The effect of some instrument operating conditions on the x-ray microanalysis of particles in the environmental scanning electron microscope

The objective of this investigation was to evaluate the practical effects of electron beam broadening in the environmental scanning electron microscope (ESEM) on particle x-ray microanalysis and to determine some of the optimum operating conditions for this type of analysis. Four sets of experiments were conducted using a Faraday cage and particles of copper, glass, cassiterite, andrutile. The accelerating voltage and chamber pressure varied from 20 to 10 kV and from 665–66 Pa (5.0 to 0.5 torr), respectively. The standard gaseous secondary electron detectors (GSED) and the long environmental secondary dectectors (ESD) for the ESEM were evaluated at different working distances. The effect of these parameters on the presence of artifact peaks was evaluated. The particles were mounted on carbon tape on an aluminum specimen mount and were analyzed individually and as a mixture. Substrate peaks were present in almost all of the spectra. The presence of neighboring particle peaks and the number of counts in these depended upon the operating conditions. In general, few of these peaks were observed with the long ESD detector at 19 mm working distance and at low chamber pressures. More peaks and counts were observed with a deviation from these conditions. The most neighboring peaks and counts were obtained with the GSED detector at 21.5 mm working distance, 10 kV accelerating voltage, and 665 Pa (5.0 torr) chamber pressure. The results of these experiments support the idea that the optimum instrumental operating conditions for EDS analysis in the ESEM occur by minimizing the gas path length and the chamber water vapor pressure, and by maximizing the accelerating voltage. The results suggest that the analyst can expect x-ray counts from the mounting materials. These tests strongly support the recommendation of the manufacturer to use the long ESD detector and a 19 mm working distance for EDS analysis. The results of these experiments indicate that neighboring particles millimeters from the target may contribute x-ray counts to the spectrum.     

Backscattered electron imaging of cultured cells: application to electron probe X-ray microanalysis using a scanning electron microscope

We report a simple method to study the elemental content in cultured human adherent cells by electron probe X-ray microanalysis with scanning electron microscopy. Cells were adapted to grow on polycarbonate tissue culture cell inserts, washed with distilled water, plunge-frozen with liquid nitrogen and freeze-dried. Unstained, freeze-dried cultured cells were visualized in the secondary and backscattered electron imaging modes of scanning electron microscopy. With backscattered electron imaging it was possible to identify unequivocally major subcellular compartments, i.e. the nucleus, nucleoli and cytoplasm. X-ray microanalysis was used simultaneously to determine the elemental content in cultured cells at the cellular level. In addition, we propose some improvements to optimize backscattered electron and X-ray signal collection. Our findings demonstrate that backscattered electron imaging offers a powerful method to examine whole, freeze-dried cultured cells for scanning electron probe X-ray microanalysis.     

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.     

Skirting: a limitation for the performance of X-ray microanalysis in the variable pressure or environmental scanning electron microscope

The variable pressure or environmental scanning electron microscope (VP-SEM; ESEM) has become the microscope of choice for many scientists and technologists. Hence, the development of robust methods for X-ray microanalysis, limited by skirting, has become critical. In this paper, two pressure variation correction methods (Doehne and Gauvin) are compared. Both of these methods appear to be effective; the results were found to be well within 10% of the values obtained at 0 Pa. The Doehne method is dependent on an empirical factor (D), therefore the accuracy of the results will depend on the accuracy of this value. Also the Doehne method is compromised by the nonlinearity of the response with pressure. The Gauvin method is more user-friendly and more precise when considering the total range of pressure.     

X-ray microanalysis of wet biological specimens in the environmental scanning electron microscope. 1. Reduction of specimen distance under different atmospheric conditions

X-ray microanalysis of non-biological and biological specimens was carried out in the environmental scanning electron microscope (ESEM) under different conditions of specimen distance (the distance travelled by the electron probe within the specimen chamber) and chamber atmosphere. Using both water vapour and argon atmospheres, it was shown that reduction in specimen distance had no effect on atmospheric gas X-ray signal in either case. Unlike water vapour, increased levels of argon (up to 10 torr) caused a marked depression of specimen P/B ratios, with a decrease in both characteristic and background (continuum) counts. These effects in argon were not altered by reduction in specimen distance. Specimen distance was important in relation to beam skirting and elemental analysis. With an extended assembly (short specimen distance), beam skirting in a water-vapour atmosphere was much reduced – leading to enhanced element detectability in a discrete biological specimen (Anabaena cyclindrica).     

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.     

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.     

An experimental model of beam broadening in the variable pressure scanning electron microscope

In the variable pressure scanning electron microscope (VP-SEM) the incident electrons pass through a gaseous environment and the beam is scattered by these interactions. We show here that the experimental intensity profile of the scattered beam can be described as Gaussian in form to a high level of accuracy. This provides a rapid means of accounting for the effects of beam scatter in imaging and microanalysis because the standard deviation of the Gaussian is a simple function of parameters such as working distance, beam energy, gas type and pressure.     

The use of helium gas to reduce beam scattering in high vapour pressure scanning electron microscopy applications

Both image quality and the accuracy of x-ray analysis invariable pressure scanning electron microscopes (VPSEMs) are often limited by the spread of the primary electronbeam due to scattering by the introduced gas. The degree of electron scattering depends partly on the atomic number Z of the gas, and the use of a low Z gas such as helium should reduce beam scattering and enhance image quality. Using anuncoated test sample of copper iron sulphide inclusions in calcium fluorite, we show that the reduction in beam scatter produced by helium is more than sufficient to compensate for its reduced efficiency of charge neutralisation. The relative insensitivity to pressure of x-ray measurements in a helium atmosphere compared with air, and the consequent ability to work over a wider range of working distances, pressures, and voltages, make helium potentially the gas of choice for many routine VPSEM applications.     

The reduction of the beam gas interactions in the variable pressure scanning electron microscope with the use of helium gas

This paper presents experimental observations on electron scattering by gases (helium and air) in the specimen chamber of a variable pressure scanning electron microscope. It shows an important reduction of the beam scattering with the use of helium gas, and the consequences for the x-ray microanalysis are discussed.     

Primary considerations for image enhancement in high-pressure scanning electron microscopy

The mechanisms of electron beam scattering are examined to evaluate its effect on contrast and resolution in high-pressure scanning electron microscopy (SEM) techniques reported in the literature, such as moist-environment ambient-temperature SEM (MEATSEM) or environmental SEM (ESEM). The elastic and inelastic scattering cross-sections for nitrogen are calculated in the energy range 5–25 keV. The results for nitrogen are verified by measuring the ionization efficiency, and measurements are also made for water vapour. The effect of the scattered beam on the image contrast was assessed and checked experimentally for a step contrast function at 20 kV beam voltage. A considerable degree of beam scattering can be tolerated in high-pressure SEM operation without a significant degradation in resolution. The image formation and detection techniques in high-pressure SEM are considered in detail in the accompanying paper.     

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.     

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.     

Absorption microanalysis with a scanning soft X-ray microscope: mapping the distribution of calcium in bone

This article describes the scanning transmission X-ray microscope operated at the National Synchroton Light Source. The application of the instrument to elemental analysis is detailed. In particular, qualitative results on the calcium distribution in human skull tissue are presented.     

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.     

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.     

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.     

Some recent experience with the scanning electron microscope in corrosion research

Corrosion and oxidation are the results of chemical reaction between a solid surface (such as of a metal or alloy) and its environment, to form a corrosion product. The product may adhere to the surface, thereby usually providing some protection from further attack, or may be removed from it (e.g. by volatilization or by dissolution in the environment). In either case this leads to changes in topography, morphology and, often, composition of the surface. Study of such changes can provide considerable information and assistance in the determination of the mechanisms of degradation. Scanning electron microscopy with associated microanalysis has thus enabled significant advances to be made in understanding many corrosion and oxidation processes. The progress attained in several areas where the technique has played a major part but has been extended to its maximum capabilities is discussed and related to the particular advantages and limitations of scanning electron microscopy in this field. One such area is the study of high-temperature corrosion processes. Here, the high resolution and depth of focus of the instrument have provided detailed information on the microstructure of high-temperature oxide and other corrosion-product scales and underlying alloy. For instance, the sizes and distributions of pores in the scales give important insight into the possibility of growth mechanisms involving gas-phase transport within the scales themselves. Associated microanalysis can assist in identification of rate-determining layers and of other localized diffusion paths which can short-circuit bulk lattice diffusion.     

A new correction method for high-resolution energy-dispersive x-ray analyses in the environmental scanning electron microscope

Spurious x-ray signals, which previously prevented high-resolution energy-dispersive x-ray analysis (EDS) in the environmental scanning electron microscope (ESEM), can be corrected using a simple method presented here. As the primary electron beam travels through the gas in the ESEM chamber, a significant fraction of the primary electrons is scattered during collisions with gas molecules. These scattered electrons form a broad skirt that surrounds the primary electron beam as it impacts the sample. The correction method assumes that changes in the width of the electron skirt with pressure are less important than changes in the skirt intensity; this method works as follows: The influence of the gas on the overall x-ray data is determined by acquiring EDS spectra at two pressures. Subtracting the two spectra provides us with a difference spectrum which is then used to correct the original data, using extrapolation, back to the x-ray spectrum expected under high-vacuum conditions. Low-noise data are required to resolve small spectral peaks; however, the principle should apply equally to x-ray maps and even to low-magnification images.     

A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g , washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 m m ammonium acetate, 300 m m sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10–11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.