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.     

‘Effects of novel root repair materials on attachment and morphological behaviour of periodontal ligament fibroblasts: Scanning electron microscopy observation’

The aim of this study was to evaluate the adhesion of periodontal ligament fibroblasts (PDLs) on newly proposed root repair materials [Biodentine, MM‐MTA, polymethylmethacrylate (PMMA) bone cement, and SDR], in comparison with contemporary root repair materials [IRM, Dyract compomer, ProRoot MTA (PMTA), and Vitrebond]. Five discs from each material were fabricated in sterile Teflon molds, and the specimens were aged and prewetted in cell culture media for 96 hours. Three material discs were used for scanning electron microscopy (SEM) for the assessment of the attachment, density, and morphological changes in the PDLs, while two samples were used for energy dispersive x‐ray spectroscopy (SEM‐EDX) to determine the elemental composition of the materials. Human PDLs were plated onto the materials at a density of 10,000/well, and incubated for 3 days. The SEM micrographs were taken at different magnifications (500× and 5000×). In the SEM, the cells were attached and well spread‐out on the surfaces of the Biodentine, PMTA, and Dyract compomer, while varied cell densities and morphological alterations were observed in the Vitrebond, IRM, MM‐MTA, SDR, and PMMA bone cement groups. The SEM‐EDX analysis revealed a maximum calcium percentage in the PMTA specimens, as well a maximum silicon percentage in the Dyract compomer specimens. This in vitro study demonstrated that the Biodentine and Dyract compomer supported PDL cell adhesion and spreading. The PMTA presented a favorable scaffold for better attachment of the PDL cell aggregates. Therefore, the calcium and silicon content of a material may enhance the PDL cell attachment.     

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.     

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.     

Investigation of mineral content of root canal dentin after the application of various antibiotic paste using energy‐dispersive X‐ray detector

The purpose of this study was to evaluate mineral content of root canal dentin after treatment with different antibiotic pastes including the mixture of metronidazole, ciprofloxacin, doxycycline, cefaclor, amoxicillin, or minocycline. Fifty extracted maxillary canine teeth were randomly divided into five groups (n = 10 teeth for each group). Root canals were prepared Reciproc rotary files. Canals were irrigated using 5 ml 5% NaOCl and 1 ml 15% EDTA. Each tooth in all groups were longitudinally splitted into two pieces as a control and experimental samples. Each experimental group received following antibiotic paste; double antibiotic paste (DAP) and triple antibiotic paste with doxycycline (TAPd), TAP with cefaclor (TAPc), TAP with amoxicillin (TAPa), and TAP with minocycline (TAPm) for 21 days. The Ca, P, Mg, Ca, and K levels, and the Ca/P ratio was analyzed by a scanning electron microscope (SEM) equipped using a Bruker energy‐dispersive X‐Ray (EDX) detector. Data were analyzed with independent samples t‐test, one‐way anova, and Duncan tests. Ca and Ca/P ratio showed a statistically significant increase TAP with amoxicillin and cefaclor (p < .05). DAP, TAPd, and TAPm did not change the mineral levels (p > .05). TAPa and TAPc with increased the Ca level and Ca/P ratio of the root canal dentin which consequently positively influences the revascularization process.     

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.     

The application of heat‐deproteinization to the morphological study of cortical bone: A contribution to the knowledge of the osteonal structure

Observation of heat‐deproteinized cortical bone specimens in incident light enabled the high definition documentation of the osteonal pattern of diaphyseal Haversian bone. This prompted a study to compare these images with those revealed by polarized light microscopy, carried out either on decalcified or thin, undecalcified, resin‐embedded sections. Different bone processing methods can reveal structural aspects of the intercellular matrix, depending on the light diffraction mode: birefringency in decalcified sections can be ascribed to the collagen fibrils orientation alone; in undecalcified sections, to both the ordered layout of collagen and the inorganic phase; in the heat‐deproteinized samples, exclusively to the hydroxyapatite crystals aggregation mode. The elemental chemical analysis documented low content of carbon and hydrogen, no detectable levels of nitrogen and significantly higher content of calcium and phosphorus in heat‐deproteinized samples, as compared with dehydrated controls. In both samples, the X‐ray diffraction (XRD) pattern did not show any significant difference in pattern of hydroxyapatite, with no peaks of any possible decomposition phases. Scanning electron microscopic (SEM) morphology of heat‐deproteinized samples could be documented with the fracturing technique facilitated by the bone brittleness. The structure of crystal aggregates, oriented in parallel and with marks of time periods, was documented. Comparative study of deproteinized and undecalcified samples showed that the matrix inorganic phase did not undergo a coarse grain thermal conversion until it reached 500°C, maintaining the original crystals structure and orientation. Incident light stereomicroscopy, combined with SEM analysis of deproteinized bone fractured surfaces, is a new enforceable technique which can be used in morphometric studies to improve the understanding of the osteonal dynamics. Microsc. Res. Tech. 79:691–699, 2016. © 2016 Wiley Periodicals, Inc.     

Dual-motion fretting behavior of mandibular cortical bone against pure titanium

Dual-motion fretting tests of flat cortical bone specimens from fresh human mandible against pure titanium (TA2) ball were carried out on a modified test rig with tilt angle of 45°. The imposed maximal loads varied from 100 to 200 N. Dynamic characteristics of dual-motion fretting tests were analyzed in combination with micro-examinations via optical microscopy (OM), laser confocal scanning microscopy (LCSM) and scanning electron microscopy (SEM) together with energy dispersive X-ray spectrum (EDX). Two types of F-D curves (the trapezoid and elliptic mode) were recorded during the tests. The examination showed that the wear scars of the dual-motion fretting were asymmetric, and the tangential component of dual-motion fretting was in the mixed fretting regime. Under the lower imposed load, only some detachment of particles and scratches without cracking were observed even after 5×10⁴ cycles. The main wear mechanisms of the dual-motion fretting damage were the abrasive and adhesive wear. Under higher imposed loads, the cracks initiated and propagated mainly at the high stress side of contact edges. The wear mechanisms of the dual-motion fretting of cortical bone under higher imposed loads were the combination of the adhesive wear, abrasive wear, cracking and lubrication of the human bone tissue debris. And the lubrication of the debris played an important role during the dual-motion fretting processes.     

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.     

Morphological and mineral analysis of dental enamel after erosive challenge in gastric juice and orange juice

This study evaluated and compared in vitro the morphology and mineral composition of dental enamel after erosive challenge in gastric juice and orange juice. Human enamel specimens were submitted to erosive challenge using gastric juice (from endoscopy exam) (n = 10), and orange juice (commercially-available) (n = 10), as follows: 5 min in 3 mL of demineralization solution, rinse with distilled water, and store in artificial saliva for 3 h. This cycle was repeated four times a day for 14 days. Calcium (Ca) loss after acid exposure was determined by atomic emission spectroscopy. The presence of carbonate (CO) and phosphate (PO) in the specimens was evaluated before and after the erosive challenge by FT-Raman spectroscopy. Data were tested using t-tests (P < 0.05). Morphology of enamel was observed in scanning electron microscopy (SEM). The mean loss of Ca was: 12.74 ± 3.33 mg/L Ca (gastric juice) and 7.07 ± 1.44 mg/L Ca (orange juice). The analysis by atomic emission spectroscopy showed statistically significant difference between erosive potential of juices (P = 0.0003). FT-Raman spectroscopy found no statistically significant difference in the ratio CO/PO after the erosive challenge. The CO/PO ratios values before and after the challenge were: 0.16/0.17 (gastric juice) (P = 0.37) and 0.18/0.14 (orange juice) (P = 0.16). Qualitative analysis by SEM showed intense alterations of enamel surface. The gastric juice caused more changes in morphology and mineral composition of dental enamel than orange juice. The atomic emission spectroscopy showed to be more suitable to analyze small mineral loss after erosive challenge than FT-Raman.     

Immunogold labeling of insulin growth factor-I receptors in elderly human skeletal muscle

Age-associated muscle wasting, or sarcopenia, can be delayed or reversed with interventions, including exercise and pharmaceutical agents. Mapping morphometric changes in the skeletal muscle insulin growth factor 1 receptor can provide valuable information regarding mechanisms controlling muscle protein metabolism. Immunocolloidal gold labeling is a powerful immunocytochemistry procedure for detecting antigens at the ultrastructural level, providing plausible biological markers of cell and tissue adaptations to stimuli. The intent here was to employ immunogold labeling to identify, localize, and quantify the insulin growth factor receptor-I (IGF-IR) in elderly human skeletal muscle. Needle biopsy specimens of the leg vastus lateralis muscle were fixed with 1% glutaraldehyde and 4% paraformaldehyde, dehydrated, and embedded in LR white resin. Pilot experiments were carried out to establish optimal dilutions of primary and secondary antibodies and to employ controls to establish staining specificity. The 6 nm gold particles were first evident when viewed at transmission electron microscopy (TEM) magnifications at 54,000x and clearest at 71,000x. Consistencies were noted in the staining patterns, with the majority of particles lying in proximity to the myofilaments. Gold particles were also found randomly along the outer membrane of the sarcolemma and the mitochondrial membranes. National Institutes of Health (NIH) Image 1.55 version software was used to measure receptor density (NIH, Bethesda, Md., USA). It appears that immunogold labeling of postembedded tissue samples is a sensitive method for detecting IGF-I receptors at the ultrastructural level.     

A comparison of structural and mechanical properties in cancellous bone from the femoral head and acetabulum

Mechanical interlock obtained by penetration of bone cement into cancellous bone is critical to the success of cemented total hip replacement (THR). Although acetabular component loosening is an important mode of THR failure, the properties of acetabular cancellous bone relevant to cement penetration are not well characterized. Bone biopsies (9 mm diameter, 10 mm long) were taken from the articular surfaces of the acetabulum and femoral head during total hip replacement. After mechanical and chemical defatting the two groups of bone specimens were characterized using flow measurement, mechanical testing and finally serial sectioning and three-dimensional computer reconstruction. The mean permeabilities of the acetabular group (1.064 x 10(-10) m2) and femoral group (1.155 x 10(-10) m2) were calculated from the flow measurements, which used saline solution and a static pressure of 9.8 kPa. The mean Young's modulus, measured non-destructively, was 47.4 MPa for the femoral group and 116.4 MPa for the acetabular group. Three-dimensional computer reconstruction of the specimens showed no significant differences in connectivity and porosity between the groups. Results obtained using femoral head cancellous bone to investigate bone cement penetration and fixation are directly relevant to fixation in the acetabulum.     

Image restoration for TV‐scan moving images acquired through a semiconductor backscattered electron detector

A semiconductor backscattered electron (BSE) detector has become popular in scanning electron microscopy session. However, detectors of semiconductor type have a serious disadvantage on the frequency characteristics. As a result, fast scan (e.g. TV‐scan) BSE image should be blurred remarkably. It is the purpose of this study to restore this degradation by using digital image processing technology. In order to improve it practically, we have to settle several problems, such as noise, undesirable processing artifacts, and ease of use. Image processing techniques in an impromptu manner like a conventional mask processing are unhelpful for this study, because a complicated degradation of output signal affects severely the phase response as well as the amplitude response of our SEM system. Hence, based on the characteristics of an SEM signal obtained from the semiconductor BSE detector, a proper inverse filter in Fourier domain is designed successfully. Finally, the inverse filter is converted to a special convolution mask, which is skillfully designed, and applied for TV‐scan moving BSE images. The improved BSE image is very effective in the work for finding important objects. SCANNING 31: 229–235, 2009. © 2010 Wiley Periodicals, Inc.     

Systemically alendronate was incorporated into dental tissues but did not cause morphological or mechanical changes in rats teeth

This study evaluated the effect of the systemic use of sodium alendronate in rats in vivo. Forty‐five Wistar rats aged 36 to 42 days and weighing 200 to 230 g were randomly assigned to a control group (n = 20), which received distilled water, and an experimental group (n = 25), which received 2 weekly doses of 1 mg/kg of chemically pure sodium alendronate. The animals were killed after 60 days of treatment. The tibias were removed for analysis of bone mineral density by dual‐energy X‐ray absorptiometry (DXA). Then, the maxillary incisors were extracted for analysis of the mineralized dental tissues using fluorescence spectroscopy (FS), scanning electron microscopy (SEM), bright field microscopy (BFM), and cross‐sectional microhardness (CSMH) testing. DXA and CSMH data were subjected to statistical analysis by Kruskal‐Wallis test (5% significance level). The experimental group presented higher bone mineral density than the control group by DXA. FS analysis revealed presence of alendronate in the mineralized dental tissues of the specimens of the experimental group. Significant morphological differences were not found by SEM and BFM. Enamel and dentin (100 and 300 μm from the dentinoenamel junction) CSMH data did not show significant difference between the control and experimental groups. Based on the obtained results, we conclude that while alendronate increased the bone mineral density and was incorporated into the mineralized dental tissues it did not cause significant alterations in the morphology and microhardness of rat incisor enamel and dentin. Microsc. Res. Tech. 75:1265–1271, 2012. © 2012 Wiley Periodicals, Inc.     

Young's modulus repeatability assessment using cycling compression loading on cancellous bone

For various applications, precision of the Young's modulus of cancellous bone specimens is needed. However, measurement variability is rarely given. The aim of this study was to assess the Young's modulus repeatability using a uniaxial cyclic compression protocol on embedded specimens of human cancellous bone. Twelve specimens from 12 human calcanei were considered. The specimens were first defatted and then 1 or 2 mm at the ends were embedded in an epoxy resin. The compression experiment consists in applying 20 compressive cycles between 0.2 per cent and 0.6 per cent strain with a 2 Hz loading frequency. The coefficient of variation of the current protocol was found to be 1.2 percent. This protocol showed variability similar to the end-cap technique (considered as a reference). It can be applied on porous specimen (especially human bone) and requires minimal bone length to limit end-artifact variability. The current method could be applied in association with noninvasive measurements (such as ultrasound) with full compatibility. This possibility opens the way for bone damage follow-up based on Young's modulus monitoring.     

Zinc as an essential trace element in the acceleration of matrix vesicles-mediated mineral deposition

BACKGROUND: Zinc (Zn) has a potent stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption. PURPOSE: The effect of Zn on the function of matrix vesicles (MVs) remains controversial. The purpose of this study was to investigate the effect of Zn on alkaline phosphatase (ALP) activity of osteoblasts and in the initial biological MVs‐mediated mineral deposition. STUDY DESIGN: Osteoblasts were treated with varying concentrations of Zn dissolved in culture medium. After three, five, and seven days of culture, ALP activity was assayed. For the detection of a low level of calcium concentration in MVs, X‐ray fluorescence (XRF) analyses were applied. The effect of Zn for the transformation of calcium phosphate was analyzed using a scanning electron microscope fitted with an energy dispersive X‐ray microanalysis (EDX) system. RESULTS: The ALP activity of osteoblasts in culture medium supplemented with 1 × 10^?5M of Zn was significantly increased at both five and seven days. XRF data demonstrated higher levels of calcium concentration over time in the Zn‐supplemented group. EDX data showed that mineral deposits beginning on day 3 were transformed from whitlockite to calcium phosphate near hydroxyapatite, and that Zn accelerated this transformation. CONCLUSIONS: The proper concentration of Zn increased the ALP activity of osteoblasts after five and seven days of incubation. The present XRF and EDX data suggest that the increase of mineral deposition with Zn exposure for one to five days might be mediated by the activation of ALP and calcium‐binding proteins. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.     

Elemental distributions in femoral bone of rat under osteoporosis preventive treatments

One of the abnormalities of bone architecture is osteoporosis as occurring in post‐menopausal women. Especially long bones, such as femur, become more fragile and more prone to fracture. The efficiency of several osteoporosis preventative treatments based on oestrogen and progestin in bone structure and mineral recovery was studied using ovariectomized Wistar rats as an osteoporosis experimental model. Diagonal cross‐sections of the proximal epiphysis of femoral bones were analysed using nuclear microscopy techniques in order to map and determine the concentration profiles of P, Ca, S, Fe and Zn from the epiphysis to diaphysis and across the cortical and trabecular bone structures. In control animals (not ovariectomized), the S and Zn contents significantly characterized differences between cortical and trabecular bone structures, whereas P and Ca showed increased gradients from the epiphyseal region to the diaphysis. After ovariectomy the differences observed were differential according to the type of hormonal supplementation. A significant decrease in P and Ca contents and depletion of minor and trace minerals, such as S, Fe and Zn, were found for both cortical and trabecular bone structures after ovariectomy relative to controls. Bone mineral contents were reversed to control levels by synthetic oestrogen supplementation, and combined oestrogen and progesterone treatment. Recovery was more evident in the femoral epiphysis and neck than in the diaphysis. The use of oestrogen alone did not lead to bone recovery after ovariectomy. Alterations in bone mineral composition observed for animals receiving synthetic oestrogen and combined oestrogen and progesterone supplement might reflect beneficial structural changes in critical regions of long bones, mostly affected in post‐menopausal osteoporosis.     

Calcium silicate cement‐induced remineralisation of totally demineralised dentine in comparison with glass ionomer cement: tetracycline labelling and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy, in combination with tetracycline labelling, was used to observe the remineralising potentials of a calcium silicate‐based restorative material (Biodentine^TM) and a glass ionomer cement (GIC:?Fuji?IX) on totally demineralised dentine. Forty demineralised dentine discs were stored with either cement in three different solutions: phosphate buffered saline (PBS) with tetracycline, phosphate‐free tetracycline, and tetracycline‐free PBS. Additional samples of demineralised dentine were stored alone in the first solution. After 8‐week storage at 37 °C, dentine samples were imaged using two‐photon fluorescence microscopy and Raman spectroscopy. Samples were later embedded in PMMA and polished block surfaces studied by 20 kV BSE imaging in an SEM to study variations in mineral concentration. The highest fluorescence intensity was exhibited by the dentine stored with Biodentine^TM in the PBS/tetracycline solution. These samples also showed microscopic features of matrix remineralisation including a mineralisation front and intra‐ and intertubular mineralisation. In the other solutions, dentine exhibited much weaker fluorescence with none of these features detectable. Raman spectra confirmed the formation of calcium phosphate mineral with Raman peaks similar to apatite, while no mineral formation was detected in the dentine stored in cement‐free or PBS‐free media, or with GIC. It could therefore be concluded that Biodentine^TM induced calcium phosphate mineral formation within the dentine matrix when stored in phosphate‐rich media, which was selectively detectable using the tetracycline labelling.     

Double-layer coating for high-resolution low-temperature scanning electron microscopy

Specimen damage caused by mass loss due to electron beam irradiation is a major limitation in low-temperature scanning electron microscopy of bulk specimens. At high primary magnifications (e.g. 100 000x) a hydrated sample is usually severely damaged after one slow scan (about 3000 e^— nm^—2). The consequences of this beam damage are significantly reduced by coating the frozen-hydrated sample with a 5–10-nm-thick carbon layer. Since this layer covers up surface details, the sample is first unidirectionally shadowed with a thin heavy metal layer (e.g. 2 nm of platinum) that is in close contact with the biological surface (double layer coating). This heavy metal layer can be visualized in field-emission scanning electron microscopy with the material-dependent backscattered electron signal. The method allows for routine observation of large frozen-hydrated samples. By use of an in-lens field-emission SEM and a sensitive backscattered electron detector, structural information comparable to that obtained with the transmission electron microscopy freeze-fracture replica technique can be achieved.     

Can X-ray spectrum imaging replace backscattered electrons for compositional contrast in the scanning electron microscope?

The high throughput of the silicon drift detector energy dispersive X-ray spectrometer (SDD-EDS) enables X-ray spectrum imaging (XSI) in the scanning electron microscope to be performed in frame times of 10-100?s, the typical time needed to record a high-quality backscattered electron (BSE) image. These short-duration XSIs can reveal all elements, except H, He, and Li, present as major constituents, defined as 0.1 mass fraction (10 wt%) or higher, as well as minor constituents in the range 0.01-0.1 mass fraction, depending on the particular composition and possible interferences. Although BSEs have a greater abundance by a factor of 100 compared with characteristic X-rays, the strong compositional contrast in element-specific X-ray maps enables XSI mapping to compete with BSE imaging to reveal compositional features. Differences in the fraction of the interaction volume sampled by the BSE and X-ray signals lead to more delocalization of the X-ray signal at abrupt compositional boundaries, resulting in poorer spatial resolution. Improved resolution in X-ray elemental maps occurs for the case of a small feature composed of intermediate to high atomic number elements embedded in a matrix of lower atomic number elements. XSI imaging strongly complements BSE imaging, and the SDD-EDS technology enables an efficient combined BSE-XSI measurement strategy that maximizes the compositional information. If 10?s or more are available for the measurement of an area of interest, the analyst should always record the combined BSE-XSI information to gain the advantages of both measures of compositional contrast.