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.     

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).     

The use of helium gas to reduce beam scattering in high vapour pressure scanning electron microscopy applications

Both image quality and the accuracy of x-ray analysis invariable pressure scanning electron microscopes (VPSEMs) are often limited by the spread of the primary electronbeam due to scattering by the introduced gas. The degree of electron scattering depends partly on the atomic number Z of the gas, and the use of a low Z gas such as helium should reduce beam scattering and enhance image quality. Using anuncoated test sample of copper iron sulphide inclusions in calcium fluorite, we show that the reduction in beam scatter produced by helium is more than sufficient to compensate for its reduced efficiency of charge neutralisation. The relative insensitivity to pressure of x-ray measurements in a helium atmosphere compared with air, and the consequent ability to work over a wider range of working distances, pressures, and voltages, make helium potentially the gas of choice for many routine VPSEM applications.     

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.     

A proposal for statistical evaluation of the detection of gunshot residues on a suspect

The possibility of accidental contamination of a suspect by gunshot residues (GSRs) is considered. If two hypotheses are taken into account ("the suspect has shot a firearm" and "the suspect has not shot a firearm"), the likelihood ratio of the conditional probabilities of finding a number n of GSRs is defined. Choosing two Poisson distributions, the parameter lambda of the first one coincides with the mean number of GSRs that can be found on a firearm shooter, while the parameter mu of the second one is the mean number of GSRs that can be found on a nonshooter. In this scenario, the likelihood ratio of the conditional probabilities of finding a number n of GSRs in the two hypotheses can be easily calculated. The evaluation of the two parameters lambda and mu and of the goodness of the two probability distributions is performed by using different sets of data: "exclusive" lead-antimony-barium GSRs have been detected in two populations of 31 and 28 police officers at diverse fixed times since firearm practice, and in a population of 81 police officers who stated that they had not handled firearms for almost 1 month. The results show that the Poisson distributions well fit the data for both shooters and nonshooters, and that the probability of detection of two or more GSRs is normally greater if the suspect has shot firearms.     

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.     

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.     

Microstructure and mineral composition of dental enamel of permanent and deciduous teeth

Purpose: This study evaluated and compared in vitro the microstructure and mineral composition of permanent and deciduous teeth's dental enamel. Methods: Sound third molars (n = 12) and second primary molars (n = 12) were selected and randomly assigned to the following groups, according to the analysis method performed (n = 4): Scanning electron microscopy (SEM), X‐Ray diffraction (XRD) and Energy dispersive X‐ray spectrometer (EDS). Qualitative and quantitative comparisons of the dental enamel were done. The microscopic findings were analyzed statistically by a nonparametric test (Kruskal‐Wallis). The measurements of the prisms number and thickness were done in SEM photomicrographs. The relative amounts of calcium (Ca) and phosphorus (P) were determined by EDS investigation. Chemical phases present in both types of teeth were observed by the XRD analysis. Results: The mean thickness measurements observed in the deciduous teeth enamel was 1.14 mm and in the permanent teeth enamel was 2.58 mm. The mean rod head diameter in deciduous teeth was statistically similar to that of permanent teeth enamel, and a slightly decrease from the outer enamel surface to the region next to the enamel‐dentine junction was assessed. The numerical density of enamel rods was higher in the deciduous teeth, mainly near EDJ, that showed statistically significant difference. The percentage of Ca and P was higher in the permanent teeth enamel. Conclusions: The primary enamel structure showed a lower level of Ca and P, thinner thickness and higher numerical density of rods. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Independent trafficking of flavocytochrome b558 and myeloperoxidase to phagosomes during phagocytosis visualised by energy‐filtering and energy‐dispersive spectroscopy‐scanning transmission electron microscopy

When polymorphonuclear leukocytes (PMNs) phagocytose opsonised zymosan particles (OPZ), free radicals and reactive oxygen species (ROS) are formed in the phagosomes. ROS production is mediated by NADPH oxidase (Nox), which transfers electrons in converting oxygen to superoxide (O₂^?). Nox‐generated O₂^? is rapidly converted to other ROS. Free radical‐forming secretory vesicles containing the Nox redox center flavocytochrome b558, a membrane protein, and azurophil granules with packaged myeloperoxidase (MPO) have been described. Presuming the probable fusion of these vesicular and granular organelles with phagosomes, the translation process of the enzymes was investigated using energy‐filtering and energy‐dispersive spectroscopy‐scanning transmission electron microscopy. In this work, the primary method for imaging cerium (Ce) ions demonstrated the localisation of H₂O₂ generated by phagocytosing PMNs. The MPO activity of the same PMNs was continuously monitored using 0.1% 3,3′‐diaminobenzidine‐tetrahydrochloride (DAB) and 0.01% H₂O₂. A detailed view of these vesicular and granular structures was created by overlaying each electron micrograph with pseudocolors: blue for Ce and green for nitrogen (N).     

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.     

Investigation of foreign substances in food

Scanning electron microscopy (SEM) together with energy-dispersive spectroscopy (EDS) and Fourier transform infrared spectroscopy (FTIR) were used to investigate foreign substances from seven categories of foreign substances in food. (1) Naturally occurring foreign substances--Using FTIR, a foreign substance was identified as a natural resin probably from the product. (2) Foreign substances introduced during food processing. Scanning electron microscopy-EDS was used to identify a foreign material found on surf clams as calcium phosphate from a product/ingredient interaction. Using SEM-EDS, a crystalline material in a meat product was identified as calcium salts of chloride and phosphate. Fourier transform infrared spectroscopy was used to identify foreign material that clogged an aerosol valve as chipboard. Using SEM-EDS, the metal in the heel of a glass bottle was identified as copper sulfide-containing metal inclusion. (3) Insects, reptiles, and rodents--Scanning electron microscopy was used to determine that a mouse found in food was not processed with the food, but entered the container after it left the factory. (4) Glass fragments--Glass from various sources can be distinguished from one another using SEM-EDS either by the level of the major elements in glass or by the presence of elements in one glass, but not in another. (5) Glass-like particles--Using SEM-EDS, glass-like particles found on beets were determined to be a fatty acid. (6) Metal foreign objects--Using SEM-EDS, metals from a variety of sources can be easily distinguished. For example, a tin-soldered container can be distinguished from a lead-soldered can. Using SEM-EDS, the metal fiber found on the bottom of a two-piece can likely enter the can during the final stage of the manufacture of the drawn and ironed food can. (7) Drug capsule identification--Fourier transform infrared spectroscopy was used to determine that a pill found in food was ibuprofen.     

Chemical and morphological evaluation of enamel and dentin near cavities restored with conventional and zirconia modified glass ionomer subjected to erosion‐abrasion

Microenergy dispersive X‐ray fluorescence (μ‐EDXRF) spectroscopy and scanning electron microscopy (SEM) were used to test the hypothesis that zirconia modified glass ionomer cement (GIC) could improve resistance to erosion‐abrasion to a greater extent than conventional cement. Bovine enamel (n = 40) and dentin (n = 40) samples were prepared with cavities, filled with one of the two restorative materials (GIC: glass‐ionomer cement or ZrGIC: zirconia‐modified GIC). Furthermore, the samples were treated with abrasion‐saliva (AS) or abrasion‐erosion cycles (AE). Erosive cycles (immersion in orange juice, three times/day for a duration of 1 min over a 5 day period) and/or abrasive challenges (electric toothbrush, three times/day for a duration of 1 min over a 5 day period) were performed. Positive mineral variation (MV%) on the enamel after erosion‐abrasion was observed for both materials (p < 0.05), whereas a negative MV% on the dentin was observed for both materials and treatments (p < 0.05). The SEM images showed clear enamel loss after erosion‐abrasion treatment and material degradation was greater in GIC_AE compared to those of the other groups. Toothbrush abrasion showed a synergistic effect with erosion on substance loss of bovine enamel, dentin, GIC, and ZrGIC restorations. Zirconia addition to the GIC powder improved the resistance to abrasive‐erosive processes. The ZrGIC materials may find application as a restorative material due to improved resistance as well as in temporary restorations and fissure sealants.     

Ultrastructure and reproduction behaviour of single CHO-K1 cells exposed to near infrared femtosecond laser pulses

In the present work, the authors investigated ultrastructural changes as well as the reproduction behaviour of preselected single CHO-K1 cells exposed to 170 femtosecond laser pulses at different power output levels in comparison with cells outside the illumination volume. The ultrashort laser pulses were provided by an 80 MHz Ti:sapphire laser at 780 nm. The cells were scanned ten times with a scan rate of 1/16 s(-1). Single CHO-K1 cells exposed to low mean power of 2 mW revealed no significant changes in ultrastructure after laser exposure. In some cases, changes of mitochondria with slight disordering of cristae were found. Cytoplasm was filled with vesicles that seemed to be released from Golgi stacks. Cells irradiated with higher powers demonstrated more dramatic changes in ultrastructure. A considerable number of swollen mitochondria in conjunction with loss of cristae was observed. The main event of mitochondrial changes was the formation of electron dense bodies in the mitochondrial matrix. In addition, lumen of endoplasmatic reticulum was enlarged. Highest applied mean laser power of 12.5 mW lead to complete destruction of mitochondria and their transformation to electron dense structures containing membrane material. Compared with cell targets irradiated with 2 mW mean power, the release of vesicles from Golgi stacks seemed to be rather moderate. Cells localised outside the laser beam revealed no ultrastructural changes. Low mean laser power at 2 mW was unable to impair the reproduction behaviour of CHO-K1 cells. At higher laser power output levels, CHO-K1 cells started to delay cell division. At 12.5 mW, no cell division occurred. The obtained results may be helpful in recommending parameters for safe femtosecond laser microscopy of living specimens.     

Mistakes encountered during automatic peak identification of minor and trace constituents in electron‐excited energy dispersive X‐ray microanalysis

Automated peak identification in electron beam‐excited X‐ray microanalysis with energy dispersive X‐ray spectrometry has been shown to be subject to occasional mistakes even on well‐separated, high‐intensity peaks arising from major constituents (arbitrarily defined as a concentration, C, which exceeds a mass fraction of 0.1). The peak identification problem becomes even more problematic for constituents present at minor (0.01≤C≤0.1) and trace (C<0.01) levels. “Problem elements” subject to misidentification as major constituents are even more vulnerable to misidentification when present at low concentrations in the minor and trace ranges. Additional misidentifications attributed to trace elements include minor X‐ray family members associated with major constituents but not assigned properly, escape and coincidence peaks associated with major constituents, and false peaks owing to chance groupings of counts in spectra with poor counting statistics. A strategy for robust identification of minor and trace elements can be based on application of automatic peak identification with careful inspection of the results followed by multiple linear least‐squares peak fitting with complete peak references to systematically remove each identified major element from the spectrum before attempting to assign remaining peaks to minor and trace constituents. SCANNING 31: 91–101, 2009. Published by Wiley Periodicals, Inc.     

Backscattered electron SEM imaging of cells and determination of the information depth

Backscattered electron imaging of HT29 colon carcinoma cells in a scanning electron microscope was studied. Thin cell sections were placed on indium‐tin‐oxide‐coated glass slides, which is a promising substrate material for correlative light and electron microscopy. The ultrastructure of HT29 colon carcinoma cells was imaged without poststaining by exploiting the high chemical sensitivity of backscattered electrons. Optimum primary electron energies for backscattered electron imaging were determined which depend on the section thickness. Charging effects in the vicinity of the SiO₂ nanoparticles contained in cell sections could be clarified by placing cell sections on different substrates. Moreover, a method is presented for information depth determination of backscattered electrons which is based on the imaging of subsurface nanoparticles embedded by the cells.     

Chemical and crystallographic study of remineralized surface on initial carious enamel treated with Galla chinensis

To investigate the morphologic, chemical and crystallographic characters of remineralized surface on initial carious enamel treated with Galla chinensis, scanning electron microscopy equipped with energy dispersive analysis spectroscopy were used, and X‐ray microdiffraction (microzone XRD) was used for the first time to analyze in situ the microzone crystallite of remineralized surface on carious enamel. Bovine sound enamel slabs were demineralized to produce initial carious lesion in vitro. Then, the lesions were exposed to a pH‐cycling regime for 12 days of remineralization. Each daily cycle included 4×1 min applications with one of the three treatments: distilled and deionized water (DDW); 1 g/L NaF; 4 g/L G. chinensis extract (GCE). After the treatments, some rod‐like deposits and many irregular prominences were found on GCE‐treated enamel surface, and the intensities of Ca and P signals showed a tendency to increase; Ca:P ratio was significantly higher than that of DDW‐treated enamel. X‐ray microdiffraction showed hydroxyapatite was still the main component of GCE‐treated enamel, and the crystallinity was increased, the crystal lattice changed gently with decreased lattice parameter a. These results indicated the potential of GCE in promoting the remineralization of initial enamel carious lesions, and supported the previous hypothesis about GCE mechanism. Combined with the anti‐bacteria and demineralization inhibition properties of GCE, the natural G. chinensis may become one more promising agent for caries prevention. SCANNING 31: 236–245, 2009. © 2010 Wiley Periodicals, Inc.     

Microscopy and microanalysis of complex nanosized strengthening precipitates in new generation commercial Al–Cu–Li alloys

Precipitates (ppts) in new generation aluminum–lithium alloys (AA2099 and AA2199) were characterised using scanning and transmission electron microscopy and atom probe tomography. Results obtained on the following ppts are reported: Guinier–Preston zones, T₁ (Al₂CuLi), β’ (Al₃Zr) and δ’ (Al₃Li). The focus was placed on their composition and the presence of minor elements. X‐ray energy‐dispersive spectrometry in the electron microscopes and mass spectrometry in the atom probe microscope showed that T₁ ppts were enriched in zinc (Zn) and magnesium up to about 1.9 and 3.5 at.%, respectively. A concentration of 2.5 at.% Zn in the δ’ ppts was also measured. Unlike Li and copper, Zn in the T₁ ppts could not be detected using electron energy‐loss spectroscopy in the transmission electron microscope because of its too low concentration and the small sizes of these ppts. Indeed, Monte Carlo simulations of EEL spectra for the Zn L_2,3 edge showed that the signal‐to‐noise ratio was not high enough and that the detection limit was at least 2.5 at.%, depending on the probe current. Also, the simulation of X‐ray spectra confirmed that the detection limit was exceeded for the Zn K_α X‐ray line because the signal‐to‐noise ratio was high enough in that case, which is in agreement with our observations.