Scanning electron microscopy backscattered-electron images of paint cross sections show the compositional contrast within the paint system. They not only give valuable information about the pigment composition and layer structure but also about the aging processes in the paint. This article focuses on the reading of backscatter images of lead white-containing samples from traditional oil paintings (17th–19th centuries). In contrast to modern lead white, traditional stack process lead white is characterized by a wide particle size distribution. Changes in particle morphology and distribution are indications of chemical/physical reactivity in the paint. Lead white can be affected by free fatty acids to form lead soaps. The dissolution of lead white can be recognized in the backscatter image by gray (less scattering) peripheries around particles and gray amorphous areas as opposed to the well-defined, highly scattering intact lead white particles. The small particles react away first, while the larger particles/lumps can still be visible. Formed lead soaps appear to migrate or diffuse through the semipermeable paint system. Lead-rich bands around particles, at layer interfaces and in the paint medium, are indications of transport. The presence of lead-containing crystals at the paint surface or inside aggregates furthermore point to the migration and mineralization of lead soaps.

We report variable resolution fluctuation electron microscopy (VRFEM) measurements on Cu64.5Zr35.5 metallic glass acquired using scanning transmission electron microscopy nanodiffraction using coherent probes 0.8 to 11 nm in diameter. The VRFEM results show that medium range atomic order structure of Cu64.5Zr35.5 bulk metallic glass at the ∼1 nm scale has large fluctuations, but the structure becomes almost completely homogeneous at the 11 nm scale. We show that our experimental VRFEM data are consistent with two different models, the pair persistent model and the amorphous/nanocrystal composite model. We also report a new way to filter VRFEM data to eliminate the effect of specimen thickness gradient using high-angle annular dark field images as references.

Detection and tracking of stem cell state are difficult due to insufficient means for rapidly screening cell state in a noninvasive manner. This challenge is compounded when stem cells are cultured in aggregates or three-dimensional (3D) constructs because living cells in this form are difficult to analyze without disrupting cellular contacts. Multiphoton laser scanning microscopy is uniquely suited to analyze 3D structures due to the broad tunability of excitation sources, deep sectioning capacity, and minimal phototoxicity but is throughput limited. A novel multiphoton fluorescence excitation flow cytometry (MPFC) instrument could be used to accurately probe cells in the interior of multicell aggregates or tissue constructs in an enhanced-throughput manner and measure corresponding fluorescent properties. By exciting endogenous fluorophores as intrinsic biomarkers or exciting extrinsic reporter molecules, the properties of cells in aggregates can be understood while the viable cellular aggregates are maintained. Here we introduce a first generation MPFC system and show appropriate speed and accuracy of image capture and measured fluorescence intensity, including intrinsic fluorescence intensity. Thus, this novel instrument enables rapid characterization of stem cells and corresponding aggregates in a noninvasive manner and could dramatically transform how stem cells are studied in the laboratory and utilized in the clinic.

A vascular corrosion cast of an entire mouse kidney was scanned with a modular multiresolution X-ray nanotomography system. Using an isotropic voxel pitch of 0.5 μm, capillary systems such as the vasa recta, peritubular capillaries and glomeruli were clearly resolved. This represents a considerable improvement over corrosion casts scanned with microcomputed tomography systems. The resolving power of this system was clearly demonstrated by the unique observation of a dense, subcapsular mat of capillaries enveloping the entire outer surface of the cortical region. Resolution of glomerular capillaries was comparable to similar models derived from laser scanning confocal microscopy. The high-resolution, large field of view and the three-dimensional nature of the resulting data opens new possibilities for the use of corrosion casting in research.

The teeth of the marine mollusk Acanthopleura hirtosa are an excellent example of a complex, organic, matrix-mediated biomineral, with the fully mineralized teeth comprising layers of iron oxide and iron oxyhydroxide minerals around a calcium apatite core. To investigate the relationship between the various mineral layers and the organic matrix fibers on which they grew, sections have been prepared from specific features in the teeth at controlled orientations using focused ion beam processing. Compositional and microstructural details of heterophase interfaces, and the fate of the organic matrix fibers within the mineral layers, can then be analyzed by a range of transmission electron microscopy (TEM) techniques. Energy-filtered TEM highlights the interlocking nature of the various mineral phases, while high-angle annular dark-field scanning TEM imaging demonstrates that the organic matrix continues to exist in the fully mineralized teeth. These new insights into the structure of this complex biomaterial are an important step in understanding the relationship between its structural and physical properties and may help explain its high strength and crack-resistance behavior.

Artifacts are the nemesis of trace element analysis in electron-excited energy dispersive X-ray spectrometry. Peaks that result from nonideal behavior in the detector or sample can fool even an experienced microanalyst into believing that they have trace amounts of an element that is not present. Many artifacts, such as the Si escape peak, absorption edges, and coincidence peaks, can be traced to the detector. Others, such as secondary fluorescence peaks and scatter peaks, can be traced to the sample. We have identified a new sample-dependent artifact that we attribute to Compton scattering of energetic X-rays generated in a small feature and subsequently scattered from a low atomic number matrix. It seems likely that this artifact has not previously been reported because it only occurs under specific conditions and represents a relatively small signal. However, with the advent of silicon drift detectors and their utility for trace element analysis, we anticipate that more people will observe it and possibly misidentify it. Though small, the artifact is not inconsequential. Under some conditions, it is possible to mistakenly identify the Compton scatter artifact as approximately 1% of an element that is not present.

Aberration-corrected scanning transmission electron microscopes (STEMs) provide sub-Angstrom lateral resolution; however, the large convergence angle greatly reduces the depth of field. For microscopes with a small depth of field, information outside of the focal plane quickly becomes blurred and less defined. It may not be possible to image some samples entirely in focus. Extended depth-of-field techniques, however, allow a single image, with all areas in focus, to be extracted from a series of images focused at a range of depths. In recent years, a variety of algorithmic approaches have been employed for bright-field optical microscopy. Here, we demonstrate that some established optical microscopy methods can also be applied to extend the ∼6 nm depth of focus of a 100 kV 5th-order aberration-corrected STEM (αmax = 33 mrad) to image Pt-Co nanoparticles on a thick vulcanized carbon support. These techniques allow us to automatically obtain a single image with all the particles in focus as well as a complimentary topography map.

Copper, gold, and tungsten thin films have been exposed to 30 kV Ga+ ion irradiation, and the resulting microstructural modifications are studied as a function of ion dose. The observed microstructural changes include texture development with respect to the easy channeling direction in the target, and in the case of Cu, an additional intermetallic phase is produced. Texture development in these target materials is a function of the starting materials grain size, and these changes are not observed in large grained materials. The accepted models of differential damage driven grain growth are not supported by the results of this study. The implications of this study to the use of focused ion beam tools for sample preparation are discussed.

Microscopy and Microanalysis would like to thank the following sponsors for their generous support of the M&M 2011 Conference and EXPO. (Sponsors as of May 16, 2011)

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Little is known about how age influences the ways in which cardiac fibroblasts interact with the extracellular matrix. We investigated the deformation of collagen substrates by neonatal and adult rat cardiac fibroblasts in monolayer and three-dimensional (3D) cultures, and quantified the expression of three collagen receptors [discoidin domain receptor (DDR)1, DDR2, and β1 integrin] and the contractile protein alpha smooth muscle actin (α-SMA) in these cells. We report that adult fibroblasts contracted 3D collagen substrates significantly less than their neonate counterparts, whereas no differences were observed in monolayer cultures. Adult cells had lower expression of β1 integrin and α-SMA than neonate cultures, and we detected significant correlations between the expression of α-SMA and each of the collagen receptors in neonate cells but not in adult cells. Consistent with recent work demonstrating age-dependent interactions with myocytes, our results indicate that interactions between cardiac fibroblasts and the extracellular matrix change with age.

In this work, we present the results of an analytical method developed for detailed pigment identification, stratigraphy, and degradation of the paint layers of mural paintings applied in the study of the 17th century frescoes from the Misericordia Church of Odemira (Southwest Portugal). In situ X-ray fluorescence spectrometry analyses were performed on three panels of the mural paintings and complemented by colorimetric measurements. The different color areas were also sampled as microfragments (approx. 1 mm2) that were studied as taken or mounted in epoxy resin to expose the different paint layers. The microfragments of paint layers and their cross sections were characterized by optical microscopy and scanning electron microscopy coupled with energy dispersive X-ray spectrometry. Furthermore, elemental analysis was obtained with spatially resolved confocal synchrotron radiation μ-X-ray fluorescence spectrometry performed at ANKA synchrotron FLUO beamline. Occasionally, phase analysis by μ-X-ray diffraction was also performed. Results from the different techniques allowed pigment identification and, in some cases, the evaluation of color changes due to degradation processes and, considering the Southern Portugal geology, the identification of their possible provenance. The pigments used were essentially yellow, brown and red ochres, smalt blue, copper green, and black earths, probably from local sources.

The quantitative scanning electron microscope–energy dispersive X-ray (SEM-EDX) analysis of a horneblende and two augite prismatic samples reduced to submicrometric particles was performed, and error due to the particle effects (“absent mass” and the “reduced absorption” effect) was minimized. Correction factors as a function of fragment size were obtained for O, Na, Mg, Si, Ca, and Fe. In addition, the influence of chemical composition of the samples used as standards (the matrix effect) on correction factors was evaluated. The results indicate that the absent mass effect is dominant for all elements except for the light elements O and Na, for which the reduced absorption effect is dominant. No significant matrix effect has been observed. By using corrected SEM-EDX data, the error on quantification of the element concentration has been estimated to be 3% relative for light elements and below 2% relative for heavy elements (notably, about 1% relative for Fe).

Methods for creating speckle patterns on mouse arteries for use in deformation and strain field measurements in a stereomicroscope digital image correlation (DIC) system are described. Both fluorescent microsphere binding and ethidium bromide (EB) nuclear staining were used to generate high contrast, random patterns on mouse carotid arteries. To quantify the quality of each pattern, several metrics are used including (a) histogram distribution for each intensity pattern and (b) pixel-level variance in intensity pattern noise. Results demonstrate that both approaches provide sufficient pattern contrast for use in image-based methods to measure deformations in soft tissue. While fluorescent nuclear staining generates higher pixel-level intensity noise, this method provides better overall pattern quality (greater spatial uniformity and broader histogram) for automated DIC analysis when used at the appropriate magnification. Using recently developed theoretical predictions, estimates for the standard deviation in image-correlation-based displacements due to the measured intensity pattern variance are presented for fluorescent microsphere binding and EB nuclear staining patterns. Results confirm that both patterning approaches provide relatively small standard deviation in displacement measurements and hence are appropriate for measurement of deformations in small artery specimens.

The detection of line-like features in images finds many applications in microanalysis. Actin fibers, microtubules, neurites, pilis, DNA, and other biological structures all come up as tenuous curved lines in microscopy images. A reliable tracing method that preserves the integrity and details of these structures is particularly important for quantitative analyses. We have developed a new image transform called the “Coalescing Shortest Path Image Transform” with very encouraging properties. Our scheme efficiently combines information from an extensive collection of shortest paths in the image to delineate even very weak linear features.

This work comprises the structural characterization of Eucalyptus and Pinus radiata pulp fibers and their corresponding fibrillated materials, based on quantitative electron microscopy techniques. Compared to hardwood fibers, the softwood fibers have a relatively open structure of the fiber wall outer layers. The fibrillation of the fibers was performed mechanically and chemi-mechanically. In the chemi-mechanical process, the pulp fibers were subjected to a TEMPO-mediated oxidation to facilitate the homogenization. Films were made of the fibrillated materials to evaluate some structural properties. The thicknesses and roughnesses of the films were evaluated with standardized methods and with scanning electron microscopy (SEM), in backscattered electron imaging mode. Field-emission SEM (FE-SEM) and transmission electron microscopy (TEM) were performed to quantify the nanofibril morphology. In this study, we give additional and significant evidences about the suitability of electron microscopy techniques for quantification of nanofibril structures. In addition, we conclude that standard methods are not suitable for estimating the thickness of films having relatively rough surfaces. The results revealed significant differences with respect to the morphology of the fibrillated material. The differences are due to the starting raw material and to the procedure applied for the fibrillation.

A series of multislice simulations to quantify the effect of various degrees of order, composition, and thickness on the electron diffracted intensities were performed using the L10 FePt system as the case study. The dynamical diffraction studies were done in both a convergent electron beam diffraction and selected area electron diffraction condition. The L10 symmetry demonstrated some peculiar challenges in the simulation, in particular between the {111} plane normal and the ⟨111⟩ direction, which are not equivalent because of tetragonality. A hybrid weighting function atom of Fe-Pt was constructed to account for S < 1 or nonequiatomic compositions. This statistical approach reduced the complexity of constructing a crystal with the probability that a particular atom was at a particular lattice site for a given order parameter and composition. Considerations of accelerating voltage, convergent angle, and thermal effects are discussed. The simulations revealed significant differences in intensity ratios between films of various compositions but equivalent unit cell numbers and degree of order.

Atom probe tomography (APT) represents a significant step toward atomic resolution microscopy, analytically imaging individual atoms with highly accurate, though imperfect, chemical identity and three-dimensional (3D) positional information. Here, a technique to retrieve crystallographic information from raw APT data and restore the lattice-specific atomic configuration of the original specimen is presented. This lattice rectification technique has been applied to a pure metal, W, and then to the analysis of a multicomponent Al alloy. Significantly, the atoms are located to their true lattice sites not by an averaging, but by triangulation of each particular atom detected in the 3D atom-by-atom reconstruction. Lattice rectification of raw APT reconstruction provides unprecedented detail as to the fundamental solute hierarchy of the solid solution. Atomic clustering has been recognized as important in affecting alloy behavior, such as for the Al-1.1Cu-1.7Mg (at. %) investigated here, which exhibits a remarkable rapid hardening reaction during the early stages of aging, linked to clustering of solutes. The technique has enabled lattice-site and species-specific radial distribution functions, nearest-neighbor analyses, and short-range order parameters, and we demonstrate a characterization of solute-clustering with unmatched sensitivity and precision.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.