Retrograde transport is a process in which proteins are trafficked from the plasma membrane and endosomes to biosynthetic and secretory organelles, namely the Golgi apparatus and endoplasmic reticulum (ER). A number of plant and bacterial toxins, including cholera toxin and ricin toxin, exploit retrograde transport to gain entry into host cells, although the specifics of this process have remained difficult to probe by laser scanning confocal microscopy (LSCM). Here we demonstrate the use of super-resolution and live-cell imaging [stimulated emission depletion (STED)] to visualize exogenously applied ricin toxin within the ER. The improved resolution obtained by STED, as compared with LSCM (0.09 versus 0.19 μm), provides a more accurate determination of the amount of ricin that had trafficked to the ER.

Despite previous research efforts in the fields of histology and cell physiology, the relationship between chromatin structural organization and nuclear shape remains unclear. The aim of this research was to test the existence and strength of correlations between mathematical parameters of chromatin microarchitecture and roundness of the nuclear envelope. On a sample of 240 nuclei of adrenal zona fasciculata cells stained using the DNA-specific Feulgen method, we quantified fractal parameters such as fractal dimension and lacunarity, as well as textural parameters such as angular second moment (ASM), entropy, inverse difference moment, contrast, and variance. Circularity of the nuclear envelope was determined from the nuclear area and perimeter. The results indicate that there is a statistically significant negative correlation between chromatin ASM and circularity. Moreover, there was a statistically significant positive correlation between chromatin fractal dimension and envelope circularity. This is the first study to demonstrate these relationships in adrenal tissue, and also one of the first studies to test the connection between circularity and fractal and gray-level co-occurrence matrix parameters in DNA-specific Feulgen stain. The results could be useful both as an addition to the current knowledge on chromatin/nuclear envelope interactions, and for design of future computer-assisted research software for evaluation of nuclear morphology.

The article explores the theory of infrared–visible sum frequency generation microscopy of phospholipid envelopes with dimensions larger than the wavelength of the nonlinear emission. The main part of the study concerns derivation and accounting for the contributions of effective nonlinear responses specific to sites on the surfaces of a bilayer envelope and their dependence on polarization condition and experimental geometry. The nonlinear responses of sites are mapped onto the image plane according to their emission directions and the numerical aperture of a sampling microscope objective. According to the simulation results, we discuss possible approaches to characterize the shape of the envelope, to extract molecular hyperpolarizabilities, and to anticipate possible heterogeneity in envelope composition and anisotropy of the environment proximal to the envelope. The modeling approach offers a promising analytic facility to assist connecting microscopy observations in engineered liposomes, cellular envelopes, and subcellular organelles of relatively large dimensions to molecular properties, and hence to chemistry and structure down to available spatial resolution.

Literature about fish kidney peroxisomes is scarce. To tackle this caveat, a stereological approach on renal peroxisome morphological parameters was performed for the first time in a fish, establishing correlations with maturation stages as it was previously done in brown trout liver. Three-year-old brown trout males and females were collected at the major seasons of their reproductive cycle. Trunk kidney was fixed and processed for catalase cytochemistry. Classical stereological methods were applied to electromicrographs to quantitate morphological parameters. Different seasonal variation patterns were observed between genders, and between renal proximal tubule segments I and II. In males, peroxisomes from proximal tubule segment II had a relatively higher volume and number in May, being individually bigger in February. Females presented similar trends, though with less marked variations. Overall, males and females did not show exactly the same seasonal patterns for most peroxisomal parameters, and no correlations were found between the latter and the gonado-somatic index (GSI). Hence, and despite the variations, the morphology of renal peroxisomes is not strictly correlated with gonad maturation kinetics, therefore suggesting that kidney peroxisome morphology is not seasonally modulated by sex steroids, like estradiol, as it seems to happen in liver peroxisomes.

Calcium regulates many intracellular events such as growth and differentiation during different stages of gamete development. The aim of this study was to localize and quantify the intracellular distribution of calcium during different developmental stages of spermatogenesis in sterlet, Acipenser ruthenus, using a combined oxalate–pyroantimonate technique. The distribution of calcium was described in spermatogonium, spermatocyte, spermatid, and spermatozoon stages. In the spermatogonium and spermatocyte, calcium deposits were mainly localized in the nucleus and cytoplasm. The spermatid had calcium in the nucleus, developing acrosomal vesicle, and cytoplasm. Intracellular calcium transformed from scattered deposits in spermatogonia and spermatocyte stages into an unbound form in spermatid and the spermatozoon. The proportion of area covered by calcium increased significantly (p<0.05) from early to late stages of spermatogenesis. The largest proportion of area covered by calcium was observed in the nucleus of the spermatozoon. In conclusion, although most of the intracellular calcium is deposited in limited areas of the spermatogonium and spermatocyte, it is present an unbound form in the larger area of spermatids and spermatozoa which probably reflects changes in its physiological function and homeostasis during the process of male gamete production in spermatogenesis.

The effect of heavy metal in fish has been the focus of extensive research for many years. However, the combined effect of heavy metals and nanomaterials is still a new subject that needs to be studied. The aim of this study was to examine histopathologic alterations in the gills of Nile tilapia (Oreochromis niloticus) to determine possible effects of lead (Pb), carbon nanotubes, and Pb+carbon nanotubes on their histological integrity, and if this biological system can be used as a tool for evaluating water quality in monitoring programs. For this, tilapia were exposed to Pb, carbon nanotubes and Pb+carbon nanotubes for 4 days. The main alterations observed were epithelial structure, hyperplasia and displacement of epithelial cells, and alterations of the structure and occurrence of aneurysms in the secondary lamella. The most severe alterations were related to the Pb+carbon nanotubes. We conclude that the oxidized multi-walled carbon nanotubes enhanced the acute lead toxicity in Nile tilapias. This work draws attention to the implications of carbon nanomaterials released in the aquatic environment and their interaction with classical pollutants.

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.

Yttria-stabilized tetragonal zirconia polycrystals (3Y-TZP) is a ceramic material used in indirect dental restorations. However, phase transformation at body temperature may compromise the material’s mechanical properties, affecting the clinical performance of the restoration. The effect of mastication on 3Y-TZP aging has not been investigated. 3Y-TZP specimens (IPS E-max ZirCAD and Z5) were aged in three different modes (n=13): no aging (control), hydrothermal aging (HA), or chewing simulation (CS). Mechanical properties and surface topography were analyzed. Analysis of variance showed that neither aging protocol (p=0.692) nor material (p=0.283) or the interaction between them (p=0.216) had a significant effect on flexural strength, values ranged from 928.8 MPa (IPSHA) to 1,080.6 MPa (Z5HA). Nanoindentation analysis showed that material, aging protocol, and the interaction between them had a significant effect (p<0.001) on surface hardness and reduced Young’s modulus. The compositional analysis revealed similar yttrium content for all the experimental conditions (aging: p=0.997; material: p=0.248; interaction material×aging: p=0.720). Atomic force microscopy showed an effect of aging protocols on phase transformation, with samples submitted to CS exhibiting features compatible with maximized phase transformation, such as increased volume of the material microstructure at the surface leading to an increase in surface roughness.

The aim of this study was to compare surface structures of laser-irradiated dental hard tissues using confocal (CFM), atomic force (AFM), and scanning electron microscopy (SEM). The general potential of the AFM in analyzing laser-irradiated surfaces was determined in this context. Specimens of human enamel and dentin were irradiated using an 8.6 W Nd:YVO4 laser with a pulse duration of 8 ps, λCenter=1,064 nm, and a pulse repetition rate of 500 kHz. Surface topology of irradiated areas (1 mm2) was investigated using AFM, CFM, and SEM. Surface roughness Rz was measured only with the AFM and the CFM. For non-irradiated enamel and dentin surfaces, roughnesses for CFM and AFM are in the nanometer range. However, major differences in roughness were determined for laser-prepared surfaces. For enamel, Rz(CFM)=2.33 μm is much higher compared with Rz(AFM)=0.09 μm; in the case of dentin, Rz(CFM)=5.35 μm is also much higher compared with Rz(AFM)=0.093 μm. Information regarding structural properties of surfaces needs real dimensions, particularly for use in dentistry. In this respect, AFM technology provides no additional results that lead to a significant improvement.

Poly[sulfur-random-(1,3-diisopropenylbenzene)] copolymers synthesized via inverse vulcanization represent an emerging class of electrochemically active polymers recently used in cathodes for Li–S batteries, capable of realizing enhanced capacity retention (1,005 mAh/g at 100 cycles) and lifetimes of over 500 cycles. The composite cathodes are organized in complex hierarchical three-dimensional (3D) architectures, which contain several components and are challenging to understand and characterize using any single technique. Here, multimode analytical scanning and transmission electron microscopies and energy-dispersive X-ray/electron energy-loss spectroscopies coupled with multivariate statistical analysis and tomography were applied to explore origins of the cathode-enhanced capacity retention. The surface topography, morphology, bonding, and compositions of the cathodes created by combining sulfur copolymers with varying 1,3-diisopropenylbenzene content and conductive carbons have been investigated at multiple scales in relation to the electrochemical performance and physico-mechanical stability. We demonstrate that replacing the elemental sulfur with organosulfur copolymers improves the compositional homogeneity and compatibility between carbons and sulfur-containing domains down to sub-5 nm length scales resulting in (a) intimate wetting of nanocarbons by the copolymers at interfaces; (b) the creation of 3D percolation networks of conductive pathways involving graphitic-like outer shells of aggregated carbons; (c) concomitant improvements in the stability with preserved meso- and nanoscale porosities required for efficient charge transport.

A systematic study has been carried out to compare the surface morphology, shell thickness, mechanical properties, and binding behavior of melamine–formaldehyde microcapsules of 5–30 μm diameter size with various amounts of core content by using scanning and transmission electron microscopy including electron tomography, in situ nanomechanical tensile testing, and electron energy-loss spectroscopy. It is found that porosities are present on the outside surface of the capsule shell, but not on the inner surface of the shell. Nanomechanical tensile tests on the capsule shells reveal that Young’s modulus of the shell material is higher than that of bulk melamine–formaldehyde and that the shells exhibit a larger fracture strain compared with the bulk. Core-loss elemental analysis of microcapsules embedded in epoxy indicates that during the curing process, the microcapsule-matrix interface remains uniform and the epoxy matrix penetrates into the surface micro-porosities of the capsule shells.

We report electron probe microanalysis measurements on nickel silicides, Ni5Si2, Ni2Si, Ni3Si2, and NiSi, which were done in order to investigate anomalies that affect the analysis of such materials by using the Ni L3-M4,5 line (Lα). Possible sources of systematic discrepancies between experimental data and theoretical predictions of Ni L3-M4,5k-ratios are examined, and special attention is paid to dependence of the Ni L3-M4,5k-ratios on mass-attenuation coefficients and partial fluorescence yields. Self-absorption X-ray spectra and empirical mass-attenuation coefficients were obtained for the considered materials from X-ray emission spectra and relative X-ray intensity measurements, respectively. It is shown that calculated k-ratios with empirical mass attenuation coefficients and modified partial fluorescence yields give better agreement with experimental data, except at very low accelerating voltages. Alternatively, satisfactory agreement is also achieved by using the Ni L3-M1 line (Lℓ) instead of the Ni L3-M4,5 line.

In the present study, nano-sized cuboid-shaped particles in Mg–Nd–Y are studied by means of Cs-corrected atomic-scale high-angle annular dark-field scanning transmission electron microscopy. The structure of the cuboid-shaped phase is identified to be yttrium (major component) and neodymium atoms in face-centered cubic arrangement without the participation of Mg. The lattice parameter a=5.15 Å. During isothermal aging at 225°C, Mg3(Nd,Y) precipitates adhere to surface (100) planes of the cuboid-shaped particles with the orientation relationship: $[100]_{{{\rm Mg}_{{\rm 3}} {\rm RE}}} \,/\,\,/\,[100]_{{{\rm Cuboid}}} $ and $[310]_{{{\rm Mg}_{{\rm 3}} {\rm RE}}} \,/\,\,/\,[012]_{{{\rm Cuboid}}} $ . The fully coherent interfaces between the precipitates and the cuboid-shaped phases are reconstructed and categorized into two types: $(400)_{{{\rm Mg}_{{\rm 3}} {\rm RE}}} $ interface and $(200)_{{{\rm Mg}_{{\rm 3}} {\rm RE}}} $ interface.

Alnico alloys have long been used as strong permanent magnets because of their ferromagnetism and high coercivity. Understanding their structural details allows for better prediction of the resulting magnetic properties. However, quantitative three-dimensional characterization of the phase separation in these alloys is still challenged by the spatial quantification of nanoscale phases. Herein, we apply a dual tomography approach, where correlative scanning transmission electron microscopy (STEM) energy-dispersive X-ray spectroscopic (EDS) tomography and atom probe tomography (APT) are used to investigate the initial phase separation process of an alnico 8 alloy upon non-magnetic annealing. STEM-EDS tomography provides information on the morphology and volume fractions of Fe–Co-rich and Νi–Al-rich phases after spinodal decomposition in addition to quantitative information of the composition of a nanoscale volume. Subsequent analysis of a portion of the same specimen by APT offers quantitative chemical information of each phase at the sub-nanometer scale. Furthermore, APT reveals small, 2–4 nm Fe-rich α1 phases that are nucleated in the Ni-rich α2 matrix. From this information, we show that phase separation of the alnico 8 alloy consists of both spinodal decomposition and nucleation and growth processes. The complementary benefits and challenges associated with correlative STEM-EDS and APT are discussed.

In this paper the potential of time-of-flight secondary ion mass spectroscopy combined with focused ion beam technology to characterize the composition of a solid oxide fuel cell (SOFC) in three-dimension is demonstrated. The very high sensitivity of this method allows even very small amounts of elements/compounds to be detected and localized. Therefore, interlayer diffusion of elements between porous electrodes and presence of pollutants can be analyzed with a spatial resolution of the order of 100 nm. However, proper element recognition and mass interference still remain important issues. Here, we present a complete elemental analysis of the SOFC as well as techniques that help to validate the reliability of obtained results. A discussion on origins of probable artifacts is provided.

Determining and acting on thermo-physical properties at the nanoscale is essential for understanding/managing heat distribution in micro/nanostructured materials and miniaturized devices. Adequate thermal nano-characterization techniques are required to address thermal issues compromising device performance. Scanning thermal microscopy (SThM) is a probing and acting technique based on atomic force microscopy using a nano-probe designed to act as a thermometer and resistive heater, achieving high spatial resolution. Enabling direct observation and mapping of thermal properties such as thermal conductivity, SThM is becoming a powerful tool with a critical role in several fields, from material science to device thermal management. We present an overview of the different thermal probes, followed by the contribution of SThM in three currently significant research topics. First, in thermal conductivity contrast studies of graphene monolayers deposited on different substrates, SThM proves itself a reliable technique to clarify the intriguing thermal properties of graphene, which is considered an important contributor to improve the performance of downscaled devices and materials. Second, SThM’s ability to perform sub-surface imaging is highlighted by thermal conductivity contrast analysis of polymeric composites. Finally, an approach to induce and study local structural transitions in ferromagnetic shape memory alloy Ni–Mn–Ga thin films using localized nano-thermal analysis is presented.

In this paper the characterization of a gypsum plaster sample from the end of the 19th century simulating imperial red porphyry using a multi-analytical approach is presented and discussed. The results of X-ray diffraction (XRD), thermogravimetric and differential thermal analysis (TGA-DTA), physical and mechanical properties are summarized. In order to have further insight into the microstructure, polarized light microscopy (PLM), scanning electron microscopy coupled with energy dispersive X-ray spectrometer (SEM-EDS), and micro Raman spectroscopy analyzes were also made. They helped to clarify the main issues raised by the other complementary analytical techniques and allowed the establishment of interrelations between the different properties, providing important information about the materials, the skills, and the technological development involved in the art of imitating noble stones with gypsum pastes. This study also contributes to our knowledge concerning the preservation of these types of elements that are important in the context of European decorative arts and rarely reported in the literature.

Jadeite was greatly appreciated by pre-Hispanic cultures in Mesoamerica. Despite its importance, knowledge of its mining sources was lost after the Spanish conquest. In the 1950s the only confirmed jadeite deposits in Mesoamerica were found in the Motagua River Fault (MRF), Guatemala. The aim of this study is to present a methodology that is appropriate for the study of archeological jadeite objects using non-destructive spectroscopic and micro-ion beam analysis techniques. This methodology has been applied to perform mineral, elemental, and luminescence characterization of five jadeite samples from the MRF, with white, lilac, and green colors. Fourier-transformed infrared spectroscopy and X-ray diffraction analysis confirmed the presence of jadeite, albite, and omphacite as the main mineral phases in the samples. Elemental maps using particle-induced X-ray emission (PIXE) with a nuclear microprobe and elemental concentration analysis from individual mineral grains using micro-PIXE coupled with micro-ionoluminescence (IL) allowed the detection of minor feldspar, titanite, and grossular mineral contents. Distinctive features from the mineral, elemental, and luminescence characterization have been found that allow the identification of these five jadeite samples.

Phase plate (PP) imaging has proven to be valuable in transmission cryo electron microscopy of unstained, native-state biological specimens. Many PP types have been described, however until the recent implementation of the “hole-free” phase plate (HFPP), imaging has been challenging. We found the HFPP to be simple to construct and to set up in the transmission electron microscopy, but care in implementing automated data collection is needed. Performance may be variable, both initially and over time, thus it is important to monitor and evaluate image quality by observing the power spectrum. We found that while some HFPPs gave transfer to high resolution without CTF oscillation, most reached high resolution when operated with modest defocus.

There is an unmet need for researchers in life sciences and clinical pathology to obtain untainted target cells with very high accuracy, which are suitable for subsequent genome and protein analysis. In this paper, an electrostatic capture laser microdissection technology (ECM) is proposed and explained. Following microscopic identification and separation of target cells from the surrounding tissues using laser cutting, the ECM uses electrostatic forces to capture target cells. Validation experiments indicate that ECM can capture a wide assortment of contamination-free homogeneous samples, ranging from very tiny pieces of a few micrometers in diameter to large pieces with a surface area of over 40,000 µm2. Evidence is also provided indicating that uncontaminated homogeneous tissue materials collected by ECM are suitable for further DNA and RNA analysis. This suggests that ECM capture causes little or no identifiable damage to the collected tissues. This technique has significant advantages compared with existing traditional capture methods, such as very low risk of biological sample damage and the fact that it can be applied to both upright and inverted microscopy. The latter allows for separating target cells in tissue culture. ECM usage provides a cost-effective alternative to more traditional laser capture microdissection techniques.