This article reviews different methods used to perform quantitative X-ray microanalysis in the electron microscope and also demonstrates the urgency of measuring the fundamental parameters of X-ray generation for the development of accurate standardless quantitative methods. Using ratios of characteristic lines acquired on the same X-ray spectrum, it is shown that the Cliff and Lorimer KA-B factor can be used in a general correction method that is appropriate for all types of specimens and electron microscopes, providing that appropriate corrections are made for X-ray absorption, fluorescence, and indirect generation. Since the fundamental parameters appear in the KA-B factor, only the ratio of the ionization cross sections needs to be known, not their absolute values. In this regard, the measurement of ratios of the KA-B factor (or intensities at different beam energies of the same material with no change of beam spreading in the material) permits the validation for the best models to compute the ratio of ionization cross sections. It is shown, using this method, that the nonrelativistic Bethe equation, to compute ionization cross section, is very close to the equation of E. Casnati et al. (J Phys B15, 155–167, 1982) and also to the equations proposed by D. Bote and F. Salvat (Phys Rev A77, 042701, 2008) for the computation of the ratio of ionization cross sections. The method is extended to show that it could be used to determine the values of the Coster-Kronig transitions factors, an important fundamental parameter for the generation of L and M lines that is mostly known with poor accuracy. The detector efficiency can be measured with specimens where their intensities were measured with an energy dispersive spectrometer detector, the efficiency of which has been measured in an X-ray synchrotron (M. Alvisi et al., Microsc Microanal12, 406–415, 2006). The spatial resolution should always be computed when performing quantitative X-ray microanalysis and the equations of R. Gauvin (Microsc Microanal13(5), 354–357, 2007) for bulk materials and the one presented in this article for thin films should be used. The effects of X-rays generated by fast secondary electrons and by Auger electrons are reviewed, and their effect can be detrimental for the spatial resolution of materials involving low-energy X-ray lines, in certain specific conditions. Finally, quantitative X-ray microanalysis of heterogeneous materials is briefly reviewed.

A mathematical framework based on singular value decomposition is used to analyze the covariance among interatomic frequency distributions in spatial distribution maps (SDMs). Using this approach, singular vectors that capture the covariance within the SDM data are obtained. The structurally relevant singular vectors (SRSVs) are identified. Using the SRSVs, we extract information from z-SDMs that not only captures the offset between the atomic planes but also captures the covariance in the atomic structure among the neighborhood atomic planes. These refined z-SDMs classify the Δ(Δz) slices in the SDMs into structurally relevant information, noise, and aberrations. The SRSVs are used to construct refined xy-SDMs that provide enhanced structural information for three-dimensional atom probe tomography.

The ability to accurately reconstruct original spatial positions of field-evaporated ions emitted from a surface is fundamental to the success of atom probe tomography. As such, a clear understanding of the evolution of specimen shape and the resultant ions' trajectories during field evaporation plays an important role in improving reconstruction accuracy. To further this understanding, field-evaporation simulations of a bilayer specimen composed of two materials having an evaporation field difference of 20% were performed. The simulated field-evaporation patterns qualitatively compare favorably with experimental data, which provides confidence in the accuracy of specimen shapes predicted by the simulation. Correlations of known original atom positions with detector hit positions as a function of lateral detector position and evaporated depth were derived from the simulation. These correlations are contrasted with the current state-of-the-art reconstruction method thus outlining limitations of the current methodology. A pair of transformations are defined that take into account field-evaporated specimen shapes, and the resulting radial magnifications, to relate recorded ion positions in detector space to reconstructed atomic positions in specimen space. This novel process, when applied to simulated data, results in approximately a factor of 2 improvement in accuracy for reconstructions of interfaces with unequal fields (most general interfaces). This method is not constrained by the fundamental assumption of a hemispherical specimen shape.

In this article the Cu-Au binding energy in Cu3Au is determined by comparing experimental atom probe tomography (APT) results to simulations. The resulting bonding energy is supported by density functional theory calculations. The APT simulations are based on the Müller-Schottky equation, which is modified to include different atomic neighborhoods and their characteristic bonds. The local environment is considered up to the fifth next nearest neighbors. To compare the experimental with simulated APT data, the AtomVicinity algorithm, which provides statistical information about the positions of the neighboring atoms, is applied. The quality of this information is influenced by the field evaporation behavior of the different species, which is connected to the bonding energies.

Recent developments in the technology of laser-pulsed local-electrode atom-probe (LEAP) tomography include a picosecond ultraviolet (UV) laser system having a 355 nm wavelength and both external and in-vacuum optics. This approach ensures focusing of the laser beam to a smaller spot diameter than has heretofore been obtained using a green (532 nm wavelength) picosecond laser. We compare the mass spectra acquired, using either green or UV laser pulsing, from nickel-based superalloy specimens prepared either electrochemically or by lifting-out from bulk material using ion-beam milling in a dual-beam focused ion beam microscope. The utilization of picosecond UV laser pulsing yields improved mass spectra, which manifests itself in higher signal-to-noise ratios and mass-resolving power (m/Δm) in comparison to green laser pulsing. We employ LEAP tomography to investigate the formation of misoriented defects in nickel-based superalloys and demonstrate that UV laser pulsing yields better accuracy in compositional quantification than does green laser pulsing. Furthermore, we show that using a green laser the quality of mass spectra collected from specimens that were lifted-out by ion milling is usually poorer than for electrochemically-sharpened specimens. Employing UV laser pulsing yields, however, improved mass spectra in comparison to green laser pulsing even for ion-milled microtips.

The TEAM 0.5 electron microscope is employed to demonstrate atomic resolution phase contrast imaging and focal series reconstruction with acceleration voltages between 20 and 300 kV and a variable dose rate. A monochromator with an energy spread of ≤0.1 eV is used for dose variation by a factor of 1,000 and to provide a beam-limiting aperture. The sub-Ångstrøm performance of the instrument remains uncompromised. Using samples obtained from silicon wafers by chemical etching, the [200] atom dumbbell distance of 1.36 Å can be resolved in single images and reconstructed exit wave functions at 300, 80, and 50 kV. At 20 kV, atomic resolution <2 Å is readily available but limited by residual lens aberrations at large scattering angles. Exit wave functions reconstructed from images recorded under low dose rate conditions show sharper atom peaks as compared to high dose rate. The observed dose rate dependence of the signal is explained by a reduction of beam-induced atom displacements. If a combined sample and instrument instability is considered, the experimental image contrast can be matched quantitatively to simulations. The described development allows for atomic resolution transmission electron microscopy of interfaces between soft and hard materials over a wide range of voltages and electron doses.

This article deals with the measurement of strain in semiconductor heterostructures from convergent beam electron diffraction patterns. In particular, three different algorithms in the field of (circular) pattern recognition are presented that are able to detect diffracted disc positions accurately, from which the strain in growth direction is calculated. Although the three approaches are very different as one is based on edge detection, one on rotational averages, and one on cross correlation with masks, it is found that identical strain profiles result for an InxGa1−xNyAs1−y/GaAs heterostructure consisting of five compressively and tensile strained layers. We achieve a precision of strain measurements of 7–9·10−4 and a spatial resolution of 0.5–0.7 nm over the whole width of the layer stack which was 350 nm. Being already very applicable to strain measurements in contemporary nanostructures, we additionally suggest future hardware and software designs optimized for fast and direct acquisition of strain distributions, motivated by the present studies.

Zach phase plates (PPs) are promising devices to enhance phase contrast in transmission electron microscopy. The Zach PP shifts the phase of the zero-order beam by a strongly localized inhomogeneous electrostatic potential in the back focal plane of the objective lens. We present substantial improvements of the Zach PP, which overcome previous limitations. The implementation of a microstructured heating device significantly reduces contamination and charging of the PP structure and extends its lifetime. An improved production process allows fabricating PPs with reduced dimensions resulting in lower cut-on frequencies as revealed by simulations of the electrostatic potential. Phase contrast with inversion of PbSe nanoparticles is demonstrated in a standard transmission electron microscope with LaB6 cathode by applying different voltages.

Amplitude modulation (AM) scanning force microscopy (SFM) is superior to frequency modulation SFM in simplicity, sensitivity, and stability, but is still replaced by the latter because it is too slow when the Q factor is high (bandwidth < 0.5 Hz for Q > 50,000 and resonant frequency ω0 < 50 kHz). We report a close-loop AM detector that has an 18 Hz bandwidth, better than 1 mHz frequency resolution and excellent response to step frequency changes even for Q ∼ 60,000 and ω0 ∼ 32 kHz. Its superiority is well shown by the comparison of magnetic force microscope images taken under the new and old AM detection modes with the tip and scan area (videotape sample) being unchanged. Also important is that shifting the driving frequency from near the resonance peak to further away from the peak does not decrease the frequency resolution as much as we expect (but can increase the response speed).

Based on the quantitative fluorescence resonance energy transfer (FRET) method named PbFRET we reported recently, we herein developed a partial acceptor photobleaching-based quantitative FRET algorithm named B-PbFRET method. B-PbFRET overcomes not only the acceptor excitation crosstalk and donor emission spectral crosstalk but also the acceptor emission spectral crosstalk that harasses previous methods including fluorescence lifetime (FLIM), fluorescence recovery of donor after acceptor photobleaching, and acceptor sensitized emission (SE)-based methods. B-PbFRET method is implemented by simultaneously measuring the fluorescence intensity of both donor and acceptor channels at donor excitation before and after partial acceptor photobleaching, and it can directly measure the FRET efficiency (E) without any verified references. Based on the theoretical analysis of B-PbFRET, we also developed a more straightforward correction method named C-PbFRET to obtain the absolute E from the value measured by PbFRET for a given donor-acceptor pair. We validated both B-PbFRET and C-PbFRET methods by measuring the E of two linked constructs, 18AA and SCAT3 proteins, in single living cells, and our data demonstrated that both B-PbFRET and C-PbFRET methods can directly measure the absolute E of the linked constructs inside living cells under different degrees of acceptor emission spectral crosstalk.

Adenosine triphosphate (ATP) is a well-known energy source for muscle contraction. In this study, to visualize localization of ATP, a luciferin-luciferase reaction (LLR) was performed in mouse skeletal muscle with an “in vivo cryotechnique” (IVCT). First, to confirm if ATP molecules could be trapped and detected after glutaraldehyde (GA) treatment, ATP was directly attached to glass slides with GA, and LLR was performed. The LLR was clearly detected as an intentional design of the ATP attachment. The intensity of the light unit by LLR was correlated with the concentration of the GA-treated ATP in vitro. Next, LLR was evaluated in mouse skeletal muscles with IVCT followed by freeze-substitution fixation (FS) in acetone-containing GA. In such tissue sections the histological structure was well maintained, and the intensity of LLR in areas between muscle fibers and connective tissues was different. Moreover, differences in LLR among muscle fibers were also detected. For the IVCT-FS tissue sections, diaminobenzidine (DAB) reactions were clearly detected in type I muscle fibers and erythrocytes in capillaries, which demonstrated flow shape. Thus, it became possible to perform microscopic evaluation of the numbers of ATP molecules in the mouse skeletal muscles with IVCT, which mostly reflect living states.

Based on a scanning electron microscope operated at 30 kV with a homemade specimen holder and a multiangle solid-state detector behind the sample, low-kV scanning transmission electron microscopy (STEM) is presented with subsequent electron tomography for three-dimensional (3D) volume structure. Because of the low acceleration voltage, the stronger electron-atom scattering leads to a stronger contrast in the resulting image than standard TEM, especially for light elements. Furthermore, the low-kV STEM yields less radiation damage to the specimen, hence the structure can be preserved. In this work, two-dimensional STEM images of a 1-μm-thick cell section with projection angles between ±50° were collected, and the 3D volume structure was reconstructed using the simultaneous iterative reconstructive technique algorithm with the TomoJ plugin for ImageJ, which are both public domain software. Furthermore, the cross-sectional structure was obtained with the Volume Viewer plugin in ImageJ. Although the tilting angle is constrained and limits the resulting structural resolution, slicing the reconstructed volume generated the depth profile of the thick specimen with sufficient resolution to examine cellular uptake of Au nanoparticles, and the final position of these nanoparticles inside the cell was imaged.

We investigated the effects of sample preparation and of the exposure to an electron beam on particles in cryo-electron tomographs. Various virus particles with icosahedral symmetry were examined, allowing a comparison of symmetrically related components that should be identical in structure but might be affected differently by these imaging artifacts. Comparison of tomographic reconstructions with previously determined structures established by an independent method showed that neither freezing nor electron beam exposure produced a significant amount of shrinkage along the z axis (thickness). However, we observed damage to regions of the particles located close to the surface of the vitreous ice.

Advances in DNA sequencing, based on fluorescent microscopy, have transformed many areas of biological research. However, only relatively short molecules can be sequenced by these technologies. Dramatic improvements in genomic research will require accurate sequencing of long (>10,000 base-pairs), intact DNA molecules. Our approach directly visualizes the sequence of DNA molecules using electron microscopy. This report represents the first identification of DNA base pairs within intact DNA molecules by electron microscopy. By enzymatically incorporating modified bases, which contain atoms of increased atomic number, direct visualization and identification of individually labeled bases within a synthetic 3,272 base-pair DNA molecule and a 7,249 base-pair viral genome have been accomplished. This proof of principle is made possible by the use of a dUTP nucleotide, substituted with a single mercury atom attached to the nitrogenous base. One of these contrast-enhanced, heavy-atom-labeled bases is paired with each adenosine base in the template molecule and then built into a double-stranded DNA molecule by a template-directed DNA polymerase enzyme. This modification is small enough to allow very long molecules with labels at each A-U position. Image contrast is further enhanced by using annular dark-field scanning transmission electron microscopy (ADF-STEM). Further refinements to identify additional base types and more precisely determine the location of identified bases would allow full sequencing of long, intact DNA molecules, significantly improving the pace of complex genomic discoveries.

Despite recent advances in hematopoietic tissue research, effects of aging on hematopoietic erythroid precursor (EP) cells are unclear. In this article we present results suggesting that chromatin textural entropy of EP cells in mouse spleen increases with age, while chromatin homogeneity decreases. The experiment was conducted on a total of 32 male Swiss white mice. Spleen tissue was acquired from four age groups: 10 days, 1 month, 4 months, and 7 months old mice. A total of 640 randomly selected, nonoverlapping EP cell nuclei (20 per animal) were analyzed using the gray level co-occurrence matrix method. There was statistically highly significant difference between the age groups, both in chromatin entropy (ANOVA, F = 12.99, p < 0.0001) and in homogeneity (ANOVA, F = 7.05, p < 0.001). When the individual groups were compared (ANOVA post hoc test), statistical difference was detected in all group pairs, except between the animals 4 months and 7 months old, either in chromatin entropy or homogeneity. The detected increase of chromatin disorder in mouse juvenile period/early adulthood suggests that cell intrinsic factors such as epigenetic dysregulation and DNA damage accumulation may have an important role in EP cell aging.

The effects of plasma lipid overload on pancreatic islet function and on mineral imbalance are issues under debate. However, the outcomes may be biased by the different metabolisms of different species. This prospective study evaluated whether a high fat diet intake changed the distribution of physiologically relevant elements within pancreatic endocrine and exocrine tissues of Sprague Dawley rats and New Zealand White rabbits. Nuclear microscopy techniques provided images of the specimen density and structure as well as the elemental distributions and quantification of P, S, Cl, K, Ca, Fe, and Zn using unstained cryosections of pancreas. Our results indicate that pancreatic islets in normal rats and rabbits had lower tissue density and higher Ca, Fe, and Zn content compared to exocrine tissue, and that rabbit islets exhibit the highest Zn content (3,300 μg/g in rabbits versus 510 μg/g in rats). Fat diet intake resulted in large deposits of fat in the pancreas, which modified the density contrast of tissues and also resulted in a twofold decrease of Ca and Zn concentrations in islets of both rats and rabbits. This result indicates that a fat diet leads to a reduction in essential trace element concentrations in pancreas, which in turn may hamper endocrine function.

The use of plants as a source of medicine is an important component of the health care system in rural India. Carex baccans (Cyperaceae) and Potentilla fulgens (Rosaceae) have been known since ancient times in northeast India for their antitumor, antidiabetic, and antihelmintic properties. The present study was designed to determine the subacute toxicity profile of the root tuber extract of C. baccans and root-peel extract of P. fulgens in Wistar rats. The subacute oral toxicity was conducted using sublethal doses of 40, 50, 100, 150, 200, and 400 mgkg−1 body weights. Surface topographical and ultrastructural observations of liver and intestinal microvilli showed remarkable deformation and disruption, accompanied by quantitative changes in the liver enzymes, i.e., aspartate aminotransferase and alanine aminotransferase in comparison to those of the control group. Apoptotic cell death was observed in the liver cells of rats exposed to both of the plant extracts. A significant increase in splenic lymphocyte count was also observed in rats exposed to the highest concentration of both extracts. The results showed that consumption of the plant extracts at higher doses may cause toxicological effect if treatment continues for a long time.

Optical and scanning electron microscopy (SEM) of red blood cells (RBCs) of common carp, Cyprinus carpio, inhabiting a polluted lake in Northeast India revealed a number of abnormalities. About 7% of the RBC showed the presence of a micronucleus, besides the presence of some bi-nucleated and abnormally shaped nuclei. RBCs, white blood cells, and hemoglobin content were found to be reduced significantly as compared to the control. SEM showed the presence of spherocytes, early stages of echinocytes, cytoplasmic blebbing, erythrocytes with contraction from one side, abnormal shape of erythrocytes (hexagonal/pentagonal/tetragonal), development of lobopodial projections, cell rupture, membrane internalization, and formation of ring-shaped RBC. Energy dispersive X-ray spectroscopy (EDS) showed the presence of a considerable percentage of silicon and lead in erythrocytes of the fish collected from the polluted lake, in contrast to a negligible concentration of the two elements in control fish. Significance of the study in relation to fish health in a polluted body of water and the importance of SEM, EDS, and light microscopy in utilizing hematological parameters as pollution indicators are discussed.

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.

The recent developments of phase-contrast synchrotron imaging techniques have been of great interest for paleontologists, providing three-dimensional (3D) tomographic images of anatomical structures, thereby leading to new paleobiological insights and the discovery of new species. However, until now, it has not been used on features smaller than 5–7 μm voxel size in fossil bones. Because much information is contained within the 3D histological architecture of bone, including an ontogenetic record, crucial for understanding the paleobiology of fossil species, the application of phase-contrast synchrotron tomography to bone at higher resolutions is potentially of great interest. Here we use this technique to provide new 3D insights into the submicron-scale histology of fossil and recent bones, based on the development of new pink-beam configurations, data acquisition strategies, and improved processing tools. Not only do the scans reveal by nondestructive means all of the major features of the histology at a resolution comparable to that of optical microscopy, they provide 3D information that cannot be obtained by any other method.