New system for secondary electron detection in variable-pressure scanning electron microscopy

A novel secondary electron detection system combining a two‐stage detector head and a differential pumping system is presented. The detector head consisted of a scintillation Everhart‐Thornley detector and a microsphere plate, separating it from the lower vacuum in the intermediate chamber (below 0.1 mbar). The system was arranged asymmetrically, which should contribute to a lower gas leakage through the plate and a longer life span of the plate. The system offered all the advantages of the scintillator detector in a wide range of gas pressures, from high vacuum to those of the order of 10 mbar, typical of high‐pressure scanning electron microscopy.     

Imaging deep holes in structures with gaseous secondary electron detection in the environmental scanning electron microscope

The gaseous secondary electron detector (GSED) in the environmental scanning electron microscope (ESEM) permits collection of electron signals from deep inside blind holes in both conducting and insulating materials. The placement of the GSED as the final pressure-limiting aperture of the ESEM creates a situation of apparent illumination along the line of sight of the observer. In principle, any point struck by the primary beam can be imaged. Image quality depends on the depth of the hole. In brass, features at the bottom of a 1.5 mm diameter hole that was 8 mm deep were successfully imaged.     

Dynamic secondary electron contrast effects in liquid systems studied by environmental scanning electron microscopy

We report an investigation into a dynamic contrast phenomenon in water-oil emulsions imaged in the environmental scanning electron microscope. Secondary electron contrast between oil and water phases is shown to change with scan rate, even inverting in extreme cases. This effect is attributed to the fact that charge carriers in liquids have intermediate mobilities compared with those in metallic conductors and solid insulators. Thus, increasing the electron energy flux density (via slower scan rates) results in the temporary accumulation of excess charge, which in turn gives rise to increased secondary electron emission. Excess charge dissipates between frames, however, such that classical charging of the specimen is not observed. The oils used here have conductivities lower than that of water, making them more susceptible to the effect. However, the material within the primary electron interaction volume is a conductive medium. We demonstrate that charging effects are not seen in regions of the oil where the interaction volume is in contact with the more conductive continuous water phase. Secondary electron emission from these regions therefore approximates to the intrinsic yield.     

Development of environmental scanning electron microscopy electron beam profile imaging with self-assembled monolayers and secondary ion mass spectroscopy

A method for demonstrating the scattering of the primary electron beam in the presence of a gas has been developed. A self-assembled decanethiol monolayer is damaged by primary beam electrons. The damaged portion of the mono-layer is exchanged with another thiol-containing molecule by immersion in solution. The resulting film is imaged using a secondary ion mass spectrometer. Three-dimensional reconstruction of the data yields a representation of scattered electrons in the gaseous environment of the environmental scanning electron microscope.     

Imaging of semiconducting polymer blend systems using environmental scanning electron microscopy and environmental scanning transmission electron microscopy

This study has investigated the potential of environmental electron microscopy techniques for studying the structure of polymer‐based electronic devices. Polymer blend systems composed of F8BT and PFB were examined. Excellent contrast, both topographical and compositional, can be achieved using both conventional environmental scanning electron microscopy (ESEM) and a transmission detector giving an environmental scanning transmission electron microscope (ESTEM) configuration. Controllable charging effects present in the ESEM were observed, giving rise to a novel voltage contrast. This shows the potential of such contrast to provide excellent images of phase structure and charge distributions.     

Conditions for imaging emulsions in the environmental scanning electron microscope

Environmental scanning electron microscopy (ESEM) is a technique capable of imaging volatile and/or insulating samples in their natural state, without prior specimen preparation. It is thus a powerful potential tool for the study of the structure and dynamics of emulsions and other complex liquid systems, at a resolution greater than that obtainable by conventional optical microscopy. We present images of a variety of liquid systems containing micron-scale and smaller features. The morphology of these systems may be clearly discerned. The contrast observed between the liquid phases was consistent with the model proposed by Stokes et al. (1998). The limits of resolution were determined by sample motion and by beam damage effects; under optimum conditions, resolution of a few tens of nanometers was obtained. This compares favourably with conventional and confocal optical microscopy. In some samples, thin films (solid or liquid) could be observed at the liquid/gas interface. Some of these films were so thin that they did not completely obscure the underlying structure of the bulk sample.     

Quantitative secondary ion mass spectrometry imaging of self-assembled monolayer films for electron beam dose mapping in the environmental scanning electron microscope

Fluorinated alkanethiol self-assembled monolayers (SAM) films immobilized on gold substrates have been used as electron-sensitive resists to map quantitatively the spatial distribution of the primary electronbeam scattering in an environmental scanning electron microscope (ESEM). In this procedure, a series of electron dose standards are prepared by exposing a SAM film to electron bombardment in well-defined regions at different levels of electron dose. Microbeam secondary ion mass spectrometry (SIMS) using Cs⁺ bombardment is then used to image the F^- secondary ion signal from these areas. From the reduction in F^- intensity as a function of increasing electron dose, a calibration curve is generated that allows conversion of secondary ion signal to electron dose on a pixel-by-pixel basis. Using this calibration, electron dose images can be prepared that quantitatively map the electron scattering distribution in the ESEM with micrometer spatial resolution. The SIMS imaging technique may also be used to explore other aspects of electron-surface interactions in the ESEM.     

Real imaging and size values of Saccharomyces cerevisiae cells with comparable contrast tuning to two environmental scanning electron microscopy modes

The combined application of electron microscopy (EM) is frequently used for the microstructural investigation of biological specimens and plays two important roles in the quantification and in gaining an improved understanding of biological phenomena by making use of the highest resolution capability provided by EM. The possibility of imaging wet specimens in their "native" states in the environmental scanning electron microscope (ESEM) at high resolution and large depth of focus in real time is discussed in this paper. It is demonstrated here that new features can be discovered by the elimination of even the least hazardous approaches in some preparation techniques, that destroy the samples. Since the analysis conditions may influence the morphology and the extreme surface sensitivity of living biological systems, the results obtained from the same cultured cell with two different ESEM modes (Lvac mode and wet mode) were compared. This offers new opportunities compared with ESEM-wet/Lvac-mode imaging, since wet-mode imaging involves a real contrast and gives an indication of the changes in cell morphology and structure required for cell viability. In this study, wet-mode imaging was optimized using the unique ability of cell quantities for microcharacterization in situ giving very fine features of topological effects. Accordingly, the progress is reported by comparing the results of these two modes, which demonstrate interesting application details. In general, the functional comparisons have revealed that the fresh unprocessed Saccharomyces cerevisiae cells (ESEM-wet mode) were essentially unaltered with improved and minimal specimen preparation timescales, and the optimal cell viability degree was visualized and also measured quantitatively while the cell size remained unchanged with continuous images.     

Charge contrast imaging of material growth and defects in environmental scanning electron miscroscopy--linking electron emission and cathodoluminescence

An electron-based technique for the imaging of crystal defect distribution such as material growth histories in non- and poorly conductive materials has been identified in the variable pressure or environmental scanning electron microscope. Variations in lattice coherence at the meso-scale can be imaged in suitable materials. Termed charge contrast imaging (CCI), the technique provides images that correlate exactly with emitted light or cathodoluminescence in suitable materials. This correlation links cathodoluminescence and an electron emission. The specific operating conditions for observation of these images reflect a complex interaction between the electron beam, the positive ions generated by electron-gas interactions in the chamber, a biased detector, and the sample. The net result appears to be the suppression of all but very near surface electron emission from the sample, probably from of the order of a few nanometres. Consequently, CCI are also sensitive to very low levels of surface contaminants. Successful imaging of internal structures in a diverse range of materials indicate that the technique will become an important research tool.     

Quality improvement of environmental secondary electron detector signal using helium gas in variable pressure scanning electron microscopy

The quality of the image signal obtained from the environmental secondary electron detector (ESED) employed in a variable pressure (VP) SEM can be dramatically improved by using helium gas. The signal-to-noise ratio (SNR) increases gradually in the range of the pressures that can be used in our modified SEM. This method is especially useful in low-voltage VP SEM as well as in a variety of SEM operating conditions, because helium gas can more or less maintain the amount of unscattered primary electrons. In order to measure the SNR precisely, a digital scan generator system for obtaining two images with identical views is employed as a precondition.     

A method to measure the effective gas path length in the environmental or variable pressure scanning electron microscope

A simple method is described to determine the effective gas path length when incident electrons scatter in the gas above the specimen. This method is based on the measurement of a characteristic x-ray line emitted from a region close to the incident beam. From various experimental measurements performed on various microscopes, it is shown that the effective gas path length may increase with the chamber pressure and that it is also often dependent of the type of x-ray bullet.     

Environmental scanning electron microscopy observation of the ultrastructure of Demodex

In this study, numbers of Demodex of hair follicles and sebaceous glands were prepared and the ultrastructure (especially the mouthparts) of Demodex was observed firstly with environmental scanning electron microscopy (ESEM). The most suitable treatment methods and optimal environmental condition for observing the genus samples were found. The samples were washed with detergent and rinsed with distilled water, and then were taken to the specimen stage, on which there was carbon adhesive tape, using special tools. When the temperature was at 5 degrees C and chamber pressure at 5 mbar respectively, the surface of the samples could be fully imaged without covering water or dehydration. The sample surfaces were plump and clear without postmortem changes and charging artifacts. Detailed information about each part of Demodex was observed by ESEM, and clear three-dimensional images were recorded. The mouthparts of D. folliculorum were composed of a complex set of structures, which included a round oral opening, a sharp oral needle, and a special hypostome that looked like a longitudinal spindle in the central position. On the end segment of palpus, there were seven strong palpal claws located on each side of the mouthparts. D. folliculorum had special piercing mouthparts, while the mouthparts of D. brevis were a simpler structure. We could not observe the oral needle of D. brevis, and there were only five pairs of palpal claws on the end segment of palpus. The offensive organs of Demodex resulted in its pathogenic effects. After studying hundreds of Demodex, we identified both female and male species of D. folliculorum, but only females of D. brevis in our sample.     

Monte Carlo simulation of secondary electron images for real sample structures in scanning electron microscopy

Monte Carlo simulation methods for the study of electron beam interaction with solids have been mostly concerned with specimens of simple geometry. In this article, we propose a simulation algorithm for treating arbitrary complex structures in a real sample. The method is based on a finite element triangular mesh modeling of sample geometry and a space subdivision for accelerating simulation. Simulation of secondary electron image in scanning electron microscopy has been performed for gold particles on a carbon substrate. Comparison of the simulation result with an experiment image confirms that this method is effective to model complex morphology of a real sample.     

Minimizing sample evaporation in the environmental scanning electron microscope

The ElectroScan environmental scanning electron microscope (ESEM) enables wet samples to be observed by eliminating air but allowing water vapour into the sample chamber. However, evaporation from, and condensation on, the sample may occur during the pumpdown sequence used to reach this state, which means that the sample may not be in its natural state when viewed if due care is not taken. In this paper, the pumping system of the ESEM is described mathematically and expressions are derived for the evaporation and condensation. This treatment is then used to calculate the optimum pumpdown sequence. The importance of using the optimized procedure is illustrated by micrographs of fat emulsions.     

An environmental scanning electron microscopy study of aqueous gibbsite suspensions

Synthetic gibbsite has been used as a model system for study in the environmental scanning electron microscopy (ESEM), to probe its utility as a tool to study clay dispersions under different conditions of aggregation. We have been able to show that we can study the nature of the platelet interactions as the pH is altered, by imaging the dispersions after water evaporation from the surface to permit the surface of the platelets to be clearly seen. It has been possible to show that at alkaline pH there are very few face-edge contacts between the platelets, consistent with what is known about the charges at high pH on the faces and edges of the gibbsite. In contrast, at lower pHs, when faces and edges have opposite sign charges, there are significantly more platelets touching with edge and faces in contact. Finally, when the salt lithium chloride is added to a dispersion at approximately neutral pH, the plates appear to stack suggesting face-face interactions in the dispersion. Thus, ESEM has been able to demonstrate the variability of packing in gibbsite dispersions and to correlate the structures observed with the known charge distribution on the gibbsite platelets.     

Some aspects of optimizing contrasts for the investigation of joint materials in the environmental scanning electron microscope

In the environmental scanning electron microscope, material joints of different atomic mass and different electrical conducting properties can easily be observed simultaneously without coating the specimen. For such heterogeneous materials, the quality of the image can be optimized with respect to contrast and resolution if the contrast types as well as their significance to the composition of the image are known.     

Charging in scanning electron microscopy "from inside and outside"

This paper is an attempt to analyse most of the complicated mechanisms involved in charging and discharging of insulators investigated by scanning electron microscopy (SEM). Fundamental concepts on the secondary electron emission (SEE) yield from insulators combined with electrostatics arguments permit to reconsider, first, the widespread opinion following which charging is minimised when the incident beam energy E0 is chosen to be equal to the critical energy E(o)2, where the nominal total yield delta(o) + eta(o) = 1. For bare insulators submitted to a defocused irradiation, it is suggested here that the critical energy under permanent irradiation EC2 corresponds to a range of primary electrons, R, and nearly equals the maximum escape depth of the secondary electrons, r. This suggestion is supported by a comparison between published data of the SEE yield delta(o) of insulators (short pulse experiments) and experimental results obtained from a permanent irradiation for EC2. New SEE effects are also predicted at the early beginning of irradiation when finely focused probes are used. Practical considerations are also developed, with specific attention given to the role of a contamination layer where a negative charging may occur at any beam energy. The role of the various time constants involved in charging and discharging is also investigated, with special attention given to the dielectric time constant, which explains the dose rate-dependent effects on the effective landing energy in the steady state. Numerical applications permit to give orders of magnitude of various effects, and several other practical consequences are deduced and illustrated. Some new mechanisms for the contrast reversal during irradiation or with the change of the primary electron (PE) energy are also suggested.     

High‐resolution,high‐throughput imaging with a multibeam scanning electron microscope

Electron–electron interactions and detector bandwidth limit the maximal imaging speed of single‐beam scanning electron microscopes. We use multiple electron beams in a single column and detect secondary electrons in parallel to increase the imaging speed by close to two orders of magnitude and demonstrate imaging for a variety of samples ranging from biological brain tissue to semiconductor wafers.     

Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium‐tin‐oxide

Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field‐of‐view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold‐labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium‐tin‐oxide was deposited by ion‐sputtering on gold‐decorated HeLa cells and neurons. Indium‐tin‐oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold‐conjugated markers. Microsc. Res. Tech. 78:433–443, 2015. © 2015 Wiley Periodicals, Inc.     

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.