Usually, in electron probe microanalysis, the X-ray diffusion range is around lμm on the sample surface. For this reason, the maximum useful magnification of X-ray image seems to be limited to a few thousand times. The X-ray diffusion on the \im order takes place in the bulk sample. However, when a sample is cut to a thin film, it is likely that the X-ray diffusion range will be made smaller, and it is also possible to observe very a narrow area X-ray image at higher magnification. The thin film method is a sampling technique used often in transmission electron microscopy. The microtome method has been applied to organic materials, and the method using focus ion beam (FIB) has been applied to inorganic materials. Takahashi et al. have reported the effectiveness of the microtome application to EPMA of soft materials. in this report, the FIB method was applied to a sampling of inorganic materials in order to obtain high resolution X-ray images of real samples.

Monte Carlo simulation is a suitable tool for the numerical generation of x-ray spectra by electron beams and, more specifically, for the quantification in electron probe microanalysis (EPMA). in this communication we describe the application of the general-purpose code PENELOPE to EPMA. This code simulates electron-photon showers in complex material structures consisting of homogeneous regions of arbitrary composition limited by quadric surfaces. It is devised to cover a wide energy range (from ∼500 eV to about 1 GeV). The interaction models implemented in PENELOPE are based on the most reliable information available. They combine results from first principles calculations (this is the case, e.g., for electron elastic scattering, photon Compton scattering), semiempirical models (in electron inelastic scattering) and information from evaluated data bases. to facilitate the random sampling, the cross sections of various interaction mechanisms are described through analytical expressions, which are adjusted to yield accurate values of relevant transport properties (mass attenuation coefficients, transport mean free paths, stopping powers, . . . ).

Monte Carlo simulation of electron scattering has been widely used in various fields such as microanalysis, microscopy and microlithography. Various simulation models have been reported so far. in applications to quantitative x-ray microanalysis the accuracy of the model has been significantly improved by introducing the Mott cross section. However, in the analyses at low energies of an electron beam or at energies near the x-ray excitation energy, the simulation accuracy becomes worse. This is probably because the discrete energy loss process is not incorporated into the simulation model. to improve this default, we developed the model which includes the discrete energy loss process[l]. The outline of the model is described in the following.

1. 1) Elastic scattering

We used the Mott cross section. The Mott cross sections for Al, Cu, Ag and Au elements are calculated at various energies. From this data base we obtain the differential elastic scattering cross section and the total elastic cross section for arbitarary elements and energies by using the interporation or the extrapolation.

The classical schemes to convert the x-ray intensity into concentration, using the ZAF or the φ(ρ z ) methods, are valid for specimens having homogeneous composition and flat surfaces. Quantitative schemes have also been developed for x-ray microanalysis of multi-layered specimens. More recently, a quantitative method has been proposed for the microanalysis of spherical inclusions embedded in a matrix.

For the case of specimens having a non-planar surface, a quantitative method based on the peak to background ratio, using photons of the same energy, has been proposed. in order to test this correction method, a Monte Carlo program has been developed in order to compute the complete x-ray spectrum, characteristic lines plus bremstrallung, emitted from materials having rough surfaces. Figure [1] shows the geometry of the rough surfaces simulated. The parameters of these surfaces are the summit semi-angle, θ, and the height of the roughness, h.

In Castaing’s classic Ph.D. dissertation he described how the limiting value of x-ray spatial resolution for x-ray microanalysis, of about 1 μm, was not imposed by the diameter of the electron beam, but by the size of the region excited inside the specimen. Fifty years later this limit still applies to the majority of measurement made in EMAs and SEMs, even though there is often a need to analyze much finer structures. When high resolution chemical analysis is required, it is generally necessary to prepare thin sections and examine them in an analytical electron microscope where the maximum diameter of the excited volume may be as small as a few nanometers. Since it is not always possible or practical, it is important to determine just what is the best spatial resolution attainable for the examination of polished or “as received” samples with an EMA or SEM and how to achieve it experimentally.

In variable pressure (VPSEM) and environmental SEM (ESEM) caution is required in the intepretation of x-ray data from non-conductive and electrically isolated samples for unique reasons. This paper reviews the documented procedures and presents data on hither-to undefined aspects.

The most widely recognised artefacts are associated with the scatter of primary electrons from their convergent trajectory to the sample surface through interactions with the chamber gas. Beam ‘skirt’ measurements have been made by various researchers and this scatter is well characterised. “Point” analyses are corrupted by addition of components from the surrounding phases and x-ray maps are effectively ‘defocussed’ or ‘blurred’. The magnitude is a function of pressure, amongst many other variables, and it has been shown that these scatter effects can be reduced through extrapolation of data collected at differing chamber pressures2. Bremsstrahlung-based corrections for primary beam variations and drift have also been shown to be effective.

A number of methods have been proposed to correct for the electron beam scattering effects on xray microanalysis in the environmental scanning electron microscope (ESEM). This paper presents an evaluation of two of these methods. The Doehne method is based on the observation that x-ray counts due to the unscattered electron beam increase with decreasing chamber pressure whereas the inverse is true for x-ray counts due to scattered electrons. The x-ray count intercept, at zero pressure, of the regression lines relating x-ray counts to chamber vapor pressure is an estimate of the high-vacuum intensity. The Gauvin method is based on the relationship between x-ray counts and the fraction of the electron beam that is unscattered, fp.The fraction of the unscattered beam is calculated using an equation derived from scattering theory and uses the accelerating voltage, the gas path length, and the chamber vapor pressure.

An inevitable consequence of the presence of the gas in the sample chamber of a low-vacuum scanning electron microscope (LV-SEM) and environmental SEM (ESEM) is the electron beam broadening due to the scattering in the gas. The electron broadening has a large impact on x-ray analysis because the fluorescent characteristic x rays generated far from the center of the electron probe form a high background, which reduces the detection sensitivity of x-ray analysis and degrades the x-ray image contrast. We report in this paper of using a polycapillary focusing x-ray optic between the sample and the energy-dispersive spectrometer as a spatial filter to filter out unwanted x-rays generated far from the specimen. As a result, the x-ray image contrast and the detection sensitivity of the system were notably improved.

A polycapillary focusing optic collects a large solid angle of x rays from an x-ray source of small area at its input focus, guide them through the curved channels by multiple external total reflections, and focus them to the output focus.

There is increasing interest in performing x-ray microanalysis of uncoated insulators while operating in unconventional SEM operating modes such as “low voltage” scanning electron microscopy (LVSEM), where the accelerating voltage is ≤ 5 kV and the pressure is low (<10-4 Pa), or variable pressure environmental SEM (VP-ESEM), where a selected gas is maintained at pressures in the range of 1 Pa -1000 Pa. LVSEM and VP-ESEM as microscopy techniques have proven to be extremely successful for imaging uncoated insulators through various charge dissipation mechanisms that are not available under conventional SEM operating conditions (accelerating voltage ≥ 10 kV and pressure < 10-3 Pa). in LVSEM, surface charging of insulators can often be controlled by careful choice of the accelerating voltage, sample tilt, and scan rate, while in VP-ESEM the charged species in the relatively dense gas (ions, secondary electrons) form a self-neutralizing plasma to provide an additional route for discharging the specimen.

Energy-dispersive X-ray (EDX) spectrum imaging, where a full spectrum is acquired at each pixel in a rastered image, combines the advantages of imaging and analysis for quantitative compositional mapping. This method has great advantages for the analysis of compositional variations, because the contrast inherent in EDX spectrum images arises from the distinctiveness of the characteristic X-ray spectra in various regions of the surveyed microstructure. EDX spectrum imaging provides for simultaneous spatial, spectral and temporal resolution of the X-rays emitted from the specimen. Many EDX manufacturers have incorporated a spectrum imaging capability within their acquisition and analysis software, and ongoing improvements in computing power will enhance the practicality of spectrum imaging methods, in particular the acquisition, manipulation and analysis of the large raw data files that are generated with this method. This paper surveys the potential role of spectrum imaging in tasks that support true quantitative X-ray microanalysis of bulk specimens: robust qualitative analysis, the construction of representative spectra that describe the specimen, and the extraction of elemental concentrations from the representative spectra.

It is a conundrum that faces microanalysts every day: how do you comprehensively survey the chemistry of a microstructure in a reasonable time? Traditional solutions in the SEM would include taking an image and then collecting x-ray spectra from a series of points. This method relies heavily on the operator’s ability to identify chemically distinct regions. in addition, for a complex microstructure, this could take a considerable amount of time to do correctly. On the positive side, if care is taken, these ‘point spectra’ can be readily quantified. An alternative method that has been used widely is x-ray mapping, where a window of intensity in the spectra is integrated and displayed as an image. This has the advantage that qualitative elemental distributions from an area of a microstructure can be visualized. The disadvantages are that maps are generally not quantitative, can be susceptible to artifacts, and rely on fore knowledge of the elements to map.

X-ray microanalysis of any type of specimen in its natural state without the use of conventional SEM specimen preparation techniques has immense potential in a wide range of scientific and industrial applications. This capability would be particularly useful in microanalysis applications where it is highly desirable to preserve the integrity of the specimen, for example in semiconductor failure analysis and forensic investigations. in principle, this X-ray microanalysis goal can be achieved in an environmental or variable pressure scanning electron microscope (VPSEM) because specimen charging and vacuum stability problems are negated by the presence of a gas in the specimen chamber. However, the accuracy and spatial resolution of X-ray microanalysis in the VPSEM is significantly degraded by the chamber gas as it scatters primary beam electrons, generating spurious X-rays far from the analysis point. to date, two different X-ray measurement strategies have been developed to facilitate X-ray microanalysis at high chamber pressure in the VPSEM.

The demand for accurate mineralogical data is increasing rapidly as exploration methods, prospect evaluation procedures and metallurgical optimisation studies become more sophisticated. in response to these needs, semi-automated and automated image processing systems which detect minerals using optical microscopy, scanning electron microscopy or electron microprobe microanalysis (EPMA) are becoming increasingly important tools in the exploration, mining and mineral processing industries. CSIRO Minerals has developed an EPMA based imaging (or mapping) method for characterising ilmenite concentrates. The method uses a JEOL 8900R EPMA to collect elemental x-ray maps which are then processed using in-house developed software, Chimage. The mapping procedure differs from traditional automated identification systems in that no detailed a priori knowledge of the mineral phases is required. in addition, Chimage software enables complete processing and interpretation of the data set off-line. Elemental data can be displayed in either scatter or ternary diagrams showing clusters which allow mineral phases to be identified.

This work proposes a new consistency check total based on measurement of bremsstrahlung intensity which can be used to validate analyses even when concentrations have been normalised to 100%.

If elemental content of a material is unknown, spectral peaks have to be identified, either interactively, or by automatic procedures. When peak overlap is severe (e.g. Si K/ Ta M / W M, Al K/Br L), it is easy for an element to be misidentified or missed altogether and at low beam kV, some elements will not even produce a characteristic line suitable for analysis. When element identification or quantification requires deconvolution of closely spaced lines, minor miscalibration or inaccurate peak modelling produces inaccurate peak area estimates, Ai, instead of true peak areas, Ai*, and consequently inaccurate concentration estimates, Ci* . A common consistency check is to confirm that the analysis total is close to unity:

The mechanism for assembly of membrane lipid bilayers in vivo is generally considered to occur on existing membranes which act as templates for the assembly process. Not presently understood is the source of the membrane template. A thermodynamic theory describing a spontaneous process for assembly of unilamellar vesicles with properties of a critical state (1,2) has been tested in this study. The spontaneous formation of large unilamellar vesicles (LUV’s) from phospholipid components of cell membranes at a critical temperature T* has now been documented by cryo-electron microscope studies using aqueous dispersions of dimyrystoylphosphatidylcholine (T* = 29°C), and the total lipid extracts of E. coli membranes cultured at 32°C (T* = 32°C). The lipids were incubated at temperatures below, at and above the critical temperatures for LUV assembly. in conformity with thermodynamic theory, and other physical measurements that indicate unique bilayer properties exist at T* (3-5), LUV’s are seen to form only at T*.

Structured latex coatings find such applications as specialty paints, micro- and ultra-filtration membranes, templates for synthesizing inorganic complementary structures, and embedding media for whole-cell based bioreactors. to control well the microstructure and other properties of the final coatings requires understanding the film formation process that transforms a deposited layer of colloidal particles of polymer into a coherent solid coating as it dries, usually in air. This process cannot be visualized by light microscopy techniques when the latex particles are a few hundred down to a few tens of nanometers in size, as is normally the case. Conventional electron microscopy requires too high vacuum for liquid samples to survive. Environmental scanning electron microscopy tolerates modest partial pressure of solvent but is limited to a coating’s top surface and at low resolution. What is needed is to observe the interior of a drying, hydrated coating at nm-resolution. Cryo-SEM is a powerful means of studying microstructure evolution throughout a coating’s thickness in the wet and moist stages of film formation.

Low temperature scanning electron microscopy (LTSEM) avoids many of the artifacts associated with chemical fixation, dehydration and critical point drying. As a result, this technique has had numerous applications ranging from biology to hydrology. Unfortunately, experiments using LTSEM are frequently compromised by the limited sample manipulations that are available on most cryo-stages and by the cost of specimen holders. Whereas the conventional SEM stage enables 360° rotation and tilts as great as 90°, the braids or hoses required for cooling a cryo-stage generally limit rotation to about 30° and tilt to less than 40° from normal. Furthermore, the specimen holders for LTSEM may cost several hundred dollars each, thereby prohibiting experiments that require numerous holders. This study describes an inexpensive specimen holder that solves these problems.

Figure la illustrates a drawing of the specimen holder supplied with the standard transfer device from the Oxford CT 1500 Cryotrans System.

Cryo-techniques have been successfully used in scanning electron microscopy (SEM). They are especially promising for high-resolution SEM to improve specimen preservation and reduce radiation damage [1, 2]. A number of cryo-preparation systems are commercially available for SEM, however, our experience has shown that modifications are needed to perform highresolution imaging (>50,000x).

Emitech K1250 system consists of a sample preparation chamber, control unit, and cryo-stage. Magnetron sputter coating is standard and electron-beam evaporation is optional. A vacuum transfer device facilitates the sample transfer between the preparation chamber and the SEM to prevent contamination. The Emitech cryo-stage replaces the Hitachi S-4700 standard stage and the cryo-stage temperature is monitored and controlled by the Emitech control unit.

The specimen is mounted on a sample holder that mounts to the cryo-stage. Therefore, the distance from the specimen to cryo-stage, the thermal capacity of the sample holder, and the thermal contact between them will affect the actual temperature of the specimen.

Creating contrast of unstained frozen hydrated cells and isolated macromolecules in order to provide high fidelity recordings present several difficulties and therefor require attempts to maximize image information for characterization purposes. Adobe Photoshop 5.0 was employed to overlay (“layer“) topographic high resolution secondary electron (SE-I) images (HRSEM) onto STEM images with and without polarity reversal employed during digital image acquisition. Cells and molecular samples precipitated from an aqueous droplet or aerosol and supported on carbon and holey carbon coated grids were plunge frozen and Chromium (Cr) coated. Since Cr-coated cryosamples on carbon-grid supports have very low mass density and because signal to noise is low in HRSEM recordings, especially in regions which overhang holey carbon expanses, then the combined contrasts obtained in a composite image containing STEM and HRSEM contrasts result in greater detail recognition.

Tandem cryo-imaging, using an in-lens field emission (FE) SEM (DS-130F) operated at 25kV and fitted with above-lens secondary electron detector and below-lens scanning transmission detector, provided digital images of frozen cells and macromolecular complexes.

The recent increase in use of Field Emission Scanning Instruments in biology has shown the potential of these systems for sub-cellular imaging, as they essentially provide transmission EM levels of resolution for surface imaging. in structures lacking inherent contrast in thin sections, such as nuclear pore complexes,(NPC) high resolution surface imaging has provided significant complementary structural information to the macromolecular architecture of the NPC. (Goldberg and Allen, 1996). A series of intermediate structural forms of the NPC during its formation has also been described using FESEM, which would not have been resolvable in TEM.(Goldberg, Weise, Allen, Wilson, 1997). All this work was performed with chemical fixation, dehydration and critical point drying, followed by high resolution Chromium coating. We now are extending these studies to a cryo-preservation approach, mindful of the requirement of rapid freezing to preserve the more labile events of membrane dynamics, and also to circumvent the requirement to expose membranes to organic solvents during conventional dehydration.