We have measured the secondary electron yield curve for liquid water using an Environmental SEM. The secondary electron emission coefficient, measured as a function of incident electron energy, is important for interpreting contrast in hydrated biological and inorganic specimens. This information is even more critical for water than other materials, as it is a factor of prime importance in understanding radiation damage in biological tissues.[1]

These measurements were taken using a Philips XL-30 field emission ,ESEM, and repeated on an Electroscan E3 ESEM, equipped with a CeB6 filament. A specially designed Faraday cup was fashioned from brass and fitted with a removable graphite cup having an inset for a platinum aperture. This assembly was placed into an electrically floating Peltier cooling stage, and connected to a KE Instruments probe current meter.

Three-dimensional reconstructions are a useful visualisation aid for understanding the volumetric relationships between features in an object. Typically, three dimensional reconstructions have evolved with regards to biological applications, where the collection and manipulation of the data has matured to a point where volumetric rendering has become almost routine. Two common data collection procedures involve either virtual sectioning using CLSM, where layers within the object are imaged successively without mechanical sectioning, or alternatively the projection of the object is imaged, via TEM while being rotated through a range of angles. This paper describes a novel application of three dimensional imaging by collecting 2 dimensional serial section images using a recently described technique for imaging materials using a VPSEM, known as Charge Contrast Imaging.

Figure 1 shows a typical image of a polished grain of gibbsite. Previous work has correlated the appearance of the concentric rings to laboratory controlled precipitation events, thus showing the technique provides information on the precipitation history

Environmental scanning electron microscopy (ESEM) has matured into a mainstream technique in many areas of microscopy. Instrumentation has evolved and our understanding of some of the imaging mechanisms has progressed. However the majority of laboratories where ESEMs are located are based around the materials sciences. Despite the fact that ESEM is the only SEM instrument that permits liquid water to be present whilst imaging, the housing of such a microscope in biological EM units has been relatively rare. This authors laboratory is a multi-user EM unit based in a School of Biological Sciences. There exists the opportunity for basic biological scientists, clinical and pre-clinical medical and dental researchers to make use of such a resource. Indeed as the ESEM is housed alongside a conventional high vacuum instrument and a cryo high vacuum instrument there exists the ideal opportunity to carry out comparative studies.

This study will examine a range of biological samples using ESEM, cryo SEM and dry high vacuum SEM.

Contrast in secondary electron images of p-n junctions has been observed using a FESEM operating at 1kV. This contrast has been attributed to energy band-bending effects and dopant level variations. In this paper, we report that contrast from the depletion layer of a p-n junction can be obtained in an ESEM using a gaseous secondary electron detector (GSED). The contrast is caused by a signal induced in the GSED by charging and discharging of the device.

A 1N4002 p+pn silicon power diode was cross-sectioned along the lead axis to expose the Ag ohmic contacts and the semiconductor device. The section was mechanically polished to an optical finish using diamond abrasives and mounted in a holder which allowed electrical connection to both the p and n sides of the diode. Imaging experiments were performed in a Philips Electroscan XL30 ESEM and a JEOL 6300F below-lens FESEM. Current images were obtained with a GW Electronics 103 specimen current amplifier.

The environmental scanning electron microscope (ESEM) is not typically used for quantitative analysis by energy dispersive x-ray spectrometry (EDS) because the electron beam is scattered by the chamber gas forming a broad tail or skirt rather than a focused spot. Scattered electrons can contribute x-rays from areas not directly under the beam, which compromises the spectrum for quantitative analysis. For specimens compatible with high vacuum, quantitative EDS analysis in a conventional electron microscope is preferred. However, the ESEM has found its niche in providing a vehicle for investigating those specimens that for any of a variety of reasons are not suitable for high vacuum electron microscopy and microanalysis. For these specimens, it is important that we find the most accurate way to perform EDS analysis in the low vacuum environment.

Research has focused on reducing, accounting for, predicting, or measuring the electron scattering on a simplified system. Instrumental tricks to reduce the scattering of the primary electron beam have been reported in the past.

The purpose of this study is to evaluate the variable pressure correction technique (VPCT) as a solution to the problem of extraneous x-ray peaks due to electron beam broadening in the chamber gas of the ESEM. The basis of VPCT is the observation that target x-ray counts decrease with increasing chamber pressure; whereas, x-ray counts due to beam broadening increase. If data are collected at two or more chamber pressures, the number of x-ray counts for an element can be corrected to that expected at zero gas pressure (high vacuum). Tests of NIST SRM 482 have shown EDS x-ray analysis in the ESEM (within the chamber pressure range of 1 to 8 torr) to have comparable accuracy and precision values to those of EDS in the traditional SEM. The samples used in the these studies, however, were quite large (ca. 500 μm in diameter) and so extraneous EDS peaks, due to the electron beam broadening effect of the chamber gas, were minimized.

The 60% Au / 40% Cu wire of SRM 482 was pressed into a hole in the surface of an Al specimen stub so as to produce a flat surface with a sharp interface between the wire and the stub. Spectra were collected at 5 and 150 μm from the junction of the wire and the Al stub at chamber pressures of 2, 4, and 8 torr.

The environmental scanning electron microscope (ESEM) allows imaging and analysis in a gaseous environment at pressures up to 20 torr. Gas phase scattering of primary electrons leads to a loss of useable probe current and produces an electron skirt around the beam that can degrade the spatial resolution in microanalytical studies. Progress in ESEM microanalysis requires accurate modeling and measurement of the beam intensity profile. Previously, x-ray techniques and beam sensitive materials have been used to measure the extent of the skirt. These investigations have yielded inconsistent skirt distances possibly due to dose dependence of the measurement techniques. We present a new, high gain method to measure the useable probe current and skirt profile using the electron beam induced current (EBIC) from a P-N junction.

The interface of an N- and a P-type semiconductor contains an electrostatic donor-acceptor dipole (“depletion layer”) created by an electron-hole recombination energy minimization mechanism.

It is well known that the interaction of the electron beam with the gas in the VP-SEM or ESEM generate the so-called skirt as a result of the elastic collisions between the electrons and the molecules. Since the electrons in the skirt hit the specimen far away from the electron beam, they degrade the resolution of the analyses performed in the VPSEM or ESEM. However, the magnitude and the shape of the skirt are still a matter of controversy despite the fundamental importance of knowing these two factors. In this context, a Monte Carlo program has been developed to simulate the interaction of the electron beam with a gas as a function of the gas composition, gas pressure, electron beam energy and working distance (in reality, we should talk of the total distance traveled by the electron beam in the gas). This Monte Carlo program used a single scattering approach considering elastic collisions only since energy loss is negligible owing to the low density of the gas. 10 millions electron trajectories have been simulated for each conditions.

One of the unique abilities of the environmental scanning electron microscope is that it permits realtime dynamic observations of materials under controlled temperature/atmosphere conditions [1]. The need for accurate and precise measurements of dimensional changes at elevated temperatures arise in many material areas including determining the longitudinal and transverse coefficient of thermal expansion of fibers, sintering studies, hydration and dehydration of absorbent polymers, and strain measurement of materials under an applied load. The purpose of this research is to evaluate the capabilities of an ElectroScan 2020 environmental scanning electron microscope (ESEM™) in performing precise in situ spatial measurements at elevated temperatures. Identification and proposed solution and/or compensation of imaging difficulties such as specimen drift, sample stability, and thermal strain of a reference grid are addressed.

In general, the accuracy of spatial measurements in a scanning electron microscope (SEM) depends on several factors including calibration of the scanned area, the effects of lens hysteresis and reproducibility in the working distance and column alignment.

A variety of porous materials are used in a number of tissue-engineering applications such as cell transplantation and drug delivery. They provide a temporary scaffolding for a large cell mass, however they must have a fully interconnected pore structure. One such material is sodium alginate, a naturally derived polysaccharide which is isolated from seaweed. Sodium alginate can de dissolved in water to form a highly viscous solution that can be cross-linked in the presence of divalent cations (e.g., Ca2+) to form what is known a as hydrogels. We have developed a method for the fabrication of porous beads of sodium alginate and have attempted to measure the pore size, density and distribution in the beads. Characterization of the pore size and distribution is necessary to determine if the material is appropriate to promote cellular in-growth. The characterization procedures have been limited to conventional scanning electron microscopy (SEM) and mercury intrusion porosimetry. There has been some concern as to the potential introduction of artifacts (spatial distortion) during dehydration and sample preparation during these characterizations.

Biological scanning electron microscopy is increasingly performed at low beam energies in order to improve image contrast, reduce charging artifacts, and minimize beam induced damage to the sample. It is natural then to wish to also use these same low accelerating voltage for X-ray microanalysis using an energy dispersive spectrometer (EDS) but a variety of fundamental physical effects affect the performance that can be achieved. An X-ray photon can only be emitted when the incident beam Eo energy exceeds the critical excitation energy Ecrit for that line. As the beam energy is reduced the number of elements that can be excited falls and it is becomes necessary to use L- and M-lines rather than the K-lines accessible at higher energies. At 5keV, the upper limit of ‘low voltage’ microscopy, K-lines can be excited from elements up to calcium, L-lines can be detected up to cesium, and the rest of the periodic table is available using M-lines. The entire periodic table is therefore, in principle, available at an overvoltage U>2, where U = E0/Ecrit But at lower energies the number of accessible excitations falls and some elements cannot, with current technology, then be analyzed and for general purposes an incident energy lower than about 3keV is probably too limiting to be useful.

Microanalysis performed at low electron beam energies (≤ 5 keV) is limited by the physics of x-ray generation and the performance of existing semiconductor energy dispersive spectrometry (EDS) and wavelength dispersive spectrometry (WDS). Low beam energy restricts the atomic shells that can be excited for elements of intermediate and high atomic number, forcing the analyst to consider using unconventional M- and N-shells for elements such as Sn and Au. Unfortunately, these shells have very low fluorescent yield, which results in inherently low spectral peak-to-background ratios. The modest energy resolution of semiconductor EDS leads to poor limits of detection for these weakly emitted photons. The situation is further complicated by the inevitable interferences with the much more strongly excited K-shell x-rays of the light elements, particularly carbon and oxygen. WDS has the spectral resolution to overcome the resolution limitations of semiconductor EDS. However, WDS has a low geometric efficiency, and because of its narrow instantaneous spectral transmission, spectral scanning is required to detect and analyze x-ray peaks. Moreover, the high resolution field-emission-gun scanning electron microscope (FEG-SEM) provides only a few nanoamperes of beam current.

The use of a high-brightness field-emission gun and a high-resolution lens system in modern SEM instruments allows us to perform high-resolution chemical analysis and atomic-number contrast imaging of bulk materials at low voltages. Low voltage microanalysis is especially useful for analyzing surface coatings or deposits, catalyst particles, small inclusions or precipitates, and biological samples. In this report, we present useful applications as well as limitations of low voltage microanalysis in studying surface-related problems.

In the study of film formation on metal coupons for inhibiting metal corrosion in drinking water supplies, highly porous films were found to form on fresh metal coupons after treatment with various corrosion inhibitors. Figure 1a shows a low voltage (1.5 kV) SE image of a copper test coupon that was exposed to flowing water for a period of two months. The SE image clearly shows the porosity of the surface film and the presence of different phases.

The observation of microstructural features smaller than 300 nm is generally performed using Transmission Electron Microscopy (TEM) because conventional Scanning Electron Microscopes (SEM) do not have the resolution to image such small phases. Since the early 1990’s, a new generation of microscopes is now available on the market. These are the Field Emission Gun Scanning Electron Microscope with a virtual secondary electron detector. The field emission gun gives a higher brightness than those obtained using conventional electron filaments allowing enough electrons to be collected to operate the microscope with incident electron energy, E0, below 5 keV, with probe diameter smaller than 2.5 nm. Furthermore, what gives FE-SEM outstanding resolution is the combination of new magnetic lenses with a virtual secondary electron (SE) detector. The new lenses are designed to reduce the spherical and chromatic aberration coefficients, giving a smaller probe size. Contrary to the conventional systems, the SE detector is located above the objective lens and it becomes a virtual or through-the-lens (TTL) detector. Therefore, the SE image is mostly made up of all SEs of type I, almost eliminating those of type II and III which are generated by the backscattered electrons inside the specimen as well as in the chamber. It has been shown recently that Nb(CN) precipitates in Fe, as small than 10 nm, can be imaged with a FE-SEM Hitachi S-4500 with the TTL detector.

Conventionally, x-ray microanalysis on scanning electron microscopes (SEM) with energy dispersive spectrometers (EDS) has been performed with relatively high primary energies (>10 kv). for most samples this results in reasonably good separation of the generated x-ray line series from different elements enabling unambiguous identification and therefore accurate qualitative analysis. Under these circumstances it is widely accepted that quantitative analysis of polished bulk samples is possible on a routine basis with relative errors around 1-5% and detection limits of the order of 0.1%.

However, in order to address the analysis requirements of new advanced materials with sub-micron features, there is growing interest in performing x-ray microanalysis at low beam energies(<5kv). this is now a more realistic goal due to the routine availability of field emission sem's which can operate with much improved beam sizes at low beam energies with sufficient beam current to perform practical microanalysis, in conjunction with the improved low energy performance of current, commercially available EDS systems.

An earlier investigation had shown that it is possible to perform quantitative energy dispersive x-ray microanalysis at primary beam voltages as low as 5kV on bulk biological material maintained in either the fully frozen hydrated or the dried state. This present study extends the earlier work and provides data which shows that the same type of analysis can be achieved at voltages as low as 3kV on organic samples composed primarily of hydrogen, carbon and oxygen which contain varying amounts of bromine, sulphur and chlorine. Such materials are equally beam sensitive as fully frozen hydrated biological samples and range in density from 1.1 to 2.2kg.m3. The samples fall into three main classes. 1. Chlorinated and/or sulphonated organic polymers. 2. A brominated organic sample. 3. Three experimental brominated plastics kindly supplied by Ms. B.S.Chuah, Melville Laboratory for Polymer Synthesis, Cambridge University. The remaining samples were purchased from Aldrich Chemicals at the highest purity available. The atomic weight percent and density was calculated for each of the eight specimens using the Electron Flight Simulation™ software programme.

The need to analyze bulk samples containing features with submicron dimensions has driven revaluation of the processes controlling the interaction of electron beams with inorganic, polymer and semiconductor materials, and to development of LVEDS analysis at lower beam energies of E0 <5kV (1,2).

It has previously been shown (1,2) that the physics is much as expected with the vertical penetration range (R) along the beam direction in many cases predicted quite accurately for beam energy E0 by the simple Bethe (e.g. in 3) power law with R = F(E0)5/3. These same factors are effective to varying degrees in all three dimensions. The strong dependence of the range on energy has practical importance for the identification of sub-micron particles, including to help to determine the root cause of a defect Fig. 1 is an example of the sequential analysis of the exact same sub-micron particle, with the very real potential for a processing disaster, on the surface of a silicon wafer. When this feature is analyzed with a 3kV electron beam we learn it is alumina (A12O3). The analysis comes only from the target particle and the data have a simple relationship to the chemistry and the sensitivity for the light element (O) is excellent, providing simple and direct qualitative identification of the oxide compound.

It has been shown recently that precipitates as small as 10 nm can be imaged with a FE-SEM using a Through-The-Lens (TTL) SE detector or the so-called Upper Detector (UD). This is a very significant finding because normally, such small precipitates are observed in the TEM where specimen preparation techniques are generally a timely process. However, from Monte Carlo simulations using CASINO, it has been shown that NbC precipitates embedded in Fe as small than 6 nm can be imaged with BE. The experimental demonstration was difficult because no good BSE detectors were available at that time for low energy work.

Recently, a new BSE detector has been developed for low energy work from the GW corporation, the Centaurus BSE detector. This detector is coupled with an HITACHI S-4700 FESEM In this work, images of Mg2Zn precipitates in Al obtained with the UD and the Centaurus BSE detector are compared.

One of the advantages of performing X-ray microanalysis at low (≤5 kV) operating voltages is that high spatial resolution (≤250 nm) chemical maps of the specimen can be acquired. Spectrum imaging, where a full spectrum is acquired for each pixel in a two-dimensional array, provides the most comprehensive method of characterization, as long as the sampling density (pixel size) is sufficiently smaller than the spatial resolution. Multivariate statistical analysis (MSA) methods are effective in reducing the large (typically ∼10 MByte) raw spectrum images to the modest (typically ∼100 kByte) data files that contain all of the statistically significant information of interest about the specimen. Preliminary analysis of a cross section of a computer chip from a major semiconductor company was previously performed using limited spectrum imaging capabilities available with the 4pi X-ray mapping module, which allowed for simultaneous acquisition of only 48 channels. MSA of images acquired with only a 960 eV portion of the spectrum containing the Al-K, Si-K and W-M lines showed that excellent contrast between the Si- and W-rich regions of the specimen could be achieved in spite of the strong overlap between Si-Kα and W-Mα (∼34 eV separation).

Telemicroscopy, sometimes called remote microscopy, is an expanding set of microscopy techniques, made possible by the advent of ubiquitous and pervasive Internet technologies such as the World Wide Web. Recently, telemicroscopy has moved from laboratory prototypes to commercially available software. This paper discusses a real-world application of telemicroscopy in terms of a microscopy consultancy service offered by ERA Technology Ltd. in Leatherhead, UK.

Many organisations require microscopical services on an occasional basis. These needs are not however, sufficiently frequent as to justify the purchase and upkeep of an SEM or the recruitment and retention of a trained instrument operator Such organisations employ consultancy firms to perform the required analyses. In many such consultancies, a sample is sent to the microscopist with appropriate instructions. Once the investigation is complete, the consultant returns sample, micrographs and other results. The SEM service run by ERA has always offered its customers the opportunity for personal participation in any investigation, by attendance at SEM sessions at the Leatherhead laboratory.