In an article published in Microscopy and Microanalysis recently (Jia et al., 2004), it was claimed that aberration-corrected high resolution transmission electron microscopy (HRTEM) allows the quantitative measurement of oxygen concentrations in ceramic materials with atomic resolution. Similar claims have recently appeared elsewhere, based on images obtained through aberration correction (Jia et al., 2003; Jia & Urban, 2004) or very high voltages (Zhang et al., 2003). Seeing oxygen columns is a significant achievement of great importance (Spence, 2003) that will doubtlessly allow some exciting new science; however, other models could provide a better explanation for some of the experimental data than variations in the oxygen concentration. Quantification of the oxygen concentrations was attempted by comparing experimental images with simulations in which the fractional occupancy in individual oxygen columns was reduced. The results were interpreted as representing nonstoichiometry within the bulk and at grain boundaries. This is plausible because previous studies have shown that grain boundaries can be nonstoichiometric (Kim et al., 2001), and it is indeed possible that oxygen vacancies are present at boundaries or in the bulk. However, is this the only possible interpretation? We show that for the thicknesses considered a better match to the images is obtained using a simple model of surface damage in which atoms are removed from the surface, which would usually be interpreted as surface damage or local thickness variation (from ion milling, for example).

Extended abstract of a paper presented at Microscopy and Microanalysis 2005 in Honolulu, Hawaii, USA, July 31--August 4, 2005

The ecdysial suture is the region of the arthropod exoskeleton that splits to allow the animal to emerge during ecdysis. We examined the morphology and composition of the intermolt and premolt suture of the blue crab using light microscopy and scanning electron microscopy. The suture could not be identified by routine histological techniques; however 3 of 22 fluorescein isothiocyanate-labeled lectins tested (Lens culinaris agglutinin, Vicia faba agglutinin, and Pisum sativum agglutinin) differentiated the suture, binding more intensely to the suture exocuticle and less intensely to the suture endocuticle. Back-scattered electron (BSE) and secondary electron observations of fracture surfaces of intermolt cuticle showed less mineralized regions in the wedge-shaped suture as did BSE analysis of premolt and intermolt resin-embedded cuticle. The prism regions of the suture exocuticle were not calcified. X-ray microanalysis of both the endocuticle and exocuticle demonstrated that the suture was less calcified than the surrounding cuticle with significantly lower magnesium and phosphorus concentrations, potentially making its mineral more soluble. The presence or absence of a glycoprotein in the organic matrix, the extent and composition of the mineral deposited, and the thickness of the cuticle all likely contribute to the suture being removed by molting fluid, thereby ensuring successful ecdysis.

Isolation and culture of thymic epithelial cells (TECs) using conventional primary tissue culture techniques under conditions employing supplemented low calcium medium yielded an immortalized cell line derived from the LDA rat (Lewis [Rt1l] cross DA [Rt1a]) that could be manipulated in vitro. Thymi were harvested from 4–5-day-old neonates, enzymically digested using collagenase (1 mg/ml, 37°C, 1 h) and cultured in low calcium WAJC404A medium containing cholera toxin (20 ng/ml), dexamethasone (10 nM), epidermal growth factor (10 ng/ml), insulin (10 μg/ml), transferrin (10 μg/ml), 2% calf serum, 2.5% Dulbecco's Modified Eagle's Medium (DMEM), and 1% antibiotic/antimycotic. TECs cultured in low calcium displayed round to spindle-shaped morphology, distinct intercellular spaces (even at confluence), and dense reticular-like keratin patterns. In high calcium (0.188 mM), TECs formed cobblestone-like confluent monolayers that were resistant to trypsinization (0.05%) and displayed keratin intermediate filaments concentrated at desmosomal junctions between contiguous cells. Changes in cultured TEC morphology were quantified by an analysis of desmosome/membrane relationships in high and low calcium media. Desmosomes were significantly increased in the high calcium medium. These studies may have value when considering the growth conditions of cultured primary cell lines like TECs.

According to statistics provided by the American Heart Association for 2002, the latest year available, cardiovascular diseases are the leading cause of death in both males and females in the United States with over 434,000 males and 494,000 females dying each year from cardiovascular-related diseases. This is 36.6% of all deaths in males and 39.8% in females and represents billions of dollars in health care costs. Cardiovascular-associated diseases run the gamut from congenital defects that manifest early in development through diseases such as stroke and infarcts frequently associated with advanced age. Investigative techniques to study these diseases are varied and include a full range of imaging, genomic, and proteomic technologies.

With this issue, Microscopy and Microanalysis begins its eleventh year of publication. Our journal is getting better year-by-year. In 2004 this journal published more scientific articles than in any previous year. The first biological special issue, on parasites, increased the prominence of the life sciences within these pages. The growing popularity of the journal is also indicated by several letters to the editor about matters arising in certain articles. Such scientific exchanges highlight for our readership the subtleties of interpretation regarding advances in science, instrumentation, technique, and theory.

The official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada / Société de, Microscopie du Canada, Mexican Microscopy Society, Brazilian Society for Microscopy and Microanalysis, Venezuelan Society of Electron Microscopy, European Microbeam Analysis Society, Australian Microscopy and Microanalysis Society.

Published in affiliation with Royal Microscopical Society, German Society for Electron Microscopy, Belgian Society for Microscopy, Microscopy Society of Southern Africa.

Editor in Chief, Editor, Microanalysis: Charles E. Lyman, Materials Science and Engineering, Lehigh University, 5 East Packer Avenue, Bethlehem, Pennsylvania 18015-3195, Phone: (610) 758-4249, Fax: (610) 758-4244, e-mail: [email protected].

Editor, Biological Applications: Ralph Albrecht, Department of Animal Sciences, University of Wisconsin-Madison, 1675 Observatory Drive, Madison, Wisconsin 53706-1581, Phone: (608) 263-3952, Fax: (608) 262-5157, e-mail: [email protected].

Editor, Materials Applications: David J. Smith, Center for Solid State Science, Arizona State University, Tempe, Arizona 85287-1704, Phone: (480) 965-4540, Fax: (480) 965-9004, e-mail: [email protected].

Editor, Materials Applications: Elizabeth Dickey, Materials Science and Engineering, Pennsylvania State University, 223 MRL Building, University Park, PA 16802-7003, Phone: (814) 865-9067, Fax: (814) 863-8561, e-mail: [email protected].

Editor, Light and Scanning Probe, Microscopies: Brian Herman, Cellular and Structural Biology, University of Texas at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7762, Phone: (210) 567-3800, Fax: (210) 567-3803, e-mail: [email protected].

Editor, Biological Applications: Heide Schatten, Veterinary Pathobiology, University of Missouri-Columbia, 1600 E. Rollins Street, Columbia, Missouri 65211-5030, Phone: (573) 882-2396, Fax: (573) 884-5414, e-mail: [email protected].

Calendar Editor, Book Review Editor: JoAn Hudson, Advanced Materials Research Labs., Clemson Univ. Research Park, Rm. 105, Anderson, SC 29625, Phone: (864) 656-7535, Fax: (864) 656-2466, e-mail: [email protected].

Special Section Editor: James N. Turner, Phone: (518) 474-2811, Fax: (518) 474-8590, e-mail: [email protected].

Expo Editor: William T. Gunning III, Phone: (419) 383-5256, Fax: (419) 383-3066, e-mail: [email protected].

Proceedings Editor: Stuart McKernan, Phone: (612) 624-6009, Fax: (612) 625-5368, e-mail: [email protected].

Quantitative dimensional measurements of micro- and nanometre-sized structures are urgently required from science and industry. Due to their very high vertical resolution (down to sub nanometres) and high lateral resolution (<10 nm) scanning probe microscopes (SPMs) are of great interest for such metrological applications. Additionally, SPM methods are able to measure surfaces in a number of modes like contact, intermittent-contact and non-contact mode. The forces between tip and sample are low during the measurement and, even in contact mode, reach only a few nanonewtons. This fact prevents scratching of the measured surface during the SPM scanning procedure even when very sharp tips are used.

The Ninth Frontiers of Electron Microscopy in Materials Science Conference (FEMMS 2003) was held October 5–10, 2003 at the Claremont Resort and Spa in Berkeley, CA. Major sponsors for this meeting included Lawrence Livermore National Laboratory, Argonne National Laboratory, Lawrence Berkeley National Laboratory, Brookhaven National Laboratory, Frederick Seitz Materials Research Laboratory, Oak Ridge National Laboratory, National Science Foundation, and University of California at Davis. Sponsors also included LEO Electron Microscopy Ltd. (Carl Zeiss SMT), E. A. Fischione, Inc., Gatan, Inc., Thermo NORAN (Thermo Electron Corp.), FEI Company, Hitachi-HHTA, JEOL USA, Inc., Seiko Instruments, and CEOS GmbH.

Atomic Force Microscopy (AFM) has become a ubiquitous tool for analyzing the topography of a wide variety of materials, especially as nanoscale features become more significant for both understanding as well as determining materials properties [1]. Many AFM variations have also been developed for measuring surface properties beyond straightforward cartography. In many of these cases, the contrast mechanisms are often either extremely complex, or not well understood, even though the principles are simple. For example, Piezo-Force Microscopy (PFM) is relatively easy to understand and use in a standard lab for measuring electromechanical properties of materials, but care must be taken in order to obtain quantitative results as described below.

To examine new cytochemical aspects of the bacterial adhesion, a strain 41452/01 of the oral commensal Streptococcus sanguis and a wild strain of Staphylococcus aureus were grown with and without sucrose supplementation for 6 days. Osmiumtetraoxyde (OsO4), uranyl acetate (UA), ruthenium red (RR), cupromeronic blue (CB) staining with critical electrolytic concentrations (CECs), and the tannic acid–metal salt technique (TAMST) were applied for electron microscopy. Cytochemically, only RR-positive fimbriae in S. sanguis were visualized. By contrast, some types of fimbriae staining were observed in S. aureus glycocalyx: RR-positive, OsO4-positive, tannophilic and CB-positive with ceasing point at 0.3 M MgCl2. The CB staining with CEC, used for the first time for visualization of glycoproteins of bacterial glycocalyx, also reveals intacellular CB-positive substances—probably the monomeric molecules, that is, subunits forming the fimbriae via extracellular assembly. Thus, glycosylated components of the biofilm matrix can be reliably related to single cells. The visualization of intracellular components by CB with CEC enables clear distinction between S. aureus and other bacteria, which do not produce CB-positive substances. The small quantities of tannophilic substances found in S. aureus makes the use of TAMST for the same purpose difficult. The present work protocol enables, for the first time, a partial cytochemical differentiation of the bacterial glycocalyx.

From the basic light microscope through high-end imaging systems such as multiphoton confocal microscopy and electron microscopes, microscopy has been and will continue to be an essential tool in developing an understanding of cardiovascular development, function, and disease. In this review we briefly touch on a number of studies that illustrate the importance of these forms of microscopy in studying cardiovascular biology. We also briefly review a number of imaging modalities such as computed tomography, (CT) Magnetic resonance imaging (MRI), ultrasound, and positron emission tomography (PET) that, although they do not fall under the realm of microscopy, are imaging modalities that greatly complement microscopy. Finally we examine the role of proper imaging system calibration and the potential importance of calibration in understanding biological tissues, such as the cardiovascular system, that continually undergo deformation in response to strain.

Microscopy and Microanalysis editorial board, editorial board representatives from affiliated societies, and founding editor.

The authors are grateful to the Editor for the opportunity to reply to the letter by Lupini et al. The authors of the above letter comment on a set of recent articles in which the novel technique of imaging at a negative value of the spherical aberration coefficient of the objective lens in an aberration-corrected transmission electron microscope (NCSI technique) is methodically described and applied to the measurement of the occupancy of atomically resolved oxygen columns in perovskites. In particular, the authors raise doubts about the possibility of inferring quantitative data from measurements of the local image intensity at the position of the oxygen atom columns. With reference to the study by Jia et al. (2003a), the letter authors present an image simulation on the basis of which it is stated that the observed effect of a reduced intensity at the oxygen atomic columns should not be interpreted in terms of reduced oxygen occupancy but can, as the authors claim, be “better” explained on the basis of the effect of surface roughness on contrast. In addition, the authors emphasize the work of Kim et al. (2001) with respect to the nonstoichiometry of the oxygen occupancy in grain boundaries of SrTiO3 and criticize our reference to the literature in which it is reported that oxygen cannot be observed by the scanning transmission electron microscopy (STEM) technique in Z-contrast. In the following, we shall demonstrate that in spite of the fact that a nonideal surface morphology can—as in the application of any (!) electron microscopic technique whether used in TEM or in STEM—have an effect on local image intensity, meaningful quantitative measurements of relative oxygen-atom site occupancies can be carried out employing the NCSI technique.

Electron tomography is a well-established technique for three-dimensional structure determination of (almost) amorphous specimens in life sciences applications. With the recent advances in nanotechnology and the semiconductor industry, there is also an increasing need for high-resolution three-dimensional (3D) structural information in physical sciences. In this article, we evaluate the capabilities and limitations of transmission electron microscopy (TEM) and high-angle-annular-dark-field scanning transmission electron microscopy (HAADF-STEM) tomography for the 3D structural characterization of partially crystalline to highly crystalline materials. Our analysis of catalysts, a hydrogen storage material, and different semiconductor devices shows that features with a diameter as small as 1–2 nm can be resolved in three dimensions by electron tomography. For partially crystalline materials with small single crystalline domains, bright-field TEM tomography provides reliable 3D structural information. HAADF-STEM tomography is more versatile and can also be used for high-resolution 3D imaging of highly crystalline materials such as semiconductor devices.

This is a report of the adaptation of microwave processing in the preparation of liver biopsies for transmission electron microscopy (TEM) to examine ultrastructural damage of mitochondria in the setting of metabolic stress. Hemorrhagic shock was induced in pigs via 35% total blood volume bleed and a 90-min period of shock followed by resuscitation. Hepatic biopsies were collected before shock and after resuscitation. Following collection, biopsies were processed for TEM by a rapid method involving microwave irradiation (Giberson, 2001). Samples pre- and postshock of each of two animals were viewed and scored using the mitochondrial ultrastructure scoring system (Crouser et al., 2002), a system used to quantify the severity of ultrastructural damage during shock. Results showed evidence of increased ultrastructural damage in the postshock samples, which scored 4.00 and 3.42, versus their preshock controls, which scored 1.18 and 1.27. The results of this analysis were similar to those obtained in another model of shock (Crouser et al., 2002). However, the amount of time used to process the samples was significantly shortened with methods involving microwave irradiation.

The nucleolus is the main site for synthesis and processing of ribosomal RNA in eukaryotes. In mammals, plants, and yeast the nucleolus has been extensively characterized by electron microscopy, but in the majority of the unicellular eukaryotes no such studies have been performed. Here we used ultrastructural cytochemical and immunocytochemical techniques as well as three-dimensional reconstruction to analyze the nucleolus of Trypanosoma cruzi, which is an early divergent eukaryote of medical importance. In T. cruzi epimastigotes the nucleolus is a spherical intranuclear ribonucleoprotein organelle localized in a relatively central position within the nucleus. Dense fibrillar and granular components but not fibrillar centers were observed. In addition, nuclear bodies resembling Cajal bodies were observed associated to the nucleolus in the surrounding nucleoplasm. Our results provide additional morphological data to better understand the synthesis and processing of the ribosomal RNA in kinetoplastids.

Extended abstract of a paper presented at Microscopy and Microanalysis 2005 in Honolulu, Hawaii, USA, July 31--August 4, 2005

In this article, two simple methods, evaporation-condensation and catalytic thermal evaporation, were used to investigate the synthesis of CdS nanostructures for nanoscale optoelectronic applications. To understand their growth mechanisms, various electron microscopy and microanalysis techniques were utilized in characterizing their morphologies, internal structures, growth directions and elemental compositions. The electron microscopy study reveals that when using the evaporation-condensation method, branched CdS nanorods and self-assembled arrays of CdS nanorods were synthesized at 800°C and 1000°C, respectively. Instead of morphological differences, both types of CdS nanorods grew along the [0001] direction. However, when using the catalytic thermal evaporation method (Au as the catalyst), patterned CdS nanowires and nanobelts were formed at the temperature region of 500–600°C and 600–750°C, respectively. Their growth direction was along the direction [1010] instead of [0001]. Based on the microscopy and microanalysis results, we propose some growth mechanisms in relation to the growth processes of those exotic CdS nanostructures.

Scanning Probe Microscopy (SPM) [1] has been an important tool to organize matter on the nanometer scale. It has been proved to be a powerful tool not only for imaging but also for nanofabrication. SPM-based nanofabrication comprises manipulation of atoms or molecules and SPM-based nanolithography. SPM-based nanolithography, referred to as scanning probe lithography (SPL) holds good promise for fabrication of nanometer-scale patterns as an emerging generic lithography technique that employs SPM to directly pattern nanometer-scale features under appropriate conditions. The water meniscus formation between the tip and the flat substrate, due to the water layer present on any surface of a material at ambient conditions, has been studied experimentally and theoretically [2-6] using SPM techniques. The water effect in the imaging process is well understood [2, 4, 6-8]. Dip pen nanolithogaphy (DPN) [9] is one example of a technique that uses the water effect to transfer material from the tip onto sample surface in direct-write fashion with nanoscopic resolution.