Aluminum-lithium alloys are widespread in the aerospace industry. The new 2099 and 2199 alloys provide improved properties, but their microstructure and texture are not well known. This article describes how state-of-the-art field-emission scanning electron microscopy (FE-SEM) can contribute to the characterization of the 2099 aluminum-lithium alloy and metallic alloys in general. Investigations were carried out on bulk and thinned samples. Backscattered electron imaging at 3 kV and scanning transmission electron microscope imaging at 30 kV along with highly efficient microanalysis permitted correlation of experimental and expected structures. Although our results confirm previous studies, this work points out possible substitutions of Mg and Zn with Li, Al, and Cu in the T1 precipitates. Zinc and magnesium are also present in “rice grain”–shaped precipitates at the grain boundaries. The versatility of the FE-SEM is highlighted as it provides information in the macro- and microscales with relevant details. Its ability to probe the distribution of precipitates from nano- to microsizes throughout the matrix makes FE-SEM an essential technique for the characterization of metallic alloys.

A sample preparation method is described for enabling direct correlation of site-specific plan-view and cross-sectional transmission electron microscopy (TEM) analysis of individual nanostructures by employing a dual-beam focused-ion beam (FIB) microscope. This technique is demonstrated using Si nanowires dispersed on a TEM sample support (lacey carbon or Si-nitride). Individual nanowires are first imaged in the plan-view orientation to identify a region of interest; in this case, impurity atoms distributed at crystalline defects that require further investigation in the cross-sectional orientation. Subsequently, the region of interest is capped with a series of ex situ and in situ deposited layers to protect the nanowire and facilitate site-specific lift-out and cross-sectioning using a dual-beam FIB microscope. The lift-out specimen is thinned to electron transparency with site-specific positioning to within ∼200 nm of a target position along the length of the nanowire. Using the described technique, it is possible to produce correlated plan-view and cross-sectional view lattice-resolved TEM images that enable a quasi-3D analysis of crystalline defect structures in a specific nanowire. While the current study is focused on nanowires, the procedure described herein is general for any electron-transparent sample and is broadly applicable for many nanostructures, such as nanowires, nanoparticles, patterned thin films, and devices.

For over a decade fluorescence microscopy has demonstrated the capacity to achieve single-molecule localization accuracies of a few nanometers, well below the ∼200 nm lateral and ∼500 nm axial resolution limit of conventional microscopy. Yet, only the recent development of new fluorescence labeling modalities, the increase in sensitivity of imaging hardware, and the creation of novel image analysis tools allow for the emergence of single-molecule-based super-resolution imaging techniques. Novel methods such as photoactivated localization microscopy and stochastic optical reconstruction microscopy can typically reach a tenfold increase in resolution compared to standard microscopy methods. Their implementation is relatively easy only requiring minimal changes to a conventional wide-field or total internal reflection fluorescence microscope. The recent translation of these two methods into commercial imaging systems has made them further accessible to researchers in biology. However, these methods are still evolving rapidly toward imaging live samples with high temporal resolution and depth. In this review, we recall the roots of single-molecule localization microscopy, summarize major recent developments, and offer perspective on potential applications.

The centrosome is the principal microtubule organization center in cells, giving rise to microtubule-based organelles (e.g., cilia, flagella). The aim was to study the osteocyte centrosome morphology at an ultrastructural level in relation to its mechanosensitive function. Osteocyte centrosomes and cilia in tibial cortical bone were explored by acetylated alpha-tubulin (AαTub) immunostaining under confocal microscopy. For the first time, fine ultrastructure and spatial orientation of the osteocyte centrosome were explored by transmission electron microscopy on serial ultrathin sections. AαTub-positive staining was observed in 94% of the osteocytes examined (222/236). The mother centriole formed a short primary cilium and was longer than the daughter centriole due to an intermediate zone between centriole and cilium. The proximal end of the mother centriole was connected with the surface of daughter centriole by striated rootlets. The mother centriole exhibited distal appendages that interacted with the cell membrane and formed a particular structure called “cilium membrane prolongation.” The primary cilium was mainly oriented perpendicular to the long axis of bone. Mother and daughter centrioles change their original mutual orientation during the osteocyte differentiation process. The short primary cilium is hypothesized as a novel type of fluid-sensing organelle in osteocytes.

In the last decade, the applicability of focus ion beam–field emission scanning electron microscopy (FIB-FESEM) in the biological field has begun to get relevance. Among the possibilities offered by FIB-FESEM, high-resolution three-dimensional (3D) reconstruction of biological structures is one of the most interesting. Using this tool, the 3D porosity of four different types of mouse oocyte zona pellucida (ZP) was analyzed. A surface analysis of the mouse oocyte ZP was first performed by SEM. Next, one oocyte per ZP type was selected, and an area of its ZP was completely milled, using the cut and view mode, in the FIB-FESEM. Through a 3D reconstruction of the milled area, a map of the distribution of the pores across the ZP was established and the number and volume of pores were quantified, thus enabling for the first time the study of the inner porosity of the mouse ZP. Differences in ZP porosity observed among the four types analyzed allowed us to outline a model to explain the changes that the ZP undergoes through immature, mature, predegenerative, and degenerative stages.

This study presents the morphological description and histochemical characterization of gill filaments of the Brazilian endemic bivalve Diplodon expansus, aiming to broaden the morphological knowledge of this species and establish the structure of the gills that will serve as control in histopathological studies applied to biomonitoring. The gill filaments are divided into three zones: frontal, intermediate, and abfrontal. In the center of the filament, haemocytes circulate through the haemolymph vessel, which is internally lined by endothelium. The frontal surface of the filament is covered with cilia, the lateral surface exhibits aquifer ducts, and the abfrontal surface presents ciliated and nonciliated cells. The epithelium of the filaments is composed of ciliated cells, nonciliated absorptive cells, and mucocytes. The support of the filaments is made by two specialized structures called skeletal rod and skeletal loop. Based on the obtained information, the gill filaments of the studied species present some peculiar characteristics that are not yet reported in detail in the literature such as the simultaneous presence of skeletal rod and skeletal loop. On the other hand, the general constitution of the filament is similar to that described for both marine and limnic bivalves and seems to be suitable for ecotoxicological studies.

The anatomy and topochemistry in normal and compression wood tracheid cell wall of Pinus bungeana Zucc. were investigated by fluorescence microscopy and confocal Raman microscopy. Using fluorescence microscopy, the severity of compression wood was classed as a mild type for the reason that it did not contain all compression wood features. Chemical imaging by confocal Raman microscopy was used for analyzing the distribution of lignin and cellulose, as well as the functional groups of lignin in tracheid cell walls. By comparison with normal wood, highly lignified outer S2 layer [S2(L)], thicker S1 layer, and obviously reduced lignification in the middle lamella were characteristic of compression wood. In addition, smaller microfibril angle was observed in the S2(L) region. The distribution of coniferyl alcohol and coniferyl aldehyde in normal and compression wood was enriched in S1 and S2 layers but lack in cell corner and/or S2L regions, which showed an opposite pattern to lignin distribution. Confocal Raman microscopy with high spatial resolution contributes to a further understanding of the differences between normal and compression wood in polymers distribution and molecules orientation in situ.

We undertook a study of Porphyra acanthophora var. brasiliensis to determine its responses under ambient conditions, photosynthetically active radiation (PAR), and PAR+UVBR (ultraviolet radiation-B) treatment, focusing on changes in ultrastructure, and cytochemistry. Accordingly, control ambient samples were collected in the field, and two different treatments were performed in the laboratory. Plants were exposed to PAR at 60 μmol photons m−2 s−1 and PAR + UVBR at 0.35 W m−2 for 3 h per day during 21 days of in vitro cultivation. Confocal laser scanning microscopy analysis of the vegetative cells showed single stellate chloroplast in ambient and PAR samples, but in PAR+UVBR-exposed plants, the chloroplast showed alterations in the number and form of arms. Under PAR+UVBR treatment, the thylakoids of the chloroplasts were disrupted, and an increase in the number of plastoglobuli was observed, in addition to mitochondria, which appeared with irregular, disrupted morphology compared to ambient and PAR samples. After UVBR exposure, the formation of carpospores was also observed. Plants under ambient conditions, as well as those treated with PAR and PAR+UVBR, all showed different concentrations of enzymatic response, including glutathione peroxidase and reductase activity. In summary, the present study demonstrates that P. acanthophora var. brasiliensis shows the activation of distinct mechanisms against natural radiation, PAR and PAR+UVBR.

The objective of this article is to evaluate the resistance to degradation of resin-dentin bonds formed with three one-step adhesives. Flat, mid-coronal dentin surfaces were bonded with the self-etching adhesives [Tokuyama Bond Force (TBF), One Up Bond F Plus (OUB), and G-Bond (GB)]. The bonded teeth were subjected to fatigue loading, chemical degradation, and stored in distilled water for four time periods (up to 12 months). Specimens were tested for microtensile bond strength and microleakage. Fractographic analysis was performed by scanning electron microscopy. Bonded interfaces were examined by light microscopy using Masson's trichrome staining. An atomic force microscope was employed to analyze phase separation and surface nanoroughness (Ra) at the polymers. Vickers microhardness and the degree of the conversion (DC) were also determined. ANOVA and multiple comparisons tests were performed. Bond strength significantly decreased after the chemical challenge, but not after load cycling. Aging decreased bond strength after 6 months in TBF and GB, in OUB after 12 months. An increase of the nonresin protected collagen zone occurred in all groups, after storing. TBF showed the highest roughness, microhardness, and DC values, and GB showed the lowest. Mild self-etch one-step adhesives (TBF/OUB) showed a higher degree of cure, lower hydrophilicity, and major resistance to degradation of resin-dentin bonds when compared to highly acidic self-etching adhesive (GB).

Confocal Raman Microscopy. Thomas Dieing, Olaf Hollricher, and Jan Toporski (Eds.). Springer, New York, 2011, 289 pages. ISBN 978-3642125218

Raman microscopy is the combination of Raman spectroscopy with optical light microscopy and was first presented in the mid-1970s. It combines the advantages of vibrational Raman spectroscopy as a noninvasive technique providing a wealth of chemical information on the properties of molecules and solids with the imaging capabilities and small sample volume requirements of an optical microscope. “Seeing is believing”: Raman microscopy can visualize the inherent chemical inhomogeneity of a sample without the need of external labels, information that cannot be obtained from simply looking at an unstained sample in the standard optical microscope. During the last decade, confocal Raman microscopy has gone through a rapid development with respect to instrumentation and software as well as the diversity of applications.