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

The process of neurogenesis and formation of neural stem cells is reported in human neurospheres (NS) and embryoid bodies (EB) derived from human embryonic stem cells, in vitro, and compared with neural tissue formed in human ectopic embryos in week 4 (stage 9), developed in vivo. This morphological study was done using digital imaging by light microscopy and routine transmission electron microscopy. Both NS and EB form neural rosettes from the surface epithelium much like the process of neural tube formation from ectoderm in the embryo. The rosette is the developmental signature of neuroprogenitors in cultures of differentiating embryonic stem cells and is a radial arrangement of columnar cells that express many of the proteins expressed in neuroepithelial cells in the neural tube. The NS produce all of the major classes of progeny of the neural tube, some of which have been documented here. Specific neural markers expressed in the NS and the clinical implications of this study in cell therapy are also discussed.

A simple plan-view sample preparation technique for transmission electron microscopy (TEM) specimens is proposed for thin films by tearing-off the film with adhesive tape. The demand for very thin samples is highest for nanostructured materials where the structure of 2–5 nm sized features (grains) needs to be resolved; therefore, overlapping of nanometer-sized features should be avoided. The method provides thin areas at the fracture edges of plan-view specimens with thickness in the range of the grain size in the film allowing for artifact free high-resolution TEM imaging. Nanostructured materials typically fracture between the grains providing areas with the thickness of the grain size. Besides the swiftness of the method, the samples are free of surface amorphization artifacts, which can occur in ion beam milling up to 1 nm depth even at low energy ion bombardment. The thin film tear-off technique is demonstrated on a CuMn alloy thin film with grain size of 2 nm.

The geometry and three-dimensional (3D) morphology of the ceria particles synthesized by spray pyrolysis (SP) from two different precursors—cerium acetate hydrate and cerium nitrate hydrate (CeA and CeN ceria particles)—were characterized by transmission electron microscopy and electron tomography. Results were compared with surface area measurements, confirming that the surface area of CeA ceria particles is twice as large as that of CeN ceria particles. This result was supported by 3D microstructural observations, which have revealed that CeA ceria particles contain open pores (connected to surfaces) and closed pores (embedded in particles), while CeN ceria particles only contained closed pores. This experimental result suggests that the type of porosity is controlled by the precursors and could be related to their melting temperature during the heating process in SP.

Strain-induced selective growth was investigated in a 1.5% temper-rolled Fe∼1%Si alloy using the electron backscatter diffraction (EBSD) technique. The EBSD technique was used to quantify the presence of orientation spreads within grains and to show that this particular case of selective growth can be directly related to differences in stored energy as reflected in the geometrically necessary dislocation content. The differences in stored energy were sufficient to give rise to selective growth as evidenced by bi-modal grain sizes.

This one-day pre-meeting congress, organized by the MSA Aberration-Corrected Electron Microscopy (ACEM) FIG, will be a forum for the discussion of the latest advances and solutions to problems associated with application of aberration correction technology. There will be platform presentations by both invited and contributed speakers, with poster presentations during a working lunch. Invited speakers will introduce innovations and issues, while contributors will highlight practical experiences and solutions to problems encountered during the application of ACEM to on-going experimental studies. This workshop includes: image collection/interpretation, new spectroscopies or other signals, artifacts and practical experiences in applications of ACEM to difficult situations such as hard/soft materials and in-situ experiments. All platform presentations will be intentionally kept short (∼15–20 minutes) to allow the maximum amount of interaction and information flow among attendees. Please send one page abstracts, including figures, to [email protected] with the subject line: M&M PMC Abstract.

It is well known that c-Jun N-terminal kinase (JNK) plays pivotal roles in various mitotic events, but its function in mammalian oocyte meiosis remains unknown. In this study, we found that no specific JNK2 signal was detected in germinal vesicle stage. JNK2 was associated with the spindles especially the spindle poles and cytoplasmic microtubule organizing centers at prometaphase I, metaphase I, and metaphase II stages. JNK2 became diffusely distributed and associated with the midbody at telophase I stage. Injection of myc-tagged JNK2α1 mRNA into oocytes also revealed its localization on spindle poles. The association of JNK2 with spindle poles was further confirmed by colocalization with the centrosomal proteins, γ-tubulin and Plk1. Nocodazole treatment showed that JNK2 may interact with Plk1 to regulate the spindle assembly. Then we investigated the possible function of JNK2 by JNK2 antibody microinjection and JNK specific inhibitor SP600125 treatment. These two manipulations caused abnormal spindle formation and decreased the rate of first polar body (PB1) extrusion. In addition, inhibition of JNK2 resulted in impaired localization of Plk1. Taken together, our results suggest that JNK2 plays an important role in spindle assembly and PB1 extrusion during mouse oocyte meiotic maturation.

We have investigated the potential of utilizing analytical electron microscopy to quantitatively examine the grounds used by van Gogh and, in particular, the absolute amount of extender employed. To determine the accuracy that can be achieved, a series of oil paint reconstructions were used as standards. The proportion of extender was measured using scanning electron microscopy and energy dispersive X-ray spectroscopy, and a relative error of 10% or better was achieved. The same method was then used to determine the ground composition of real samples from van Gogh paintings. The results obtained in this work are part of a more quantitative method of comparing and classifying paint cross sections, which will supplement the more traditional qualitative approach. The information obtained from this study is being used to add to our knowledge of the methods and materials used by van Gogh, which is helping in the reconstruction of van Gogh's oeuvre and attribution.

Tumors consist of a heterogeneous population of neoplastic cells infiltrated by an equally heterogeneous collection of nonneoplastic cells that comprise the tumor microenvironment. Tumor growth, invasion, and metastasis depend on multiple interactions between these cells. To assess their potential as therapeutic targets or vehicles for tumor specific delivery of therapeutic agents, we examined the contribution of bone marrow derived cells (BMDCs) to the intestinal tumor microenvironment. Hematopoietic stem cells expressing the enhanced green fluorescent protein (eGFP) were transplanted into lethally irradiated ApcMin/+ mice, and their engraftment was analyzed by confocal microscopy. The results showed abundant infiltration of eGFP cells into the small intestine, colon, and spleen compared to heart, muscle, liver, lung, and kidney. Within the intestine, there was a pronounced gradient of engraftment along the anterior to posterior axis, with enhanced infiltration into adenomas. Immunofluorescence analysis showed that osteopontin was expressed in tumor stromal cells but not in nontumor stromal populations, suggesting that gene expression in these cells is distinct. Tumor vasculature in ApcMin/+ mice was chaotic compared to normal intestinal regions. Our data suggest that BMDCs can be harnessed for tumor-targeted therapies to enhance antitumor efficacy.

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

These full-day courses run from 8:30 AM to 5:00 PM on Sunday, August 7, 2011 (additional fees are required).

The ability to prepare multiple cross-section transmission electron microscope (XTEM) samples from one XTEM sample of specific sub-10 nm features was demonstrated. Sub-10 nm diameter Si nanowire (NW) devices were initially cross-sectioned using a dual-beam focused ion beam system in a direction running parallel to the device channel. From this XTEM sample, both low- and high-resolution transmission electron microscope (TEM) images were obtained from six separate, specific site Si NW devices. The XTEM sample was then re-sectioned in four separate locations in a direction perpendicular to the device channel: 90° from the original XTEM sample direction. Three of the four XTEM samples were successfully sectioned in the gate region of the device. From these three samples, low- and high-resolution TEM images of the Si NW were taken and measurements of the NW diameters were obtained. This technique demonstrated the ability to obtain high-resolution TEM images in directions 90° from one another of multiple, specific sub-10 nm features that were spaced 1.1 μm apart.

Infrared reflectography is a well-known technique based on wideband imaging in the near-infrared (NIR) range used for painting diagnostics in conservation laboratories.. This work is focused on the application of multiband reflectography for analysis of pictorial layers and differentiated detection of painting features. This technique generates a set of narrowband NIR images of the painting. Starting from a dataset that is registered, metrically correct, and calibrated, the capability of collecting both spectral and spatial information has been exploited by processing the image cube with interplane techniques. Examples on artworks by Caravaggio, Veronese, Bronzino, and Schiavone are presented.

In characterization of metal nanoparticle doped spherical composites, the two-dimensional nature of transmission electron microscopy (TEM) images leads to ambiguities about the true location of the nanoparticles. Walking-in of simulated projections in comparison with actual TEM images leads to quantitative results such as location-dependent particle sizes and particle number density. This method takes advantage of the strength of fuzzy neural network computations via the human hunter-gatherer's visual system's evolved superiority while still allowing quantitative results by use of exact numerical simulations.

Electron backscatter diffraction (EBSD) techniques were used to characterize “hot-rolled” quenched and partitioned microstructures produced via Gleeble thermal simulations representing a hot-strip cooling practice for steel. In particular, EBSD was utilized to positively identify the morphology and location of retained austenite, to qualitatively distinguish martensite from ferrite, and in an attempt to identify transition carbides. Large pools of retained austenite and some thin films were accurately indexed; however, there was some disparity between austenite volume fractions measured by EBSD and those measured by X-ray diffraction. Due to similarities between the crystal structures of martensite and ferrite (body centered tetragonal versus body centered cubic, respectively), martensite could not be distinguished from ferrite by indexing of diffraction patterns; however, martensite could qualitatively be distinguished from ferrite by regions of low image quality based on the very high dislocation density of martensite.

Quantification of very low density molecular coatings on large (60 nm) gold nanoparticles (AuNPs) is demonstrated via the use of antibody-mediated self-limiting self-assembly of small and large AuNPs into raspberry-like structures subsequently imaged by atomic force microscopy (AFM). AFM imaging is proposed as an automated, lower-cost, higher-throughput alternative to immunostaining and imaging by transmission electron microscopy. Synthesis of large AuNPs, containing one of three ligand molecules in one of three stoichiometries (1, 2, or 10 ligands per AuNP), and small probe AuNPs with one of three antibody molecules in a one antibody per AuNP ratio, enabled a range of predicted self-limiting self-assembled structures. A model predicting the probability of observing a given small to large AuNP ratio based on a topography measurement such as AFM is described, in which random orientational deposition is assumed and which accounts for the stochastic synthesis method of the library AuNPs with varied ligand ratios. Experimental data were found to agree very well with the predictive models when using an established AFM sample preparation method that avoids drying-induced aggregation.

The quantification of three classes of graphite inclusions in cast iron, namely, nodular, flake, and irregular, is the most important process in the foundry industry. This classification is based on the ISO 945 proposed morphology of graphite inclusions. This work presents a novel solution for automatic quantitative analysis of graphite inclusions into the three mentioned classes. The proposed work comprises three stages, namely, preprocessing of micrographs, classification of graphite inclusions, and then quantification of inclusions in each class. An effort has been made in this work to propose a minimum set of features to represent graphite inclusion morphology. The method employs just two geometric shape descriptors: the diameter ratio and the area ratio. A fuzzy rule based classifier is built using known feature values that are efficient in the classification of the three classes of graphite inclusions. The proposed method is automatic, fast, and provides the basis for determining many more morphological parameters that can be determined with the least effort. The results obtained by the proposed method are compared with the manual method. It is observed that the results obtained from the proposed method are useful in the optimization of cast iron manufacturing in the foundry industry.

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

An open source software package dedicated to processing stored electron backscatter patterns is presented. The package gives users full control over the type and order of operations that are performed on electron backscatter diffraction (EBSD) patterns as well as the results obtained. The current version of EBSD-Image (www.ebsd-image.org) offers a flexible and structured interface to calculate various quality metrics over large datasets. It includes unique features such as practical file formats for storing diffraction patterns and analysis results, stitching of mappings with automatic reorganization of their diffraction patterns, and routines for processing data on a distributed computer grid. Implementations of the algorithms used in the software are described and benchmarked using simulated diffraction patterns. Using those simulated EBSD patterns, the detection of Kikuchi bands in EBSD-Image was found to be comparable to commercially available EBSD systems. In addition, 24 quality metrics were evaluated based on the ability to assess the level of deformation in two samples (copper and iron) deformed using 220 grit SiC grinding paper. Fourteen metrics were able to properly measure the deformation gradient of the samples.

Microscopy & Microanalysis (M&M) stands as the premier meeting of the Microscopy Society of America, the Microanalysis Society, and the International Metallographic Society, Inc. We are pleased welcome to all the physical, life, and analytical scientists whose work encompasses the full range of microscopic and analytical techniques. In particular, we extend a warm welcome to those researchers who are new to our meeting, and are confident that you will find areas of specific interest in our meeting this year and for many years to come.