In the present research long-term pulmonary toxicity of lead was investigated in rats treated by intraperitoneal administration of lead acetate for three consecutive days (25 mg/kg per day). Five weeks after treatment average lead content in the whole blood was 0.41 μg/dL ± 0.05, in the lung homogenates it measured 3.35 μg/g ± 0.54, as compared to the control values of 0.13 ± 0.07 μg/dL and 1.03 μg/g ± 0.59, respectively. X-ray microanalysis of lung specimens displayed lead localized mainly within type II pneumocytes and macrophages. At the ultrastructural level the effects of lead toxicity were found in lung capillaries, interstitium, epithelial cells, and alveolar lining. Alveolar septa showed intense fibrosis, consisting of collagen, elastin, and fibroblasts. Thinned alveolar septa had emphysematous tissue with some revealing signs of angiogenesis. Type II pneumocytes contained lamellar bodies with features of laminar destruction. Fragments of the surfactant layer were often detached from the alveolar epithelium. These findings indicate that 5 weeks after exposure, lead provokes reconstruction of the alveolar septa including fibrosis and emphysematous changes in the lung tissue.

The Registration Desk at M&M 2013 is located on the Exhibit (Street) Level outside Hall D in the Indiana Convention Center (See Floor Plan/Map). Pick up your badge and Sunday Social Reception tickets at the Registration desk before 8:00 pm on Sunday. The Sunday Social starts at 6:30 pm in the Convention Center's Sagamore Ballroom.

A quantitative and automated phase analysis of dual-phase (DP) steel using electron backscatter diffraction (EBSD) was attempted. A ferrite–martensite DP microstructure was produced by intercritical annealing and quenching. An EBSD map of the microstructure was obtained and post-processed for phase discrimination. Band slope (BS), which was a measure of pattern quality, exhibited much stronger phase contrast than another conventional one, band contrast. Owing to high sensitivity to lattice defect and little orientation dependence, BS provided handiness in finding a threshold for phase discrimination. Its grain average gave a superior result on the discrimination and volume fraction measurement of the constituent phases in the DP steel.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

In order to enhance micro-computer tomography (micro-CT) imaging of corrosion casts of fine vasculature, metals can be added to the casting resin before perfusion. However, perfused metals lead to vasoconstriction or vessel damage resulting in nonphysiologic vascular casts. A novel method for coating methyl methacrylate vascular casts with osmium tetroxide has been developed in order to increase micro-CT contrast without affecting the vascular structure. This technique was verified using corrosion casts of the lung vasculature of New Zealand white rabbits. Osmium tetroxide coating of methyl methacrylate vascular corrosion casts resulted in an increase in overall sample contrast that translated into an increase in the resolution of the vasculature. This method can therefore lead to increased resolution in the characterization of fine vascular structures.

This year's social event in the grand Indiana Convention Center Sagamore Ballroom will let you maximize your social time with your friends and colleagues without having to worry about the weather or get on a bus. The Sunday Social Event is a great opportunity to catch up with all your friends and colleagues, make some new ones, and enjoy some wine or local beer and a delicious supper buffet. It's all under one roof!

In ferritic stainless steel (FSS), undesirable surface defects of ridging appear during deep drawing. The formation of these defects is attributed to the inhomogeneous distribution of orientations of individual grains. In the present work, a new electron backscattered diffraction R(α)-value map was introduced, and the dependence of the tensile directions on the formation of ridging in an FSS sheet was discussed using this map. The results showed that large grain colonies in the R(α)-value maps lead to the formation of severe ridging in an FSS sheet.

It is important to know the detailed DNA structure on carbonaceous surfaces for further application of DNA-functionalized carbonaceous materials in diverse research areas. In this study, the topographic and structural characteristics of the separated single DNA molecules and their assembly on highly oriented pyrolytic graphite (HOPG) surfaces have been investigated by atomic force microscopy (AFM). AFM results indicate that both circular and linear DNA molecules tend to form hexagonal patterns along with some unusual structures that include node, protrusion, cruciform, parallel single-stranded DNA (ssDNA), and compact zigzag. Furthermore, parallel ssDNA patterns and their crossed structures have been obtained under high-temperature conditions. Our AFM results reveal that a bare HOPG surface can induce DNA molecules to form various unusual structures. This finding is helpful for understanding the adsorption behavior of DNA on other carbonaceous surfaces such as carbon nanotubes and graphene. In addition, the hexagonal DNA patterns in this study are similar to those formed on the alkylamine-modified HOPG surface, which implies that a bare HOPG, without any chemical modification, has a strong ability to align biomolecules. This study could expand our knowledge of the diversities of DNA structures and the aligning ability of carbonaceous surfaces.

Selective hydrogenation is an important process in petrochemistry to purify feedstock for polymer synthesis. For this process, catalysts containing metallic palladium deposited with an eggshell distribution on porous alumina are usually employed. For this kind of catalyst, the activity is known to be in close relation with the thickness of the palladium crust. As palladium oxide is brown and alumina is white, the palladium distribution in a catalyst bead before the reduction step can be characterized by optical microscopy. We propose an original and automatic procedure of optical image analysis to obtain a fast and robust method to measure the mean crust thickness of a catalyst batch and the corresponding standard deviation. The approach is validated by two different methods. First, we compared the crust thickness with those obtained by electron probe microanalysis. Then, catalytic tests of four samples with varying palladium crust thicknesses were performed and confirmed the expected correlation between activity and crust thickness measured by optical microscopy coupled with image analysis.

Nanoparticle tracking analysis (NTA) was first applied to biologically active nanocomplexes to obtain concurrent information on their size, state of aggregation, concentration, and antigenic specificity in liquid. The subject of the NTA was an immunogenic complex (a candidate nanovaccine) comprised of spherical particles (SPs) generated by thermal remodeling of the tobacco mosaic virus and Rubella virus tetraepitopes exposed on the surface of SP.

Major Society Awards for MSA and MAS, along with other awards, will be presented at the Plenary Session immediately following the Keynote Symposium (Monday morning). IMS Awards are presented at the IMS Awards Banquet on Wednesday evening (ticket purchase required). For detailed listings of all awards, criteria, and award winners, please see individual Society information in the Meeting and Program Guide.

A long time objective of ophthalmologists is to diagnose corneal cells dysfunction prior to its pathologic expression. With this motivation, we are currently developing a new instrument for in vivo metabolic imaging of corneal tissues.

Metabolic alterations are known to be the first sign of several corneal pathologies and can be assessed through non-invasive monitoring of metabolic co-factors flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). The quantification of the relative proportions between free and protein-bound NADH and FAD can be achieved using fluorescence lifetime-resolved methods. This approach has already been applied in age-related macular degeneration, diabetic retinopathy and epithelial cancer.

FAD and NADH imaging can be performed by one-photon excitation (1PE) and two-photon excitation (2PE) techniques. The latest has the advantage of allowing simultaneous excitation of both metabolic co-factors. However, there are still safety concerns when considering in vivo ocular studies in humans using 2PE.

Due to these concerns we used, as a first approach, a 1PE system for evaluating the feasibility of corneal FAD imaging. The use of FAD has advantages over NADH. It can be excited over longer excitation wavelengths, is more resistant to photo-bleaching and is located exclusively in the mitochondrial space.

A PicoQuant MicroTime 100 (PicoQuant GmbH, Berlin, Germany) coupled to an Olympus BX51 Microscope (Olympus Corporation, Tokyo, Japan) was used to monitor FAD autofluorescence. The instrument uses a 440 nm pulsed diode laser (330 ps) running at a pulse rate of 40 MHz. The instrument was modified by us to allow the acquisition of both fluorescence lifetime and reflectance images and to enhance scattered light rejection.

Intensity decay curves were processed with SymPhoTime v5.3 Software (PicoQuant GmbH, Berlin, Germany). The fluorescence decay times were obtained after applying a non-linear least square fit to the decay data and the goodness of fit was evaluated by the analysis of the residuals and the chi-squared (χ2).

We have acquired fluorescence lifetime images of ex vivo healthy Wistar rat corneas (Fig.1) using two different instrument setups: 1- using the emission filters provided by the manufacturer; 2- placing extra emission filters to fully reject the scattered excitation light. In both setups, FAD fluorescence data presented a bi-exponential decay with a short (protein-bound FAD) and a longer (free FAD) lifetime component.

While both setups provide FAD fluorescence decays, only the second retrieves valid metabolic information. We obtained two lifetime components, one of 0.118 (0.028) ns and another of 2.11 (0.16) ns, with a relative contributions of 39.4 (2.2) and 60.6 (2.2), respectively. These values are in accordance with the literature.

Corneal layer discrimination is possible based on morphologic characteristics. However, the fluorescence lifetime images do not provide morphological detail (Fig.1), possibly because FAD is only present in the mitochondria. These organelles are small and tend to accumulate around the nuclei.

So, we modified the instrument’s optical setup to allow the acquisition of both fluorescence lifetime images and reflectance images. Figure 2 shows an example of the corneal epithelial layer.

The image resolution and depth penetration are still not ideal. Since the assessment of corneal endothelial layer metabolic function is also within our goals, we are currently implementing further modifications to improve both the instrument’s resolution and depth penetration.

The characterization of FAD fluorescence lifetime in unhealthy corneas is important to detect corneal dysfunctions prior to its pathologic expression. Therefore, we intend to study metabolic altered Wistar rat corneas. The alterations will be induced by potassium cyanide, which is a reversible inhibitor of the fourth complex of the mitochondrial electron transport chain.

Financial support received from the Fundação para a Ciência e a Tecnologia under the research projects PTDC/SAU-BEB/104183/2008 and PTDC/SAU-ENB/122128/2010.

Radiation damage is often an issue during high-resolution imaging, making low-dose focusing and stigmation essential, in particular when no part of the sample can be “sacrificed” for this. An example is serial block-face electron microscopy, where the imaging resolution must be kept optimal during automated acquisition that can last months. Here, we present an algorithm, which we call “Maximum-A-Posteriori Focusing and Stigmation (MAPFoSt),” that was designed to make optimal use of the available signal. We show that MAPFoSt outperforms the built-in focusing algorithm of a commercial scanning electron microscope even at a tenfold reduced total dose. MAPFoSt estimates multiple aberration modes (focus and the two astigmatism coefficients) using just two test images taken at different focus settings. Using an incident electron dose density of 2,500 electrons/pixel and a signal-to-noise ratio of about one, all three coefficients could be estimated to within <7% of the depth of focus, using 19 detected secondary electrons per pixel. A generalization to higher-order aberrations and to other forms of imaging in both two and three dimensions appears possible.

Azelaic acid (AzA) is used in the treatment of acne. However, side effects and low compliance have been associated with several topical treatments with AzA. Nanotechnology presents a strategy that can overcome these problems. Polymeric nanoparticles can control drug release and targeting and reduce local drug toxicity. The aim of this study was to produce and evaluate an innovative topical treatment for acne with AzA-loaded poly-dl-lactide/glycolide copolymer nanoparticles. A soft white powder of nanoparticles was prepared. The mean size of loaded nanoparticles was <400 nm and zeta potential was negative. Spherical nanoparticles were observed by scanning electron microscopy. Encapsulation efficiency was around 80% and a strong interaction between the polymer and the drug was confirmed by differential scanning calorimetric analysis. In vitro drug release studies suggested a controlled and pulsatile release profile. System efficacy tests suggested similar results between the loaded nanoparticles and the nonencapsulated drug against the most common bacteria associated with acne. Cytotoxicity of AzA-loaded nanoparticles was concentration dependent, although not pronounced. The occluded patch test seemed to indicate that the formulation excipients were safe and thus AzA-loaded nanoparticles appear to be an efficient and safe treatment for acne.

A Monte Carlo simulation was used to determine geometry correction factors that increase accuracy of quantitative X-ray microanalysis of laterally semithick biological materials. A model composed of cellulose with homogeneously distributed biological elements and lateral dimensions between 0.5–25 μm was chosen. The specimen was exposed to 5, 10, and 15 keV electrons, the net intensities of characteristic X-rays registered for the elements, and presented as a function of the lateral dimensions of the model. This showed the double decay exponential function fitted the distribution of X-ray intensities in relation to the model size. The applicability of the function as a correction method was successfully tested for 30 specimens with varying composition and dimensions. The value of relative error decreased from ±60% to ±5% when the correction was applied. Moreover, the minimal lateral size of the material was defined, below which the correction is not required. The simulation also revealed that the difference of the weighted sum of Z2/A between the unknown and the standard could reach 25% without significant influence on the quantitative results. The correction method could be helpful for accurate assessment of elemental composition in biological or organic matrices, when their lateral dimensions are smaller than the distribution range.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Recent studies have shown that ghrelin increases pancreatic exocrine secretion. However, the potential effects of ghrelin on the morphology of exocrine pancreas (EP) remain unknown. In this work, using fractal analysis, we demonstrate that centrally administered ghrelin increases structural complexity and tissue disorder in rat EP. The study was carried out on a total of 40 male Wistar rats divided into four groups (n = 10): ghrelin-treated animals (average age, 1.5 months), ghrelin-treated animals (8.5 months), and controls (1.5 and 8.5 months). The pancreas tissue sections were stained with hematoxylin/eosin and visualized by light microscopy. For each animal, the average values of tissue fractal dimension, lacunarity, as well as parameters of co-occurrence matrix texture, were determined using tissue digital micrographs. The results indicate that ghrelin administration increases EP fractal dimension and textural entropy, and decreases lacunarity, regardless of the age. To our knowledge, this is the first study to investigate the effects of ghrelin on the morphological properties of pancreatic tissue, and also the first to apply fractal and textural analysis methods in quantification of EP tissue architecture.

The urinary bladder is a unique organ in that its normal function is storage and release of urine, and vasculature in its wall exhibits specialized features designed to accommodate changes in pressure with emptying and filling. Although we have previously described the fine details of the microvasculature of the urinary bladder of the rabbit and dog, information on the fine details of the microvasculature of the mouse bladder were deemed to be of value because of the increasing use of this species in developing genetic models for studying human disorders. The present study shows that many of the special features of the microvasculature of the mouse urinary bladder are similar to those described in the rabbit and dog, including vessel coiling, abundant collateral circulation, arterial sphincters, and a dense mucosal capillary plexus.

The therapeutic usefulness of anticancer agents relies on their ability to kill cancer cells while sparing normal cells and tissues. Ruthenium complexes hold great potential as suitable alternative drugs to the current cisplatin in the treatment of cancer. Our approach in this field has been the study of organoruthenium complexes, [RuII(η5-Cp)] family, namely [RuII(η5-Cp)(bipy)(PPh3)]+ (PPh3 = triphenylphosphine; bipy = 2,2’-bipyridine), TM34. This compound was tested for antitumor activity against tumor cells representative of human cancer diseases. A large spectrum of activity was found, largely surpassing cisplatin in efficacy (Fig 1).

How a drug distributes and localizes within cells is of fundamental importance as the drug must concentrate in the compartment that houses its target(s). The importance of cellular enzymes on the effects of anticancer drugs for which multiple biological pathways have been proposed led us to address the involvement of the lysosomes and lysosomal enzymes (AcPases) in the mechanism of cell death.

The cytotoxicity of TM34 was tested using the MTT assay. TM34 cellular distribution was measured by ICP-MS. The effect of TM34 on the activity of acid phosphatase (AcPase) was evaluated by the hydrolysis of p-nitrophenyl phosphate to p-nitrophenol. AcPase localization was investigated by TEM using the Record and Griffing (1988) cerium-based method.

Results showed that the sensitivity of AcPase assay is higher in comparison to cell proliferation assays based on the reduction of tetrazolium salts (MTT) (Fig 2). From TEM images AcPase reaction product appears as dark electron dense deposits in the lysosomes and dictiosomes. After 3h treatment TM34 provokes disruption and vesiculation of the Golgi apparatus, although some reaction product is present in the lysosomes (Fig 2).

The lysosome could be considered a possible target for TM34. Morphological evidence was observed for the participation of the Golgi apparatus in the vesiculation induced by the compound.

Supported by the Fundação para a Ciência e Tecnologia (FCT): PTDC/QUI-QUI/101187/2008.