Arterial physiology relies on a delicate three-dimensional (3D) organization of cells and extracellular matrix, which is remarkably altered by vascular diseases like abdominal aortic aneurysms (AAA). The ability to explore the micro-histology of the aorta wall is important in the study of vascular pathologies and in the development of vascular constitutive models, i.e., mathematical descriptions of biomechanical properties of the wall. The present study reports and validates a fast image processing sequence capable of quantifying collagen fiber organization from histological stains. Powering and re-normalizing the histogram of the classical fast Fourier transformation (FFT) is a key step in the proposed analysis sequence. This modification introduces a powering parameter w, which was calibrated to best fit the reference data obtained using classical FFT and polarized light microscopy (PLM) of stained histological slices of AAA wall samples. The values of w = 3 and 7 give the best correlation (Pearson's correlation coefficient larger than 0.7, R2 about 0.7) with the classical FFT approach and PLM measurements. A fast and operator independent method to identify collagen organization in the arterial wall was developed and validated. This overcomes severe limitations of currently applied methods like PLM to identify collagen organization in the arterial wall.

Lymphatic stomata are small openings of lymphatic capillaries on the surface of the mesothelium that lines the serous cavity and have the function of active absorption. They play an important role in physiological and pathological conditions. The cavity of the tunica vaginalis is a typical serous cavity of the testis, but the lymphatic stomata of the tunica vaginalis of humans have never been reported. Here, we studied their ultrastructure by scanning and transmission electron microscopy. The submesothelial connective tissue with foramina was investigated after the mesothelial cells were digested using NaOH solution. We found the lymphatic stomata in cuboidal mesothelial cell regions of the parietal layer of the tunica vaginalis of humans with a diameter of about 1–2 μm. Sometimes, closed lymphatic stomata could be observed. Our study is the first to report the existence of lymphatic stomata of the tunica vaginalis of humans. We found that the tunica vaginalis cavity is connected with the lymphatic system through the stomata, which might provide a morphological basis for the drainage of hydrocele and tumor metastasis of the tunica vaginalis of humans.

In this talk the utility of atomic force microscopy (AFM) for research into infectious parasites will be discussed. AFM has grown from relatively recent beginnings to become an extremely powerful technique in the life sciences, coupling high resolution imaging with a range of non-imaging experiments. Importantly, these experiments can be performed in situ, even on individual molecules or on live cells.

The two examples discussed relate to the important diseases leishmaniasis and malaria. Leishmaniasis is a disease caused by the protozoan parasite of the Leishmania genera, and causes approximately 60,000 deaths per year. Despite the high death toll, the disease has been the subject of relatively little research and little treatment is available, probably because the most severe cases are confined to developing nations. The most severe form, visceral leishmaniasis is caused by the species known as Leishmania infantum (syn. L. chagasi). A promising new anti-leishmania drug, DS01 has been recently isolated from amphibian secretions and can kill L. infantum in low concentrations. We were able to culture and prepare for microscopy L. infantum promastigotes for the first time, as well as to study the effects of DS01 on cell morphology and membrane integrity. The results from both AFM and SEM are highly complementary and illustrate the possibility of membrane-focussed activity as well as the possibility of attack on the flagella (figure 1).

Malaria is one of the most deadly diseases in the world, killing more than 600,000 people per year, mostly in low-income countries. It is caused by Plasmodium parasites, and the most commonly studied stage is that in which the parasite invades the blood. Prior to blood invasion, the parasites infect hepatocytes in the liver, with formation of a parasitophorous vacuole, where they develop into exoerythrocytic forms and multiply to generate thousands of merozoites, later released into the bloodstream and causing disease. However, infection of liver cells, which is clinically silent, is required for disease progression. We studied infection of liver cells by Plasmodium using combined epifluorescence and atomic force microscopy. We observed significant changes in cell morphology as infection progressed (figure 2). Furthermore we made nanoindentation measurements with the AFM, to determine cellular stiffness. We observed stiffening of the cells after 48 hours of infection compared to uninfected cells. This was a cellular response to the Plasmodium infection, rather than a result of the stiffness of the invading parasites themselves. This stiffening may be caused by reinforcement of cytoskeletal structures, and we believe this may reflect a self-defence mechanism by the cell itself.

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.

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

Here we present a novel laboratory-based cryogenic soft X-ray microscope for whole cell tomography of frozen hydrated samples. We demonstrate the capabilities of this compact cryogenic microscope by visualizing internal subcellular structures of Saccharomyces cerevisiae cells. The microscope is shown to achieve better than 50 nm half-pitch spatial resolution with a Siemens star test sample. For whole biological cells, the microscope can image specimens up to 5 μm thick. Structures as small as 90 nm can be detected in tomographic reconstructions following a low cumulative radiation dose of only 7.2 MGy. Furthermore, the design of the specimen chamber utilizes a standard sample support that permits multimodal correlative imaging of the exact same unstained yeast cell via cryo-fluorescence light microscopy, cryo-soft X-ray microscopy, and cryo-transmission electron microscopy. This completely laboratory-based cryogenic soft X-ray microscope will enable greater access to three-dimensional ultrastructure determination of biological whole cells without chemical fixation or physical sectioning.

The present study investigates the effect of the solidification strategy for AA 6063 alloy on the surface appearance of anodized extrusions. The microstructure of the samples was analyzed using both light optical microscopy and scanning electron microscopy. Results show that if heavy segregation occurs from rapid solidification, coarse Mg2Si particles form, thus reducing the potential for precipitation strengthening by the finer β-Mg2Si developed in the solid state. Differentially-strained regions formed during hot extrusion induce differences in particle size for magnesium silicide (Mg2Si) precipitates. Anodizing generates surface roughness due to Mg2Si particle dissolution and AlFeSi decohesion, which is related to both particle size and deformation. During anodizing, an oxide layer forms on the surface of the extruded products, which can lead to streak formation, usually a subject of rejection due to unacceptable heterogeneous reflectivity.

ZK60 (Mg–Zn–Zr) alloys exhibited a variation in precipitates with aging, and their mechanical properties also changed. Microindentation tests were carried out on two types of ZK60 alloys of solid solution (T4) and peak aging (T6). Microstructure and texture evolution during indentation was investigated using electron backscatter diffraction. Twinning occurred near the indentation marks in most grains. It was found that tensile twinning was dominant, and two twin variants were usually observed. Texture and microstructure evolution by twinning and slip activation was further examined by uniaxial compression test with strain. The initial random orientation gradually changed into basal fibers with strain. Some grains with nonbasal orientations aligned with the loading direction easily underwent twinning followed by slip deformation. Other grains near basal orientations revealed only slip deformation.

Microscopy and Microanalysis would like to thank the following sponsors for their generous support of the M&M 2013. (Sponsors as of June 18, 2013).

Previous studies have described promising antitumor activity of an organometallic Ru(II) complex, η5-Cyclopentadienyl(2,2′-bipyridyl)(triphenylphosphane) Ruthenium(II) triflate ([(η5-C5H5)Ru(2,2′-bipyridyl)(PPh3)][CF3SO3]) herein designated as TM34. Its broad spectrum of activity against a panel of human tumor cell lines and high antiproliferative efficiency prompted us to focus on its mode of action. We present herein results obtained with two human tumor cell lines A2780 and MDAMB231 on the compound distribution within the cell, the mechanism of its activity, and its cellular targets. The prospective metallodrug TM34 revealed: (a) fast antiproliferative effects even at short incubation times for both cell lines; (b) preferential localization at the cell membrane and cytosol; (c) cellular activity by a temperature-dependent process, probably macropinocytosis; (d) inhibition of a lysosomal enzyme, acid phosphatase, in a dose-dependent mode; and (e) disruption and vesiculation of the Golgi apparatus, which suggest the involvement of the endosomal/lysosomal system in its mode of action. These results are essential to elucidate the basis for the cytotoxic activity and mechanism of action of this RuII(η5-cyclopentadienyl) complex.

A clinically compatible fluorescence lifetime imaging microscopy (FLIM) system was developed. The system was applied to intraoperative in vivo imaging of head and neck squamous cell carcinoma (HNSCC). The endoscopic FLIM prototype integrates a gated (down to 0.2 ns) intensifier imaging system and a fiber-bundle endoscope (0.5-mm-diameter, 10,000 fibers with a gradient index lens objective 0.5 NA, 4-mm field of view), which provides intraoperative access to the surgical field. Tissue autofluorescence was induced by a pulsed laser (337 nm, 700 ps pulse width) and collected in the 460 ± 25 nm spectral band. FLIM experiments were conducted at 26 anatomic sites in ten patients during head and neck cancer surgery. HNSCC exhibited a weaker florescence intensity (~50% less) when compared with healthy tissue and a shorter average lifetime (τHNSCC = 1.21 ± 0.04 ns) than the surrounding normal tissue (τN = 1.49 ± 0.06 ns). This work demonstrates the potential of FLIM for label-free head and neck tumor demarcation during intraoperative surgical procedures.

Quercus suber L. has been investigated with special attention to the stem bark and its cork formation layer, but excluding the roots. Roots are the location of infection by pathogens such as Phytophthora cinnamomi responsible for the tree's sudden death. It is widely accepted that suberin establishes boundaries within tissues, serves as a barrier against free water and ion passage, and works as a shield against pathogen attacks. We followed the suberization of young secondary roots of cork oak. The first suberin deposition detectable by transmission electron microscopy (TEM) and neutral red (NR) was in the endoderm Casparian strips. Casparian strips are not detected by Sudan red 7B and Fluorol yellow (FY) that specifically stain lamellae suberin. Reaction to Sudan was verified in the endodermis and later on in phellem cells that resulted from the phellogen. Under TEM, the Sudan and FY-stained cells showed clear suberin lamellae while the newer formed phellem cells displayed a distinct NR signal compared to the outermost phellem cells. We concluded that suberin chemical components are arranged differently in the cell wall according to the physiological role or maturation stage of a given tissue.

The toxicity of titanium dioxide nanoparticles (TIO2 NPs) and oxidative stress effects were studied in two freshwater fish species (Carassius auratus and Danio rerio) exposed for 21 days to different concentrations (0.01, 0.1, 1, 10, 100/mgL) of TiO2 NPs and to a control (tap water). Additional fish were transferred to clean water for 14 days to assess the ability to recover from exposure to TiO2 NPs. Activities of the enzyme glutathione-S-transferase (GST) and lipid peroxidation (LPO) (malondialdheyde) were measured as indicators of oxidative stress. Histological and ultra-structural changes in livers from bothspecies of fish were evaluated by light and electron microscopy. Results show a general GST activity increase according to TiO2 NPs concentrations, which is in agreement with data from LPO. After 21 days, GST activities decreased possibly caused by suppression of GST synthesis as a result of severe stress. Histological and ultra-structural analysis of livers from exposed fish show degeneration of the hepatic tissue and alterations in hepatocytes such as glycogen depletion and an increase in lipofucsin lysosome-like granules. After a depuration period a partial recovery for biochemical markers and cells was observed. The results suggest that TiO2 promotes alterations in hepatic tissues compatible with oxidative stress.

The Slc26 family proteins, with one possible exception, transport anions across membranes in a wide variety of tissues in vertebrates, invertebrates, and plants. Mutations in human members of the family are a significant cause of disease. Slc26 family proteins are thought to be oligomers, but their stoichiometry of association is in dispute. A recent study, using sequential bleaching of single fluorophore-coupled molecules in membrane fragments, demonstrated that mammalian Slc26a5 (prestin) is a tetramer. In this article, the stoichiometry of two nonmammalian prestins and three human SLC26 proteins has been analyzed by the same method, including the evolutionarily-distant SLC26A11. The analysis showed that tetramerization is common and likely to be ubiquitous among Slc26 proteins, at least in vertebrates. The implication of the findings is that tetramerization is present for functional reasons.

An analysis of the nucleation mechanism of pyramids formed in (100) silicon in Na2CO3/NaHCO3 solution has been carried out. This texturization process of silicon by means of Na2CO3/NaHCO3 solutions is of special interest because it can be applied to the silicon solar cell industry to texture solar cell surfaces to decrease the front reflection and enhance light trapping in the cells. For this purpose, two microscopy techniques—scanning electron microscopy and atomic force microscopy—have been used to study the different stages of pyramidal nucleation and formation. The different aspects and factors involved in the texturization process require different analysis conditions and microscopy resolution. Tracing the transformation of determined surface areas and structures has been achieved, contributing clarification of the mechanism of pyramid nucleation in Na2CO3/NaHCO3 solutions.

Environmental scanning electron microscopy has been extensively used for studying the wetting properties of different materials. For some types of investigation, however, the traditional ways of conducting in situ dynamic wetting experiments do not offer sufficient control over the wetting process. Here, we present a novel method for controlled wetting of materials in the environmental scanning electron microscope (ESEM). It offers improved control of the point of interaction between the water and the specimen and renders it more accessible for imaging. It also enables the study of water transport through a material by direct imaging. The method is based on the use of a piezo-driven nanomanipulator to bring a specimen in contact with a water reservoir in the ESEM chamber. The water reservoir is established by local condensation on a Peltier-cooled surface. A fixture was designed to make the experimental setup compatible with the standard Peltier cooling stage of the microscope. The developed technique was successfully applied to individual cellulose fibers, and the absorption and transport of water by individual cellulose fibers were imaged.

Candidatus Magnetoglobus multicellularis is an uncultured magnetotactic multicellular prokaryote composed of 17-40 Gram-negative cells that are capable of synthesizing organelles known as magnetosomes. The magnetosomes of Ca. M. multicellularis are composed of greigite and are organized in chains that are responsible for the microorganism's orientation along magnetic field lines. The characteristics of the microorganism, including its multicellular life cycle, magnetic field orientation, and swimming behavior, and the lack of viability of individual cells detached from the whole assembly, are considered strong evidence for the existence of a unique multicellular life cycle among prokaryotes. It has been proposed that the position of each cell within the aggregate is fundamental for the maintenance of its distinctive morphology and magnetic field orientation. However, the cellular organization of the whole organism has never been studied in detail. Here, we investigated the magnetosome organization within a cell, its distribution within the microorganism, and the intercellular relationships that might be responsible for maintaining the cells in the proper position within the microorganism, which is essential for determining the magnetic properties of Ca. M. multicellularis during its life cycle. The results indicate that cellular interactions are essential for the determination of individual cell shape and the magnetic properties of the organism and are likely directly associated with the morphological changes that occur during the multicellular life cycle of this species.

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

The coccolithophores are a marine phytoplankton group that can play an important role in selective uptake of Cadmium (Cd) in neritic environments. Under project Cd-ToxCoN (PTDC/MAR/102800/2008) studies were conducted to investigate the in vitro reaction of Emiliania huxleyi to Cd exposure, namely changes in the crystal lattice of (cocco)liths, the calcite elements of the exoskeleton (coccosphere).

Uni-algal cultures of E. huxleyi were obtained by multiple cell isolation from the same water sample collected from Portuguese offshore waters, during opportunity cruises (Portuguese Marine Navy). Several morphotypes of E. huxleyi are currently recognized. The identification of the morphotype of E. huxleyi from Portuguese coastal waters was done by morphological analysis of the exoskeleton using scanning electron microcopy (JEOL JSM-5200LV and FEG-SEM JEOL 7001F) and by DNA sequencing of tufA gene following.

Experiments were conducted in batch cultures grown in enriched sea-water medium (K/10) under constant environmental conditions (14h L: 10h D, 15ºC, 40 µmol photons m-2 s-1). At the exponential growth phase, 3 replicate E. huxleyi cultures were subject for 48h to Cd concentrations of 10 µgL-1 and 100 µgL-1, two and three orders of magnitude above average marine concentration values respectively. In each case three additional replicate cultures with no Cd added were used as control.

The effect of Cd was evaluated by measuring in vivo fluorescence (ratio of variable (Fv) to maximum (Fm) fluorescence) (Water PAM fluorometer – Walz) and by detailed morphometric analysis of coccospheres and liths performed on SEM micrographs. The parameters measured are those presented in Figure 1. Malformed, damaged or tilted liths were not measured.

Morphological observations of the coccosphere and liths and DNA sequencing allowed the identification of the isolated strains of E. huxleyi as morphotype A. Results on the Cd exposure experiments indicate that E. huxleyi type A seems to tolerate high concentrations of Cd. Despite the very high concentration of Cd tested no lethal limit was reached and Fv/Fm values recorded after 48h at 10 µgL-1 Cd (0.607±0.008) and 100 µgL-1 Cd (0.603±0.008), very similar to the values recorded in the control cultures (0.642±0.016 and 0.636±0.018, respectively) and before Cd addition (0.642±0.012 and 0.636±0.007, respectively).

Regarding the different morphometric parameters analysed on coccospheres and liths, no significant measurable effects were observed. However, we observed, in response to increasing Cd concentration, an increasing number of liths with fused or partially fused elements (Fig. 2) suggesting a higher calcification of liths. This interpretation is supported by the results of the analysis of coccosphere calcium content. Cultures exposed to Cd presented a higher Ca content compared to control cultures. The highest values were recorded in coccospheres submitted to 100 µg.L-1 of Cd representing a 65% increase in Ca content in comparison to control coccospheres.

E. huxleyi is ubiquitous in present day oceans and usually very tolerant to culture conditions and is thus frequently used as a model species in the study of coccolithophores. These same characteristics may also justify the observed high tolerance to Cd. Studies with other species are needed to clarify if the surprising resistance to Cd is unique to E. huxleyi or characteristic of other unicellular algae with calcium carbonate shells.

This work was funded by projects PEst-OE/Mar/UI0199/2011, Pest/OE/CTE/UI/0263 and PTDC/MAR/102800/2008

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