We have developed the focused ion beam (FIB) fold-out technique, for transmission electron microscopy (TEM) sample preparation in which there is no fine polishing or dimpling, thus saving turnaround time. It does not require a nanomanipulator yet is still site specific. The sample wafer is cut to shape, polished down, and then placed in a FIB system. A tab containing the area of interest is created by ion milling and then “folded out” from the bulk sample. This method also allows a plan-view of the sample by removing material below the wafer's surface film or device near the polished edge. In the final step, the sample is thinned to electron transparency, ready to be analyzed in the TEM. With both a cross section and plan-view, our technique gives microscopists a powerful tool in analyzing multiple zone axes in one TEM session. The nature of the polished sample edge also includes the ability to sample many areas, allowing the user to examine a very large device or sample. More importantly, this technique could make multiple site-specific e-beam transparent specimens in one polished sample, which is difficult to do when prepared by other methods.

A method is described for quantitatively analyzing the level of interconnectivity of solid-oxide fuel cell electrode phases. The method was applied to the three-dimensional microstructure of a Ni–Y2O3-stabilized ZrO2 (Ni-YSZ) anode active layer measured by focused ion beam scanning electron microscopy. Each individual contiguous network of Ni, YSZ, and porosity was identified and labeled according to whether it was contiguous with the rest of the electrode. It was determined that the YSZ phase was 100% connected, whereas at least 86% of the Ni and 96% of the pores were connected. Triple-phase boundary (TPB) segments were identified and evaluated with respect to the contiguity of each of the three phases at their locations. It was found that 11.6% of the TPB length was on one or more isolated phases and hence was not electrochemically active.

Electrical interconnects in integrated circuits have shrunk to sizes in the range of 20–100 nm. Accurate measurements of the dimensions of these nanowires are essential for identifying the dominant electron scattering mechanisms affecting wire resistivity as they continue to shrink. We report a systematic study of the effect of line edge roughness on the apparent cross-sectional area of 90 nm Cu wires with a TaN/Ta barrier measured by conventional two-dimensional projection imaging and three-dimensional electron tomography. Discrepancies in area measurements due to the overlap of defects along the wire's length lead to a 5% difference in the resistivities predicted by the two methods. Tomography of thick cross sections is shown to give a more accurate representation of the original structure and allows more efficient sampling of the wire's cross-sectional area. The effect of roughness on measurements from projection images is minimized for cross-section thicknesses less than 50 nm, or approximately half the spatial frequency of the roughness variations along the length of the investigated wires.

Caliciviruses cause rabbit haemorrhagic disease (RHD) that kills more than 90% of the infected adult animals within 1 a 3 days of infection. The virus replicates in the liver and causes a fulminant hepatitis in adult rabbits leading to RHD. A mystery of the calicivirus infection is that young rabbits (less than 8-weeks old) are resistant to the infection, in spite of undergoing viral replication in the liver and of expressing transient hepatitis. Heterophils were the predominant inflammatory cells seen in hepatic tissue of infected adult rabbits, whereas mononuclear cells dominated the inflammatory infiltrates of the infected young rabbits (4-weeks-old). In order to define the role of inflammation in the pathogenesis of the calicivirus infection, we have studied the cellular inflammatory response in young rabbits experimentally infected by calicivirus. For this, we have used transmission electron microscopy (TEM) and flow cytometry to identify the inflammatory cells that infiltrate the hepatic tissue of young rabbits at 48 hours of calicivirus infection. In same infected rabbits, lymphoid organs (spleen and thymus) were used to quantify by flow cytometry the total number of leukocytes seen inside these organs.

Quantification of immunostaining is a widely used technique in pathology. Nonetheless, techniques that rely on human vision are prone to inter- and intraobserver variability, and they are tedious and time consuming. Digital image analysis (DIA), now available in a variety of platforms, improves quantification performance: however, the stability of these different DIA systems is largely unknown. Here, we describe a method to measure the reproducibility of DIA systems. In addition, we describe a new image-processing strategy for quantitative evaluation of immunostained tissue sections using DAB/hematoxylin-stained slides. This approach is based on image subtraction, using a blue low pass filter in the optical train, followed by digital contrast and brightness enhancement. Results showed that our DIA system yields stable counts, and that this method can be used to evaluate the performance of DIA systems. The new image-processing approach creates an image that aids both human visual observation and DIA systems in assessing immunostained slides, delivers a quantitative performance similar to that of bright field imaging, gives thresholds with smaller ranges, and allows the segmentation of strongly immunostained areas, all resulting in a higher probability of representing specific staining. We believe that our approach offers important advantages to immunostaining quantification in pathology.

Abundant charcoalified seed fern (pteridosperm) pollen organs and ovules have been recovered from Late Viséan (Mississippian 330 Ma) limestones from Kingswood, Fife, Scotland. To overcome limitations of data collection from these tiny, sometimes unique, fossils, we have combined low vacuum scanning electron microscopy on uncoated specimens with backscatter detector and synchrotron radiation X-ray tomographic microscopy utilizing the Materials Science and TOMCAT beamlines at the Swiss Light Source of the Paul Scherrer Institut. In combination these techniques improve upon traditional cellulose acetate peel sectioning because they enable study of external morphology and internal anatomy in multiple planes of section on a single specimen that is retained intact. The pollen organ Melissiotheca shows a basal parenchymatous cushion bearing more than 100 sporangia on the distal face. Digital sections show the occurrence of pollen in some sporangia. The described ovule is new and has eight integumentary lobes that are covered in spirally arranged glandular hairs. Virtual longitudinal sections reveal the lobes are free above the pollen chamber. Results are applied in taxonomy and will subsequently contribute to our understanding of the former diversity and evolution of ovules, seeds, and pollen organs in the seed ferns, the first seed-bearing plants to conquer the land.

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009

The Presidents of the Microscopy Society of America, the Microbeam Analysis Society and the International Metallographic Society invite you to join them in Richmond for the 2009 Microscopy & Microanalysis meeting. This EXPO issue of Microscopy and Microanalysis provides you with full details of the scientific meeting and its ancilliary events, as well as giving valuable information about the many vendors who will be exhibiting their products at the meeting. Attendance at the meeting will provide you with a useful snapshot of the diverse activities taking place across the many scientific disciplines that currently utilize microscopy and microanalysis.

Clitoria ternatea is known for its antimicrobial activity but the antifungal effects of leaf extract on growth and morphogenesis of Aspergillus niger have not been observed. The extract showed a favorable antifungal activity against A. niger with a minimum inhibition concentration 0.8 mg/mL and minimum fungicidal concentration 1.6 mg/mL, respectively. The leaf extract exhibited considerable antifungal activity against filamentous fungi in a dose-dependent manner with 0.4 mg/mL IC50 value on hyphal growth of A. niger. The main changes observed under scanning electron microscopy after C. ternatea extract treatment were loss of cytoplasm in fungal hyphae and the hyphal wall and its diameter became markedly thinner, distorted, and resulted in cell wall disruption. In addition, conidiophore alterations were also observed when A. niger was treated with C. ternatea leaf extract.

Erectile dysfunction (ED) is considered equivalent to endothelial dysfunction and thus an early manifestation of atherosclerosis and cardiovascular disease (CVD). Atherosclerosis that is frequently provoked by regular consumption of high-fat diet (HFD) has been recently recognized as a chronic inflammation condition associated to vessel lipid deposition and vascular smooth cells proliferation that together impair endothelium-dependent relaxation of corpus cavernosum (CC) smooth muscle. It is also well established that HFD regular intake by its own induces endothelial dysfunction. The main objective of this study was to evaluate effects of HFD continuous intake on atherosclerosis progression of CC vessels of the rat. Due to their intervention on atherosclerosisrelated vascular remodelling pathways, angiopoietins (Ang1, Ang2) expression in erectile tissue of this rat experimental model was characterized.

The 67th Annual Meeting of the Microscopy Society of America, held in conjunction with our co-sponsoring societies, will take place in Richmond, Virginia in late July. Now widely known as Microscopy and Microanalysis, this meeting provides the MSA membership with a useful overview of many diverse activities involving microscopy and microanalysis that are of topical interest. In addition to symposia devoted to a range of contemporary topics in the biological and physical sciences, as well as advances in instrumentation, the meeting program will include a set of “Back to Basics” Tutorials, and continuations of the highly successful In-Meeting Intensive Workshops, and the special “Family Affair”, which provides hands-on activities and demonstrations aimed at children of all ages. MSA Council is concerned to reach out to more people in the wider community who utilize microscopes of many shapes and forms in their daily activities, and these developments are just part of our ongoing educational and outreach efforts in this direction.

Two purely carbon-based functional polymer systems were investigated by bright-field conventional transmission electron microscopy (CTEM) and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM). For a carbon black (CB) filled polymer system, HAADF-STEM provides high contrast between the CB agglomerates and the polymer matrix so that details of the interface organization easily can be revealed and assignment of the CB phase is straightforward. For a second system, the functional polymer blend representing the photoactive layer of a polymer solar cell, details of its nanoscale organization could be observed that were not accessible with CTEM. By varying the camera length in HAADF-STEM imaging, the contrast can be enhanced between crystalline and amorphous compounds due to diffraction contrast so that nanoscale interconnections between domains are identified. In general, due to its incoherent imaging characteristics HAADF-STEM allows for reliable interpretation of the data obtained.

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009