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.     

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.     

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.     

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.     

Mineral content changes in bone associated with damage induced by the electron beam

Energy-dispersive x-ray (EDX) spectroscopy and backscattered electron (BSE) imaging are finding increased use for determining mineral content in microscopic regions of bone. Electron beam bombardment, however, can damage the tissue, leading to erroneous interpretations of mineral content. We performed elemental (EDX) and mineral content (BSE) analyses on bone tissue in order to quantify observable deleterious effects in the context of (1) prolonged scanning time, (2) scan versus point (spot) mode, (3) low versus high magnification, and (4) embedding in poly-methylmethacrylate (PMMA). Undemineralized cortical bone specimens from adult human femora were examined in three groups: 200x embedded, 200x unembedded, and 1000x embedded. Coupled BSE/EDX analyses were conducted five consecutive times, with no location analyzed more than five times. Variation in the relative proportions of calcium (Ca), phosphorous (P), and carbon (C) were measured using EDX spectroscopy, and mineral content variations were inferred from changes in mean gray levels ("atomic number contrast") in BSE images captured at 20 keV. In point mode at 200x, the embedded specimens exhibited a significant increase in Ca by the second measurement (7.2%, p < 0.05); in scan mode, a small and statistically nonsignificant increase (1.0%) was seen by the second measurement. Changes in P were similar, although the increases were less. The apparent increases in Ca and P likely result from decreases in C: -3.2% (p < 0.05) in point mode and -0.3% in scan mode by the second measurement. Analysis of unembedded specimens showed similar results. In contrast to embedded specimens at 200x, 1000x data showed significantly larger variations in the proportions of Ca, P, and C by the second or third measurement in scan and point mode. At both magnifications, BSE image gray level values increased (suggesting increased mineral content) by the second measurement, with increases up to 23% in point mode. These results show that mineral content measurements can be reliable when using coupled BSE/EDX analyses in PMMA-embedded bone if lower magnifications are used in scan mode and if prolonged exposure to the electron beam is avoided. When point mode is used to analyze minute regions, adjustments in accelerating voltages and probe current may be required to minimize damage.     

Use of backscattered electron detector arrays for forming backscattered electron images in the scanning electron microscope

The backscattered electron (BSE) signal in the scanning electron microscope (SEM) can be used in two different ways. The first is to give a BSE image from an area that is defined by the scanning of the electron beam (EB) over the surface of the specimen. The second is to use an array of small BSE detectors to give an electron backscattering pattern (EBSP) with crystallographic information from a single point. It is also possible to utilize the EBSP detector and computer-control system to give an image from an area on the specimen--for example, to show the orientations of the grains in a polycrystalline sample ("grain orientation imaging"). Some further possibilities based on some other ways for analyzing the output from an EBSP detector array, are described.     

Monte Carlo simulation of secondary electron and backscattered electron images in scanning electron microscopy for specimen with complex geometric structure

A new Monte Carlo technique for the simulation of secondary electron (SE) and backscattered electron (BSE) of scanning electron microscopy (SEM) images for an inhomogeneous specimen with a complex geometric structure has been developed. The simulation is based on structure construction modeling with simple geometric structures, as well as on the ray-tracing technique for correction of electron flight-step-length sampling when an electron trajectory crosses the interface of the inhomogeneous structures. This correction is important for the simulation of nanoscale structures of a size comparable with or even less than the electron scattering mean free paths. The physical model for electron transport in solids combines the use of the Mott cross section for electron elastic scattering and a dielectric function approach for electron inelastic scattering, and the cascade SE production is also included.     

Surface roughness of preparations for backscattered electron-scanning electron microscopy: the image differences and their Monte Carlo simulation

Patterns and levels of mineralisation in the biological hard tissues have been studied using the backscattered electron (BSE) mode in the scanning electron microscope (SEM). To prevent gross topographic detail overwhelming changes in signal from composition, samples are embedded in polymethylmethacrylate (PMMA) and a flat block surface produced by polishing or micromilling. This study was undertaken to establish the degree of residual topography achieved in these finishing processes. A sample of human rib was embedded in PMMA and prepared, as for examination in the SEM, by polishing on graded abrasives and pre- and, finally, ultramilling. After each preparation step, the block face was imaged using a confocal reflection microscope surface mapping facility. The recorded topographies were used in a Monte Carlo simulation to model the surface interface and thus, for each of the sample preparation techniques, to calculate predicted variations in BSE signal. The latter were compared with experimental data derived under standard operating conditions in the SEM. Micromilling produced block faces with typical peak-trough relief of 80 nm, while hand polishing left occasional scratches 1.5 microns deep with a general undulation of 150-250 nm. Monte Carlo simulations of a rough surface of bone using these data predicted that additional contrast levels of 5% could be expected from micromilled surfaces and > 10% for hand polished samples of bone. Thus, micromilling is the best preparation method for bone, since this tissue develops a collagen orientation-related relief on polishing, which may be largely responsible for the (incorrect) supposition that lamellation in bone is related to changes in net degree of mineralisation.     

A method of direct particle deposition on a carbon planchet to study mineral dust in human lung by scanning electron microscopy

Following Na-hypochlorite digestion of lung tissue, mineral particles extracted in the chloroform layer were deposited directly on a pre-smoothed carbon planchet for combined scanning electron microscopy and X-ray energy dispersive spectrometry (SEM and XEDS). Total mineral particle counts were obtained, and detailed physical characteristics of the fibrous particles were documented at 600, 1,500, 4,500 and 9,000 x in three lungs without, and one lung with, histories of occupational exposure. This preparation method was simple, collected more than 99% of identifiable mineral particles in the chloroform layer, gave excellent object to background contrast without heavy metal coatings, and was suitable for XEDS. Comparable fibrous particles from the chloroform layer could also be studied by selected-area electron diffraction to complement the results of XEDS. By this method, we found particles or fibers larger than 0.1 μm were readily counted and measured at 4,500 x. At 600 x, ferruginous bodies were found to be more than twice in number than when sought for by light microscopy. It was determined that 4,500 x is the most efficient magnification to examine and diagnose this type of specimen. The present study illustrates the importance of determining the most efficient magnification to be utilized in particle counts.     

Registration of confocal scanning laser microscopy and quantitative backscattered electron images for the temporospatial quantification of mineralization density in 18-month old thoroughbred racehorse articular calcified cartilage

Combined backscattered electron scanning electron microscopy (BSE SEM) and confocal scanning laser microscopy (CSLM) have been used to put tissue mineralization data into the context of soft tissue histology and fluorescent label information. Mineralization density (Dm) and linear accretion rate (LAR) are quantifiable parameters associated with mineralizing fronts within calcified tissues. Quantitative BSE (qBSE) may be used to determine Dm, while CSLM may be used to detect label fluorescence from which LAR is calculated. Eighteen-month old Thoroughbred horses received single calcein injections 19 and 8 days prior to euthanasia, labeling sites of active mineralization with fluorescent bands. Confocal scanning laser microscopy images of articular calcified cartilage (ACC) from distal third metacarpal condyles were registered to qBSE images of the same sites using an in-house program. ImageJ and Sync Windows enabled the simultaneous collection of LAR and Dm data. The repeatability of the registration and measurement protocols was determined. Dm profiles between calcein labels were explored for an association with time. Dm was 119.7 +/- 24.5 (mean +/- standard deviation) gray levels (where 0 = backscattering from monobrominated and 255 from monoiodinated dimethacrylate standards, respectively), while modal and maximum LAR were 0.45 and 3.45 microm/day, respectively. Coefficients of variation (CV) for Dm were 0.70 and 0.77% with and without repeat registration, respectively; CVs for LAR were 1.90 and 2.26% with and without repeat registration, respectively. No relationship was identified between Dm and time in the 11-day interlabel interval. Registration of CSLM to qBSE images is sufficiently repeatable for quantitative studies of equine ACC.     

Monte Carlo modeling of secondary x-ray fluorescence across phase boundaries in electron probe microanalysis

Secondary fluorescence induced by photoelectric absorption of x-rays generated by an electron beam can occur when the characteristic x-ray energy of material “A” exceeds the critical excitation energy of material “B.” An expression is developed to calculate secondary fluorescence across a planar boundary from a discrete source placed at any (X, Y, Z) coordinates relative to the boundary. The expression can be incorporated into a Monte Carlo electron trajectory simulation which calculates the discrete distribution of primary x-ray generation.     

Identification of growth increments in the shell of the bivalve mollusc Arctica islandica using backscattered electron imaging

Annually resolved growth increments in the shell of the bivalve mollusc Arctica islandica have previously been used in combination with geochemical measurements to successfully construct high-resolution proxy records of past marine environmental conditions. However, to ensure the accuracy of these paleoenvironmental reconstructions it is essential that the annual growth series of increments within the examined shells are reliably identified, and can be distinguished from spurious lines caused by nonannual perturbations such as those resulting from storm disturbance. The current methods used for identifying the growth increment series are sometimes compromised because of ambiguity that results from the employed preparation methods. Here it is shown that backscattered electron imaging of polished shell cross sections may be used to clearly discriminate between the two compositionally and structurally distinct increments that comprise 1 year of outer shell growth. This method, involving minimal specimen preparation, is likely to be primarily useful as a validation technique of particular value in cases where increment identification using existing methods is difficult or ambiguous.     

Theoretical explanation of the relationship between backscattered electron and x-ray linear attenuation coefficients in calcified tissues

X-ray absorption and backscattered electron (BSE) microscopies are two commonly used techniques for estimating mineral contents in calcified tissues. The resolution in BSE images is usually higher than in x-ray images, but due to the previous lack of good standards to quantify the grey levels in BSE images of bones and teeth, x-ray microtomog-raphy (XMT) images of the same specimens have been used for calibration. However, the physics of these two techniques is different: for a specimen with a given composition, the x-ray linear attenuation coefficient is proportional to density, but there is no such relation with the BSE coefficient. To understand the reason that this calibration appears to be valid, the behaviour of simulated bone samples was investigated. In this, the bone samples were modelled as having three phases: hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂), protein, and void (either empty or completely filled with polymethylmethacrylate (PMMA), a resin which is usually used for embedding bones and teeth in microscopic studies). The x-ray linear attenuation coefficients (calculated using published data) and the BSE coefficients (calculated using Monte Carlo simulation) were compared for samples of various phase proportions. It was found that the BSE coefficient correlated only with the x-ray attenuation coefficient for samples with PMMA infiltration. This was attributed to the properties of PMMA (density and mean atomic number) being very similar to those of the protein; therefore, the sample behaves like a two-phase system which allows the establishment of a monotonic relation between density and BSE coefficient. With the newly developed standards (brominated and iodinated dimethacrylate esters) for BSE microscopy of bone, grey levels can be converted to absolute BSE coefficients by linear interpolation, from which equivalent densities can be determined.     

Backscattered electron imaging of the undersurface of resin-embedded cells by field-emission scanning electron microscopy

In this study backscattered electron (BSE) imaging was used to display cellular structures stained with heavy metals within an unstained resin by atomic number contrast in successively deeper layers. Balb/c 3T3 fibroblasts were cultured on either 13-mm discs of plastic Thermanox, commercially pure titanium or steel. The cells were fixed, stained and embedded in resin and the disc removed. The resin block containing the cells was sputter coated and examined in a field-emission scanning electron microscope. The technique allowed for the direct visualization of the cell undersurface and immediately overlying areas of cytoplasm through the surrounding embedding resin, with good resolution and contrast to a significant depth of about 2 μm, without the requirement for cutting sections. The fixation protocol was optimized in order to increase heavy metal staining for maximal backscattered electron production. The operation of the microscope was optimized to maximize the number of backscattered electrons produced and to minimize the spot size. BSE images were collected over a wide range of accelerating voltages (keV), from low values to high values to give ‘sections' of information from increasing depths within the sample. At 3–4 keV only structures a very short distance into the material were observed, essentially the areas of cell attachment to the removed substrate. At higher accelerating voltages information on cell morphology, including in particular stress fibres and cell nuclei, where heavy metals were intensely bound became more evident. The technique allowed stepwise ‘sectional’ information to be acquired. The technique should be useful for studies on cell morphology, cycle and adhesion with greater resolution than can be obtained with any light-microscope-based system.     

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.     

A new method of compositional image formation by a four-element backscattered electron detector in an SEM

A special mixing procedure for signals from a four element backscattered electron (BSE) detector is proposed for compositional image formation when a sample with a rough surface is examined by a scanning electron microscope (SEM). The new method allows appreciable suppression of the influence of the sample surface topography in a compositional mode for take-off angles less than about 30°, relative to the microscope axis. The theoretical approach based on the analysis of BSE angular distribution is compared with the experiment. The mixing procedure uses a dimensionless parameter, which depends mainly on take-off angle. Photographs of the Ge-Zn structure with its rough surface were taken in conventional and proposed compositional modes for take-off angle 11° and electron energy 20 keV and show a considerable suppression of the topographic effect when the new method is used.     

Multiphase analysis of Bi-Sr-Ca-Cu-O high Tc superconductors with x-ray compositional mapping

Ba-Sr-Ca-Cu-O (BSCCO) high Tc superconductor specimens were prepared as part of a solid-solubility study in the region of the 2223 phase. The identification and determination of the amount of each phase in the final products were necessary. Quantitative digital x-ray compositional mapping with the electron microprobe was done with five wavelength spectrometers, one for each of the cations. To facilitate phase identification and quantification, image processing rules were developed for the nine possible phases, with each assigned a unique color. Using this procedure, at least six different phases were identified. Examples are cited in color.     

Analysis of sections of implanted macroporous calcium phosphate bone substitutes by proton-induced x-emission method and energy-dispersive spectrometry

The osseointegration of porous calcium phosphate ceramics once implanted evolves in several stages. The mechanism of integration of such material usually is evaluated by histologic analysis. The trace elements present in bone can be detected in the ceramic and help to provide a semiquanti-tative evaluation of osseointegration. Two different methods of microanalysis, energy-dispersive spectrometry (EDS) and proton induced x-emission (PIXE) were used in this study to determine the appearance of trace elements (Zn, Sr, and Fe) present in bone at the implantation site containing the ceramic. Porous HA-ceramic cylinders were implanted in the cortical bone of sheep femurs for periods ranging from 2 to 36 weeks. Thick sections of the implant-containing bone were made at the end of the implantation period. A scanning line with proton or electron impacts 0.5 mm apart was plotted from the edges of the cortical bone across the implanted ceramic and the resulting x-ray spectra were determined. Following EDS analysis, the sections were surface-stained, observed under a light microscope, and the pore volume occupied by bone tissue was measured. The spectra obtained by PIXE method showed two regions for each element characterising either the bone tissue or the ceramic. Zinc and strontium present in the bone tissue, but absent from the ceramic, appeared 8 and 12 weeks after implantation, respectively. The concentration of iron present in the implant decreased with time. EDS showed no significant level of either element in the bone or the ceramic. Histologic observation revealed that immature bone invaded the pores of the outer layer of the ceramic as early as 2 weeks after implantation. The ceramics were totally osseoin-tegrated 20 weeks after implantation, although ceramic degradation continued for longer. In this experiment, the PIXE method was apparently sufficiently sensitive for monitoring the amount of trace element appearing in bone-implanted material.