X-ray spectrometry and spectrum image mapping at output count rates above 100 kHz with a silicon drift detector on a scanning electron microscope

A third-generation silicon drift detector (SDD) in the form of a silicon multicathode detector (SMCD) was tested as an analytical x-ray spectrometer on a scanning electron microscope. Resolution, output count rate, and spectral quality were examined as a function of the detector peaking time from 8 micros to 250 ns and over a range of input count rate (dead time). The SDD-SMCD (50 mm2 active area) produced a resolution of 134 eV with a peaking time of 8 micros. The peak width and peak channel were nearly independent of the input count rate (at 8 micros peaking time, the peak width degradation was 0.003 eV/percent dead time and peak position change was -0.7 eV over the dead time range tested). Maximum output count rates as high as 280 kHz were obtained with a 500 ns peaking time (188 eV resolution) and 500 kHz with a 250 ns peaking time (217 eV resolution). X-ray spectrum imaging was achieved with a pixel dwell time as short as 10 ms (with 1.3 ms overhead) in which a 2048 channel (10 eV/channel) spectrum with 2-byte intensity range was recorded at each pixel (scanned at 128 x 128). With a 220 kHz output count rate, a minor constituent of iron (present at a concentration of 0.04 mass fraction or 4 weight %) in an aluminum-nickel alloy could be readily detected in the x-ray maps derived from the x-ray spectrum image database accumulated in 185 s.     

Particle‐induced x‐ray analysis using focused ion beams

In addition to the production of secondary electrons and secondary ions, characteristic x‐ray emission may also result from ion/solid interactions and is the basis for the well‐known analysis technique referred to as particle‐induced x‐ray emission. Characteristic x‐rays may be emitted by either bombardment by MeV protons or heavy ions of a few keV. The advantage to heavy ions is that the x‐ray yield is confined to the region near the surface defined by the collision cascade. An advantage of heavy ion‐induced x‐ray emission over electron‐induced x‐ray emission is that the Bremsstrahlung is potentially orders of magnitude lower. Thus, ion‐induced x‐ray spectra may provide for superior peak‐to‐noise ratios, and there‐fore, offers trace element sensitivity compared with elec‐tron‐induced x‐ray emission. In addition, the near surface ion/solid interactions also allow for the possibility of surface analysis or depth profiling. A Dual Beam instrument was used to collect focused ion beam‐induced x‐ray (FIBIX) spectra. The acquisition of characteristic x‐rays from targets via FIBIX is demonstrated and compared with scanning electron microscopy‐induced x‐ray energy dispersive spectroscopy spectra and is consistent with the theory described above.     

Quantitative electron probe microanalysis of rough targets: testing the peak-to-local background method

Rough samples with topography on a scale that is much greater than the micrometer dimensions of the electron interaction volume present an extreme challenge to quantitative electron beam x-ray microanalysis with energy-dispersive x-ray spectrometry. Conventional quantitative analysis procedures for flat, bulk specimens become subject to large systematic errors due to the action of geometric effects on electron scattering and the x-ray absorption path compared with the ideal flat sample. The best practical approach is to minimize geometric effects through specimen reorientation using a multiaxis sample stage to obtain the least compromised spectrum. When rough samples must be analyzed, corrections for geometric factors are possible by the peak-to-local background (P/B) method. Correction factors as a function of photon energy can be determined by the use of reference background spectra that are either measured locally or calculated from pure element spectra and estimated compositions. Significant improvements in accuracy can be achieved with the P/B method over conventional analysis with simple normalization.     

Errors in quantitative backscattered electron analysis of bone standardized by energy-dispersive x-ray spectrometry

Backscattered electron (BSE) imaging has proven to be a useful method for analyzing the mineral distribution in microscopic regions of bone. However, an accepted method of standardization has not been developed, limiting the utility of BSE imaging for truly quantitative analysis. Previous work has suggested that BSE images can be standardized by energy-dispersive x-ray spectrometry (EDX). Unfortunately, EDX-standardized BSE images tend to underestimate the mineral content of bone when compared with traditional ash measurements. The goal of this study is to investigate the nature of the deficit between EDX-standardized BSE images and ash measurements. A series of analytical standards, ashed bone specimens, and unembedded bone specimens were investigated to determine the source of the deficit previously reported. The primary source of error was found to be inaccurate ZAF corrections to account for the organic phase of the bone matrix. Conductive coatings, methyl-methacrylate embedding media, and minor elemental constituents in bone mineral introduced negligible errors. It is suggested that the errors would remain constant and an empirical correction could be used to account for the deficit. However, extensive preliminary testing of the analysis equipment is essential.     

A spectroscopic scanning electron microscope design

This paper describes a proposal to improve the design of scanning electron microscopes (SEMs). The design is based upon using an SEM column similar to the conventional one, magnetic sector plates and a mixed field immersion objective lens. The optical axis of the SEM column lies in the horizontal direction and the primary beam is turned through 90 degrees before it reaches the specimen. This arrangement allows for the efficient collection, detection and spectral analysis of the scattered electrons on a hemispherical surface that is located well away from the rest of the SEM column. The proposed SEM design can also be easily extended to incorporate time multiplexed columns and multi-column arrays.     

New pathways for improved quantification of energy‐dispersive X‐ray spectra of semiconductors with multiple X‐ray lines from thin foils investigated in transmission electron microscopy

Theoretical approaches to quantify the chemical composition of bulk and thin‐layer specimens using energy‐dispersive X‐ray spectroscopy in a transmission electron microscope are compared to experiments investigating (In)GaAs and Si(Ge) semiconductors. Absorption correctors can be improved by varying the take‐off angle to determine the depth of features within the foil or the samples thickness, or by definition of effective k‐factors that can be obtained from plots of k‐factors versus foil thickness or, preferably, versus the K/L intensity ratio for a suitable element. The latter procedure yields plots of self‐consistent absorption corrections that can be used to determine the chemical composition, iteratively for SiGe using a set of calibration curves or directly from a single calibration curve for InGaAs, for single X‐ray spectra without knowledge of sample thickness, density or mass absorption coefficients.     

Fundamental problems of imaging subsurface structures in the backscattered electron mode in scanning electron microscopy

In‐depth imaging of subsurface structures in scanning electron microscopy (SEM) is usually obtained by detecting backscattered electrons (BSE). For a layer‐by‐layer imaging in BSE microtomography, it is preferable to use an energy filtering of BSE. A simple approach is used to estimate the contrast by using backscattering coefficients of bulk materials and the maximum escape depths of the BSE. The contrast obtained by BSE energy filtering is about twice that of the standard BSE method by varying the acceleration voltage. The contrast decreases with increasing information depth. The information depth is about four times smaller than the electron range. The transmission of the spectrometer influences the minimum current of the order of 10^?8 A that is needed to get a contrast of 1%, for example.     

Microscopic diagnosis of the leaf and stem of Piper solmsianum C.DC

Piper solmsianum C.DC., which is popularly known as pariparoba, is a shrub that measures 1–3 m in height and it inhabits areas with wet tropical soils. The objective of this study was to analyze the leaf and stem anatomy using light microscopy, scanning electron micrographs, and energy‐dispersive X‐ray spectroscopy in order to provide information for species identification. The anatomical profile showed the following main microscopic markers: hypostomatic leaf; hypodermis layer on both sides; pearl glands; biconvex midrib shape; five collateral vascular bundles in open arc with the central bundle larger than the others; circular stem shape; collateral vascular bundles arranged in two rings; sinuous sclerenchymatic sheath in the pith; secretory idioblasts; and starch grains in the mesophyll, in the ground parenchyma of the midrib, petiole, and in the stem; and six morphotypes of calcium oxalate crystals (styloids, cuneiform, tabular crystal rosettes, cuneiform crystal rosettes, elongated square dipyramids, as well as very elongated square dipyramids).     

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.     

Accuracy and precision of quantitative energy-dispersive x-ray spectrometry in the environmental scanning electron microscope

The accuracy and precision of quantitative energy-dispersive x-ray spectrometry in the environmental scanning electron microscope have been estimated using a series of copper / gold alloys of known composition. The mean values (five to six replicate experiments) had relative errors within +/- 5%, and most were within +/- 3.5%. All relative standard deviations were < 5% and most were < 3%. Since the standard specimens were large (approximately 500 microm) in diameter, electron scattering in the 2 torr of water vapor above the specimen did not affect the results. This level of accuracy and precision was possible only by using a novel specimen surface charge neutralization scheme.     

Influence of topography and specimen preparation on backscattered electron images of bone

Backscattered electron (BSE) images of bone exhibit graylevel contrast between adjacent lamellae. Mathematical models suggest that interlamellar contrast in BSE images is an artifact due to topographic irregularities. However, little experimental evidence has been published to support these models, and it is not clear whether submicron topographical features will alter BSE graylevels. The goal of this study was to determine the effects of topography on BSE image mean graylevels and graylevel histogram widths using conventional specimen preparation techniques. White-light interferometry and quantitative BSE imaging were used to investigate the relationship between the BSE signal and specimen roughness. Backscattered electron image graylevel histogram widths correlated highly with surface roughness in rough preparations of homogeneous materials. The relationship between BSE histogram width and surface roughness was specimen dependent. Specimen topography coincided with the lamellar patterns within the bone tissue. Diamond micromilling reduced average surface roughness when compared with manual polishing techniques but did not significantly affect BSE graylevel histogram width. The study suggests that topography is a confounding factor in quantitative BSE analysis of bone. However, there is little quantitative difference between low-to-moderate magnification BSE images of bone specimens prepared by conventional polishing or diamond micromilling.     

Secondary and backscattered electron imaging of weathered chromian spinel

Secondary (SE) and backscattered electron (BSE) imaging as well as x-ray microanalysis have demonstrated that the weathering of chromian spinel occurs as a progressive form of alteration. Numerous chemical discriminant analysis methods based on the composition of chromian spinel are used to locate valuable deposits of minerals. These methods will be misleading if the correct interpretation of the weathering of chromian spinel and the subsequent pattern of changes in its mineral chemistry are not properly assessed using scanning electron microscopy. This assessment is vital in understanding the geological processes involved and the economic potential of any indicated deposit. Minerals such as chromian spinel, pyrope garnet, and picroilmenite are considered to be highly resistant to weathering and abrasion and are therefore useful in the search for associated valuable deposits of diamond, nickel, platinum, and gold. Known as indicator minerals, they are usually present in relatively large concentrations compared with the target mineral (e.g., diamond) and form large and often subtle dispersion anomalies adjacent to the deposit. Chromian spinel has long been regarded as a stable indicator mineral; however, detailed SE and BSE imaging indicates that many of the chromian spinels that are routinely examined using scanning electron microscopes (SEM) and microprobes are extensively altered. Secondary electron and BSE imaging of weathered chromian spinel in a normal SEM provides valuable data on the form and chemical style of the alteration. Secondary electron imaging of weathered chromian spinel in the environmental SEM (ESEM) not only enhances the difference in atomic number between unaltered and altered areas but also allows high-resolution imaging of these very fine replacement textures.     

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.     

Thermal effects of the electron beam and implications of surface damage in the analysis of bone tissue

Electron beam interactions with specimens in the scanning electron microscope (SEM) can lead to increased surface temperatures and damage. These changes may have significant consequences in the analysis of bone tissue. An investigation was performed to measure the surface temperature changes associated with the electron beam on a thermocouple with systematic variations in operating conditions. Probe currents, magnifications, and accelerating voltages were incrementally adjusted to measure the temperature changes and to make assessments for determining optimal operating conditions for the SEM in future analyses of bone tissue. Results from this study suggest that thermal effects were minimal at lower accelerating voltages (< 20 kV), lower probe currents (< 10 nA), and lower magnifications, but surface damage may still occur during the analysis of bone tissue.     

New developments in electron energy loss spectroscopy

In the electron microscope, spectroscopic signals such as the characteristic X-rays or the energy loss of the incident beam can provide an analysis of the local composition or electronic structure. Recent improvements in the energy resolution and sensitivity of electron spectrometers have improved the quality of spectra that can be obtained. Concurrently, the calculations used to simulate and interpret spectra have made major advances. These developments will be briefly reviewed. In recent years, the focus of analytical electron microscopy has moved away from single spectrum acquisition to mapping and imaging. In particular, the use of spectrum imaging (SI), where a full spectrum is acquired and stored at each pixel in the image is becoming widespread. A challenge for the application of spectrum imaging is the processing of such large datasets in order to extract the significant information. When we go beyond the mapping of composition and look to map bonding and electronic structure this becomes both more important and more difficult. Approaches to processing spectrum imaging data sets acquired using electron energy loss spectroscopy (EELS) will be explored in this paper.     

Imaging connected porosity of crystalline rock by contrast agent‐aided X‐ray microtomography and scanning electron microscopy

We set out to study connected porosity of crystalline rock using X‐ray microtomography and scanning electron microscopy with energy dispersive X‐ray spectroscopy (SEM‐EDS) with caesium chloride as a contrast agent. Caesium is an important radionuclide regarding the final deposition of nuclear waste and also forms dense phases that can be readily distinguished by X‐ray microtomography and SEM‐EDS. Six samples from two sites, Olkiluoto (Finland) and Grimsel (Switzerland), where transport properties of crystalline rock are being studied in situ, were investigated using X‐ray microtomography and SEM‐EDS. The samples were imaged with X‐ray microtomography, immersed in a saturated caesium chloride (CsCl) solution for 141, 249 and 365 days and imaged again with X‐ray microtomography. CsCl inside the samples was successfully detected with X‐ray microtomography and it had completely penetrated all six samples. SEM‐EDS elemental mapping was used to study the location of caesium in the samples in detail with quantitative mineral information. Precipitated CsCl was found in the connected pore space in Olkiluoto veined gneiss and in lesser amounts in Grimsel granodiorite. Only a very small amount of precipitated CsCl was observed in the Grimsel granodiorite samples. In Olkiluoto veined gneiss caesium was found in pinitised areas of cordierite grains. In the pinitised areas caesium was found in notable excess compared to chloride, possibly due to the combination of small pore size and negatively charged surfaces. In addition, elevated concentrations of caesium were found in kaolinite and sphalerite phases. The findings concerning the location of CsCl were congruent with X‐ray microtomography.     

Mistakes encountered during automatic peak identification in low beam energy X-ray microanalysis

Automated peak identification in electron beam excited X-ray microanalysis with energy dispersive X-ray spectrometry (EDS) is subject to occasional mistakes even on well-separated, high-intensity peaks arising from major constituents. The problem is exacerbated when analysis conditions are restricted to operation in the "low beam energy scanning electron microscopy" (i.e. "low voltage scanning electron microscopy" or LVSEM) regime where the incident beam energy is 5 keV or less. These low beam energy microanalysis conditions force the analyst to use low fluorescence yield L-shell and M-shell peaks rather than higher yield K-shell and L-shell peaks typically selected for elements of intermediate and high atomic number under conventional high beam energy (>10 keV) conditions. Misidentifications can arise in automated peak identification procedures when only a single energy channel is used to characterize an EDS peak. The effect of the EDS measurement process is to convolve the closely spaced Lalpha-Lbeta and Malpha-Mbeta peaks into a single peak with a peak channel shift of 20 eV or more from the Lalpha or Malpha value, which is typically sought in an X-ray database. An extensive list of problem situations encountered in low beam energy microanalysis is presented based upon observed peak identification mistakes as well as likely troublesome situations based upon proximity in peak energy. Robust automatic peak identification requires implementation of peak fitting that utilizes the full peak shape.     

Transformation reactions of partially gold-coated bioactive Ca-P glass granules investigated by analytical scanning electron microscopy

In the present work, we have clarified the detail of the surface transformation reactions of bioactive calcium-phosphate (Ca-P) glass granules induced by in vivo implantation in rabbit dorsal muscle sites. To this aim we have compared the behaviour, during the same implantation, between the as-prepared and gold-coated only-on-one-side glass granules. The deposited gold layer enabled us to determine very precisely the initial position of the surface of the glass before the transformation took place. In addition, since the gold layer acts as a diffusion barrier, it allowed the study of the direction and the mechanism of crystal growth which occurred at the glass surface. Lapped and polished cross-sections of the samples were examined by backscattered electron (BSE) imaging and quantitative (with standards) x-ray energy dispersive spectroscopy (EDS) in a scanning electron microscope (SEM). The observations showed the presence of an interlayered structure. Quantitative EDS microanalysis performed by profiling the electron beam across the samples indicated the presence of hydrated calcium phosphate in the external layer, an inhomogeneous silica-rich gel-type layer in the middle layer, and an unaffected original Ca-P glass in the centre. From the comparison with those granules gold-coated on one side, we deduced that the hydrated calcium phosphate layer grew towards the interior of the granules at the expense of the starting glass. A simple model, based on the balance of the concentrations of the elements which have diffused in the different layers, is proposed to explain the contribution of the elements constituting the original glass to the formation of the different layers. This result agrees with the experimental data obtained from image analysis and the microstructural behaviour of this type of glass is discussed.     

A scanning electron microscope based new method for determining degree of substitution of sodium carboxymethyl cellulose

Na-CMC or sodium carboxylmethyl cellulose is a water soluble anionic polymer obtained by introducing carboxymethyl groups along the cellulose chain. Na-CMC is usually synthesized by the alkali catalyzed reaction of cellulose with monochloroacetic acid. The functional properties of Na-CMC depend on the degree of substitution of the cellulose structure (i.e. how many of the hydroxyl groups are substituted per monomer unit), and also on the chain length of the cellulose backbone. The degree of substitution of Na-CMC is usually determined according to ASTM D1439 which evolves the conversion of the Na-CMC to free acid then again forming Na-CMC by adding excess alkali and finally titrating the excess alkali with standard hydrochloric acid (0.3 N). The used volume of the standard alkali determines the degree of substitution. These existing chemical methods for determining the degree of substitution are not very convenient and very time-consuming involving the use of hazardous chemicals. In this research, we have evaluated that the scanning electron microscope equipped with Energy Dispersive X-Ray Analysis can be used to directly determine the degree of substitution.     

Preserving elemental content in adherent mammalian cells for analysis by synchrotron‐based x‐ray fluorescence microscopy

Trace metals play important roles in biological function, and x‐ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side‐by‐side comparison shows that plunge‐freezing‐based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with fresh media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. When chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x‐ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.