Protein Microcrystals in Crystallization Buffer by Atmospheric Scanning Electron Microscopy

X-ray crystallography requires high quality crystals above a given size. This requirement not only limits the proteins to be analyzed, but also reduces the speed of the structure determination. Indeed, the tertiary structures of many physiologically important proteins remain elusive because of the so-called “crystallization bottleneck”. Once microcrystals have been obtained, crystallization conditions can be optimized to produce bigger and better crystals. However, the identification of microcrystals can be difficult due to the resolution limit of optical microscopy. Electron microscopy has sometimes been utilized instead, with the disadvantage that the microcrystals usually must be observed in vacuum, which precludes the usage for crystal screening. The atmospheric scanning electron microscope (ASEM) allows samples to be observed in solution. Here, we report the use of this instrument in combination with a special thin-membrane dish with a crystallization well. It was possible to observe protein crystals of lysozyme, lipase B and a histone chaperone TAF-Iβ in crystallization buffers, without the use of staining procedures. The smallest crystals observed with ASEM were a few μm in width, and ASEM can be used with non-transparent solutions. Furthermore, the growth of salt crystals could be monitored in the ASEM, and the difference in contrast between salt and protein crystals made it easy to distinguish between these two types of microcrystals. These results indicate that the ASEM could be an important new tool for the screening of protein microcrystals.

Rapid imaging of mycoplasma in solution using Atmospheric Scanning Electron Microscopy (ASEM)

Mycoplasma is a genus of bacterial pathogen that causes disease in vertebrates. In humans, the species Mycoplasma pneumoniae causes 15% or more of community-acquired pneumonia. Because this bacterium is tiny, corresponding in size to a large virus, diagnosis using optical microscopy is not easy. In current methods, chest X-rays are usually the first action, followed by serology, PCR amplification, and/or culture, but all of these are particularly difficult at an early stage of the disease. Using Mycoplasma mobile as a model species, we directly observed mycoplasma in buffer with the newly developed Atmospheric Scanning Electron Microscope (ASEM). This microscope features an open sample dish with a pressure-resistant thin film window in its base, through which the SEM beam scans samples in solution, from below. Because of its 2–3μm-deep scanning capability, it can observe the whole internal structure of mycoplasma cells stained with metal solutions. Characteristic protein localizations were visualized using immuno-labeling. Cells were observed at low concentrations, because suspended cells concentrate in the observable zone by attaching to sialic acid on the silicon nitride (SiN) film surface within minutes. These results suggest the applicability of the ASEM for the study of mycoplasmas as well as for early-stage mycoplasma infection diagnosis.