Advantages of stereo imaging of metallic surfaces with low voltage backscattered electrons in a field emission scanning electron microscope

High emission current backscattered electron (HC-BSE) stereo imaging at low accelerating voltages (≤ 5 keV) using a field emission scanning electron microscope was used to display surface structure detail. Samples of titanium with high degrees of surface roughness, for potential medical implant applications, were imaged using the HC-BSE technique at two stage tilts of + 3° and − 3° out of the initial position. A digital stereo image was produced and qualitative height, depth and orientation information on the surface structures was observed. HC-BSE and secondary electron (SE) images were collected over a range of accelerating voltages. The low voltage SE and HC-BSE stereo images exhibited enhanced surface detail and contrast in comparison to high voltage (> 10 keV) BSE or SE stereo images. The low voltage HC-BSE stereo images displayed similar surface detail to the low voltage SE images, although they showed more contrast and directional sensitivity on surface structures. At or below 5 keV, only structures a very short distance into the metallic surface were observed. At higher accelerating voltages a greater appearance of depth could be seen but there was less information on the fine surface detail and its angular orientation. The combined technique of HC-BSE imaging and stereo imaging should be useful for detailed studies on material surfaces and for biological samples with greater contrast and directional sensitivity than can be obtained with current SE or BSE detection modes.     

Diffraction effects and inelastic electron transport in angle‐resolved microscopic imaging applications

We analyse the signal formation process for scanning electron microscopic imaging applications on crystalline specimens. In accordance with previous investigations, we find nontrivial effects of incident beam diffraction on the backscattered electron distribution in energy and momentum. Specifically, incident beam diffraction causes angular changes of the backscattered electron distribution which we identify as the dominant mechanism underlying pseudocolour orientation imaging using multiple, angle‐resolving detectors. Consequently, diffraction effects of the incident beam and their impact on the subsequent coherent and incoherent electron transport need to be taken into account for an in‐depth theoretical modelling of the energy‐ and momentum distribution of electrons backscattered from crystalline sample regions. Our findings have implications for the level of theoretical detail that can be necessary for the interpretation of complex imaging modalities such as electron channelling contrast imaging (ECCI) of defects in crystals. If the solid angle of detection is limited to specific regions of the backscattered electron momentum distribution, the image contrast that is observed in ECCI and similar applications can be strongly affected by incident beam diffraction and topographic effects from the sample surface. As an application, we demonstrate characteristic changes in the resulting images if different properties of the backscattered electron distribution are used for the analysis of a GaN thin film sample containing dislocations.     

The effect of subsurface topography on secondary electron images from chromate pretreated aluminium surfaces

In secondary and scanning transmission electron microscopes, secondary electron images of surface films can be dominated by an image derived from electrons back-scattered from the interface between the film and the substrate. The extent of the domination has been established by studying the variation in image obtained using primary beams of different energy and by platinum coating to enhance surface secondary electron emission. Studies of thicker films also established that chemical or structural difference within a film also lead to imaging effects. In general, 5 keV electrons are the most effective in producing subsurface and structural or chemical imaging effects.     

Utilization of integrated correlative light and electron microscopy (iCLEM) for imaging sedimentary organic matter

We report here a new microscopic technique for imaging and identifying sedimentary organic matter in geologic materials that combines inverted fluorescence microscopy with scanning electron microscopy and allows for sequential imaging of the same region of interest without transferring the sample between instruments. This integrated correlative light and electron microscopy technique is demonstrated with observations from an immature lacustrine oil shale from the Eocene Green River Mahogany Zone and mid‐oil window paralic shale from the Upper Cretaceous Tuscaloosa Group. This technique has the potential to allow for identification and characterization of organic matter in shale hydrocarbon reservoirs that is not possible using either light or electron microscopy alone, and may be applied to understanding the organic matter type and thermal regime in which organic nanoporosity forms, thereby reducing uncertainty in the estimation of undiscovered hydrocarbon resources.     

Possibility of charge contrast imaging of polymeric materials

Preliminary results illustrate the possibility of charge contrast imaging (CCI) of polymeric materials. Possible CCI images of low-density polyethylene and polyvinyl chloride reveal details that may aid in the characterization of the microstructure of polymeric materials. These pictures were obtained with a Hitachi S-3000N variable pressure scanning electron microscope with the environmental secondary electron detector (ESED).     

Charge Contrast Imaging of Nonconductive Samples in the High‐Vacuum Field Emission Scanning Electron Microscope

The charge contrast images (CCI) on insulating or poorly conducting samples were observed under steady‐state charging conditions with a thermal field emission scanning electron microscope under high vacuum by using an Everhart‐Thornley detector. The charge contrast on plumbous titanate‐nickel composite particles and patterned sapphires could be the indicators of near‐surface features, compositional variations and conductivity distributions. Optimum imaging conditions for observing the CCI include the electron energy, the electron flux density and the electron dose. Contrast characteristics associated with surface and near‐surface secondary electron emission yield enhanced above the trapped charge‐up regions, as charge trapping selectively enhanced the poorly conductive phase and lattice distorted area. SCANNING 29: 230‐237, 2007. © 2007 Wiley Periodicals, Inc.     

An automated image alignment system for the scanning electron microscope

We have developed an automated image alignment system for the scanning electron microscope (SEM). This system enables specific locations on a sample to be located and automatically aligned with submicron accuracy. The system comprises a sample stage motorization and control unit together with dedicated imaging electronics and image processing software. The standard SEM sample stage is motorized in the X and Y axes with stepping motors which are fitted with rotary optical encoders. The imaging electronics are interfaced to beam deflection electronics of the SEM and provide the image data for the image processing software. The system initially moves the motorized sample stage to the area of interest and acquires an image. This image is compared with a reference image to determine the required adjustments to the stage position or beam deflection. This procedure is repeated until the area imaged by the SEM matches the reference image. A hierarchical image correlation technique is used to achieve submicron alignment accuracy in a few seconds. The ability to control the SEM beam deflection enables the images to be aligned with an accuracy far exceeding the positioning ability of the SEM stage. The alignment system may be used on a variety of samples without the need for registration or alignment marks since the features in the SEM image are used for alignment. This system has been used for the automatic inspection of devices on semiconductor wafers, and has also enabled the SEM to be used for direct write self-aligned electron beam lithography.     

Nanotip electron gun for the scanning electron microscope

Experimental nanotips have shown significant improvement in the resolution performance of a cold field emission scanning electron microscope (SEM). Nanotip electron sources are very sharp electron emitter tips used as a replacement for the conventional tungsten field emission (FE) electron sources. Nanotips offer higher brightness and smaller electron source size. An electron microscope equipped with a nanotip electron gun can provide images with higher spatial resolution and with better signal-to-noise ratio. This could present a considerable advantage over the current SEM electron gun technology if the tips are sufficiently long-lasting and stable for practical use. In this study, an older field-emission critical dimension (CD) SEM was used as an experimental test platform. Substitution of tungsten nanotips for the regular cathodes required modification of the electron gun circuitry and preparation of nanotips that properly fit the electron gun assembly. In addition, this work contains the results of the modeling and theoretical calculation of the electron gun performance for regular and nanotips, the preparation of the SEM including the design and assembly of a measuring system for essential instrument parameters, design and modification of the electron gun control electronics, development of a procedure for tip exchange, and tests of regular emitter, sharp emitter and nanotips. Nanotip fabrication and characterization procedures were also developed. Using a "sharp" tip as an intermediate to the nanotip clearly demonstrated an improvement in the performance of the test SEM. This and the results of the theoretical assessment gave support for the installation of the nanotips as the next step and pointed to potentially even better performance. Images taken with experimental nanotips showed a minimum two-fold improvement in resolution performance than the specification of the test SEM. The stability of the nanotip electron gun was excellent; the tip stayed useful for high-resolution imaging for several hours during many days of tests. The tip lifetime was found to be several months in light use. This paper summarizes the current state of the work and points to future possibilities that will open when electron guns can be designed to take full advantage of the nanotip electron emitters.     

Application of the scatter diagram technique to the scanning electron microscope: preliminary results from diamond

Using primary beam energies E0 ranging from 0.2 to 15 keV and an in-lens detector, a series of images of the same region of an artificial microstructured diamond sample have been acquired in scanning electron microscopy. Next, the images were analysed by using a scatter diagram technique to underline the topographic contrast change and contrast reversal. The results obtained from 0.5 to 15 keV are discussed with the help of an expression derived from the constant loss model for the secondary electron yield delta of diamond, but including the respective roles of the angle of incidence, i, and of the angle of detection, alpha. More surprising is the quality of images obtained at a beam energy as low as 0.2 keV, and more difficult to explain is the significant contrast change between 0.2 keV and 0.5 keV. For the first time, scatter diagrams are used as a diagnostic tool in scanning electron microscopy, and after some improvements it is hoped that the experimental approach followed here may lead to quantitative estimates of the local tilts of a specimen surface.     

A method for imaging single clay platelets by scanning electron microscopy

A method for preparing and observing clay platelets for size and shape analysis using scanning electron microscopy (SEM) was developed. Samples of the clay platelets were prepared by polyelectrolyte-assisted adsorption onto a pyrolytic graphite surface. The use of graphite as a substrate was advantageous because of the low number of secondary electrons emitted from it during imaging by SEM. The resulting low background noise allowed the emission from the approximately 1 nm thick clay sheets to be clearly visualized. Images of centrifuged montmorillonite showed large exfoliated platelets with lateral dimensions between 200 and 600 nm. In contrast, uncentrifuged montmorillonite appeared to contain a large amount of unexfoliated clusters. Although it was not possible to obtain high-quality images of the smaller sheets of Laponite RD, the images of this material did contain size features comparable to the approximately 30 nm2 size reported previously using light scattering, as well as transmission electron and atomic force microscopies.     

Quantitative detection of gold nanoparticles on individual, unstained cancer cells by scanning electron microscopy

Gold nanoparticles are rapidly emerging for use in biomedical applications. Characterization of the interaction and delivery of nanoparticles to cells through microscopy is important. Scanning electron microscopes have the intrinsic resolution to visualize gold nanoparticles on cells. A novel sample preparation protocol was developed to enable imaging of cells and gold nanoparticles with a conventional below lens scanning electron microscopes. The negative influence of 'charging' on the quality of scanning electron microscopes' images could be limited by deposition of biological cells on a conductive (gold) surface. The novel protocol enabled high-resolution scanning electron microscopes' imaging of small clusters and individual gold nanoparticles on uncoated cell surfaces. Gold nanoparticles could be counted on cancer cells with automated routines.     

Atomic imaging using secondary electrons in a scanning transmission electron microscope: experimental observations and possible mechanisms

We report detailed investigation of high-resolution imaging using secondary electrons (SE) with a sub-nanometer probe in an aberration-corrected transmission electron microscope, Hitachi HD2700C. This instrument also allows us to acquire the corresponding annular dark-field (ADF) images both simultaneously and separately. We demonstrate that atomic SE imaging is achievable for a wide range of elements, from uranium to carbon. Using the ADF images as a reference, we studied the SE image intensity and contrast as functions of applied bias, atomic number, crystal tilt, and thickness to shed light on the origin of the unexpected ultrahigh resolution in SE imaging. We have also demonstrated that the SE signal is sensitive to the terminating species at a crystal surface. A possible mechanism for atomic-scale SE imaging is proposed. The ability to image both the surface and bulk of a sample at atomic-scale is unprecedented, and can have important applications in the field of electron microscopy and materials characterization.     

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 (9×10⁸ 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.     

Choosing the optimum accelerating voltage (EO) to visualize submicron precipitates with a field emission scanning electron microscope

With the advent of field emission scanning electron microscopes (FESEM), the observation of small phases in the 5 to 50 nm range seems to be possible at low accelerating voltage using backscattered electron imaging mode. In this context, it is important to understand the contrast of multiphased materials at such low energy. A Monte Carlo program to simulate electron trajectories of multiphased materials (CASINO) was used to compute electron backscattering images. Simulations of images for various compositions of spherical precipitates embedded in a homogeneous matrix as a function of precipitate size and accelerating voltage are presented. These simulations show the concept of an optimum accelerating voltage to maximize the contrast of electron backscattering images. The results presented in this paper show that the contrast of backscattering images of multiphased images in the scanning electron microscope is not only a function of the atomic number difference, but that it is also strongly related to the geometry and the size of the phases.     

Backscattered electron imaging of the undersurface of resin-embedded cells by field-emission scanning electron microscopy

In this study backscattered electron (BSE) imaging was used to display cellular structures stained with heavy metals within an unstained resin by atomic number contrast in successively deeper layers. Balb/c 3T3 fibroblasts were cultured on either 13-mm discs of plastic Thermanox, commercially pure titanium or steel. The cells were fixed, stained and embedded in resin and the disc removed. The resin block containing the cells was sputter coated and examined in a field-emission scanning electron microscope. The technique allowed for the direct visualization of the cell undersurface and immediately overlying areas of cytoplasm through the surrounding embedding resin, with good resolution and contrast to a significant depth of about 2 μm, without the requirement for cutting sections. The fixation protocol was optimized in order to increase heavy metal staining for maximal backscattered electron production. The operation of the microscope was optimized to maximize the number of backscattered electrons produced and to minimize the spot size. BSE images were collected over a wide range of accelerating voltages (keV), from low values to high values to give ‘sections' of information from increasing depths within the sample. At 3–4 keV only structures a very short distance into the material were observed, essentially the areas of cell attachment to the removed substrate. At higher accelerating voltages information on cell morphology, including in particular stress fibres and cell nuclei, where heavy metals were intensely bound became more evident. The technique allowed stepwise ‘sectional’ information to be acquired. The technique should be useful for studies on cell morphology, cycle and adhesion with greater resolution than can be obtained with any light-microscope-based system.     

The fine structure of fenestrated adrenocortical capillaries revealed by in-lens field-emission scanning electron microscopy and scanning transmission electron microscopy

Cell biologists probing the physiologic movement of macromolecules and solutes across the fenestrated microvascular endothelial cell have used electron microscopy to locate the postulated pore within the fenestrae. Prior to the advent of in-lens field-emission high-resolution scanning electron microscopy (HRSEM) and ultrathin m et al coating technology, quick-freeze, platinum-carbon replica and grazing thin-section transmission electron microscopy (TEM) methods provided two-dimensional or indirect imaging methods. Wedge-shaped octagonal channels composed of fibrils interwoven in a central mesh were depicted as the filtering structures of fenestral diaphragms in images of platinum replicas enhanced by photographic augmentation. However, image accuracy was limited to replication of the cell surface. Subsequent to this, HRSEM technology was developed and provided a high-fidelity, three-dimensional topographic image of the fenestral surface directly from a fixed and dried bulk adrenal specimen coated with a 1 nm chromium film. First described from TEM replicas, the “flower-like” structure comprising the fenestral pores was readily visualized by HRSEM. High-resolution images contained particulate ectodomains on the lumenal surface of the endothelial cell membrane. Particles arranged in a rough octagonal shape formed the fenestral rim. Digital acquisition of analog photographic recordings revealed a filamentous meshwork in the diaphragm, thus confirming and extending observations from replica and grazing section TEM preparations. Endothelial cell pockets, first described in murine renal peritubular capillaries, were observed in rhesus and rabbit adrenocortical capillaries. This report features recent observations of fenestral diaphragms and endothelial pockets fitted with multiple diaphragms utilizing a Schottky field-emission electron microscope. In-lens staging of bulk and thin section specimens allowed tandem imaging in HRSEM and scanning TEM modes at 25 kV.     

Imaging of thermal and elastic surface properties by scanning electron acoustic microscopy

Scanning electron acoustic microscopy is a new technique for imaging the thermal and elastic properties of surfaces and detecting subsurface flaws. It can be carried out in a modified scanning electron microscope. The effects of electron beam energy and phase angle on scanning electron acoustic images of the thermal and elastic properties of surfaces were studied with an alumina fiber/aluminum matrix composite for fiber directions both transverse and coaxial to the surface. Images produced with 10- and 30-keV electrons at beam modulation frequencies of 80–1200 kHz appeared to be identical, with the exception of a lower signal-to-noise ratio for the lower electron energy. This observation suggests that the energy input from the beam can be considered to occur at the surface for electron energies below 30 keV and frequencies below 1200 kHz. Images recorded at 0° phase angle mapped regions of different thermal and elastic properties. Images recorded at 90° phase angle highlighted the boundaries between such regions. Scanning electron acoustic microscopy can image features of different thermal and elastic properties at greater depth than traditional imaging with backscattered electrons. The practical application of the technique to the study of surfaces is illustrated by the imaging of grain structure and subsurface particles for an extruder barrel.     

Signal components in the environmental scanning electron microscope

We demonstrate that the gas-amplified secondary electron signal obtained in the environmental scanning electron microscope has both desired and spurious components. In order to isolate the contributions of backscattered and secondary electrons, two sets of samples were examined. One sample consisted of a pair of materials having similar secondary emission coefficients but different backscatter coefficients, while the other sample had a pair with similar backscatter but different secondary emission coefficients. Our results show how the contribution of the two electron signals varies according to the pressure of the amplifying gas. Backscatter contributions, as well as background due to gas ionization from the primary beam, become significant at higher pressure. Furthermore, we demonstrate that the relative amplification efficiencies of various electron signals are dependent upon the chemistry of the gas.     

High-pressure scanning electron microscopy of insulating materials: A new approach

A new SEM technique for imaging uncoated non-conducting specimens at high beam voltages is described which employs a high-pressure environment and an electric field to achieve charge neutralization. During imaging, the specimen surface is kept at a stable low voltage, near earth potential, by directing a flow of positive gas ions at the specimen surface under the action of an electric bias field at a pressure of about 200 Pa. In this way charge neutrality is continuously maintained to obtain micrographs free of charging artefacts. Images are formed by specimen current detection containing both secondary electron and backscattered electron signal information. Micrographs of geological, ceramic, and semiconductor materials obtained with this method are presented. The technique is also useful for the SEM examination of histological sections of biological specimens without any further preparation. A simple theory for the charge neutralization process is described. It is based on the interaction of the primary and emissive signal components with the surrounding gas medium and the resulting neutralizing currents. Further micrographs are presented to illustrate the pressure dependence of the charge neutralization process in two glass specimens which show clearly identifiable charging artefacts in conventional microscopy.