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

Mouse oocyte microfilaments (MF) were perturbed by depolymerization (cytochalasin B) or stabilization (jasplakinolide) and correlated meiotic defects examined by confocal microscopy. MF, microtubules, and mitochondria were vitally stained; centrosomes (γ-tubulin), after fixation. MF depolymerization by cytochalasin in culture medium did not affect central migration of centrosomes, mitochondria, or nuclear breakdown (GVBD); some MF signal was localized around the germinal vesicle (GV). In maturation-blocking medium (containing IBMX), central movement was curtailed and cortical MF aggregations made the plasma membrane wavy. Occasional long MF suggested that not all MF were depolymerized. MF stabilization by jasplakinolide led to MF aggregations throughout the cytoplasm. GVBD occurred (unless IBMX was present) but no spindle formed. Over time, most oocytes constricted creating a dumbbell shape with MF concentrated under one-half of the oocyte cortex and on either side of the constriction. In IBMX medium, the MF-containing half of the dumbbell over time sequestered the GV, MF, mitochondria, and one to two large cortical centrosomes; the non-MF half appeared empty. Cumulus processes contacted the oocyte surface (detected by microtubule content) and mirrored MF distribution. Results demonstrated that MF play an essential role in meiosis, primarily through cortically mediated events, including centrosome localization, spindle (or GV) movement to the periphery, activation of (polar body) constriction, and establishment of oocyte polarity. The presence of a cortical “organizing pole” is hypothesized.

Progress in structural biology very much depends upon the development of new high-resolution techniques and tools. Despite decades of study of viruses, bacteria and bacterial spores and their pressing importance in human medicine and biodefense, many of their structural properties are poorly understood. Thus, characterization and understanding of the architecture of protein surface and internal structures of pathogens is critical to elucidating mechanisms of disease, immune response, physicochemical properties, environmental resistance and development of countermeasures against bioterrorist agents. Furthermore, even though complete genome sequences are available for various pathogens, the structure-function relationships are not understood. Because of their lack of symmetry and heterogeneity, large human pathogens are often refractory to X-ray crystallographic analysis or reconstruction by cryo-electron microscopy (cryo-EM). An alternative high-resolution method to examine native structure of pathogens is atomic force microscopy (AFM), which allows direct visualization of macromolecular assemblies at near-molecular resolution. The capability to image single pathogen surfaces at nanometer scale in vitro would profoundly impact mechanistic and structural studies of Progress in structural biology very much depends upon the development of new high-resolution techniques and tools. Despite decades of study of viruses, bacteria and bacterial spores and their pressing importance in human medicine and biodefense, many of their structural properties are poorly understood. Thus, characterization and understanding of the architecture of protein surface and internal structures of pathogens is critical to elucidating mechanisms of disease, immune response, physicochemical properties, environmental resistance and development of countermeasures against bioterrorist agents. Furthermore, even though complete genome sequences are available for various pathogens, the structure-function relationships are not understood. Because of their lack of symmetry and heterogeneity, large human pathogens are often refractory to X-ray crystallographic analysis or reconstruction by cryo-electron microscopy (cryo-EM). An alternative high-resolution method to examine native structure of pathogens is atomic force microscopy (AFM), which allows direct visualization of macromolecular assemblies at near-molecular resolution. The capability to image single pathogen surfaces at nanometer scale in vitro would profoundly impact mechanistic and structural studies of pathogenesis, immunobiology, specific cellular processes, environmental dynamics and biotransformation.

The M&M 2005 meeting will begin on Saturday, July 30, with a pre-meeting congress entitled “Biophotonics and Live Cell Imaging.” The pre-meeting congress will continue on Sunday along with a number of topical Short Courses addressing digital imaging, image analysis and specimen preparation. Examples of biological topics that will be addressed during the week of the meeting include Tracking and Tagging of Stem Cells, Use of Green and Other Fluorescent Proteins, Cell Communication and Pathology, and the use of microscopy in the study of Viruses, Infectious Diseases and Their Associated Pathologies. Topical symposia in the physical sciences will examine the role of microscopy and microanalysis in the study of Extraterrestrial Materials, Catalysts, and Metallographic Techniques, Applications and Failure Analysis. In joint sessions for biological and materials scientists, several symposia will address the development and imaging of a variety of Nanoparticles, Biopolymers and Biomembranes. Instrumentation and Techniques Development sessions will also address a number of state-of the-art instruments and techniques including Aberration Corrected Electron Microscopy, ESEM, FIB, Advanced Detectors, Spectral Imaging Techniques, FRET, and Tomography. Of special interest will be the Presidential Symposium, “The Golden Anniversary of Imaging Atoms” commemorating the 50th anniversary of the first atomic images using field-ion microscopy, which will provide keynote addresses concerning the historical and advanced technology that has grown from the first application of atomic imaging. This year we will also have a series of topical sessions organized by the Focused Interest Groups of MSA. Supplementing the topical sessions will be a number of Tutorials and Ask the-Experts sessions that will provide the opportunity to learn the basics behind many popular techniques. As always, the commercial exhibits will provide the unique opportunity for hands-on learning with the largest variety of state-of-the-art instrumentation found at any microscopy meeting worldwide. The poster sessions will again provide the best venue for discussions and exchange of scientific information. To facilitate communication, we have made arrangements to have appropriate kegs of beverages available on the exhibit floor during the 3:30–5:00 PM afternoon poster sessions. To promote this interaction, posters should be put up Monday morning and not removed until after the Wednesday afternoon Poster Session ends. It is the sincere hope of the Executive Program Committee that the Scientific Program will be of interest to all microscopists in the Pacific Rim countries. We look forward to meeting and interacting with as many of you as possible in Hawaii.

The gap junction (GJ) is an aggregate of intercellular channels that facilitates cytoplasmic interchange of ions, second messengers, and other molecules of less than 1000 Da between cells. In excitable organs such as heart and brain, GJs configure extended intercellular pathways for stable and long-term propagation of action potential. In a previous study in adult rat heart, we have shown that the Drosophila disks-large related protein ZO-1 shows low to moderate colocalization at myocyte borders with the GJ protein Cx43. In the present study, we detail a protocol for characterizing the pattern and level of colocalization of ZO-1 with Cx43 in cultures of neonatal myocytes at the level of individual GJ plaques. The data indicate that ZO-1 shows on average a partial 26.6% overlap (SD = 11.3%) with Cx43 GJ plaques. There is a strong positive correlation between GJ plaque size and area of ZO-1 colocalization, indicating that the level of associated ZO-1 scales with the area of the GJ plaque. Qualitatively, the most prominent colocalization occurs at the plaque perimeter. These studies may provide insight into the presently unknown biological function of ZO-1 interaction with Cx43.

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

Spores of the biocontrol agent, Streptomyces melanosporofaciens EF-76, were entrapped by complex coacervation in beads composed of a macromolecular complex (MC) of chitosan and polyphosphate. A proportion of spores entrapped in beads survived the entrapment procedure as shown by treating spores from chitosan beads with a dye allowing the differentiation of live and dead cells. The spore-loaded chitosan beads could be digested by a chitosanase, suggesting that, once introduced in soil, the beads would be degraded to release the biocontrol agent. Spore-loaded beads were examined by optical and scanning electron microscopy because the release of the biological agent depends on the spore distribution in the chitosan beads. The microscopic examination revealed that the beads had a porous surface and contained a network of inner microfibrils. Spores were entrapped in both the chitosan microfibrils and the bead lacuna.

This work demonstrates the possibility of using the Duane–Hunt limit of the bremsstrahlung to determine E2 values of Si3N4 and AlN ceramics. The EDHL versus E0 graph demonstrates that for conductive materials, the experimental curve is parallel to the theoretical (EDHL = E0), but both curves cross in the case of insulators. The intersection points (E2 value), are 3.01 keV for Si3N4 and 2.67 keV for AlN. Imaging of ceramic grain structure at high magnification was performed to demonstrate the validity of the calculated E2 values.

Physical, chemical, and isotopic analyses of individual radioactive and other particles in the micron-size range, key tools in environmental research and in nuclear forensics, require the ability to precisely relocate particles of interest (POIs) in the secondary ion mass spectrometer (SIMS) or in another instrument, after having been located, identified, and characterized in the scanning electron microscope (SEM). This article describes the implementation, testing, and evaluation of the triangulation POIs re-location method, based on microscopic reference marks imprinted on or attached to the sample holder, serving as an inherent coordinate system. In SEM-to-SEM and SEM-to-SIMS experiments re-location precision better than 10 μm and 20 μm, respectively, is readily attainable for instruments using standard specimen stages. The method is fast, easy to apply, and facilitates repeated analyses of individual particles in different instruments and laboratories.

The surfaces of crystalline samples of 3d-metals (Mn, Fe, Co, Ni, and Cu) and their stoichiometric oxides have been studied by Auger spectroscopy. A correlation between the change in the LVV (L-inner level-valence-valence electron transition) Auger intensities and the change of the squares of the corresponding atomic-magnetic moments has been observed. This is because of the complicated nature of the Auger process. That is, the Auger electron emission is a result of the inner atomic level excitation by electron impact and Auger annihilation of the inner-level hole. Therefore, the Auger process has been considered a second-order process, and spin polarization of the valence states has been taken into account for the LMM (L-inner level-M-inner level-M-inner level electron transition) Auger spectra of 3d-metals.

Analytical transmission electron microscopy (TEM) and scanning electron microscopy (SEM) have been applied for the characterization of evolution, lateral arrangements, orientations, and the microscopic nature of nanostructures formed during the early stages of ultrahigh vacuum electron beam evaporation of Cu onto surfaces of VSe2 layered crystals. Linear nanostructure of relatively large lateral dimension (100–500 nm) and networks of smaller nanostructures (lateral dimension: 15–30 nm; mesh sizes: 500–2000 nm) are subsequently formed on the substrate surfaces. Both types of nanostructures are not Cu nanowires but are composed of two strands of crystalline substrate material elevating above the substrate surface. For the large nanostructures a symmetric roof structure with an inclination angle of ∼30° with respect to the substrate surface could be deduced from detailed diffraction contrast experiments. In addition to the nanostructure networks a thin layer of a Cu-VSe2 intercalation phase of 3R polytype is observed at the substrate surface. A dense network of interface dislocations indicates that the phase formation is accompanied by in-plane strain. We present a model that explains the formation of large and small nanostructures as consequences of compressive layer strains that are relaxed by the formation of rooflike nanostructures, finally evolving into the observed networks with increasing deposition time. The dominating contributions to the compressive layer strains are considered to be an electronic charge transfer from the Cu adsorbate to the substrate and the formation of a Cu-VSe2 intercalation compound in a thin surface layer.

diFiore's Atlas of Histology with Functional Correlations. Victor P. Eroschenko. Lippincott Williams and Wilkins, Philadelphia, PA; 10th edition, 2005, 448 pages (Softbound and Software CD (interactive), $57.95), ISBN 0-7817-5021-0

Histology (or microscopic anatomy) is an important discipline of the biological sciences that is concerned with the structure of tissues of organisms. Light microscopy and electron microscopy in conjunction with various histochemical and immunocytochemical staining techniques are typically used today by histologists to visualize, describe, and identify tissues. Histology is a fundamental discipline in the curriculum for medical, dental, and veterinary students as well as for allied health students and scholars interested in the field of biomedical research. All these students need to have a thorough knowledge of the structure and function of cells, tissues, and organs. Recently, I came across a histology book (diFiore's Atlas of Histology) that I believe is worth the investment. This book provides a selection of high-quality, full-color illustrations of various tissues of the human body and is supplemented with sections of structural/functional correlations. This 10th edition is unique in that it includes a CD that contains an interactive electronic version of the histology atlas.

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

In this article, the secondary electron-emission properties of both vertically and laterally inhomogeneous samples are discussed. To study the effect of surface coverage, the total electron-emission yield of tungsten and niobium samples was measured as a function of primary electron energy and oxide thickness. A method is suggested to avoid charging difficulties during AES measurements of samples that consist of both metal and various insulator parts.

Biophotonics and live cell imaging (Saturday & Sunday), and Pre-meeting Congress sponsors.

A method for automatic classification of the shape of graphite particles in cast iron is proposed. In a typical supervised classification procedure, the standard charts from the ISO-945 standard are used as a training and validation population. Several shape and size parameters are described and used as discriminants. A new parameter, the average internal angle, is proposed and is shown to be relevant for accurate classification. The ideal parameter sets are determined, leading to validation success rates above 90%. The classifier is then applied to real cast iron samples and provides results that are consistent with visual examination. The method provides classification results per particle, different from the traditional per field chart comparison methods. The full procedure can run automatically without user interference.

Cardiac myocytes and fibroblasts are essential elements of myocardial tissue structure and function. In vivo, myocytes constitute the majority of cardiac tissue volume, whereas fibroblasts dominate in numbers. In vitro, cardiac cell cultures are usually designed to exclude fibroblasts, which, because of their maintained proliferative potential, tend to overgrow the myocytes. Recent advances in microstructuring of cultures and cell growth on elastic membranes have greatly enhanced in vitro preservation of tissue properties and offer a novel platform technology for producing more in vivo-like models of myocardium. We used microfluidic techniques to grow two-dimensional structured cardiac tissue models, containing both myocytes and fibroblasts, and characterized cell morphology, distribution, and coupling using immunohistochemical techniques. In vitro findings were compared with in vivo ventricular cyto-architecture. Cardiac myocytes and fibroblasts, cultured on intersecting 30-μm-wide collagen tracks, acquire an in vivo-like phenotype. Their spatial arrangement closely resembles that observed in native tissue: Strands of highly aligned myocytes are surrounded by parallel threads of fibroblasts. In this in vitro system, fibroblasts form contacts with other fibroblasts and myocytes, which can support homogeneous and heterogeneous gap junctional coupling, as observed in vivo. We conclude that structured cocultures of cardiomyocytes and fibroblasts mimic in vivo ventricular tissue organization and provide a novel tool for in vitro research into cardiac electromechanical function.

Off-axis electron holography is used to measure electrostatic potential profiles across a silicon p-n junction, which has been prepared for examination in the transmission electron microscope (TEM) in two different specimen geometries using focused ion beam (FIB) milling. Results are obtained both from a conventional unbiased FIB-milled sample and using a novel sample geometry that allows a reverse bias to be applied to an FIB-milled sample in situ in the TEM. Computer simulations are fitted to the results to assess the effect of TEM specimen preparation on the charge density and the electrostatic potential in the thin sample.

Formal definitions of nanotechnological devices for drug delivery typically feature the requirements that the device itself or its essential components be man-made, and in the 1-1000 nm range in at least one dimension [1]. The known nanovectors or nanostructures can be filled with drugs for different therapies and for diagnostical aims. Targeting moieties can also be attached to their surface. Polymeric nanovectors are generally made from biodegradable polymers such as polyesters, for example, poly-e-caprolactone (PCL). The drug delivery system known as nanocapsules (NCs) can be defined as a complex nanovector that is composed by a polymeric wall surrounding an oil core, where lipophilic drugs can be encapsulated. The advantages of NCs compared to other nanovectors are the high entrapment efficiencies of lipophilic drugs, low polymer content and low inherent toxicity. On the other hand, because of its complex blend of components NCs suspension allow several forms of nanovectors to be present at the same time, such as nanospheres, liposomes and nanoemulsions [2]. These ‘contaminants’ would be present in accordance with the type of formulation and method of preparation. Atomic force microscopy (AFM) has been used as a method for imaging the surfaces of liposomes [3] and nanospheres [4] allowing information in nanoscaled dimensions. In the present work, the NCs were prepared loading two different drugs, the antifungal albaconazole (ABZ), showing a crystalline drug structure and the antimalarial halofantrine (Hf) free base, having an amorphous form. These drugs possess high lipophilic character, which favours the association of the drug with the oily core, with drug loadings above 94%. Herein we studied the behavior of ABZ-loaded and Hf-loaded NCs through the AFM technique, searching to analyze and understand possible alterations induced by the drug inclusion in these nanostructures.