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

The use of multifocal-plane, time-lapse recordings of living specimens has allowed investigators to visualize dynamic events both within ensembles of cells and individual cells. Recordings of such four-dimensional (4D) data from digital optical sectioning microscopy produce very large data sets. We describe a wavelet-based data compression algorithm that capitalizes on the inherent redunancies within multidimensional data to achieve higher compression levels than can be obtained from single images. The algorithm will permit remote users to roam through large 4D data sets using communication channels of modest bandwidth at high speed. This will allow animation to be used as a powerful aid to visualizing dynamic changes in three-dimensional structures.

This study investigated the testicular changes in the rat induced by the nonspecific phosphodiesterase inhibitor, theophylline using magnetic resonance microscopy (MRM) and ubiquitin immunostaining techniques. In vivo T1- and T2-weighted images were acquired at 2 T under anesthesia. Increased signal observed in the theophylline-treated rats suggests that leakage of MRM contrast was occurring. In vivo MRM results indicate that day 16 testis displayed an increased T1-weighted water signal in the area of the seminiferous tubule that decreased by day 32. These findings were validated by histopathology, suggesting that in vivo MRM has the sensitivity to predict changes in testis and epididymal tissues. The participation of the ubiquitin system was investigated, using probes for various markers of the ubiquitin-proteasome pathway. MRM can be used to detect subtle changes in the vascular perfusion of organ systems, and the up-regulation/mobilization of ubiquitin-proteasome pathway may be one of the mechanisms used in theophylline-treated epididymis to remove damaged cells before storage in the cauda epididymis. The combined use of in vivo MRM and subsequent tissue or seminal analysis for the presence of ubiquitin in longitudinal studies may become an important biomarker for assessing testis toxicities drug studies.

Trypanosoma cruzi is the ethiological agent of Chagas disease. New compounds are being developed based on the biosynthesis and function of sterols, because T. cruzi has a requirement for specific endogenous sterols for growth and survival. Sterol biosynthesis inhibitors (SBIs) are drugs commonly used against fungal diseases. These drugs act by depleting essential and specific membrane components and/or inducing the accumulation of toxic intermediary or lateral products of the biosynthetic pathway. In this work we present the effects of WSP488, WSP501, and WSP561, specific inhibitors of Δ24(25)-sterol methyl transferase, on the ultrastructure of T. cruzi epimastigotes. All three drugs inhibited parasite multiplication at low concentrations, with IC50 values of 0.48, 0.44, and 0.48 μM, respectively, and induced marked morphological changes including (a) blockage of cell division; (b) swelling of the mitochondrion, with several projections and depressions; (c) swelling of the perinuclear space; (d) presence of autophagosomes and myelin-like figures; (e) enlargement of the flagellar pocket and of a cytoplasmic vacuole located in close association with the flagellar pocket; (f) detachment of the membrane of the cell body; and (g) formation of a vesicle at the surface of the parasite between the flagellar pocket and the cytostome. Our results show that these drugs are potent in vitro inhibitors of growth of T. cruzi.

Indirect high resolution electron microscopy using one of several possible data-set geometries offers advantages over conventional high-resolution imaging in enabling the recovery of the complex wavefunction at the specimen exit plane and simultaneously eliminating the aberrations present in the objective lens. This article discusses results obtained using this method from structures formed by inorganic materials confined within the bores of carbon nanotubes. Such materials are shown to be atomically regulated due to their confinement, leading to integral layer architectures that we have termed “Feynman crystals.” These one-dimensional (1D) crystals also show a wide range of structural deviations from the bulk, including unexpected lattice distortions, and in some cases entirely new forms have been observed.

The anterior–posterior and dorsal–ventral progression of heart organogenesis is well illustrated by the patterning and activity of two members of different families of cell adhesion molecules: the calcium-dependent cadherins, specifically N-cadherin, and the extracellular matrix glycoproteins, fibronectin. N-cadherin by its binding to the intracellular molecule β-catenin and fibronectin by its binding to integrins at focal adhesion sites, are involved in regulation of gene expression by their association with the cytoskeleton and through signal transduction pathways. The ventral precardiac mesoderm cells epithelialize and become stably committed by the activation of these cell–matrix and intracellular signaling transduction pathways. Cross talk between the adhesion signaling pathways initiates the characteristic phenotypic changes associated with cardiomyocyte differentiation: electrical activity and organization of myofibrils. The development of both organ form and function occurs within a short interval thereafter. Mutations in any of the interacting molecules, or environmental insults affecting either of these signaling pathways, can result in embryonic lethality or fetuses born with severe heart defects. As an example, we have defined that exposure of the embryo temporally to lithium during an early sensitive developmental period affects a canonical Wnt pathway leading to β-catenin stabilization. Lithium exposure results in an anterior–posterior progression of severe cardiac defects.

An energy dispersive x-ray microanalysis study was performed throughout the total length of cementum on five impacted human teeth. Mineral content of calcium, phosphorous, and magnesium were determined with an electron probe from the cemento-enamel junction to the root apex on the external surface of the cementum. The concentration profiles for calcium, phosphorous, and magnesium were compared by using Ca/P and Mg/Ca atomic percent ratio. Our findings demonstrated that the Ca/P ratio at the cemento-enamel junction showed the highest values (1.8–2.2). However, the area corresponding to the acellular extrinsic fiber cementum (AEFC) usually located on the coronal one-third of the root surface showed a Ca/P media value of 1.65. Nevertheless, on the area representing the fulcrum of the root there is an abrupt change in the Ca/P ratio, which decreases to 1.3. Our results revealed that Mg2+ distribution throughout the length of human cementum reached its maximum Mg/Ca ratio value of 1.3–1.4 at.% around the fulcrum of the root and an average value of 0.03%. A remarkable finding was that the Mg/Ca ratio pattern distribution showed that in the region where the Ca/P ratio showed a decreasing tendency, the Mg/Ca ratio reached its maximum value, showing a negative correlation. In conclusion, this study has established that clear compositional differences exist between AEFC and cellular mixed stratified cementum varieties and adds new knowledge about Mg2+ distribution and suggests its provocative role regulating human cementum metabolism.

Nanophase aluminum powder was characterized in a field-emission-gun transmission electron microscope (TEM). Different techniques were used to investigate the structure of the particles, including conventional bright-field and dark-field imaging, scanning transmission electron microscopy (STEM), high-resolution lattice imaging, diffraction studies, energy dispersive X-ray spectroscopy (EDS) analysis and mapping, and electron energy loss spectroscopy (EELS) analysis and mapping. It has been established that the particle cores consist of aluminum single crystals that sometimes contain crystal lattice defects. The core is covered by a passivating layer of aluminum oxide a few nanometers thick. The alumina is mostly amorphous, but evidences of partial crystallinity of the oxide were also found. The thickness of this layer was measured using different techniques, and the results are in good agreement with each other. The particles are agglomerated in two distinct ways. Some particles were apparently bonded together during processing before oxidation. These mostly form dumbbells covered by a joint oxide layer. Also, oxidized particles are loosely assembled into relatively large clusters.

The obtention of hydroxyapatite (HAp) is reported using brushite (CaHPO4·2H2O) and the skeleton of a starfish (Mellita eduardobarrosoi sp. nov.), primarily composed of magnesian calcite ((Ca,Mg)CO3) as precursors. Stoichiometric amounts of both were reacted under hydrothermal conditions: a pressure of 5.8 MPa and a temperature of 200°C for 2, 4, 6, 8, 10, and 20 h of reaction times. The samples obtained were characterized by means of scanning electron microscopy, X-ray diffraction, infrared spectroscopy, and transmission electron microscopy. Two defined populations of HAp fibers were found: A bundle of fibers 75 μm in length and 1–13 μm in diameter, and a second bundle of fibers 5 μm in length and less than 0.5 μm in diameter. Furthermore, an increase in HAp formation and a Ca/P ratio as a function of reaction time were observed. The growth mechanism of HAp is also discussed.

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

Specialized conduction tissues mediate coordinated propagation of electrical activity through the adult vertebrate heart. Following activation of the atria, the activation wave is slowed down in the atrioventricular canal or node, after which it spreads rapidly into the left and right ventricles via the His-Purkinje system (HPS). This results in the ventricles being activated from the apex toward the base, which is a hallmark of HPS function. The development of mature HPS function follows significant phases of cardiac morphogenesis. Initially, the cardiac impulse propagates in a slow, linear, and isotropic fashion from the sinus venosus at the most caudal portion of the tubular heart. Although the speed of impulse propagation gradually increases as it travels toward the anterior regions of the heart tube, the actual sequence of ventricular activation in the looped heart proceeds in the same direction as blood flow. Eventually, the immature base-to-apex sequence of ventricular activation undergoes an apparent reversal, changing to the mature apex-to-base pattern. Using an optical mapping approach, we demonstrate that the timing of this last transition shows striking dependence on hemodynamic loading of the ventricle, being accelerated by pressure overload and delayed in left ventricular hypoplasia. Comparison of chick and mammalian hearts revealed some striking similarities as well as key differences in the timing of such events during cardiac organogenesis.

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Historically, the decapod crustacean circulatory system has been classed as open. However, recent work on the blue crab, Callinectes sapidus, suggests the circulatory system may be more complex than previously described. Corrosion casting techniques were refined and used to map the circulatory system of a variety of crab species (order: Decapoda; family: Cancridae) to determine if the complexity observed in the blue crab was present in other species. Seven arteries arose from the single chambered heart. The anterior aorta, the paired anterolateral arteries, and the paired hepatic arteries exited from the anterior aspect of the heart. The small-diameter posterior aorta exited posteriorly from the heart. Exiting from the ventral surface of the heart, the sternal artery branched to supply the legs and mouthparts of the crab. These arteries were more complex than previously described, with arterioles perfusing all areas of the body. The arterioles split into fine capillary-like vessels. Most of these capillaries were blind ending. However, in several areas (antennal gland, supraesophageal ganglion) complete capillary beds were present. After passing through the capillary-like vessels, blood drained into a series of sinuses. However, rather than being arbitrary spaces as previously described, scanning electron micrographs showed the sinuses to be distinct units. Most of the sinuses formed a series of flattened membrane-bound lacunae. This complexity may qualify the decapod crustacean circulatory system as one that is “partially closed” rather than open.

Evaporation rate and subsequent exposure to humid air affect the surface morphology and composition of cesium iodide (CsI) films and, in turn, their photoemissive efficiency when used as photocathodes. The surface morphology and elemental composition of 300-nm-thick CsI films grown at two different rates (1 nm/s and 0.04 nm/s), both freshly evaporated and after 24-h exposure to humid air were investigated by means of atomic force microscopy and scanning electron microscopy/electron diffraction spectroscopy. The CsI film freshly evaporated at a slow rate exhibited a granular surface presenting circular holes or craters where the CsI material was moved from the center to the boundaries. After 24-h exposure to humid air, this film coalesced in large grain showing a marked increase of surface roughness. Conversely, the CsI film grown at a fast rate mostly retained its original surface uniformity and homogeneity with no presence of holes and craters after 24-h exposure to humid air. Further, surface roughness and average peak height decreased, but the surface coalesced in large grains spaced by small fractures where the CsI coverage was almost lost. In conclusion, the films grown at a fast evaporation rate were affected by 24-h exposure to humid air less than those grown at a slow rate, and are thus expected to possess a greater long-term stability.

Environmental transmission electron microscopy (ETEM) is opening an important window for in situ studies of interaction of water with oxides. Studies of MgO smoke nanocrystals under partial pressures of water ranging from 10 mTorr to 10 Torr found their {100} neutral surfaces to be extremely resistant to dissociative adsorption of water and hydroxylation, in agreement with recent theoretical predictions. ETEM observations of electron irradiation driven MgO smoke nanocrystal hydroxylation displayed the anticipated volume expansion, but revealed complex shape changes with elongations toward oxide corners. The reaction rate was found to increase with electron flux at constant water pressure. In situ selected area diffraction studies of MgO single crystals showed that the hydroxide grows with its basal (0001) plane parallel to the polar MgO (111) planes. This is the same crystallographic relationship as in dehydroxylation experiments, but with four variants. Electron energy loss spectroscopy found oxygen K-edge changes consistent with bulk hydroxylation.

Solid phase crystallization of plasma-enhanced chemical-vapor-deposited (PECVD) amorphous silicon (α-Si:H) in α-Si:H/Al and Al/α-Si:H structures has been investigated using transmission electron microscopy (TEM) and X-ray diffraction (XRD). Radiative heating has been used to anneal films deposited on carbon-coated nickel (Ni) grids at temperatures between 200 and 400°C for TEM studies. α-Si:H films were deposited on c-Si substrates using high vacuum (HV) PECVD for the XRD studies. TEM studies show that crystallization of α-Si:H occurs at 200°C when Al film is deposited on top of the α-Si:H film. Similar behavior was observed in the XRD studies. In the case of α-Si:H deposited on top of Al films, the crystallization could not be observed at 400°C by TEM and even up to 500°C as seen by XRD.

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

The mammalian tongue has evolved for specialized functions in different species. The structure of its papillae tells about the animal's diet, habit, and taxonomy. The opossum has four kinds of lingual papillae (filiform, conical, fungiform, vallate). Scanning electron microscopy of the external features, connective tissue cores, and corrosion casts of the microvasculature show the filiform papillae have a spearhead-like main process and spiny accessory processes around the apical part of the main process. The shape and number of both processes depend on their position on the tongue. On the apex, the main processes have shovel-like capillary networks and the accessory processes have small conical networks. On the lingual radix, the processes have small capillary loops. In the patch region, conical papillae have capillaries arranged as a full sail curving posteriorly. The fungiform papillae are scattered among the filiform papillae and have capillary baskets beneath each taste bud. Giant fungiform papillae on the tongue tip are three to four times larger than the ones on the lingual body. Capillaries of giant papillae form a fan-shaped network. The opossum has three vallate papillae arranged in a triangle. Their tops have secondary capillary loops but not their lateral surfaces. Mucosal folds on the posterolateral border have irregular, fingerlike projections with cylindrical capillary networks. These findings and the structure of the rest of the masticatory apparatus suggest the lingual papillae of opossum have kept their ancestral carnivorous features but also developed the herbivore characteristics of other marsupials.