Transmission electron microscopy (TEM) in combination with electron tomography is widely used to obtain nanometer scale three-dimensional (3D) structural information about biological samples. However, studies of whole eukaryotic cells are limited in resolution and/or contrast on account of the effect of chromatic aberration of the TEM objective lens on electrons that have been scattered inelastically in the specimen. As a result, 3D information is usually obtained from sections and not from whole cells. Here, we use chromatic aberration-corrected TEM to record bright-field TEM images of nanoparticles in a whole mount macrophage cell. Tilt series of images are used to generate electron tomograms, which are analyzed to assess the spatial resolution that can be achieved for different vertical positions in the specimen. The uptake of gold nanoparticles coated with low-density lipoprotein (LDL) is studied. The LDL is found to assemble in clusters. The clusters contain nanoparticles taken up on different days, which are joined without mixing their nanoparticle cargo.

Get visitor and tourist information on Indianapolis at the M&M 2013 Booth in the main registration area just outside the exhibit hall.

Get visitor and tourist information on Indianapolis at the M&M 2013 Booth in the main registration area just outside the exhibit hall.

Cast AZ31B-H24 magnesium alloy, comprising Mg with 3.27 wt% Al and 0.96 wt% Zn, was cold rolled and subsequently annealed. Global texture evolutions in the specimens were observed by X-ray diffractometry after the thermomechanical processing. Image-based microstructure and texture for the deformed, recrystallized, and grown grains were observed by electron backscattered diffractometry. Recrystallized grains could be distinguished from deformed ones by analyzing grain orientation spread. Split basal texture of ca. ±10–15° in the rolling direction was observed in the cold-rolled sample. Recrystallized grains had widely spread basal poles at nucleation stage; strong {0001} basal texture developed with grain growth during annealing.

A hybrid nanobeam diffraction/imaging method, which combines well-developed diffraction imaging with nanobeam diffraction (NBD) pattern analysis, is described for structural mapping of disordered materials. Spatially resolved crystallographic information is obtained by NBD imaging by collecting NBD patterns at predefined intervals within a field of interest. The resulting dataset of NBD patterns is preprocessed to produce a spectral-imaging-like dataset and is further analyzed via multivariate statistical analysis methods in order to extract the relevant structural components and their distribution within the area of the sample under study without prior knowledge. Additional radial distribution function analysis of either the principal components or averaged data provides real-space maps of short-range order within the field of interest. This technique is demonstrated for two systems, one with multiple amorphous phases and one with multiple phases (amorphous and nanocrystalline) with similar chemistry.

The taxonomy of fungal species, similar to that of many other microorganisms, suffers frequent revisions due to the discovery of new species and to the development and gathering of characterization data and morphological information. Morpho-taxonomy helps in the identification of many species. This work presents the macro, micro-morphological, and spectral mass analyses for phenotypic characterization of 13 species of Aspergillus section Nigri, showing that the characterization of spores (conidia) by scanning electron microscopy can be used as a tool to discriminate key morphological characteristics and separate closely related fungi. These results were corroborated by colony plates, stereomicroscopy, light microscopy, and spectral mass data.

Given an unknown multicomponent alloy, and a set of standard compounds or alloys of known composition, can one improve upon popular standards-based methods for energy dispersive X-ray (EDX) spectrometry to quantify the elemental composition of the unknown specimen? A method is presented here for determining elemental composition of alloys using transmission electron microscopy–based EDX with appropriate standards. The method begins with a discrete set of related reference standards of known composition, applies multivariate statistical analysis to those spectra, and evaluates the compositions with a linear matrix algebra method to relate the spectra to elemental composition. By using associated standards, only limited assumptions about the physical origins of the EDX spectra are needed. Spectral absorption corrections can be performed by providing an estimate of the foil thickness of one or more reference standards. The technique was applied to III-V multicomponent alloy thin films: composition and foil thickness were determined for various III-V alloys. The results were then validated by comparing with X-ray diffraction and photoluminescence analysis, demonstrating accuracy of approximately 1% in atomic fraction.

Nanoparticles composed of a magnetic iron oxide core surrounded by a metal shell have utility in a broad range of biomedical applications. However, the presence of surface energy differences between the two components makes wetting of oxide with metal unfavorable, precluding a “core–shell” structure of an oxide core completely surrounded by a thin metal shell. Three-dimensional island growth followed by island coalescence into thick shells is favored over the two-dimensional layer-by-layer growth of a thin, continuous metal coating of a true core–shell. Aqueous synthesis of gold-coated magnetite nanoparticles with analysis by infrared, energy-dispersive X-ray, and electron energy loss spectroscopies; high-resolution transmission electron microscopy; selected area electron diffraction; and high-angle annular dark-field scanning transmission electron microscopy showed two distinct morphologies that are inconsistent with an idealized core–shell. The majority were isolated ~16–22-nm-diameter nanoparticles consisting of ~7-nm-diameter magnetite and a thick deposition of gold, most often discontinuous, with some potentially “sandwiched” morphologies. A minority were aggregates of agglomerated magnetite decorated with gold but displaying significant bare magnetite. Both populations were successfully conjugated to fibrinogen and targeted to surface-activated platelets, demonstrating that iron oxide–gold nanoparticles produced by aqueous synthesis do not require an ideal core–shell structure for biological activity in cell labeling and targeting applications.

During mitosis nucleation-promoting factors (NPFs) bind to the Arp2/3 complex and activate actin assembly. JMY and WAVE2 are two critical members of the NPFs. Previous studies have demonstrated that NPFs promote multiple processes such as cell migration and cytokinesis. However, the role of NPFs in development of mammalian embryos is still unknown. Results of the present study show that the NPFs JMY and WAVE2 are critical for cytokinesis during development of mouse embryos. Both JMY and WAVE2 are expressed in mouse embryos. After injection of JMY or WAVE2 siRNA, all embryos failed to develop to the morula or blastocyst stages. Moreover, using fluorescence intensity analysis, we found that the expression of actin decreased, and multiple nuclei were observed within a single cell indicating that NPFs-induced actin reduction caused the failure of cell division. In addition, injection of JMY and WAVE2 siRNA also caused ARP2 degradation, indicating that involvement of NPFs in development of mouse embryos is mainly through regulation of ARP2/3-induced actin assembly. Taken together, these data suggested that WAVE2 and JMY are involved in development of mouse embryos, and their regulation may be through a NPFs-Arp2/3-actin pathway.

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

A single layer of LaAlO3 with a nominal thickness of one unit cell, which is sandwiched between a SrTiO3 substrate and a SrTiO3 capping layer, is quantitatively investigated by high-resolution transmission electron microscopy. By the use of an aberration-corrected electron microscope and by employing sophisticated numerical image simulation procedures, significant progress is made in two aspects. First, the structural as well as the chemical features of the interface are determined simultaneously on an atomic scale from the same specimen area. Second, the evaluation of the structural and chemical data is carried out in a fully quantitative way on the basis of the absolute image contrast, which has not been achieved so far in materials science investigations using high-resolution electron microscopy. Considering the strong influence of even subtle structural details on the electronic properties of interfaces in oxide materials, a fully quantitative interface analysis, which makes positional data available with picometer precision together with the related chemical information, can contribute to a better understanding of the functionality of such interfaces.

Tuberculosis (TB) is one of the major causes of mortality and morbidity worldwide accounting for 3.1 million deaths per year. This disease, caused by Mycobacterium tuberculosis (M. tuberculosis) made a deadly comeback, during the 1990’s, triggered mainly by the emergence of acquired immunodeficiency syndrome (AIDS). More recently, the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) M. tuberculosis strains, uncovered the most freighting face of this disease an incurable infection with the currently available therapeutic tools. Although Portugal is considered a medium incidence setting, annually are reported MDR and even XDR TB cases. The majority of these cases occur in the Lisbon area and the strains involved are genetically related being known as Lisboa family.

In the present work a group of 283 M. tuberculosis isolates collected in a Lisbon hospital during a two years period (2008-2009) were studied. The morphology of colonies grown on Lowenstein-Jensen slants was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) using previously described procedures. The aim of the study was the establishment of a link between mycobacteria drug susceptibility and structure. In the first part of the study approximately 20 isolates, with different drug susceptibility profiles ranging from pan-susceptible to XDR, were grown on Lowenstein-Jensen slants and their morphology was compared. Although all mycobacteria originated rough colonies their size differ with the drug susceptibility profile. The pan-susceptible strains generated larger colonies than drug resistant strains as shown in figure 1.These colonies were then processed for SEM analysis. The results obtained show that mycobacteria surface are distinct in susceptible and drug resistant strains as shown in figure 2.A and B. While drug susceptible mycobacteria have a homogenous surface (Figure 1A), drug resistant bacteria present a heterogeneous surface (Figure 2B) with small protrusions (Fig. 2B inset). In order to evaluate the existence of differences in the ultrastructure of circulating M. tuberculosis strains the colonies were processed and analysed by TEM. For this approach were selected only two isolates: the pan-susceptible R188/09 and the XDR HPV108/09.

The results obtained by the analysis of at least 300 bacteria present in non consecutive sections show that mycobacteria cell width (0 350 nm) is similar for both bacteria (Table 1). Nevertheless, their cell length and cell envelope width are significantly different. The XDR strain is shorter (p=0.009) and has a ticker cell envelope (p=0.004) than the pan-susceptible strain. These results are in agreement with those published in the literature.

Altogether our data clearly shows the existence of a link between mycobacteria ultrastructure and drug susceptibility. In order to better evaluate these differences a larger number of isolates must be studied. The use of other electron microscopy techniques, such as CEMOVIS, will avoid the formation of undesirable artefacts (e.g. mesosome) produced by dehydration and room temperature sectioning allowing a better characterization of mycobacteria ultrastructure.

The authors acknowledge the funding by Fundação para a Ciência e Tecnologia (SFRH/BD/73579/2010, C2008-C2008_P2 and PEst-OE/CTM-UI0084/2011 grants.)

Two kinds of Sr2FeReO6 (SFRO) samples, pristine SFRO and Re-excess SFRO, were prepared and we visualized the local atomic structure in terms of cationic ordering in the prepared SFRO samples via high-angle annular dark-field (HAADF) scanning transmission electron microscopy (STEM). HAADF-STEM results demonstrated the high degree of cationic ordering maintains in both the pristine SFRO and Re-excess SFRO samples. On the other hand, defective structures such as antiphase boundary and Re-deficient phase were observed dominantly in the pristine SFRO, and thus the poor magnetic property in the pristine SFRO is attributed to those defective structures related with the frustrated Fe/Re ordering.

Degranulation of intestinal mast cells in rainbow trout was studied ex vivo by means of texture analysis and related to the maximal intestinal contraction elicited by degranulation itself. Two strips from the same intestinal segment from ten trout were sampled, processed for light microscopy and stained with Giemsa solution. One of the two strips was exposed to an incremental dose of compound 48/80 in an isolated organ bath before processing. Gray-level RGB channel equivalent and 8-bit gray-level images of five granular cytoplasm areas of mast cells for each section were analyzed for texture features and to evaluate discrimination possibility between treatment groups by means of linear discriminant analysis according to feature selection methods and RGB stacks. Differential mean values (after–before compound 48/80) of the green (r2 = 0.84, p < 0.01) and blue (r2 = 0.83, p < 0.01) RGB channels and 8-bit grayscale (r2 = 0.76, p < 0.05) image correlated significantly with the respective value of maximal intestinal contraction. A possible acidic (anionic) nature for the putative pro-contractile basophil agonist can be inferred.

Welcome to the 71st Microscopy & Microanalysis meeting, M&M 2013 in Indianapolis, Indiana! The Microscopy Society of America, the Microanalysis Society, and the International Metallographic Society have excelled again bringing the latest and most innovative applications and instrumental developments from investigators in the biological and physical sciences using microscopy and microanalysis techniques. M&M 2013 features more than 35 symposia covering a broad range of topics, ample educational opportunities in the form of courses and tutorials and pre-meeting events including courses, a congress and a workshop.

Tuberculosis (TB) is a major health problem. The emergence of multidrug resistant (MDR) Mycobacterium tuberculosis (Mtb) isolates confounds treatment strategies. In Portugal, cases of MDR-TB are reported annually with an increased incidence noted in Lisbon. The majority of these MDR-TB cases are due to closely related mycobacteria known collectively as the Lisboa family and Q1 cluster. Genetic determinants linked to drug resistance have been exhaustively studied resulting in the identification of family and cluster specific mutations. Nevertheless, little is known about other factors involved in development of mycobacteria drug resistance. Here, we complement genetic analysis with the study of morphological and structural features of the Lisboa family and Q1 cluster isolates by using scanning and transmission electron microscopy. This analysis allowed the identification of structural differences, such as cell envelope thickness, between Mtb clinical isolates that are correlated with antibiotic resistance. The infection of human monocyte derived macrophages allowed us to document the relative selective advantage of the Lisboa family isolates over other circulating Mtb isolates.

Recombinant ferritin is an excellent template for the synthesis of magnetic nanoparticles. This paper describes carefully performed experiments both to identify ironoxides within nanoparticles and to measure the number of iron atoms in the cores of recombinant human H-chain ferritin (HFn), based on spectroscopy techniques. Using electron energy-loss spectroscopy (EELS) analysis, magnetite (Fe3O4) has been unequivocally identified as the ironoxide formed within HFn cores under special preparation conditions. Atom counting analysis by EELS and high-angle annular dark-field imaging further allowed the correlation of the particle sizes to the real Fe atom numbers in a quantitative manner. These results help clarify some structural confusion between magnetite and maghemite (γ-Fe2O3), and also provide standard data for the number of Fe atoms within Fe3O4 particles of a given size, whose use is not limited to cases of magnetite synthesized in the cores of recombinant human ferritin.

The correction of the aberrations of electron lenses is the long story of many seemingly fruitless efforts to improve the resolution of electron microscopes by compensating for aberrations of round electron lenses over a period of 50 years. The problem started in 1936 when Scherzer demonstrated that the chromatic and spherical aberrations of rotationally symmetric electron lenses are unavoidable. Moreover, the coefficients of these aberrations cannot be made sufficiently small. As a result, the resolution limit of standard electron microscopes equals about one hundred times the wavelength of the electrons, whereas modern light microscopes have reached a resolution limit somewhat smaller than the wavelength. In 1947, Scherzer found an ingenious way for enabling aberration correction. He demonstrated in a famous article that it is in theory possible to eliminate chromatic and spherical aberrations by lifting any one of the constraints of his theorem, either by abandoning rotational symmetry or by introducing time-varying fields, or space charges. Moreover, he proposed a multipole corrector compensating for the spherical aberration of the objective lens.

We previously used synchrotron infrared microspectroscopy to describe the biochemical signature of skeletal muscle (biceps brachii) from the frozen ancient remains of a young man. In this current paper, we use light microscopy to assess the state of preservation of cellular components in the trapezius muscle from these same ancient remains and then use mid-infrared analysis at the Canadian Light Source synchrotron facility to further analyze the tissue. We compare spectra between the trapezius samples from the ancient remains and a recently deceased cadaver (control). Infrared spectra indicate preservation of secondary structure, with the α-helix being the principal component, along with triple helical portions of the protein backbone. Our mid-infrared analysis indicates an energy reserve in the skeletal muscle in the ancient remains.