Clathrin heavy chain 1 (CLTC) has been considered a “moonlighting protein” which acts in membrane trafficking during interphase and in stabilizing spindle fibers during mitosis. However, its roles in meiosis, especially in mammalian oocyte maturation, remain unclear. This study investigated CLTC expression and function in spindle formation and chromosome congression during mouse oocyte meiotic maturation. Our results showed that the expression level of CLTC increased after germinal vesicle breakdown (GVBD) and peaked in the M phase. Immunostaining results showed CLTC distribution throughout the cytoplasm in a cell cycle-dependent manner. Appearance and disappearance of CLTC along with β-tubulin (TUBB) could be observed during spindle dynamic changes. To explore the relationship between CLTC and microtubule dynamics, oocytes at metaphase were treated with taxol or nocodazole. CLTC colocalized with TUBB at the enlarged spindle and with cytoplasmic asters after taxol treatment; it disassembled and distributed into the cytoplasm along with TUBB after nocodazole treatment. Disruption of CLTC function using stealth siRNA caused a decreased first polar body extrusion rate and extensive spindle formation and chromosome congression defects. Taken together, these results show that CLTC plays an important role in spindle assembly and chromosome congression through a microtubule correlation mechanism during mouse oocyte maturation.

Digital quantification of a two-dimensional structure was applied to a GDC(Gd2O3-doped CeO2)/LSM(La0.85Sr0.15MnO3) composite cathode employed for solid oxide fuel cells. With the aid of high-resolution imaging capability based on secondary and backscattered electron images, two-dimensional electron micrographs were converted to digital binary files using an image processing tool combined with the line intercept method. Statistical analysis combined with a metallurgical tool was employed to determine microstructural factors, i.e., volume fraction, size distribution, and interconnectivity. The current work reports the quantification of the two-dimensional structural images of GDC/LSM composites applicable to solid oxide fuel cells, with the aim of obtaining the volume fraction, size distribution, and interconnectivity as functions of composite composition. The volume fractions of the solid constituent phases exhibit compositional dependence in cathodes; however, LSM interconnectivity increases gradually as a function of LSM composition, whereas that of GDC decreases significantly at 50 wt% LSM.

The aberration-corrected scanning transmission electron microscope has great sensitivity to environmental or instrumental disturbances such as acoustic, mechanical, or electromagnetic interference. This interference can introduce distortions to the images recorded and degrade both signal noise and resolution performance. In addition, sample or stage drift can cause the images to appear warped and leads to unreliable lattice parameters being exhibited. Here a detailed study of the sources, natures, and effects of imaging distortions is presented, and from this analysis a piece of image reconstruction code has been developed that can restore the majority of the effects of these detrimental image distortions for atomic-resolution data. Example data are presented, and the performance of the restored images is compared quantitatively against the as-recorded data. An improvement in apparent resolution of 16% and an improvement in signal-to-noise ratio of 30% were achieved, as well as correction of the drift up to the precision to which it can be measured.

Arbuscular mycorrhizal fungi (AMF) are ubiquitous underground symbiotic associations between most plants and fungi from the phylum Glomeromycota. From this symbiosis plants generally improve their capacity to obtain less mobile soil nutrients (like phosphorus) and increase resistance against biotic and abiotic stresses. Concomitantly the fungus has access to host carbon photosynthates. As obligate symbionts these fungi require always a host to survive, hence dual cultures is an obligatory laboratory requisite to maintain and multiply live Glomeromycota fungi. These cultures are mainly of three sorts: 1) Open pot cultures, 2) in vitro cultures with Ti DNA transformed root systems, 3) and in vitro cultures with L. cruciata, presented here. The internal mycelium of AMF usually assumes one or both of the morph-types named Arum (first described on Arum maculatum) and Paris (observed in Paris quadrifolia). The Paris-type morphology is characterized by hyphae with an intra-cellular growth, from cell-to-cell, forming coils and arbusculate coils. The Arum-type shows mainly intercellular hyphae growth, longitudinally between cells, with the arbuscules mounting upright on short intra-cellular branches.

This study presents the first comparative morphologic characterization of 4 species of AMF grown in vitro with Lunularia cruciata (L.) Dumortier ex. Lindberg. Glomus clarum Nicol. & Schenck (CNPAB 005), G. intraradices Schenck & Smith (MUCL 43204), G. proliferum Dalpé & Declerck (MUCL 41827); and Gigaspora margarita Becker & Hall (CNPAB 001) internal hyphae share the same morph-type of L. cruciata colonization: the Paris-type. Stable and viable monoxenic cultures of AMF with L. cruciata were first presented by Fonseca and co-workers in 2006 for G. intraradices and G. proliferum on Petri dish (Fig. 1.1). In this work we bring two more species (G. clarum and Gi. margarita) cultured in vitro with L. cruciata, on bi-layer medium in flask containers (Fig. 1.2). The production of spores occurred mainly, among the rhizoids, between overlapping thallus and over the thallus (Fig. 1.3). Only the Glomus species produced spores within the medium.

L. cruciata belongs to a group of complex thalloid liverworts with an internally differentiated anatomy: an upper epidermis forming air chambers where the chlorophyllous cells are located, a conspicuous vacuolated parenchyma, and the lower epidermis with its scales and rhizoids (Fig. 1.4). All four species show highly similar architecture of colonization with most fungi distributed in the thallus’ midrib parenchyma, and with internal mycelium architecture compatible with the Paris-type colonization (Figures 1.5 to 1.8). Although consistent the occurrence of Paris-type colonization among these four species, more species needed to be examined before a generalization can be given. The availability of AMF isolates for research at international fungi collections and laboratories is limited, and from these axenic isolates for in vitro culture are very few, which hinders the prove of the hypotheses that Paris-type colonization is the main pattern of hyphae colonization in L. cruciata mycothallus.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

A new mathematical algorithm is reported for the accurate and efficient analysis of pore properties of nanoporous anodic alumina (NAA) membranes using scanning electron microscope (SEM) images. NAA membranes of the desired pore size were fabricated using a two-step anodic oxidation process. Surface morphology of the NAA membranes with different pore properties was studied using SEM images along with computerized image processing and analysis. The main objective was to analyze the SEM images of NAA membranes quantitatively, systematically, and quickly. The method uses a regularized shock filter for contrast enhancement, mathematical morphological operators, and a segmentation process for efficient determination of pore properties. The algorithm is executed using MATLAB, which generates a statistical report on the morphology of NAA membrane surfaces and performs accurate quantification of the parameters such as average pore-size distribution, porous area fraction, and average interpore distances. A good comparison between the pore property measurements was obtained using our algorithm and ImageJ software. This algorithm, with little manual intervention, is useful for optimizing the experimental process parameters during the fabrication of such nanostructures. Further, the algorithm is capable of analyzing SEM images of similar or asymmetrically porous nanostructures where sample and background have distinguishable contrast.

The growth of high-quality indium (In)-rich InXGa1−XN alloys is technologically important for applications to attain highly efficient green light-emitting diodes and solar cells. However, phase separation and composition modulation in In-rich InXGa1−XN alloys are inevitable phenomena that degrade the crystal quality of In-rich InXGa1−XN layers. Composition modulations were observed in the In-rich InXGa1−XN layers with various In compositions. The In composition modulation in the InXGa1−XN alloys formed in samples with In compositions exceeding 47%. The misfit strain between the InGaN layer and the GaN buffer retarded the composition modulation, which resulted in the formation of modulated regions 100 nm above the In0.67Ga0.33N/GaN interface. The composition modulations were formed on the specific crystallographic planes of both the {0001} and {0114} planes of InGaN.

Giardia duodenalis is a protozoan parasite that causes intestinal disorders. The trophozoites present four pairs of flagella. Here we further analyze the structural organization of the anterior flagella associated structures of G. duodenalis. High resolution scanning electron microscopy of detergent-extracted trophozoites revealed novel aspects of the interaction of the anterior flagella axonemes with the marginal plates. Images of the marginal plates showed that it was located in the anterior region of the parasite, above the crossing point of the anterior flagella axonemes toward the periphery of the cell. Two well distinguished structures were seen associated with the anterior flagella. The first one corresponds to the “dense rods”, located just below the axoneme. The second one is a system of filaments located in the upper portion of the flagellum, facing the marginal plates and connecting these two structures. The thickness of the filaments is around 18 nm and they are spaced at intervals of 4–32 nm (average 18 nm). The length of the filaments may vary from 33 to 240 nm. We suggest that this filamentous structure of Giardia may help the dynamics and behavior of the anterior flagella of trophozoites during protozoan motility and adhesion, providing favorable conditions for the establishment of parasitism.

Though three-dimensional (3D) imaging gives deep insight into the inner structure of complex materials, the stereological analysis of 2D snapshots of material sections is still necessary for large-scale industrial applications for reasons related to time and cost constraints. In this paper, we propose an original framework to estimate the orientation distribution of generalized cylindrical structures from a single 2D section. Contrary to existing approaches, knowledge of the cylinder cross-section shape is not necessary. The only requirement is to know the area distribution of the cross-sections. The approach relies on minimization of a least squares criterion under linear equality and inequality constraints that can be solved with standard optimization solvers. It is evaluated on synthetic data, including simulated images, and is applied to experimental microscopy images of fibrous composite structures. The results show the relevance and capabilities of the approach though some limitations have been identified regarding sensitivity to deviations from the assumed model.

Quality control of the inertial confinement fusion (ICF) target in the laser fusion program is vital to ensure that energy deposition from the lasers results in uniform compression and minimization of Rayleigh–Taylor instabilities. The technique of nuclear microscopy with ion beam analysis is a powerful method to provide characterization of ICF targets. Distribution of elements, depth profile, and density image of ICF targets can be identified by particle-induced X-ray emission, Rutherford backscattering spectrometry, and scanning transmission ion microscopy. We present examples of ICF target characterization by nuclear microscopy at Fudan University in order to demonstrate their potential impact in assessing target fabrication processes.

Several approaches for cell image segmentation have been proposed and applied with success in the biomedical sciences field, but cell content estimation is still a not solved problem. This paper presents a novel formed classifier that combine appropriately “intelligent scissors” (livewire), object’s pixel information (RGB values of colors) and probabilistic artificial neural networks (ANN) techniques for segmenting and estimating precisely the cytoplasm composition in relation to protein, lipid, and carbohydrates. Based on this it is possible to estimate the gonad’s state of development and the maturation stage of oocytes in adult ovigerous females gonads.

The ovigerous mature females (identifiable by their brood pouch – empty, with eggs, with eyeless or eyed embryos) were selected under a dissecting microscope in the laboratory. A total of 170 gonadic masses from females containing oocytes in different developmental stages were analyzed. A total of 100 sections were observed by light microscopy in order to record the arrangement of the ovaries and determine the maturity stage of the various germ cells.

With the ANN network (Fig. 1) trained is possible to carry out the classification step. Classification aims to associate each cytoplasm pixel to one of the selected classes. In this process each cytoplasm pixel is simulated (Figure 2 A). As result a new image containing the classified cytoplasm is produced, where each class is represented by a different colour level (Figure 2 B). Figure 2 C show from left to right the full “classification flow process” including ROI selection, cytoplasm and nucleus contour extraction, cytoplasm segmentation and cytoplasm content classification. The proposed classifier was tested successfully in a representative image dataset, demonstrating the effectiveness and robustness of this new method facilitating the histochemical analysis of cell contents, based on a gonads model.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

A forescatter electron detector (FSED) was used to acquire dark-field micrographs (DF-FSED) on thin specimens with a scanning electron microscope. The collection angles were adjusted with the detector distance from the beam axis, which is similar to the camera length of the scanning transmission electron microscope annular DF detectors. The DF-FSED imaging resolution was calculated with SMART-J on an aluminum alloy and carbon nanotubes (CNTs) decorated with platinum nanoparticles. The resolution was three to six times worse than with bright-field imaging. Measurements of nanometer-size objects showed a similar feature size in DF-FSED imaging despite a signal-to-noise ratio 12 times smaller. Monte Carlo simulations were used to predict the variation of the contrast of a CNT/Fe/Pt system as a function of the collection angles. It was constant for very high collection angles (>450 mrad) and confirmed experimentally. The reverse contrast between carbon black particles and the smallest titanium dioxide (TiO2) nanoparticles was predicted by Monte Carlo simulations and observed in the DF-FSED micrograph of a battery electrode coating. However, segmentation of the micrograph was not able to isolate the TiO2 nanoparticle phase because of the close contrast of small TiO2 nanoparticles compared to the C black particles.

The microstructural analysis of the dehydrogenation products of the Ca(BH4)2–MgH2 composite was performed using transmission electron microscopy. It was found that nanocrystalline CaB6 crystallites formed as a dehydrogenation product throughout the areas where the signals of Ca and Mg were simultaneously detected, in addition to relatively coarse Mg crystallites. The uniform distribution of the nanocrystalline CaB6 crystallites appears to play a key role in the rehydrogenation of the dehydrogenation products, which implies that microstructure is a crucial factor determining the reversibility of reactive hydride composites.

Conventional electron-probe microanalysis has an X-ray analytical spatial resolution on the order of 1–4 μm width/depth. Many of the naturally occurring Fe–Si compounds analyzed in this study are smaller than 1 μm in size, requiring the use of lower accelerating potentials and nonstandard X-ray lines for analysis. Problems with the use of low-energy X-ray lines (soft X-rays) of iron for quantitative analyses are discussed and a review is given of the alternative X-ray lines that may be used for iron at or below 5 keV (i.e., accelerating voltage that allows analysis of areas of interest <1 μm). Problems include increased sensitivity to surface effects for soft X-rays, peak shifts (induced by chemical bonding, differential self-absorption, and/or buildup of carbon contamination), uncertainties in the mass attenuation coefficient for X-ray lines near absorption edges, and issues with spectral resolution and count rates from the available Bragg diffractors. In addition to the results from the traditionally used Fe Lα line, alternative approaches, utilizing Fe Lβ, and Fe Ll-η lines, are discussed.