We report the first application of multiphoton microscopy (MPM) to generate three-dimensional (3D) images of natural minerals (micron-sized sulfides) in thick (∼120 μm) rock sections. First, reflection mode (RM) using confocal laser scanning microscopy (CLSM), combined with differential interference contrast (DIC), was tested on polished sections. Second, two-photon fluorescence (TPF) and second harmonic signal (SHG) images were generated using a femtosecond-laser on the same rock section without impregnation by a fluorescent dye. CSLM results show that the silicate matrix is revealed with DIC and RM, while sulfides can be imaged in 3D at low resolution by RM. Sulfides yield strong autofluorescence from 392 to 715 nm with TPF, while SHG is only produced by the embedding medium. Simultaneous recording of TPF and SHG images enables efficient discrimination between different components of silicate rocks. Image stacks obtained with MPM enable complete reconstruction of the 3D structure of a rock slice and of sulfide morphology at submicron resolution, which has not been previously reported for 3D imaging of minerals. Our work suggests that MPM is a highly efficient tool for 3D studies of microstructures and morphologies of minerals in silicate rocks, which may find other applications in geosciences.

Three-dimensional (3D) analysis is an essential tool to obtain quantitative results from 3D datasets. Considerable progress has been made in 3D imaging techniques, resulting in a growing need for more flexible, complete analysis packages containing advanced algorithms. At the Centre for X-ray Tomography of the Ghent University (UGCT), research is being done on the improvement of both hardware and software for high-resolution X-ray computed tomography (CT). UGCT collaborates with research groups from different disciplines, each having specific needs. To meet these requirements the analysis software package, Morpho+, was developed in-house. Morpho+ contains an extensive set of high-performance 3D operations to obtain object segmentation, separation, and parameterization (orientation, maximum opening, equivalent diameter, sphericity, connectivity, etc.), or to extract a 3D geometrical representation (surface mesh or skeleton) for further modeling. These algorithms have a relatively short processing time when analyzing large datasets. Additionally, Morpho+ is equipped with an interactive and intuitive user interface in which the results are visualized. The package allows scientists from various fields to obtain the necessary quantitative results when applying high-resolution X-ray CT as a research tool to the nondestructive investigation of the microstructure of materials.

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

To understand diffusion processes occurring inside Fe catalysts during multiwall carbon nanotube (MWCNT) growth, catalysts were studied using atomic-resolution scanning transmission electron microscopy combined with electron energy-loss spectroscopy. Nanotube walls emanate from structurally modified and chemically complex catalysts that consist of cementite and a 5 nm amorphous FeOx cap separated by a 2–3 nm thick carbon-rich region that also contains Fe and O (a-C:FexOy). Nonuniform distribution of carbon atoms throughout the catalyst base reveals that carbon molecules from the gas phase decompose near the catalyst multisection junction, where the MWCNT walls terminate. Formation of the a-C:FexOy region provides the essential carbon source for MWCNT growth. Two different carbon diffusion mechanisms are responsible for the growth of the inner and outer walls of each MWCNT.

We demonstrate that single-pass Kelvin force microscopy (KFM) and dC/dz measurements in different environments expand the compositional imaging with atomic force microscopy. The KFM and dC/dz studies were performed in the intermittent contact mode with force gradient detection of tip-sample electrostatic interactions. Both factors contribute to sensitive measurements of the surface potential and capacitance gradient with nanometer-scale spatial resolution as it was verified on a broad range of materials: metal alloys, polymers, organic layers, and liquid-like objects. For many samples the surface potential and dC/dz variations complement each other in identification of individual components of heterogeneous materials. In situ imaging in different humidity or vapors of various organic solvents further facilitate recognition of the constituents of multicomponent polymer samples due to selective swelling of components.

Ndc80 (called Hec1 in human), the core component of the Ndc80 complex, is involved in regulation of both kinetochore-microtubule interactions and the spindle assembly checkpoint in mitosis; however, its role in meiosis remains unclear. Here, we report Ndc80 expression, localization, and possible functions in mouse oocyte meiosis. Ndc80 mRNA levels gradually increased during meiosis. Immunofluorescent staining showed that Ndc80 was restricted to the germinal vesicle and associated with spindle microtubules from the Pro-MI to MII stages. Ndc80 was localized on microtubules and asters in the cytoplasm after taxol treatment, while Ndc80 staining was diffuse after disruption of microtubules by nocodazole treatment, confirming its microtubule localization. Disruption of Ndc80 function by either siRNA injection or antibody injection resulted in severe chromosome misalignment, spindle disruption, and precocious polar body extrusion. Our data show a unique localization pattern of Ndc80 in mouse oocytes and suggest that Ndc80 may be required for chromosome alignment and spindle organization, and may regulate spindle checkpoint activity during mouse oocyte meiosis.

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

Previous efficacy studies found that many insecticides used by growers could be having an adverse effect on egg parasitoids (Telenomus podisi) developing in the eggs of the brown stink bug (Euschistus servus), while unhatched stink bugs experienced lower levels of mortality. One plausible explanation for this was that insecticides might enter parasitized eggs more readily via oviposition wounds. Parasitized E. servus eggs, as well as nonparasitized stink bug (Acrosternum hilare, E. servus, Murgantia histrionica, and Podisus maculiventris) eggs, were examined using electron microscopy. Egg response to perforation by a tungsten probe served as a control. Microscopy images depicted the chorion surface as characterized by a matrix of ridges and micropylar processes in a ring around the margin of the operculum. Observations of oviposition sites showed a “scab” formed where the ovipositor penetrated the chorion, and at sites penetrated by the probe. These formations appeared to be the result of fluids from inside the egg leaking out, drying, and hardening after oviposition or probe perforation, suggesting that the response was not due to substances secreted by the parasitoid. Further, no open wounds or holes were seen to increase the possibility of insecticides entering parasitized eggs.

Structure and chemistry across the rare earth oxide-Ge interfaces of a Gd2O3-Ge-Gd2O3 heterostructure grown on p-Si (111) substrate using encapsulated solid phase epitaxy method have been studied at nanoscale using various transmission electron microscopy methods. The structure across both the interfaces was investigated using reconstructed phase and amplitude at exit plane. Chemistry across the interfaces was explored using elemental mapping, high-angle annular dark-field imaging, electron energy loss spectroscopy, and energy dispersive X-ray spectrometry. Results demonstrate the structural and chemical abruptness of both the interfaces, which is most essential to maintain the desired quantum barrier structure.

Various porous titania photocatalysts are analyzed three-dimensionally in real space by electron tomography. Shapes and three-dimensional (3D) distributions of fine pores and silver (Ag) particles (2 nm in diameter) within the pores are successfully reconstructed from the 3D data. Electron tomography is applied for measuring the specific surface area of the porous structures including open and closed porosity. Calculated specific surface areas of 22.8 m2/g for a conventional sol-gel TiO2 sample and 366 m2/g for a highly porous TiO2 sample prepared using the Pluronic P-123 self-assembly process are compared with those measured by the general BET method. The real-space surface measurement indicates that the highly porous TiO2 produced by the present method using block copolymers has a greater number of effective reaction sites for the degradation of methylene blue. Electron tomography shows a great potential to contribute considerably to the nanostructural analysis and design of such catalyst materials for photocatalysis.

Atomic force microscopy probe-induced large-area ultrathin SiOx (x ≡ O/Si content ratio and x > 2) protrusions only a few nanometers high on a SiO2 layer were characterized by scanning photoemission microscopy (SPEM) and X-ray photoemission spectroscopy (XPS). SPEM images of the large-area ultrathin SiOx protrusions directly showed the surface chemical distribution and chemical state specifications. The peak intensity ratios of the XPS spectra of the large-area ultrathin SiOx protrusions provided the elemental quantification of the Si 2p core levels and Si oxidation states (such as the Si4+, Si3+, Si2+, and Si1+ species). The O/Si content ratio (x) was evidently determined by the height of the large-area ultrathin SiOx protrusions.

On behalf of the membership of the Microanalysis Society (MAS), welcome to Microscopy and Microanalysis 2011 and to Nashville. The Microscopy and Microanalysis (M&M) meeting was established in 1996 as the joint annual meeting of the Microscopy Society of America (MSA) and MAS. Since 2002, M&M has also served as the annual meeting of the International Metallographic Society (IMS), and it is now the premier meeting in our field, spanning the full spectrum of activities in microanalysis and the varied disciplines of microscopy. I thank my counterparts in the joining societies, MSA President Nestor Zaluzec and IMS President Nat Saenz, for all the time and effort they have invested to make this meeting a success.

The differences in artifacts associated with voltage-pulsed and laser-pulsed (wavelength = 532 or 355 nm) atom-probe tomographic (APT) analyses of nanoscale precipitation in a high-strength low-carbon steel are assessed using a local-electrode atom-probe tomograph. It is found that the interfacial width of nanoscale Cu precipitates increases with increasing specimen apex temperatures induced by higher laser pulse energies (0.6–2 nJ pulse−1 at a wavelength of 532 nm). This effect is probably due to surface diffusion of Cu atoms. Increasing the specimen apex temperature by using pulse energies up to 2 nJ pulse−1 at a wavelength of 532 nm is also found to increase the severity of the local magnification effect for nanoscale M2C metal carbide precipitates, which is indicated by a decrease of the local atomic density inside the carbides from 68 ± 6 nm−3 (voltage pulsing) to as small as 3.5 ± 0.8 nm−3. Methods are proposed to solve these problems based on comparisons with the results obtained from voltage-pulsed APT experiments. Essentially, application of the Cu precipitate compositions and local atomic density of M2C metal carbide precipitates measured by voltage-pulsed APT to 532 or 355 nm wavelength laser-pulsed data permits correct quantification of precipitation.

Labeling of organelles for microscopy is achieved generally by specific dyes that either accumulate in a cellular compartment such as cyanine dyes in mitochondria or are only fluorescent under specific conditions such as the low pH in the lysosome. Here we demonstrate that Cy5—a fluorescent molecule that does not enter cells by itself—can be loaded into cells by attaching a short oligonucleotide. This very inexpensive labeling procedure can be done in the presence of serum. Therefore, very sensitive cell types should also be amenable to this procedure, and longer observations can be achieved compared to other commercially available dyes as the labeling reagent does not need to be washed out. This also points to the pitfall of using fluorescently labeled oligonucleotides for live cell imaging where the oligonucleotide is supposed to detect a specific target sequence in its subcellular distribution.

On behalf of the International Metallographic Society I would like to welcome all of you to Microscopy and Microanalysis 2011 in Nashville, TN. IMS is pleased to have contributed to the exciting technical program for the tenth year. We thank MSA Program Chair David Giovannucci, MSA Program Vice-Chair Luke Brewer, MAS Co-Chair Masashi Watanabe, and especially IMS Co-Chair Don Susan for developing an excellent roster of presentations.

The days of being able to ascertain instrument performance by simply peering through the eye pieces at a specimen are gone. However, users and granting agencies need to be confident that data collected on these instruments is uniform and quantifiable both over time and between instruments. Ideally, a LASER should not fluctuate, illumination should be completely uniform, and colors should be perfectly aligned. To check the current performance of imaging equipment, we conducted a worldwide research study utilizing three image-based tests: long-/short-term illumination stability, co-registration of signals across various wavelengths, and field illumination uniformity. To differentiate between “acceptable” and “unacceptable” performance, the deviation in illumination power could not exceed 10% (long term) or 3% (short term), the difference in the center-of-mass of imaged multicolored beads could not exceed >1 pixel between different wavelengths, and field illumination values could not exceed 10% (horizontal) or 20% (diagonal) deviation. This study established the current state of microscope performance through simple, efficient, and robust tests, while defining relative standards to assist cores in maintaining their instruments in optimal operating conditions. We developed cross-platform performance standards that will improve the validity of quantitative measurements made using various light microscopes.

High-resolution imaging and quantitative surface analysis of epicuticular waxes and epistomatal chambers of pine species were performed by field emission scanning electron microscopy and white light scanning interferometry. Both juvenile and adult needles were collected from the two-year-old seedlings of Pinus rigida and Pinus densiflora and subjected to surface observations. Epicuticular wax structures developed on the cuticle layer as well as in the epistomatal chambers and appeared to occlude the cavities in the two pine species. The stomata of P. densiflora were characterized by more distinctly raised rings around openings than P. rigida. The most common epicuticular wax structures of the two pine species included tubules with terminal openings and coiled rodlets. Wax platelets were deposited on epistomatal chambers. Either rodlets or tubules seemed to be longer and thicker in P. rigida than those in P. densiflora. White light scanning interferometry revealed quantitative surface profiles, demonstrating more ridged (ca. 4 μm high) stomatal apertures and nearly twofold deeper (ca. 20 μm deep) epistomatal chambers of P. densiflora than those of P. rigida. These results suggest that white light scanning interferometry can be applied to unravel the quantitative surface features of epicuticular sculptures on plant leaves.

The surface morphology of hollow silica microspheres has influence on their applications. After a thorough investigation of the deposition of silica nanoparticles on polystyrene (PS) beads and the surface morphology and texture of the resultant hollow silica shells with scanning electron microscopy, transmission electron microscopy, and N2-sorption measurements, the influence of surfactant [cetyltrimethylammonium bromide (CTAB)] concentration on the surface morphology of hollow silica microspheres templated by PS beads is explained. Previously, CTAB was believed to turn the surface charge of PS beads from negative into positive so that negatively charged silica could be deposited on the PS template. Here, we show CTA+ cations preferentially assemble with silica species to form silica-CTA+ composite nanoparticles. Since the zeta potential of silica-CTA+ composite nanoparticles is smaller than that of pure silica nanoparticles, these composite nanoparticles encounter less repulsion when they are deposited on the surface of PS beads and close to each other. As more CTAB is added, the silica-CTA+ nanoparticles are less negatively charged, and more compact and smooth hollow silica microspheres are obtained.

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

We report the evolution of titanium dioxide nanostructures when Au nanoparticles, supported on single crystal TiO2 substrates, were heated under ∼260 Pa of flowing O2 in an environmental transmission electron microscope. Nanostructures with different morphologies were first observed around 500°C. Our measurements show that temperature, oxygen pressure, and the electron beam control the nanostructure growth. We propose a reaction-controlled growth mechanism where mobile Ti atoms generated by the electron- beam-induced reduction of TiO2 are preferentially reoxidized at the Au-TiO2 interface.