Aspergillus fumigatus has become a leading cause of fungal morbidity and mortality, especially in immunocompromised patients. This fungus is able to grow as a multicellular community and produce a hydrophobic extracellular matrix (ECM), mainly composed of galactomannan and α-1,3 glucans, to protect itself from host defenses and antimicrobial drugs. This matrix envelops the fungus hyphae, binding them into a contiguous sheath on the colony surface, forming a biofilm and increasing the fungal resistance to adverse environmental factors. Adherence to host cells and resistance to physical removal play a key role in fungal colonization and invasion of the host and in a wide range of infections. Here we show that, by using atomic force spectroscopy, it is possible to exploit the peculiar hydrophobicity of the biofilm components (i.e., cell walls, ECM) to detect the biofilm spread, its growth, and lysis on rough surfaces. By means of this approach, we demonstrate that alginate lyase, an enzyme known to reduce negatively charged alginate levels in microbial biofilms, reduces the biofilm adhesion forces suggesting a loss of ECM from the biofilm, which could be used to enhance pharmacological treatments.

In scattering specimens, multiphoton excitation and nondescanned detection improve imaging depth by a factor of 2 or more over confocal microscopy; however, imaging depth is still limited by scattering. We applied the concept of clearing to deep tissue imaging of highly scattering specimens. Clearing is a remarkably effective approach to improving image quality at depth using either confocal or multiphoton microscopy. Tissue clearing appears to eliminate the need for multiphoton excitation for deep tissue imaging.

Vascular corrosion casting has been used for about 40 years to produce replicas of normal and abnormal vasculature and microvasculature of various tissues and organs that could be viewed at the ultrastructural level. In combination with scanning electron microscopy (SEM), the primary application of corrosion casting has been to describe the morphology and anatomical distribution of blood vessels in these tissues. However, such replicas should also contain quantitative information about that vasculature. This report summarizes some simple quantitative applications of vascular corrosion casting. Casts were prepared by infusing Mercox resin or diluted Mercox resin into the vasculature. Surrounding tissues were removed with KOH, hot water, and formic acid, and the resulting dried casts were observed with routine SEM. The orientation, size, and frequency of vascular endothelial cells were determined from endothelial nuclear imprints on various cast surfaces. Vascular volumes of heart, lung, and avian salt gland were calculated using tissue and resin densities, and weights. Changes in vascular volume and functional capillary density in an experimentally induced emphysema model were estimated from confocal images of casts. Clearly, corrosion casts lend themselves to quantitative analysis. However, because blood vessels differ in their compliances, in their responses to the toxicity of casting resins, and in their response to varying conditions of corrosion casting procedures, it is prudent to use care in interpreting this quantitative data. Some of the applications and limitations of quantitative methodology with corrosion casts are reviewed here.

In this article, we show that nanometer-sized precipitates of atomic numbers higher than those of the surrounding crystalline matrix can be clearly revealed in a conventional transmission electron microscope by high-angle, centered dark-field imaging after minimizing the diffraction contrast. The effect is similar to that of Z-contrast STEM, albeit with a spatial resolution limited to 1 nm. Its sensitivity to atomic number differences between precipitates and matrix is about 10, which is demonstrated for precipitates formed after Er, Ge, Cr, and Si ion implantation into SiC.

Aganaspis daci and Aganaspis pelleranoi (Hymenoptera: Figitidae) are important parasitoids of fruit flies. Here we studied, with light and scanning electron microscopy, aspects of their morphology that could help with plans to mass rear and thus contribute to improved pest control (preimaginal phases) and to shed light on parasitoid-pest relationships (sensillar equipment). The two species present a stalked egg, eucoiliform first and second-instar larvae and hymenopteriform third instar and mature larvae. The first instar presents tegumental differentiations in the mesoma and first metasomal segment in A. daci, but not in A. pelleranoi, while unlike other figitids, neither species displays setae in the mesosomal processes. Second and third instar and mature larvae present tegumental differentiations in A. daci, but not in A. pelleranoi. The moniliform (female) and filiform (male) antennae of A. daci and A. pelleranoi harbor seven types of sensilla, four of them (sensilla campaniformia, sensilla coeloconica type II, and two types of sensilla trichoidea) described here for the first time in Cynipoidea. The largest sensilla were the multiporous placoid sensilla, which were smaller and more numerous in A. pelleranoi. Species also differed to some extent in morphology of sensilla coeloconica. Observations on the ovipositor revealed the presence of coeloconic sensilla on Valva I in both species.

The aim of the present study was to compare dynamics of the bone healing process after different types of osteotomies. In total, 24 Wistar rats were subjected to different types of osteotomy performed with standard steel bur, piezosurgery, contact, and non-contact Erbium:yttrium-aluminum-garnet (Er:YAG) laser ablation. The animals were randomly divided into four groups, to be euthanized immediately after the procedure, or at 1, 2, or 3 weeks after surgery. The obtained bone samples were analyzed by scanning electron microscopy (SEM). Immediately after surgery, there were significant differences in the appearance of the bone defects, with presence of bone fragments and debris after standard steel bur preparation, compared with the clean smooth walls and relatively sharp edges in all other groups. The initial bone formation in defects prepared by piezosurgery was observed to be the most rapid. After 3 weeks, all bone defects were completely restored; although, differences in the healing pattern were noted, with a modest initial delay in healing after laser preparation. The first stage of the bone healing process was delayed when contact and non-contact Er:YAG laser modes were used and accelerated by piezosurgery; however, the results after 3 weeks demonstrated similar restitution of defects in all tested groups.

Focused ion-beam milling has been applied to prepare needle-shaped atom probe tomography specimens from mechanically alloyed powders without the use of embedding media. The lift-out technique known from transmission electron microscopy specimen preparation was modified to cut micron-sized square cross-sectional blanks out of single powder particles. A sequence of rectangular cuts and annular milling showed the highest efficiency for sharpening the blanks to tips. First atom probe results on a Fe95Cu5 powder mechanically alloyed in a high-energy planetary ball mill for 20 h have been obtained. Concentration profiles taken from this powder sample showed that the Cu distribution is inhomogeneous on a nanoscale and that the mechanical alloying process has not been completed yet. In addition, small clusters of oxygen, stemming from the ball milling process, have been detected. Annular milling with 30 keV Ga ions and beam currents ≥50 pA was found to cause the formation of an amorphous surface layer, whereas no structural changes could be observed for beam currents ≤10 pA.

The evolution of Guinier-Preston zones in an Al-2.7 at.% Ag alloy was studied using atom probe tomography. The composition and morphology of the GP zones are time dependent, explaining discrepancies in previous work. This result requires the metastable miscibility gap for GP zones to be reevaluated, highlighting the importance of the temporal evolution of the GP zones. Preliminary results on the composition of γ′ and γ plates are also presented.

In this article a method for determining errors of the strain values when applying strain mapping techniques has been devised. This methodology starts with the generation of a thickness/defocus series of simulated high-resolution transmission electron microscopy images of InAsxP1−x/InP heterostructures and the application of geometric phase. To obtain optimal defocusing conditions, a comparison of different defocus values is carried out by the calculation of the strain profile standard deviations among different specimen thicknesses. Finally, based on the analogy of real state strain to a step response, a characterization of strain mapping error near an interface is proposed.

The microstructure of a 55 μm diameter, cold-worked Ni-Ti microwire is investigated by different transmission electron microscopy techniques. The surface consists of a few hundred nanometer thick oxide layer composed of TiO and TiO2 with a small fraction of inhomogeneously distributed Ni. The interior of the wire has a core-shell structure with primarily B2 grains in the 1 μm thick shell, and heavily twinned B19′ martensite in the core. This core-shell structure can be explained by a concentration gradient of the alloying elements resulting in a structure separation due to the strong temperature dependence of the martensitic start temperature. Moreover, in between the B2 part of the metallic core-shell and the oxide layer, a Ni3Ti interfacial layer is detected.

The skeletal muscle extracellular matrix (ECM) supports muscle’s passive mechanical function and provides a unique environment for extracellular tissues such as nerves, blood vessels, and a cadre of mononuclear cells. Within muscle ECM, collagen is thought to be the primary load-bearing protein, yet its structure and organization with respect to muscle fibers, tendon, and mononuclear cells is unknown. Detailed examination of extracellular collagen morphology requires high-resolution electron microscopy performed over relatively long distances because multinucleated muscle cells are very long and extend from several millimeters to several centimeters. Unfortunately, there is no tool currently available for high resolution ECM analysis that extends over such distances relevant to muscle fibers. Serial block face scanning electron microscopy is reported here to examine skeletal muscle ECM ultrastructure over hundreds of microns. Ruthenium red staining was implemented to enhance contrast and utilization of variable pressure imaging reduced electron charging artifacts, allowing continuous imaging over a large ECM volume. This approach revealed previously unappreciated perimysial collagen structures that were reconstructed via both manual and semi-automated segmentation methods. Perimysial collagen structures in the ECM may provide a target for clinical therapies aimed at reducing skeletal muscle fibrosis and stiffness.

In this study, the cross-section of electron-selective titanium oxide (TiO2) contacts for n-type crystalline silicon solar cells were investigated by transmission electron microscopy. It was revealed that the excellent cell efficiency of 21.6% obtained on n-type cells, featuring SiO2/TiO2/Al rear contacts and after forming gas annealing (FGA) at 350°C, is due to strong surface passivation of SiO2/TiO2 stack as well as low contact resistivity at the Si/SiO2/TiO2 heterojunction. This can be attributed to the transformation of amorphous TiO2 to a conducting TiO2−x phase. Conversely, the low efficiency (9.8%) obtained on cells featuring an a-Si:H/TiO2/Al rear contact is due to severe degradation of passivation of the a-Si:H upon FGA.

Electron-excited X-ray mapping is a key operational mode of the scanning electron microscope (SEM) equipped with energy dispersive X-ray spectrometry (EDS). The popularity of X-ray mapping persists despite the significant time penalty due to the relatively low output count rates, typically less than 25 kHz, that can be processed with the conventional EDS. The silicon drift detector (SDD) uses the same measurement physics, but modifications to the detector structure permit operation at a factor of 5–10 times higher than conventional EDS for the same resolution. Output count rates as high as 500 kHz can be achieved with 217 eV energy resolution (at MnKα). Such extraordinarily high count rates make possible X-ray mapping through the method of X-ray spectrum imaging, in which a complete spectrum is captured at each pixel of the scan. Useful compositional data can be captured in less than 200 s with a pixel density of 160 × 120. Applications to alloy and rock microstructures, ultrapure materials with rare inclusions, and aggregate particles with complex chemistry illustrate new approaches to characterization made practical by high-speed X-ray mapping with the SDD.Note: The Siegbahn notation for characteristic X-rays is commonly used in the field of electron beam X-ray spectrometry and will be used in this article. The equivalent IUPAC notation is indicated in parentheses at the first use.In this article, the following arbitrary definitions will be used when referring to concentration (C) ranges: major: C > 0.1 (10 wt%), minor: 0.01 ≤ C ≤ 0.1 (1–10 wt%), and trace: C < 0.01 (1 wt%).

Sea urchin embryos have served as a model system for the investigation of many concepts in developmental biology. Their gastrulation consists of two stages; primary invagination, where part of the epithelium invaginates into the blastocoel, and secondary invagination, where the archenteron elongates to completely traverse the blastocoel. Primary invagination involves proliferation of cells within the vegetal plate during primary invagination, but until recently, it was assumed that the larval gastrointestinal (GI) tract developed without further involution of epithelial cells. To investigate rigorously the contribution of epithelial cell involution during archenteron and GI tract development in the sea urchin, Lytechinus variegatus, we developed a new method for cell tracking based on two-photon excited photorelease of caged fluorophores. Single-cell embryos were injected with caged dye and two-photon excitation uncaging was employed to mark small groups of cells throughout gastrulation. Two-photon excitation allowed for noninvasive, three-dimensionally resolved uncaging inside living cells with minimal biological damage. Cellular involution into the archenteron was observed throughout primary and secondary invagination, and the larval intestine was formed by further involution of cells following secondary invagination, which is inconsistent with the traditional model of sea urchin gastrulation. Further, as two-photon excitation microscopy becomes accessible to many researchers, the novel techniques described here will be broadly applicable to development of other invertebrate and vertebrate embryos.

In situ environmental transmission electron microscopy (ETEM) experiments require specimen heating holders to study material behavior in gaseous environments at elevated temperatures. In order to extract meaningful kinetic parameters, such as activation energies, it is essential to have a direct and accurate measurement of local sample temperature. This is particularly important if the sample temperature might fluctuate, for example when room temperature gases are introduced to the sample area. Using selected-area diffraction (SAD) in an ETEM, the lattice parameter of Ag nanoparticles was measured as a function of the temperature and pressure of hydrogen gas to provide a calibration of the local sample temperature. SAD permits measurement of temperature to an accuracy of ±30°C using Ag lattice expansion. Gas introduction can cause sample cooling of several hundred degrees celsius for gas pressures achievable in the ETEM.

We evaluate the usefulness of digital volume data produced with the high-resolution episcopic microscopy (HREM) method for visualizing the three-dimensional (3D) arrangement of components of human skin, and present protocols designed for processing skin biopsies for HREM data generation. A total of 328 biopsies collected from normally appearing skin and from a melanocytic nevus were processed. Cuboidal data volumes with side lengths of ~2×3×6 mm3 and voxel sizes of 1.07×1.07×1.5 µm3 were produced. HREM data fit ideally for visualizing the epidermis at large, and for producing highly detailed volume and surface-rendered 3D representations of the dermal and hypodermal components at a structural level. The architecture of the collagen fiber bundles and the spatial distribution of nevus cells can be easily visualized with volume-rendering algorithms. We conclude that HREM has great potential to serve as a routine tool for researching and diagnosing skin pathologies.

A comprehensive electron energy-loss spectroscopy (EELS) study of niobium (Nb) and stable Nb-oxide phases (NbO, NbO2, Nb2O5) was carried out. Part II of this work is devoted to quantitative EELS by means of experimental k-factors derived from the intensity ratio of the O-K edge and the Nb-M4,5 or Nb-M2,3 edges for all three stable Nb-oxides. The precision and accuracy of the quantification are investigated with respect to the influence of intensity-measurement energy windows, background subtraction, and sample thickness. Integration-window widths allowing optimum accuracy are determined. Owing to background-subtraction errors, the Nb-M4,5 edges rather than Nb-M2,3 are preferred for quantification. Different approaches are applied to improve the precision with regard to thickness-related errors. Thus, a precision up to ±1.5% is achieved by averaging spectra from all three reference oxides to determine a k-factor using Nb-M4,5. Using the experimental k-factor, the determination of atomic concentration ratios CNb/CO in the range of 0.4 (Nb2O5) to 1 (NbO) was found to be possible with an accuracy of 0.6% (relative deviation between measured and nominal composition), whereas ratios of calculated partial ionization cross sections lead to less accurate results.

Through a correlative analytical approach encompassing backscattered electron scanning electron microscopy (BSE-SEM), energy dispersive X-ray spectroscopy (EDX), and micro-Raman spectroscopy, the composition of the mineralized biofilm around a dental implant, retrieved due to peri-implantitis, was investigated. The mineralized biofilm contains two morphologically distinct regions: (i) bacteria-containing zones (Bact+), characterized by aggregations of unmineralized and mineralized bacteria, and intermicrobial mineralization, and (ii) bacteria-free zones (Bact−), comprised mainly of randomly oriented mineral platelets. Intramicrobial mineralization, within Bact+, appears as smooth, solid mineral deposits resembling the morphologies of dental plaque bacteria. Bact− is associated with micrometer-sized Mg-rich mineral nodules. The Ca/P ratio of Bact+ is higher than Bact−. The inorganic phase of Bact+ is carbonated apatite (CHAp), while that of Bact− is predominantly octacalcium phosphate (OCP) and whitlockite (WL) inclusions. Compared with native bone, the inorganic phase of Bact+ (i.e., CHAp) exhibits higher mineral crystallinity, lower carbonate content, and lower Ca/P, C/Ca, Mg/Ca, and Mg/P ratios. The various CaPs found within the mineralized dental biofilm (CHAp, OCP, and WL) are related to the local presence/absence of bacteria. In combination with BSE-SEM and EDX, micro-Raman spectroscopy is a valuable analytical tool for nondestructive investigation of mineralized dental biofilm composition and development.