The cellular oxygen sensing system of the body ensures appropriate adaptation of cellular functions toward hypoxia by regulating gene expression and ion channel activity. Two-photon laser microscopy is an ideal tool to study and prove the relevance of the molecular mechanisms within oxygen sensing pathways on the cellular and complex tissue or organ level. Images of hypoxia inducible factor 1 (HIF-1) subunit nuclear mobility and protein-protein interaction in living cells, of hypoxia-induced changes in membrane potential and intracellular calcium of live ex vivo carotid bodies as well as of rat kidney proximal tubulus function in vivo, will be shown.

Correlative labeling permits colocalization of molecular species for observation of the same sample in light (LM) and electron microscopy (EM). Myosin bands in ultrathin cryosections were labeled using both fluorophore conjugated to secondary antibody (IgG) and colloidal gold (cAu) particles conjugated to primary IgG as reporters for LM and transmission electron microscopy (TEM), respectively. This technique allows rapid evaluation of labeling via LM, prior to more time-consuming observations with TEM and also yields two complementary data sets in one labeling procedure. Quenching of the fluorescent signal was inversely related to the distance between fluorophore and cAu particles. The signal from fluorophore conjugated to secondary antibody was inversely proportional to the size of cAu conjugated to primary antibody. Where fluorophore and cAu were bound to the same antibody, the fluorescence signal was nearly completely quenched regardless of fluorophore excitation or emission wavelength and regardless of particle size, 3 nm and larger. Colloidal metal particles conjugated to primary antibody provide high spatial resolution for EM applications. Fluorophore conjugated to secondary antibody provides spatial resolution well within that of conventional fluorescence microscopy. Use of fluorescent secondary antibody moved the fluorophore a sufficient distance from the cAu particles on the primary antibody to limit quenching of fluorescence.

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

A radioactive particle embedded in a sediment collected from the Irish Sea was examined with spectrometric methods to understand the potential bioavailability of its constituents. Images of the particle surface were acquired in the backscattering mode by scanning electron microscopy. The elemental composition of the particle surface layer was measured using energy dispersive (SEM-EDX) and wavelength dispersive (SEM-WDX) X-ray spectrometers. The investigation showed that the sample consists of a calcite matrix in which uranium is present in the form of separate inclusions. The diameter of U inclusions was less than 10 μm. Synchrotron radiation based X-ray fluorescence in confocal geometry (confocal μ-XRF) was used to determine the spatial distribution of elements in the particle. Three-dimensional reconstructions of the Ca, Cr, Mn, Fe, Zn, Sr, Ba, Pb, and U distributions were performed. The oxidation state of uranium in the different inclusions was determined by synchrotron radiation based X-ray absorption in confocal geometry (confocal μ-XANES). The isotopic composition of uranium was measured by secondary ion mass spectrometry. The results revealed that uranium was depleted in 235U. Pu and other actinides were not detected.

The use of a high-brightness field-emission gun (FEG) in scanning electron microscopy (SEM) is a powerful technique to examine microstructures at very high spatial resolution down to nanometer level and has significantly enhanced our ability to solve challenging materials problems, allowing studies of nanoprecipitates.

Continuously miniaturization of electronic components using PCB's technology for the manufacturing of SDRAM's uses a solder ball attach process, producing a joint which robustness and electrical response are crucial factors to success. At present, near-eutectic Sn-Ag-Cu alloys are the leading candidates for Pb-free solders. According to Chun et al., the rapid formation of Cu-Sn intermetallic compounds at the interface affects the reliability of this solder joint and represents a major concern. In this study we characterize the interface of an attachment between the copper layer conductors of a memory and a solder ball which allows further bonding to printed circuit modules, see figure 1.a.

A real-space description of inelastic scattering in scanning transmission electron microscopy is derived with particular attention given to the implementation of the projected potential approximation. A hierarchy of approximations to expressions for inelastic images is presented. Emphasis is placed on the conditions that must hold in each case. The expressions that justify the most direct, visual interpretation of experimental data are also the most approximate. Therefore, caution must be exercised in selecting experimental parameters that validate the approximations needed for the analysis technique used. To make the most direct, visual interpretation of electron-energy-loss spectroscopic images from core-shell excitations requires detector improvements commensurate with those that aberration correction provides for the probe-forming lens. Such conditions can be relaxed when detailed simulations are performed as part of the analysis of experimental data.

An energy dispersive X-ray spectrometry (EDXS) method is developed to evaluate the composition of alloyed nanoparticles (NPs) where one of the alloying elements is removed under the electron beam during microanalysis with a transmission electron microscope (TEM). The method is demonstrated for alloyed Au-Ag NPs of a diameter ranging from 6 to 20 nm produced by laser evaporation of a water-suspended Ag-Au powder mixture of varying composition. Series of EDXS spectra are recorded for 30 NPs from samples with five different Ag:Au ratios revealing Ag depletion from NPs during electron irradiation. By studying the evolution of NPs composition as a function of dose, the initial Ag content for each NP is extrapolated. The rate of Ag depletion is discussed in terms of sputtering and knock-on damage. On average, approximately one Ag atom is lost from the NP for each Ag L X-ray detected. To assess the limitations of microanalysis in these sensitive nanoscale structures, the concept of detectability limit is adapted to our method. This benchmark is then evaluated for Ag in Au-Ag NPs of various sizes and acquisition times. This study should be regarded as a guide for the design of analytical TEM measurements of beam-sensitive NPs.

Second-harmonic generation (SHG) can be used for imaging membrane potential in neurons, but poor signal-to-noise (S/N) limits accurate measurements of small voltage transients. We use photon counting to improve the S/N of weak SHG signal detection. Photon counting generates shot-noise limited and integrable signals, eliminates pulse-to-pulse variation, and built-in discriminators reduces the background to practically zero. In single trials, by using photon counting, we obtain a more than a twofold S/N increase over analog voltage detection. Trial-to-trial variability is also reduced by 50%. Finally, we show that, using photon counting, the kinetics of fast events such as action potentials can be recorded more accurately.

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

We previously reported that translocation of mitochondria from the oocyte cortex to the perinuclear area indicates positive developmental potential that was reduced in porcine somatic cell nuclear transfer (SCNT) embryos compared to in vitro–fertilized (IVF) embryos (Katayama, M., Zhong, Z.-S., Lai, L., Sutovsky, P., Prather, R.S. & Schatten, H. (2006). Dev Biol299, 206–220.). The present study is focused on distribution of donor cell mitochondria in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using either pig fibroblasts with mitochondria-stained MitoTracker CMXRos or YFP-mitochondria 3T3 cells (pPhi-Yellow-mito) as donor cells. Transmission electron microscopy was employed for ultrastructural analysis of pig oocyte and donor cell mitochondria. Our results revealed donor cell mitochondrial clusters around the donor nucleus that gradually dispersed into the ooplasm at 3 h after SCNT. Donor-derived mitochondria distributed into daughter blastomeres equally (82.8%) or unequally (17.2%) at first cleavage. Mitochondrial morphology was clearly different between donor cells and oocytes in which various complex shapes and configurations were seen. These data indicate that (1) unequal donor cell mitochondria distribution is observed in 17.2% of embryos, which may negatively influence development; and (2) complex mitochondrial morphologies are observed in IVF and SCNT embryos, which may influence mitochondrial translocation and affect development.

Cold gas dynamic spray (cold spray) is a rapid deposition technology in which particles deposit at velocities above the speed of sound (∼340 ms−1). Generally, porosity forms in cold spray deposits due to insufficient deformation of particles. In this study, the unique capability of the X-ray microscopy and microtomography is utilized to visualize the internal structure of deposited material. The results show that this characterization technique successfully reveals porosities in the cold spray commercial purity (CP) titanium structure. Furthermore, microtomography images confirmed the experimental results for porosity measurements in which helium (compared with nitrogen) as carrier gas significantly decreases porosity in cold spray CP titanium.

Layer- and tunnel-structured manganese oxide nanomaterials are important because of their potential applications in industrial catalysis. A novel soft chemistry method was developed for the synthesis of inorganic cryptomelane nanomaterials with high surface area. Bright field transmission electron microscopy (BF-TEM) and high-resolution transmission electron microscopy (HRTEM) techniques were employed to characterize this nanomaterial. A nanosized material with fibrous texture comprised of 140–160 nm striations was identified by BF-TEM imaging. HRTEM images show multiple atomic morphologies such as “helix-type,” “doughnut-like,” and tunnel structures lying on different crystallographic planes. The crystallographic parameters of this material were analyzed and measured by X-ray powder diffraction (XRD) showing that the synthesized nanomaterial is single phased and corresponds to cryptomelane with major diffraction peaks (for 10° < 2θ < 60°) at d-spacing values of 6.99, 4.94, 3.13, 2.40, 2.16, 1.84, 1.65, and 1.54 Å. A “doughnut-like” crystal structure was confirmed based on the crystallographic data. Structure and lattice parameters refinement was performed by XRD/Rietveld analysis. Simple simulation of HRTEM images and selected area diffraction patterns were applied to interpret the HRTEM images as observed.

SiAlON ceramics have found applications in many different areas due to their excellent engineering properties such as high hardness, fracture toughness, good thermal shock and oxidation resistance. SiAlON exist mainly in two different polymorphs: a (MxSi12-(m+n)Al(m+n)OnN16-n; M: metal and rare earth cations, x≈0,35 and n≤1,35) and β (β-Si6-zAlzOzN8-z; 0≤z≤4). In general, stable alpha and beta phases separately as well as in combination of α and β are obtained by incorporation of metal and rare earth cations as sintering additives. The metal cations such as Li, Mg, Ca, Y, and most lanthanide cations with the exception of La, Ce, Pr and Eu are able to stabilise α-SiAlON structure. Ekstrom et al. 1991 found that cerium can not occupy interstitial sites in α-SiAlON structure due to the fact that ionic radius of Ce3+ (0.103 nm) is too large, whereas ionic radius of Ce4+ (0.080 nm) is too small to stabilise α-SiAlON structure. After this work, several studies carried out to incorporate cerium cations into α-SiAlON structure. It was shown that cerium cation alone can be incorporated into α-SiAlON if the samples are either fast cooled after sintering, or when the samples are spark plasma sintered. On the other hand, cerium can also be incorporated into the α-SiAlON structure when it is used as a sintering additive together with a smaller α-SiAlON stabiliser cation such as Yb or Ca. Similar results were observed in other multi-cation doped SiAlONS that non α-SiAlON stabiliser cations like Sr2+ (0.112 nm) and La3+ (0.106 nm) are able to stabilise α-SiAlON when used together with α-SiAlON stabiliser cations such as Ca or Yb. Although it was shown that cerium existed in mixed valance state at domain boundaries in Ce-doped and spark plasma sintered α-SiAlON, there is no work on the valance determination of cerium in sintered α-SiAlON which has no domain boundaries. Therefore, in this study; it was aimed to incorporate cerium into α-SiAlON structure by combining with Yb3+ and the determination of possible cerium valence states (Ce3+/Ce4+) in both α-SiAlON grains and secondary phases.

Nonlinear optical imaging techniques such as multiphoton and second harmonic generation (SHG) microscopy used in conjunction with novel signal analysis techniques such as spectroscopic and fluorescence excited state lifetime detection have begun to be used widely for biological studies. This is largely due to their promise to noninvasively monitor the intracellular processes of a cell together with the cell's interaction with its microenvironment. Compared to other optical methods these modalities provide superior depth penetration and viability and have the additional advantage in that they are compatible technologies that can be applied simultaneously. Therefore, application of these nonlinear optical approaches to the study of breast cancer holds particular promise as these techniques can be used to image exogeneous fluorophores such as green fluorescent protein as well as intrinsic signals such as SHG from collagen and endogenous fluorescence from nicotinamide adenine dinucleotide or flavin adenine dinucleotide. In this article the application of multiphoton excitation, SHG, and fluorescence lifetime imaging microscopy to relevant issues regarding the tumor-stromal interaction, cellular metabolism, and cell signaling in breast cancer is described. Furthermore, the ability to record and monitor the intrinsic fluorescence and SHG signals provides a unique tool for researchers to understand key events in cancer progression in its natural context.

Growth of bismuth oxide (most probably Bi2O3) was observed in situ in a transmission electron microscope. Bi liquid particles were dispersed on the substrates of diamond or SiO2. Introduction of oxygen up to ∼5 × 10−4 Pa resulted in formation of bismuth oxide (most probably Bi2O3) whiskers. The growth mechanism of the whisker was discussed in terms of a vapor-liquid-solid (VLS) mechanism. It is suggested that the liquid droplet of Bi acts as a physical catalyst for growth of bismuth oxide (most probably Bi2O3) whiskers.

Aberration correction leads to a substantial improvement in the directly interpretable resolution of transmission electron microscopes. Correction of the aberrations has been achieved electron-optically through a hexapole-based corrector and also indirectly by computational analysis of a focal or tilt series of images. These direct and indirect methods are complementary, and a combination of the two offers further advantages. Materials characterization has benefitted from the reduced delocalization and higher resolution in the corrected images. It is now possible, for example, to locate atomic columns at surfaces to higher accuracy and reliability. This article describes the JEM-2200FS in Oxford, which is equipped with correctors for both the image-forming and probe-forming lenses. Examples of the use of this instrument in the characterization of nanocrystalline catalysts are given together with initial results combining direct and indirect methods. The double corrector configuration enables direct imaging of the corrected probe, and a potential confocal imaging mode is described. Finally, modifications to a second generation instrument are outlined.

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

The development of a remotely operated scanning electron microscopy (SEM) system and its use by high school students and the public as an outreach program are reported. The SEM and the server are located in the National Institute for Materials Science, Tsukuba, Japan, with client computers installed at a science museum and high schools. Using a secure virtual private network system and scheduling/management groupware, observation of SEM images and energy dispersive X-ray analysis are widely and frequently performed throughout Japan.