This paper reports an unusual green pigment, brochantite (Cu4(SO4)(OH)6), on 16th-century Portuguese-Flemish paintings, attributed to the Master Frei Carlos workshop. This green mineral is usually identified as an impurity or alteration product in the green pigments verdigris (Cu(CH3COO)2nCu(OH)2) or malachite (CuCO3 Cu(OH)2). However, after thorough investigation with a broad range of analytical techniques, it became clear that, in this case, brochantite was applied as a pigment. The abundance, pigment granulometry, and pigment morphology suggest intentional use by this Portuguese-Flemish Master as a natural pigment rather than its accidental use as an alteration product. This seems to be a distinguishable feature to other painters (Flemish and Portuguese) working in Portugal at the beginning of the 16th century.

The multi-analytical study of these easel paintings was first performed by physical imaging techniques and material characterization was carried out by optical microscopy, micro-Fourier-transform infrared-spectroscopy, micro-Raman spectroscopy, scanning electron microscopy coupled with energy dispersive X-ray spectrometry, and micro-X-ray diffraction analysis (XRD).

Electron correlation microscopy (ECM) is a new technique that utilizes time-resolved coherent electron nanodiffraction to study dynamic atomic rearrangements in materials. It is the electron scattering equivalent of photon correlation spectroscopy with the added advantage of nanometer-scale spatial resolution. We have applied ECM to a Pd40Ni40P20 metallic glass, heated inside a scanning transmission electron microscope into a supercooled liquid to measure the structural relaxation time τ between the glass transition temperature Tg and the crystallization temperature, Tx. τ determined from the mean diffraction intensity autocorrelation function g2(t) decreases with temperature following an Arrhenius relationship between Tg and Tg+25 K, and then increases as temperature approaches Tx. The distribution of τ determined from the g2(t) of single speckles is broad and changes significantly with temperature.

A resolution metric intended for resolution analysis of arbitrary spatially calibrated images is presented. By fitting a simple sigmoidal function to pixel intensities across slices of an image taken perpendicular to light–dark edges, the mean distance over which the light–dark transition occurs can be determined. A fixed multiple of this characteristic distance is then reported as the image resolution. The prefactor is determined by analysis of scanning transmission electron microscope high-angle annular dark field images of Si<110>. This metric has been applied to optical, scanning electron microscope, and helium ion microscope images. This method provides quantitative feedback about image resolution, independent of the tool on which the data were collected. In addition, our analysis provides a nonarbitrary and self-consistent framework that any end user can utilize to evaluate the resolution of multiple microscopes from any vendor using the same metric.

Structural organization of the plant cell wall is a key parameter for understanding anisotropic plant growth and mechanical behavior. Four imaging platforms were used to investigate the cell wall architecture of Miscanthus sinensis cv. internode tissue. Using transmission electron microscopy with potassium permanganate, we found a great degree of inhomogeneity in the layering structure (4–9 layers) of the sclerenchymatic fiber (Sf). However, the xylem vessel showed a single layer. Atomic force microscopy images revealed that the cellulose microfibrils (Mfs) deposited in the primary wall of the protoxylem vessel (Pxv) were disordered, while the secondary wall was composed of Mfs oriented in parallel in the cross and longitudinal section. Furthermore, Raman spectroscopy images indicated no variation in the Mf orientation of Pxv and the Mfs in Pxv were oriented more perpendicular to the cell axis than that of Sfs. Based on the integrated results, we have proposed an architectural model of Pxv composed of two layers: an outermost primary wall composed of a meshwork of Mfs and inner secondary wall containing parallel Mfs. This proposed model will support future ultrastructural analysis of plant cell walls in heterogeneous tissues, an area of increasing scientific interest particularly for liquid biofuel processing.

On account of its excellent resolution and high throughput, cryoSEM imaging has recently seen resurgence. In this work, we report on the development of cryogenic triple ion gun milling (CryoTIGM™), a broad ion beam milling technique for cryo-planing of vitrified, “frozen-hydrated” specimens. We find that sections prepared with CryoTIGM™ are smooth over exceptionally large areas (~700,000 µm2), and reveal ultrastructural details in similar or better quality than freeze-fractured samples.

The adaptive significance of variation in sperm phenotype is still largely unknown, in part due to the difficulties of observing and measuring sperm movement in its natural, selective environment (i.e., within the female reproductive tract). Computer-assisted sperm analysis systems allow objective and accurate measurement of sperm velocity, but rely on being able to track individual sperm, and are therefore unable to measure sperm movement in species where sperm move in trains or bundles. Here we describe a newly developed computational method for measuring sperm movement using Fourier analysis to estimate sperm tail beat frequency. High-speed time-lapse videos of sperm movement within the female tract of the neriid fly Telostylinus angusticollis were recorded, and a map of beat frequencies generated by converting the periodic signal of an intensity versus time trace at each pixel to the frequency domain using the Fourier transform. We were able to detect small decreases in sperm tail beat frequency over time, indicating the method is sensitive enough to identify consistent differences in sperm movement. Fourier analysis can be applied to a wide range of species and contexts, and should therefore facilitate novel exploration of the causes and consequences of variation in sperm movement.

Agrowastes are produced worldwide in huge quantities and they contain interesting elements for producing inorganic cementing binders, especially silicon. Conversion of agrowastes into ash is an interesting way of yielding raw material used in the manufacture of low-CO2 binders. Silica-rich ashes are preferred for preparing inorganic binders. Sugarcane leaves (Saccharum officinarum, SL) and bamboo leaves (Bambusa vulgaris, BvL and Bambusa gigantea, BgL), and their corresponding ashes (SLA, BvLA, and BgLA), were chosen as case studies. These samples were analyzed by means of optical microscopy, Cryo-scanning electron microscopy (SEM), SEM, and field emission scanning electron microscopy. Spodograms were obtained for BvLA and BgLA, which have high proportions of silicon, but no spodogram was obtained for SLA because of the low silicon content. Different types of phytoliths (specific cells, reservoirs of silica in plants) in the studied leaves were observed. These phytoliths maintained their form after calcination at temperatures in the 350–850°C range. Owing to the chemical composition of these ashes, they are of interest for use in cements and concrete because of their possible pozzolanic reactivity. However, the presence of significant amounts of K and Cl in the prepared ashes implies a limitation of their applications.

The ex situ lift out (EXLO) adhesion forces are reviewed and new applications of EXLO for focused ion beam (FIB)-prepared specimens are described. EXLO is used to manipulate electron transparent specimens on microelectromechanical systems carrier devices designed for in situ electron microscope analysis. A new patented grid design without a support film is described for EXLO. This new slotted grid design provides a surface for holding the specimen in place and also allows for post lift out processing. Specimens may be easily manipulated into a backside orientation to reduce FIB curtaining artifacts with this slotted grid. Large EXLO specimens can be manipulated from Xe+ plasma FIB prepared specimens. Finally, applications of EXLO and manipulation of FIB specimens using a vacuum probe lift out method are shown. The vacuum probe provides more control for placing specimens on the new slotted grids and also allows for easy manipulation into a backside configuration.

This study examines the use of reflectivity-height transformation in full-field angle-deviation microscopes (ADM). In such microscopes, two light intensity distribution images of a prism’s total internal reflection and critical angle are obtained separately with two charge-coupled devices (CCDs), and are converted into a reflectivity profile point-to-point and then into angle of deviation matrix after the beam is reflected by the test sample; finally, the surface height of the sample is found through the triangular geometrical relationship. This method obtains the image through the effective imaging area of CCD. Once the two-dimensional (2D) image is obtained, the third dimension, height, is added to create a full-field 3D surface profile. Its conversion process is nonlinear; therefore, compensation must be made to reduce measurement errors. The optical magnification of high vertical resolution full-field 3D reflection-type ADM could reach >250 times, thus providing submicron measurements with nanometer vertical resolution and allowing for the simultaneous measurement of 2D and 3D images. Small defects on both transparent and nontransparent surfaces can be rapidly detected.

This paper presents new developments on the provenance study of lapis lazuli started by our group in 2008: during the years a multi-technique approach has been exploited to obtain minero-petrographic characterization and creation of a database considering only rock samples of known provenance. Since the final aim of the study is to develop a method to analyze archeological findings and artworks made with lapis lazuli in a completely non-invasive way, ion beam analysis techniques were employed to trace the provenance of the raw material used for the production of artifacts. Continuing this goal and focusing the analysis on determination of more significant minero-chemical markers for the provenance study of trace elements in different minerals, the method was extended with the use of micro X-ray fluorescence (µ-XRF), to test the potential of the technique for this application. The analyzes were focused on diopside and pyrite in lapis lazuli samples of known provenance (Afghanistan, Tajikistan, and Siberia). In addition, µ-XRF data were compared with micro proton-induced X-ray emission (µ-PIXE) results to verify the agreement between the two databases and to compare the analytical performance of both techniques for this application.

In situ environmental transmission electron microscopy (ETEM) experiments require specimen heating holders to study material behavior in gaseous environments at elevated temperatures. In order to extract meaningful kinetic parameters, such as activation energies, it is essential to have a direct and accurate measurement of local sample temperature. This is particularly important if the sample temperature might fluctuate, for example when room temperature gases are introduced to the sample area. Using selected-area diffraction (SAD) in an ETEM, the lattice parameter of Ag nanoparticles was measured as a function of the temperature and pressure of hydrogen gas to provide a calibration of the local sample temperature. SAD permits measurement of temperature to an accuracy of ±30°C using Ag lattice expansion. Gas introduction can cause sample cooling of several hundred degrees celsius for gas pressures achievable in the ETEM.