Interfacing cell-based assays in environmental scanning electron microscopy using dielectrophoresis

Development of the dielectrophoretic (DEP) live cell trapping technology and its interfacing with the environmental scanning electron microscopy (ESEM) is described. DEP microelectrode arrays were fabricated on glass substrate using photolithography and lift-off. Chip-based arrays were applied for ESEM analysis of DEP-trapped human leukemic cells. This work provides proof-of-concept interfacing of the DEP cell retention and trapping technology with ESEM to provide a high-resolution analysis of individual nonadherent cells.     

The use of environmental scanning electron microscopy for imaging wet and insulating materials

The environmental scanning electron microscope (ESEM) is a direct descendant of the conventional SEM, but also permits wet and insulating samples to be imaged without prior specimen preparation. A low pressure (up to around 10 torr) of a gas can be accommodated around the sample. When this gas is water, hydrated samples can be maintained in their native state. Whether the gas is water or some other gas, ions formed on collisions between electrons emitted from the sample and the gaseous molecules drift back towards the sample surface helping to reduce charge build up. This eliminates the need for insulators to be subjected to a conductive surface coating. These two key advantages of ESEM open up a wide range of materials to the power of scanning electron microscopy.     

环境扫描电子显微镜的成像特点

环境扫描电子显微镜(ESEM)中所指的环境并非真正意义上的大气环境(760 Torr),与传统扫描电子显微镜(SEM)样品室高真空度相比,ESEM样品室的真空度可以很低(约达20 Torr)。ESEM是在传统的SEM样品室中多一个GSED探头,因此,它在传统的SEM基础上增添了新的功能。其主要的特点是,可以观察含适量水分的样品和非导体材料样品,比如植物的叶片、动物中的昆虫、作物的籽粒、含结晶水的固体材料等。     

Nanostructure fabrication by ultra-high-resolution environmental scanning electron microscopy

Electron beam induced deposition (EBID) is a maskless nanofabrication technique capable of surpassing the resolution limits of resist-based lithography. However, EBID fabrication of functional nanostructures is limited by beam spread in bulk substrates, substrate charging, and delocalized film growth around deposits. Here, we overcome these problems by using environmental scanning electron microscopy (ESEM) to perform EBID and etching while eliminating charging artifacts at the nanoscale. Nanostructure morphology is tailored by slimming of deposits by ESEM imaging in the presence of a gaseous etch precursor and by pre-etching small features into a deposit (using a stationary or a scanned electron beam) prior to a final imaging process. The utility of this process is demonstrated by slimming of nanowires deposited by EBID, by the fabrication of gaps (between 4 and 7 nm wide) in the wires, and by the removal of thin films surrounding such nanowires. ESEM imaging provides a direct view of the slimming process, yielding process resolution that is limited by ESEM image resolution ( approximately 1 nm) and surface roughening occurring during etching.     

Ion bombardment in electron microscopy investigations

Transmission electron microscopy, transmission high energy electron diffraction, reflection high energy electron diffraction and scanning electron microscopy were employed to evaluate the possibilities of ion bombardment used in the preparation of electron microscope specimens. The experimental results show that the uncontrolled use of ion bombardment for thinning of metal foils, cleaning layers or crystal surfaces etc. may often lead to incorrect interpretation of the electron microscopy investigations. In contrast, it is stressed that controlled ion bombardment directly in the column of the electron microscope is a very useful method for the observation and study of some very interesting phenomena which occur to the structure of the specimens during ion bombardment.     

In situ high-temperature electron microscopy of 3DOM cobalt,iron oxide,and nickel

High-temperature electron microscopy was used to follow how the structure of two specimens of three-dimensionally ordered macroporous (3DOM) materials, also known as inverse opals, and one specimen of a precursor to a 3DOM material changed with temperature. The change in grain size with temperature of 3DOM cobalt and 3DOM iron oxide (as magnetite) was monitored in situ in the TEM by heating in stages to 900 and 1,000 °C, respectively. The two materials studied by TEM showed contrasting grain growth behavior. For 3DOM cobalt, carbon surrounding the nanometer-size grains led to slower grain growth in thinner sample areas than in areas in closer contact with other grains; a bimodal grain-size distribution was observed after heating above 700 °C for 90 min. The grains of the 3DOM iron oxide had no carbon coating and coarsened more evenly to give a unimodal size distribution. Line scans from selected-area diffraction (SAD) patterns were used for phase analysis and showed that traces of cobalt oxide present in the 3DOM cobalt sample at room temperature disappeared when the sample was heated above 500 °C. The transformation of a 3DOM precursor material, nickel(II) oxalate–polystyrene (PS) latex composites, was followed in situ by variable-temperature environmental scanning electron microscopy (ESEM) from room temperature to ca. 700 °C in 0.5–0.7 kPa O₂. The ESEM examination of the 3DOM precursors permitted real-time observation of the polymer template decomposition and the shrinkage that occurs upon calcination of these precursor materials.     

Electron beam induced chemical dry etching and imaging in gaseous NH(3) environments

We report the use of ammonia (NH(3)) vapor as a new precursor for nanoscale electron beam induced etching (EBIE) of carbon, and an efficient imaging medium for environmental scanning electron microscopy (ESEM). Etching is demonstrated using amorphous carbonaceous nanowires grown by electron beam induced deposition (EBID). It is ascribed to carbon volatilization by hydrogen radicals generated by electron dissociation of NH(3) adsorbates. The volatilization process is also effective at preventing the buildup of residual hydrocarbon impurities that often compromise EBIE, EBID and electron imaging. We also show that ammonia is a more efficient electron imaging medium than H(2)O, which up to now has been the most commonly used ESEM imaging gas.     

Quantitative characterization of pores in transparent ceramics by coupling electron microscopy and confocal laser scanning microscopy

This work is dedicated to the implementation of a new characterization method of porosity in transparent ceramics. This quantitative method couples scanning electron microscopy with confocal laser scanning microscopy. From this original method, the volume fraction of pores has been determined for different sintered Nd:YAG specimens with an accuracy of about 10%. This technique appears to be promising because it leads to both the pore size distribution and the residual porosity for fully dense samples. For example, it becomes possible to reach porosity levels ranging from 0.09% to 0.0004% for transparent Nd:YAG specimens which could not be measured by using conventional techniques. Finally, correlations between the residual porosity of these full dense samples and their optical properties could be established.     

环境扫描电子显微镜的特性及应用概况

简要叙述了环境扫描电子显微镜的原理，着重介绍了它的特性和在国内外应用的概况。与普通的扫描电子显微镜相比，它具有能直接检测不导电样品和含油含水样品的独特性能，为扫描电子显微镜的应用开拓了新的领域。     

Recent developments in lorentz electron microscopy

Several different methods of obtaining contrast for electron microscopy of magnetic specimens are reviewed. For the investigation of magnetization ripple and the structure of domain walls in magnetic films, two questions must be considered: the correct wave optical interpretation of Lorentz micrographs and the experimental attainment of coherence of the electron beam. Both questions have been sufficiently explored so that experimental results on magnetic film microstructure may be expected in the near future.     

Analysis of the structural changes of a phosphate glass during its dissolution in simulated body fluid

The structural changes of two calcium–sodium metaphosphate glasses during its dissolution in simulated body fluid (SBF) have been analyzed by Fourier transform Raman spectroscopy (FT-Raman), scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM). The results showed that no structural changes could be detected during the first week of dissolution. However, after approximately 4 weeks of dissolution the analysis of the glass surface revealed the presence of a thin orthophosphate layer. The elemental analysis of this layer by X-ray dispersive energy showed the presence of calcium phosphate ions, while almost no sodium was detected. The observation of the glasses by ESEM showed the formation of small crystals when the water vapor pressure of the chamber was decreased. This indicates that the layer analyzed by SEM and FT-Raman was related to a hydrated calcium phosphate layer.     

Metallurgical applications of scanning transmission electron microscopy

It is shown that scanning transmission electron microscopy (STEM) has followed two main lines of development, the pure STEM based upon a field emission electron source in which the emphasis is given to high resolution, and a combined system in which STEM is an attachment to a conventional transmission microscope (TEM + STEM). When used in combination with an energy dispersive X-ray spectrometer, the combined TEM + STEM system is shown to be extremely versatile and possibly the more useful for the applied metallurgist. The high vacuum requirements of pure STEM, however, make this system suitable to be used in conjunction with an Auger spectrometer. Examples of the various microanalysis facilities of STEM are given in the article, including micro-diffraction, rocking-beam channelling patterns, qualitative and quantitative X-ray spectroscopy analysis, particle analysis and in situ experimentation. The controversial subject of whether thicker specimens can be studied in STEM compared with conventional TEM is also discussed.     

Contributions of the environmental scanning electron microscope and X-ray diffraction in investigating the structural evolution of a SiO2 aggregate attacked by alkali-silica reaction

The structural changes of a flint aggregate attacked by alkali-silica reaction (ASR) were investigated using an environmental scanning electron microscope (ESEM) equipped with a X-ray microanalysis system. Compared to the conventional SEM, the ESEM enables the direct imaging of the samples in their natural state. The results obtained are compared to those acquired using X-ray diffraction (XRD). It appears that when calcium is present in the aggregate, the attack takes place from outside and progresses towards the inside of the aggregate. It has also been shown that calcium has an influence in slowing down the reaction by preventing the fast degradation of the aggregate. In addition, it supports the formation of new internal phases in the aggregate. The ESEM and XRD data were found to be consistent with each other.     

Processing and characterization of chitosan microspheres to be used as templates for layer-by-layer assembly

Chitosan (Ch) microspheres have been developed by precipitation method, cross-linked with glutaraldehyde and used as a template for layer-by-layer (LBL) deposition of two natural polyelectrolytes. Using a LBL methodology, Ch microspheres were alternately coated with hyaluronic acid (HA) and Ch under mild conditions. The roughness of the Ch-based crosslinked microspheres was characterized by atomic force microscopy (AFM). Morphological characterization was performed by environmental scanning electron microscopy (ESEM), scanning electron microscopy (SEM) and stereolight microscopy. The swelling behaviour of the microspheres demonstrated that the ones with more bilayers presented the highest water uptake and the uncoated cross-linked Ch microspheres showed the lowest uptake capability. Microspheres presented spherical shape with sizes ranging from 510 to 840 μm. ESEM demonstrated that a rougher surface with voids is formed in multilayered microspheres caused by the irregular stacking of the layers. A short term mechanical stability assay was also performed, showing that the LBL procedure with more than five bilayers of HA/Ch over Ch cross-linked microspheres provide higher mechanical stability.     

Morphology of a deformed rubber toughened poly(methyl methacrylate) film under tensile strain

The morphology of rubber toughened poly(methyl methacrylate) (PMMA) films was studied during deformation using environmental scanning electron microscopy (ESEM) combined with real time small angle X-ray scattering (SAXS). These two methods provide two different approaches to an in situ study of the morphology of the deforming polymer film. The ESEM study shows that the polymer film exhibits multiple craze-like streaks under tensile strain. Every streak runs through several apparently cavitated rubber particles. Further study by SAXS reveals that these streaks are not crazes. Instead they appear to be lines of cavitated rubber particles which form a particular type of dilatation band known as a “croid”. The ESEM study also shows that with increasing strain the rubber particles gradually increase in size, and that both the number and size of the croids increase. Finally the croids evolve into cracks just before the breakdown of the sample. This revised version was published online in November 2006 with corrections to the Cover Date.     

A new scanning electron microscopy approach to image aerogels at the nanoscale

A new scanning electron microscopy (SEM) technique to image poor electrically conductive aerogels is presented. The process can be performed by non-expert SEM users. We showed that negative charging effects on aerogels can be minimized significantly by inserting dry nitrogen gas close to the region of interest. The process involves the local recombination of accumulated negative charges with positive ions generated from ionization processes. This new technique made possible the acquisition of images of aerogels with pores down to approximately 3?nm in diameter using a positively biased Everhart-Thornley (ET) detector.     

氧环境扫描电子显微分析:消除绝缘材料荷电效应的新方法

在环境扫描电镜(ESEM)中注入氧气,减少和消除绝缘样品表面在电子束辐照下产生的荷电效应.二次电子像的观察显示,在压力为130Pa～600Pa的ESEM中,氧气对Al2O3、Al(OH)3等氧化物、氢氧化物及生物样品的荷电补偿效果,优于常用的水蒸汽环境.通过吸收电流Ia的实时测试,评价了氧环境的荷电补偿效果.采用氧气减少表面荷电基于一个新的概念:在电子束的辐照下,电子受激解吸可造成表面氧亏损,使能带产生畸变,形成捕获电子的势阱.氧环境提供的氧离子可实现对氧空位的修复,从而消除了荷电效应.     

In situ mechanical testing of dry and hydrated breadcrumb in the environmental scanning electron microscope (ESEM)

The environmental scanning electron microscope (ESEM) has the ability to image both dry and hydrated materials, without the need for a conductive coating, unlike conventional SEM. This presents a unique opportunity to explore the structure and dynamic mechanical characteristics of food systems, including those in a moist state. We have developed a technique in which quantitative stress-strain relationships can be obtained whilst allowing simultaneous imaging, by ESEM, of the mechanical response of a sample. The results of in situ compression tests on dry and hydrated wheat flour breadcrumbs are presented and discussed. It was found that a maximum in the critical fracture stress occurred at intermediate moisture content (16%). ESEM micrographs demonstrate the differences in mechanical behaviour at three different moisture contents (nominally dry, 16% and 30%). Our findings suggest that 'voids' in cell walls, along with discontinuities between starch granules and gluten in the crumb, play an important role in fracture initiation. Further evidence shows that voids may be bridged by 'struts' and 'strings' of matrix material, which may be factors in the control of fracture propagation.     

Intact collagen and atelocollagen sponges: Characterization and ESEM observation

In this study we have investigated the chemical–physical and morphological properties of intact and atelocollagen sponges used for tissue engineering. The porous sponges were prepared by lyophilization and their physico-chemical characteristics (water binding capacity, denaturing temperature, amino group content) were investigated. Considering the importance of the “in vivo” interactions between these sponges and the tissue, our attention was addressed (a) to clarify the relationships between the morphology and the amount of water absorbed and (b) to evaluate the influence of pepsin-alkaline treatment on the reorganization of the atelocollagen fibres. Conventional scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) were employed to study the morphology and wetting behaviour of the intact and atelocollagen sponges. The observations by SEM indicated remarkable differences both in the structure and dimension of the pores between intact and atelocollagen sponges. At the data are related to a different water binding capacity. However, the ESEM observations, achieved by changing the relative humidity in the operative chamber, demonstrated that the water adsorbed can be removed with major difficulty from atelocollagen sponges than from intact ones.