Fabrication of specimens of metamorphic magnetite crystals for field ion microscopy and atom probe microanalysis.

Field ion specimens have been successfully fabricated from samples of metamorphic magnetite crystals (Fe3O4) extracted from a polymetamorphosed, granulite-facies marble with the use of a focused ion beam. These magnetite crystals contain nanometer-scale, disk-shaped inclusions making this magnetite particularly attractive for investigating the capabilities of atom probe field ion microscopy (APFIM) for geological materials. Field ion microscope images of these magnetite crystals were obtained in which the observed size and morphology of the precipitates agree with previous results. Samples were analyzed in the energy compensated optical position-sensitive atom probe. Mass spectra were obtained in which peaks for singly ionized 16O, 56Fe and 56FeO and doubly ionized 54Fe, 56Fe and 57Fe peaks were fully resolved. Manganese and aluminum were observed in a limited analysis of a precipitate in an energy compensated position sensitive atom probe.     

Diffraction patterns of artificial two-dimensional crystals synthesized in situ in an environmental scanning transmission electron microscope

In this study, we demonstrated the use of electron‐beam‐induced deposition for synthesis of artificial two‐dimensional crystals with an in situ scanning transmission electron microscope. The structures were deposited from W(CO)₆ in an environmental scanning transmission electron microscope on a 30‐nm‐thick Si₃N₄ substrate. We present clear electron beam diffraction patterns taken from those structures. The distance between the diffraction peaks corresponded to the dot spacing in the self‐made surface crystal. We propose using these arrays of dots as anchor points for making artificial crystals for diffraction analysis of weakly scattering or beam‐sensitive molecules such as proteins.     

温度对液晶5CB拉曼光谱影响的初探

随着对光折变材料的深入研究,液晶的光折变性能受到了广泛的关注。通过测量不同温度下的向列相液晶5CB的拉曼散射光谱发现,随着温度的变化,5CB分子的几个主要振动峰基本上没有发生移动,只是在散射强度上有一些变化,各向同性相和向列相的拉曼散射强度变化比较明显。     

Cryopreparation of mammalian tissue for X-ray microanalysis in STEM

A cryopreparation technique for studies of ultrastructure and distribution of diffusible elements in biological tissue is described. Electron microscopical contrast and characteristic X-ray spectra are found to be poor in completely frozen-hydrated ultrathin cryosections of fresh chemically untreated tissue. Both STEM contrast and detection of characteristic X-rays are enhanced by careful freeze-drying in the microscope. Although the ultrastructure is affected by ice crystals, intracellular compartments can be identified by STEM without staining and studied by X-ray microanalysis.     

X-ray spectral simulation and experimental detection of phosphorus segregation to grain boundaries in the presence of molybdenum.

A fundamental limitation of X-ray energy-dispersive (EDS) spectrometry is the detection and quantitative analysis of characteristic X-rays from one element, in the presence of an overlapping peak from another element. This problem is particularly acute when the unambiguous detection of one element is crucial to ensuring the mechanical performance of the material, such as the presence of an embrittling species. This paper addresses the specific problem of defining the conditions of specimen composition and thickness under which phosphorus can be detected in the presence of molybdenum, since the Mo L1 and the P Kalpha elemental peaks are separated by 2eV. This separation is significantly below the resolution limit of X-ray spectrometry in the electron microscope. Simulations of the X-ray spectra from low-alloy steels have been performed via Desk-Top Spectrum Analyzer and compared with experimental measurements on a field-emission gun VG HB 603 dedicated scanning transmission electron microscope.     

Direct imaging of paracrystalline phospholipid structure in the electron microscope

A long chain amphiphilic molecule—the phospholipid 1,2-dihexadecyl sn glycerophosphoethanolamine—has been crystallized epitaxially so that the interlamellar molecular periodicity is parallel to the substrate and hence normal to the electron beam in the electron microscope. This has permitted the direct resolution of the 55·6 Å lamellae in unstained crystals at room temperature. The lattice images have shown the presence of line dislocations and lenticular cracks in the crystals. Of significance to their biological properties is that the lattice is undulating with a periodicity of 0·1–0·5 μm. This would also account for the difficulties encountered by X-ray and electron diffraction techniques when examining these crystals.     

Energy-dispersive X-ray microanalysis of aqueous biologic samples on bulk supports

The present investigation describes a modification of the liquid droplet technique that allows for the quantitative elemental analysis of small volumes (< 100 picoliters) of aqueous biologic samples using a scanning transmission electron microscope (Philips 400 HTG-STEM) equipped with an EDAX energy dispersive detector. Aliquots of samples and standards were micropipetted onto solid beryllium supports under paraffin oil. The oil was washed with organic solvents and the samples frozen and freeze-dried. The samples were excited in a Philips 400-HTG-STEM by scanning a 1-μm, 20-kV electron beam over the surface of the droplets, and the X-ray spectra were collected. Measured X-ray intensities in characteristic peaks were found to be linearly related to the concentration of various elements in the sample. This work demonstrates the feasibility of performing quantitative elemental analysis of minute samples and cells in a scanning transmission electron microscope equipped with an energy dispersive X-ray detector.     

The detection of low-intensity peaks in energy dispersive x-ray spectra from particles

A critical step in the processing of energy dispersive (EDS) x-ray spectra from the automated scanning electron microscopy (ASEM) analysis of particles is the detection and identification of elemental peaks. Since there are often several hundred to several thousand spectra for each ASEM analysis, it is important that this step operate rapidly and with a minimum of interaction between analyst and the program. For peaks with large peak-to-background (P/B) ratios, most peak-find or peak-fitting methods do a reasonable job even when the spectra have low signal-to-noise (S/N) ratios. The detection of peaks with small P/B ratios is much more problematical. Peak identification and fitting procedures may not work well on low-intensity peaks, particularly in spectra with low S/N ratios. In this study, three procedures for identifying x-ray peaks, with small P/B ratios in spectra with varying S/N ratios, were evaluated. The first procedure was the identification of peaks by human analysts. The results from the analysts were then used to set a benchmark for the performance of two computer-based procedures that included three different qualitative peak identification methods, and one quantitative analysis procedure. The success of the qualitative methods in finding small peaks varied widely. In general, the quantitative analysis procedure performed as well as the best human analysts and was better than the qualitative methods.     

Irregular snow crystals: structural features as revealed by low temperature scanning electron microscopy

For nearly 50 years, investigators using light microscopy have vaguely alluded to a unique type of snow crystal that has become known as an irregular snow crystal. However, the limited resolution and depth-of-field of the light microscope has prevented investigators from characterizing these crystals. In this study, a field-emission scanning electron microscope, equipped with a cold stage, was used to document the structural features, physical associations, and atmospheric metamorphosis of irregular snow crystals. The crystals appear as irregular hexagons, measuring 60 to 90 mm across, when viewed from the a-axis. Their length (c-axis) rarely exceeds the diameter. The irregular crystals are occasionally found as secondary particles on other larger forms of snow crystals; however, they most frequently occur in aggregates consisting of more than 100 irregular crystals. In the aggregates, the irregular crystals have their axes oriented parallel to one another and, collectively, tend to form columnar structures. Occasionally, these columnar structures exhibit rounded faces along one side, suggesting atmospheric metamorphoses during formation and descent. In extreme cases of metamorphoses, the aggregates would be difficult to distinguish from graupel. Frost, consisting of irregular crystals, has also been encountered, suggesting that atmospheric conditions that favor their growth can also occur terrestrially.     

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.     

The electron microscope: The British contribution

The unpredictable development of the electron microscope is reviewed in the context of the 150th Anniversary of the Royal Microscopical Society. Abbe convinced himself that an electron microscope could never be constructed. Later, J. J. Thomson established the minute corpuscular nature of the electron and Rutherford realized that such beams can reveal the nature of the atom; his student H. G. Moseley invented electron probe analysis, while looking for X-ray spectra, but the technique did not seem very practical. G. P. Thomson invented the electron diffraction camera, but it was of limited use without an electron lens. In 1931, Knoll and Ruska built a two-stage TEM with magnetic lenses and understood where Abbe went wrong. Although Ernst Ruska surpassed the resolving power of the light microscope in 1933, the first commercial TEM was manufactured in the U.K., leading to many further commercial developments. The realization of a digital computer with a stored program, now indispensible in microscopy, was also achieved first in the U.K. Gabor's electron beam holography, invented in Rugby in 1949, was not a challenge to the conventional TEM at first, but recently atomic resolution holography has been achieved in Abbe's native country, using a digital computer to process the hologram, ironically making use of the very principles laid down by Abbe in the last century for the light microscope.     

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.     

Environmental scanning electron microscopy of marine aggregates

Marine aggregates were examined for the first time in the hydrated state using an environmental scanning electron microscope (ESEM). Sample preparation consisted of fixation followed by rinsing with distilled water to remove excess salts and fixative. Aggregates were continuously observed at resolutions comparable to conventional scanning electron microscopy through stages of hydration, from completely immersed to desiccated. Because no metallic coating is required, energy-dispersive X-ray spectroscopy (EDXS) can be used to analyse rapidly constituent elements occurring at low concentrations with no spectral interference. Subtle differences in mineral particles were seen in both EDXS spectra and in direct observation of relative hydration, reflecting apparent differences in mineralogy. ESEM enabled examination of effects of desiccation and rehydration on individual particles composed primarily of hydrated polymer and eliminated dehydration artefacts in delicate organisms.     

Separation of emission lines of multiply charged ions against background of a very noisy spectrum of femtosecond laser plasma

At high intensities of interaction of femtosecond laser pulses with various targets, numerous high-energy particles emitted from plasma in all directions lead to the appearance of parasitic peaks in the X-ray spectra of femtosecond laser plasma (FLP) during detection of these peaks with a detector based on a charge-coupled device (CCD). The algorithm proposed in this study allows identification of parasitic peaks in a series of experimental spectra, which are recorded for each pixel of the CCD detector independently of other pixels, and removal of these peaks. The algorithm is able to detect peaks in the presence of both variations in the intensities of spectral lines and very intense noise. The efficiency of the algorithm has been confirmed via testing with the use of a series of model spectra and spectra of Fe and Al ions recorded in experiments devoted to studies of the interaction of ultrahigh-intensity and ultrashort laser pulses with solid targets.     

Reduction of charging in protein electron cryomicroscopy

Charging causes a loss of resolution in electron cryomicroscopy with biological specimens prepared without a continuous carbon support film. Thin conductive films were deposited onto catalase crystals prepared across holes using ion-beam sputtering and thermal evaporation and evaluated for the effectiveness of charge reduction. Deposits applied by ion-beam sputtering reduced charging but concurrently resulted in structural damage. Coatings applied by thermal evaporation also reduced charging, and preserved the specimen structure beyond 5 Å resolution as judged from electron diffraction patterns and images of glucose-embedded catalase crystals tilted to 45° in the microscope. This study demonstrates for the first time the feasibility of obtaining high-resolution data from unstained, unsupported protein crystals with a conductive surface coating.     

Image comparisons of snow and ice crystals photographed by light (video) microscopy and low-temperature scanning electron microscopy

Light (video) microscopy and low-temperature scanning electron microscopy (SEM) were used to examine and record images of identical precipitated and metamorphosed snow crystals as well as glacial ice grains. Collection procedures enabled numerous samples from distant locations to be shipped to a laboratory for storage and/or observation. The frozen samples could be imaged with a video microscope in the laboratory at ambient temperatures or with the low-temperature SEM. Stereo images obtained by video microscopy or low-temperature SEM greatly increased the ease of structural interpretations. The preparation procedures that were used for low-temperature SEM did not result in sublimation or melting. However, this technique did provide far greater resolution and depth of focus over that of the video microscope. The advantage of resolution was especially evident when examining the small particles associated with rime and graupel (snow crystals encumbered with frozen water droplets), whereas the greater depth of focus provided clearer photographs of large crystals such as depth hoar, and ice. Because the SEM images contained only surface information while the video images were frequently confounded by surface and internal information, the SEM images also clarified the structural features of depth hoar crystals and ice grains. Low-temperature SEM appears to have considerable promise for future investigations of snow and ice.     

Determining the orientations of ice crystals using electron backscatter patterns

Knowing the orientations of the ice crystals in a polycrystalline aggregate is essential for understanding and modeling the flow of naturally occurring ice. Here we show, for the first time, that the orientation of crystals in polycrystalline ice can be determined with a higher angular and spatial resolution and more rapidly than any currently used method by using electron backscatter patterns (EBSPs) in a cold-stage equipped scanning electron microscope. We also present an orientation image map constructed from EBSPs, and discuss possible applications of the technique for ice. The results indicate that obtaining EBSPs and orientation images from other frozen water-containing materials, such as clathrate hydrates, may also be possible.     

Electron diffraction from micro- and nanoparticles of hydroxyapatite

Hydroxyapatite (HAP) obtained from aqueous solutions under different conditions has been examined by high-resolution transmission electron microscopy (HRTEM) and electron diffraction, including selected-area electron diffraction (SAED) and microdiffraction. A Philips CM300 field-emission gun electron microscope with a Schottky W/ZrO field-emission tip and a spherical aberration constant of 0.65 mm was used at 300 kV. The HAP crystals had different sizes, ranging from a few nanometres to a few micrometres. Single-crystal diffraction patterns have been obtained from the largest microcrystals using the conventional SAED technique. Assemblies of nanoparticles gave only broad diffuse rings. Nevertheless, microdiffraction with electron microprobes 3.5–10 nm in diameter clearly indicated the crystalline character of the nanoparticles in these assemblies. Experimental HRTEM images, Fourier transforms and calculated images exhibited the fine structure of the HAP crystals.     

Coherent Bremsstrahlung effect observed during STEM analysis of dopant distribution in silicon devices using large area silicon drift EDX detectors and high brightness electron source

In this paper, during dopant analysis of silicon devices, we have observed a phenomenon generally neglected in EDX analysis: the coherent Bremsstrahlung (CB). We discussed the reason why and came to the conclusion that the analytical TEM used for these experiments presents a configuration and performances, which makes this equipment very sensitive to the CB effect. This is due to large collection solid angle and high counting rate of the four silicon drift EDX detectors (SDD), a high brightness electron source providing large probe current and moreover a geometry favorable to on axis crystal observations. We analyzed silicon devices containing Si [110] and Si [100] crystal areas at different energies (80–120–200 keV). We also observed relaxed SiGe (27 and 40 at% of Ge). The CB effect, whose intensity is maximum near zone axis beam alignment, manifests as characteristic broad peaks present in the X-ray spectrum background. The peak energies are predicted by a simple formula deduced for the CB models found in the literature and that we present simply. We evaluate also the CB peak intensities and discuss the importance of this effect on the detection and quantification traces of impurities. The CB peaks also give information on the analyzed crystal structure (measurement of the periodicity along the zone axis) and allow, in every particular experiment or system, to determine the median take off angle of the EDX detectors.     

Advances in ALCHEMI analysis

The ALCHEMI method for locating the sites of foreign atoms within crystals is known to be sensitive to the delocalized emission of X-rays. This can result in large errors in some cases through differences in delocalization for different excitations or by error amplification in the ratio method of analysis. An alternative approach to the analysis of ALCHEMI data, using multivariate statistical analysis, is extended to the case of multiple impurities. Initial results from zone-axis channelling experiments for a Yb-doped zirconolite (CaZrTi₂O₇) are shown to confirm the improved accuracy of this method, especially for axial orientations. Data were collected using a 400-keV analytical electron microscope fitted with an intrinsic Ge X-ray detector. The potential advantages for ALCHEMI analysis of Ge detectors are considered.