The benefits of using kinetic-energy information to aid ion discrimination in atom probe tomography (APT) are explored. Ion peak interferences in time-of-flight (TOF) mass spectra are categorized by difficulty of discrimination using TOF and kinetic-energy information. Several of these categories, which are intractable interferences when only TOF information is available, may be discriminated when kinetic-energy information also is available. Furthermore, many opportunities for removing noise from composition determinations and three-dimensional images are enabled. Modest kinetic-energy resolving powers (KRPs) of 10 or so should be sufficient to have a major impact on APT. With KRP of about 100, the energy deficits in voltage pulsing may be resolved to enable peak discrimination in straight-flight-path instruments. Real examples and simulated mass spectra are used to illustrate the benefits of kinetic-energy discrimination. Many of the conclusions are applicable generally in TOF spectroscopy. Current detectors do not provide the kinetic energy of incoming ions, but there are realistic prospects for building such detectors and these are discussed. A program to develop these detectors should be pursued.

As a major improvement in three-dimensional (3D) atom probe, the range of applicable material classes has recently been broadened by the establishment of laser-assisted atom probes (LA-3DAP). Meanwhile, measurements of materials of low conductivity, such as dielectrics, ceramics, and semiconductors, have widely been demonstrated. However, besides different evaporation probabilities, heterogeneous dielectric properties are expected to give rise to additional artifacts in the 3D volume reconstruction on which the method is based. In this article, these conceivable artifacts are discussed based on a numeric simulation of the field evaporation. Sample tips of layer- or precipitate-type geometry are considered. It is demonstrated that dielectric materials tend to behave similarly to metals of reduced critical evaporation field.

An Fe–1 at.% Cu model alloy was examined by atom probe (3DAP) and small-angle neutron scattering (SANS) to verify the accordance of the gained results. The Fe-Cu alloy was heat-treated for various times at 500°C, forming Cu-rich precipitates within the Fe matrix. The chemical compositions of the precipitates and matrix found by 3DAP were used to calculate the magnetic scattering contrast. Additionally, a magnetic moment of the precipitates that contain a significant amount of Fe was taken into account for the calculation of magnetic scattering contrast. This in turn is used for the evaluation of the magnetic scattering curves gained by SANS. Both the 3DAP data as well as the scattering curves were analyzed with regard to radius, number density, and volume fraction of the precipitates as a function of aging time. The results yielded by both techniques are in good agreement and correspond to the development of the hardness of the alloy. Minor differences can be related to the cluster search algorithm used for the analysis of the 3DAP data as well as Fe overestimation based on different field phases.

Complementary state-of-the-art optical, scanning electron, and X-ray microscopy techniques have been used to study the morphology of Apollo 11 lunar soil particles (10084-47). The combination of innovative lighting geometries with image processing of a through focal series of images has allowed us to obtain a unique collection of high-resolution light micrographs of these fascinating particles. Scanning electron microscopy (SEM) stereo-pair imaging has been exploited to illustrate some of the unique morphological properties of lunar regolith. In addition, for the first time, X-ray micrographs with submicron resolution have been taken of individual particles using X-ray ultramicroscopy (XuM). This SEM-based technique lends itself readily to the imaging of pores, cracks, and inclusions and allows the internal structure of an entire particle to be viewed. Rotational SEM and XuM movies have also been constructed from a series of images collected at sequential angles through 360°. These offer a new and insightful view of these complex particles providing size, shape, and spatial information on many of their internal features.

Nanosized incubational domain was observed in 10 at.% gadolinium-doped ceria (GDC) using high-resolution transmission electron microscopy. Dislocations were extensively observed in 10 at.% GDC instead of heavily doped 25 at.% GDC. By Fast Fourier Transform and Inverse Fast Fourier Transform analysis, it was noticed that the incubational domain existing in 10 at.% GDC has different lattice spacing and orientation from the neighboring ceria matrix. Furthermore, dislocations were usually observed in the interface region between the incubational domain and the ceria matrix. Based on experimental results, the formation mechanism of dislocation associated incubational domain in lightly gadolinium-doped ceria is rationalized.

The geometry and three-dimensional (3D) morphology of the ceria particles synthesized by spray pyrolysis (SP) from two different precursors—cerium acetate hydrate and cerium nitrate hydrate (CeA and CeN ceria particles)—were characterized by transmission electron microscopy and electron tomography. Results were compared with surface area measurements, confirming that the surface area of CeA ceria particles is twice as large as that of CeN ceria particles. This result was supported by 3D microstructural observations, which have revealed that CeA ceria particles contain open pores (connected to surfaces) and closed pores (embedded in particles), while CeN ceria particles only contained closed pores. This experimental result suggests that the type of porosity is controlled by the precursors and could be related to their melting temperature during the heating process in SP.

In characterization of metal nanoparticle doped spherical composites, the two-dimensional nature of transmission electron microscopy (TEM) images leads to ambiguities about the true location of the nanoparticles. Walking-in of simulated projections in comparison with actual TEM images leads to quantitative results such as location-dependent particle sizes and particle number density. This method takes advantage of the strength of fuzzy neural network computations via the human hunter-gatherer's visual system's evolved superiority while still allowing quantitative results by use of exact numerical simulations.

We report variable resolution fluctuation electron microscopy (VRFEM) measurements on Cu64.5Zr35.5 metallic glass acquired using scanning transmission electron microscopy nanodiffraction using coherent probes 0.8 to 11 nm in diameter. The VRFEM results show that medium range atomic order structure of Cu64.5Zr35.5 bulk metallic glass at the ∼1 nm scale has large fluctuations, but the structure becomes almost completely homogeneous at the 11 nm scale. We show that our experimental VRFEM data are consistent with two different models, the pair persistent model and the amorphous/nanocrystal composite model. We also report a new way to filter VRFEM data to eliminate the effect of specimen thickness gradient using high-angle annular dark field images as references.

Aberration-corrected scanning transmission electron microscopes (STEMs) provide sub-Angstrom lateral resolution; however, the large convergence angle greatly reduces the depth of field. For microscopes with a small depth of field, information outside of the focal plane quickly becomes blurred and less defined. It may not be possible to image some samples entirely in focus. Extended depth-of-field techniques, however, allow a single image, with all areas in focus, to be extracted from a series of images focused at a range of depths. In recent years, a variety of algorithmic approaches have been employed for bright-field optical microscopy. Here, we demonstrate that some established optical microscopy methods can also be applied to extend the ∼6 nm depth of focus of a 100 kV 5th-order aberration-corrected STEM (αmax = 33 mrad) to image Pt-Co nanoparticles on a thick vulcanized carbon support. These techniques allow us to automatically obtain a single image with all the particles in focus as well as a complimentary topography map.

Methods for creating speckle patterns on mouse arteries for use in deformation and strain field measurements in a stereomicroscope digital image correlation (DIC) system are described. Both fluorescent microsphere binding and ethidium bromide (EB) nuclear staining were used to generate high contrast, random patterns on mouse carotid arteries. To quantify the quality of each pattern, several metrics are used including (a) histogram distribution for each intensity pattern and (b) pixel-level variance in intensity pattern noise. Results demonstrate that both approaches provide sufficient pattern contrast for use in image-based methods to measure deformations in soft tissue. While fluorescent nuclear staining generates higher pixel-level intensity noise, this method provides better overall pattern quality (greater spatial uniformity and broader histogram) for automated DIC analysis when used at the appropriate magnification. Using recently developed theoretical predictions, estimates for the standard deviation in image-correlation-based displacements due to the measured intensity pattern variance are presented for fluorescent microsphere binding and EB nuclear staining patterns. Results confirm that both patterning approaches provide relatively small standard deviation in displacement measurements and hence are appropriate for measurement of deformations in small artery specimens.

Extracellular matrix (ECM) plays essential signaling and structural roles required for the proper function of cardiac valves. Cardiac valves initially form as jelly-like cushions, which must adapt to withstand the increased circulation hemodynamics associated with fetal development and birth. This increased biomechanical stability of the developing valves is largely imparted by ECM proteins, which form a highly organized fibrous meshwork. Since heart valve defects contribute to most congenital heart diseases, understanding valve development will provide insight into the pathogenesis of various congenital valve anomalies. Thus, the goal of this study is to describe the spatiotemporal deposition of fibrous ECM proteins during cardiac valve development. Chick embryonic and fetal atrioventricular and semilunar valves were examined by light, confocal, and transmission electron microscopy (TEM). Our data demonstrate that fibrous ECM proteins are deposited when the leaflets are adopting an elongated and compacted phenotype. A general pattern of increased fibrotic ECM deposition was detected in valve tissues. Also, each ECM protein examined displayed a unique pattern of organization, suggesting that regulation of fibrous protein deposition is complex and likely involves both genetic and mechanical factors. In addition, the TEM study revealed the presence of membrane protrusions from valvular endocardium, indicating a potential mechanism for mechanical force transduction.

Lizard erythrocytic viruses (LEVs) have previously been described in Lacerta monticola from Serra da Estrela, Portugal. Like other known erythrocytic viruses of heterothermic vertebrates, these viruses have never been adapted to cell cultures and remain uncharacterized at the molecular level. In this study, we made attempts to adapt the virus to cell cultures that resulted instead in the isolation of a previously undetected Ranavirus closely related to FV3. The Ranavirus was subsequently detected by polymerase chain reaction (PCR) in the blood of infected lizards using primers for a conserved portion of the Ranavirus major capsid protein gene. Electron microscopic study of the new Ranavirus disclosed, among other features, the presence of intranuclear viruses that may be related to an unrecognized intranuclear morphogenetic process. Attempts to detect by PCR a portion of the DNA polymerase gene of the LEV in infected lizard blood were successful. The recovered sequence had 65.2/69.4% nt/aa% homology with a previously detected sequence from a snake erythrocytic virus from Florida, which is ultrastructurally different from the studied LEV. These results further support the hypothesis that erythrocytic viruses are related to one another and may represent a new group of nucleo-cytoplasmic large deoxyriboviruses.

Procedures to study the molecular and morphological characteristics of microscopic organisms are often incompatible with each other. Therein, the realization of alternatives that make the characterization of these features compatible and simultaneously permit the deposition of the original material as a voucher sample into a reference collection is one of the foremost goals of biodiversity studies. In this study, we show that genomic extraction does not necessarily compromise the detailed study of the external morphology of microscopic organisms, and to do so, we used a group of aquatic mites (Acari, Hydrachnidia) as a test group. Hydrachnidia morphology is difficult to study when specimens have been stored in pure ethanol; however, proteinase K extraction leaves them flexible and easy to dissect, while, at the same time, maintaining all of their diagnostic features intact. Furthermore, autofluorescence is significantly enhanced after proteinase extraction. Our study was conducted with aquatic mites that were stored in absolute ethanol in the field and processed for DNA extraction using a Qiagen QIAamp minikit. Before and after molecular extraction, a laser scanning confocal microscopy morphological examination was carried out.

Previous efficacy studies found that many insecticides used by growers could be having an adverse effect on egg parasitoids (Telenomus podisi) developing in the eggs of the brown stink bug (Euschistus servus), while unhatched stink bugs experienced lower levels of mortality. One plausible explanation for this was that insecticides might enter parasitized eggs more readily via oviposition wounds. Parasitized E. servus eggs, as well as nonparasitized stink bug (Acrosternum hilare, E. servus, Murgantia histrionica, and Podisus maculiventris) eggs, were examined using electron microscopy. Egg response to perforation by a tungsten probe served as a control. Microscopy images depicted the chorion surface as characterized by a matrix of ridges and micropylar processes in a ring around the margin of the operculum. Observations of oviposition sites showed a “scab” formed where the ovipositor penetrated the chorion, and at sites penetrated by the probe. These formations appeared to be the result of fluids from inside the egg leaking out, drying, and hardening after oviposition or probe perforation, suggesting that the response was not due to substances secreted by the parasitoid. Further, no open wounds or holes were seen to increase the possibility of insecticides entering parasitized eggs.

High-resolution imaging and quantitative surface analysis of epicuticular waxes and epistomatal chambers of pine species were performed by field emission scanning electron microscopy and white light scanning interferometry. Both juvenile and adult needles were collected from the two-year-old seedlings of Pinus rigida and Pinus densiflora and subjected to surface observations. Epicuticular wax structures developed on the cuticle layer as well as in the epistomatal chambers and appeared to occlude the cavities in the two pine species. The stomata of P. densiflora were characterized by more distinctly raised rings around openings than P. rigida. The most common epicuticular wax structures of the two pine species included tubules with terminal openings and coiled rodlets. Wax platelets were deposited on epistomatal chambers. Either rodlets or tubules seemed to be longer and thicker in P. rigida than those in P. densiflora. White light scanning interferometry revealed quantitative surface profiles, demonstrating more ridged (ca. 4 μm high) stomatal apertures and nearly twofold deeper (ca. 20 μm deep) epistomatal chambers of P. densiflora than those of P. rigida. These results suggest that white light scanning interferometry can be applied to unravel the quantitative surface features of epicuticular sculptures on plant leaves.

Microscope and fiber-optic spectrophotometry of transmittance and backscattering both showed moss leaves to be capable of casting strong shadows, with a single leaf blocking approximately 90% of incident light from a point source. In leaves with only one layer of cells, the transmittance through the cytoplasm of single cells was similar to that for whole leaves. Analysis of cell wall birefringence by polarized-light interferometry indicated that cell walls might normally scatter rather than transmit light. Spectra transmitted through, or backscattered from, the upper green layers of moss were dominated by selective absorbance from chlorophyll, but there was also evidence of wavelength-dependent scattering, as detected in the lower layers of brown, dead moss. Specular reflectance from moss leaves was detected by polarimetry and may have contributed to the relatively high macroscopic transmittance of stationary moss in water. Shadowing by moss leaves was confirmed by dynamic measurements of mosses in turbulent water without bubbles. Flicker patterns from leaves were superimposed on the underwater flicker pattern created at the air-water interface, thus flecks of light were reduced in intensity, increased in frequency, and decreased in duration. This was detected with both point source and diffuse illumination of samples.

Following is a list of microscopy-related meetings and courses. The editors would greatly appreciate input to this list via the electronic submission form found in the MSA World-Wide Web page at www.msa.microscopy.com. We will gladly add hypertext links to the notice on the web and insert a listing of the meeting in the next issue of the Journal. Send comments and questions to Nan Yao, [email protected].