Beyond simple imaging with energetic beams – nanopatterning, correlative microscopy and statistical analysis of inclusions

Electron microscopy (EM) has advanced in an exponential way since the first transmission electron microscope (TEM) was built in the 1930’s. The urge to ‘see’ things is an essential part of human nature (talk of ‘seeing is believing’) and apart from scanning tunnel microscopes which give information about the surface, EM is the only imaging technology capable of really visualising atomic structures in depth down to single atoms. With the development of nanotechnology the demand to image and analyse small things has become even greater and electron microscopes have found their way from highly delicate and sophisticated research grade instruments to key-turn and even bench-top instruments for everyday use in every materials research lab on the planet. The semiconductor industry is as dependent on the use of EM as life sciences and pharmaceutical industry. With this generalisation of use for imaging, the need to deploy advanced uses of EM has become more and more apparent. The combination of several coinciding beams (electron, ion and even light) to create DualBeam or TripleBeam instruments for instance enhances the usefulness from pure imaging to manipulating on the nanoscale. And when it comes to the analytic power of EM with the many ways the highly energetic electrons and ions interact with the matter in the specimen there is a plethora of niches which evolved during the last two decades, specialising in every kind of analysis that can be thought of and combined with EM. In the course of this study the emphasis was placed on the application of these advanced analytical EM techniques in the context of multiscale and multimodal microscopy – multiscale meaning across length scales from micrometres or larger to nanometres, multimodal meaning numerous techniques applied to the same sample volume in a correlative manner. In order to demonstrate the breadth and potential of the multiscale and multimodal concept an integration of it was attempted in two areas: I) Biocompatible materials using polycrystalline stainless steel and II) Semiconductors using thin multiferroic films. I) The motivation to use stainless steel (316L medical grade) comes from the potential modulation of endothelial cell growth which can have a big impact on the improvement of cardio-vascular stents – which are mainly made of 316L – through nano-texturing of the stent surface by focused ion beam (FIB) lithography. Patterning with FIB has never been reported before in connection with stents and cell growth and in order to gain a better understanding of the beam-substrate interaction during patterning a correlative microscopy approach was used to illuminate the patterning process from many possible angles. Electron backscattering diffraction (EBSD) was used to analyse the crystallographic structure, FIB was used for the patterning and simultaneously visualising the crystal structure as part of the monitoring process, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were employed to analyse the topography and the final step being 3D visualisation through serial FIB/SEM sectioning. II) The motivation for the use of thin multiferroic films stems from the ever-growing demand for increased data storage at lesser and lesser energy consumption. The Aurivillius phase material used in this study has a high potential in this area. Yet it is necessary to show clearly that the film is really multiferroic and no second phase inclusions are present even at very low concentrations – ~0.1vol% could already be problematic. Thus, in this study a technique was developed to analyse ultra-low density inclusions in thin multiferroic films down to concentrations of 0.01%. The goal achieved was a complete structural and compositional analysis of the films which required identification of second phase inclusions (through elemental analysis EDX(Energy Dispersive X-ray)), localise them (employing 72 hour EDX mapping in the SEM), isolate them for the TEM (using FIB) and give an upper confidence limit of 99.5% to the influence of the inclusions on the magnetic behaviour of the main phase (statistical analysis).

Deformation of stem cell nuclei by nanotopographical cues.

Cells sense cues in their surrounding microenvironment. These cues are converted into intracellular signals and transduced to the nucleus in order for the cell to respond and adapt its function. Within the nucleus, structural changes occur that ultimately lead to changes in the gene expression. In this study, we explore the structural changes of the nucleus of human mesenchymal stem cells as an effect of topographical cues. We use a controlled nanotopography to drive shape changes to the cell nucleus, and measure the changes with both fluorescence microscopy and a novel light scattering technique. The nucleus changes shape dramatically in response to the nanotopography, and in a manner dependent on the mechanical properties of the substrate. The kinetics of the nuclear deformation follows an unexpected trajectory. As opposed to a gradual shape change in response to the topography, once the cytoskeleton attains an aligned and elongation morphology on the time scale of several hours, the nucleus changes shape rapidly and intensely.

3D refraction correction and extraction of clinical parameters from spectral domain optical coherence tomography of the cornea.

Capable of three-dimensional imaging of the cornea with micrometer-scale resolution, spectral domain-optical coherence tomography (SDOCT) offers potential advantages over Placido ring and Scheimpflug photography based systems for accurate extraction of quantitative keratometric parameters. In this work, an SDOCT scanning protocol and motion correction algorithm were implemented to minimize the effects of patient motion during data acquisition. Procedures are described for correction of image data artifacts resulting from 3D refraction of SDOCT light in the cornea and from non-idealities of the scanning system geometry performed as a pre-requisite for accurate parameter extraction. Zernike polynomial 3D reconstruction and a recursive half searching algorithm (RHSA) were implemented to extract clinical keratometric parameters including anterior and posterior radii of curvature, central cornea optical power, central corneal thickness, and thickness maps of the cornea. Accuracy and repeatability of the extracted parameters obtained using a commercial 859nm SDOCT retinal imaging system with a corneal adapter were assessed using a rigid gas permeable (RGP) contact lens as a phantom target. Extraction of these parameters was performed in vivo in 3 patients and compared to commercial Placido topography and Scheimpflug photography systems. The repeatability of SDOCT central corneal power measured in vivo was 0.18 Diopters, and the difference observed between the systems averaged 0.1 Diopters between SDOCT and Scheimpflug photography, and 0.6 Diopters between SDOCT and Placido topography.

Characterization of topographical effects on macrophage behavior in a foreign body response model.

Current strategies to limit macrophage adhesion, fusion and fibrous capsule formation in the foreign body response have focused on modulating material surface properties. We hypothesize that topography close to biological scale, in the micron and nanometric range, provides a passive approach without bioactive agents to modulate macrophage behavior. In our study, topography-induced changes in macrophage behavior was examined using parallel gratings (250 nm-2 mum line width) imprinted on poly(epsilon-caprolactone) (PCL), poly(lactic acid) (PLA) and poly(dimethyl siloxane) (PDMS). RAW 264.7 cell adhesion and elongation occurred maximally on 500 nm gratings compared to planar controls over 48 h. TNF-alpha and VEGF secretion levels by RAW 264.7 cells showed greatest sensitivity to topographical effects, with reduced levels observed on larger grating sizes at 48 h. In vivo studies at 21 days showed reduced macrophage adhesion density and degree of high cell fusion on 2 mum gratings compared to planar controls. It was concluded that topography affects macrophage behavior in the foreign body response on all polymer surfaces examined. Topography-induced changes, independent of surface chemistry, did not reveal distinctive patterns but do affect cell morphology and cytokine secretion in vitro, and cell adhesion in vivo particularly on larger size topography compared to planar controls.

Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells.

The growth of stem cells can be modulated by physical factors such as extracellular matrix nanotopography. We hypothesize that nanotopography modulates cell behavior by changing the integrin clustering and focal adhesion (FA) assembly, leading to changes in cytoskeletal organization and cell mechanical properties. Human mesenchymal stem cells (hMSCs) cultured on 350 nm gratings of tissue-culture polystyrene (TCPS) and polydimethylsiloxane (PDMS) showed decreased expression of integrin subunits alpha2, alpha , alpha V, beta2, beta 3 and beta 4 compared to the unpatterned controls. On gratings, the elongated hMSCs exhibited an aligned actin cytoskeleton, while on unpatterned controls, spreading cells showed a random but denser actin cytoskeleton network. Expression of cytoskeleton and FA components was also altered by the nanotopography as reflected in the mechanical properties measured by atomic force microscopy (AFM) indentation. On the rigid TCPS, hMSCs on gratings exhibited lower instantaneous and equilibrium Young's moduli and apparent viscosity. On the softer PDMS, the effects of nanotopography were not significant. However, hMSCs cultured on PDMS showed lower cell mechanical properties than those on TCPS, regardless of topography. These suggest that both nanotopography and substrate stiffness could be important in determining mechanical properties, while nanotopography may be more dominant in determining the organization of the cytoskeleton and FAs.

Dietary inference from upper and lower molar morphology in platyrrhine primates.

The correlation between diet and dental topography is of importance to paleontologists seeking to diagnose ecological adaptations in extinct taxa. Although the subject is well represented in the literature, few studies directly compare methods or evaluate dietary signals conveyed by both upper and lower molars. Here, we address this gap in our knowledge by comparing the efficacy of three measures of functional morphology for classifying an ecologically diverse sample of thirteen medium- to large-bodied platyrrhines by diet category (e.g., folivore, frugivore, hard object feeder). We used Shearing Quotient (SQ), an index derived from linear measurements of molar cutting edges and two indices of crown surface topography, Occlusal Relief (OR) and Relief Index (RFI). Using SQ, OR, and RFI, individuals were then classified by dietary category using Discriminate Function Analysis. Both upper and lower molar variables produce high classification rates in assigning individuals to diet categories, but lower molars are consistently more successful. SQs yield the highest classification rates. RFI and OR generally perform above chance. Upper molar RFI has a success rate below the level of chance. Adding molar length enhances the discriminatory power for all variables. We conclude that upper molar SQs are useful for dietary reconstruction, especially when combined with body size information. Additionally, we find that among our sample of platyrrhines, SQ remains the strongest predictor of diet, while RFI is less useful at signaling dietary differences in absence of body size information. The study demonstrates new ways for inferring the diets of extinct platyrrhine primates when both upper and lower molars are available, or, for taxa known only from upper molars. The techniques are useful in reconstructing diet in stem representatives of anthropoid clade, who share key aspects of molar morphology with extant platyrrhines.

Intelligent design assistant (IDA): a case base reasoning system for material and design

This paper describes the approach to the modelling of experiential knowledge in an industrial application of Case-Based Reasoning (CBR). The CBR involves retrieval techniques in conjunction with a relational database. The database is especially designed as a repository of experiential knowledge, and includes qualitative search indices. The system is intended to help design engineers and material engineers in the submarine cable industry. It consists of three parts: a materials database; a database of experiential knowledge; and a CBR system used to retrieve similar past designs based upon component and material qualitative descriptions. The system is currently undergoing user testing at the Alcatel Submarine Networks site in Greenwich.

Natural iron fertilization of the Atlantic sector of the Southern Ocean by continental shelf sources of the Antarctic Peninsula

In large parts of the Southern Ocean, primary production is limited due to shortage of iron (Fe). We measured vertical Fe profiles in the western Weddell Sea, Weddell-Scotia Confluence, and Antarctic Circumpolar Current (ACC), showing that Fe is derived from benthic Fe diffusion and sediment resuspension in areas characterized by high turbulence due to rugged bottom topography. Our data together with literature data reveal an exponential decrease of dissolved Fe (DFe) concentrations with increasing distance from the continental shelves of the Antarctic Peninsula and the western Weddell Sea. This decrease can be observed 3500 km eastward of the Antarctic Peninsula area, downstream the ACC. We estimated DFe summer fluxes into the upper mixed layer of the Atlantic sector of the Southern Ocean and found that horizontal advection dominates DFe supply, representing 54 ± 15% of the total flux, with significant vertical advection second most important at 29 ± 13%. Horizontal and vertical diffusion are weak with 1 ± 2% and 1 ± 1%, respectively. The atmospheric contribution is insignificant close to the Antarctic continent but increases to 15 ± 10% in the remotest waters (>1500 km offshore) of the ACC. Translating Southern Ocean carbon fixation by primary producers into biogenic Fe fixation shows a twofold excess of new DFe input close to the Antarctic continent and a one-third shortage in the open ocean. Fe recycling, with an estimated “fe” ratio of 0.59, is the likely pathway to balance new DFe supply and Fe fixation.

Basin scale distribution of zooplankton in the Ligurian Sea (north-western Mediterranean) in late autumn

Mesozooplankton biomass and abundance were evaluated in epipelagic waters at 59 stations covering the Italian sector of the Ligurian Sea (north-western Mediterranean) in December 1990. This region is characterised by a cyclonic circulation which encloses a central divergence zone and is associated with a main thermohaline front offshore the western Ligurian coast. At the end of autumn, mesozooplankton biomass (range: 0.80–4.24 mg DW m−3) and the abundance (range: 83.8–932 ind. m−3) were lower in the divergence zone. On the contrary, in the Ligurian frontal zone at the periphery of the divergence and on the eastern continental shelf the greatest values of biomass and abundance were recorded. Copepods and appendicularians dominated the mesozooplankton community, the main taxa being the copepods Clausocalanus spp. (46% of total zooplankton) and Oithona spp. (15%) and the appendicularian Fritillaria spp. (12%). Three hydrological sub-regions, i.e. the divergence, the eastern continental shelf and the periphery of the divergence, were characterised by different zooplankton communities and characteristic species. Environmental differences between the three zones were mainly related to changes in bottom topography, sea surface temperatures and quantity of particulate organic matter. Vertical mesozooplankton abundance and taxa distribution from the surface to 1,900 m depth were also examined in one station. The results showed that the bulk of the community was concentrated in the upper 200 m, small copepods being dominant particularly in the upper 50 m. The copepod community was more diversified in sub-superficial waters, with a maximum observed in the 200–400 m layer. The distributions of main zooplankton taxa described in epipelagic waters in the eastern Ligurian Sea in autumn were compared with their distribution at surface in the north-western Mediterranean obtained by sampling performed with the Continuous Plankton Recorder in 1997–1999. The analysis of the zooplankton community in CPR samples confirms the dominance of small copepods (Paracalanus spp., Clausocalanus spp., Oithona spp.) and appendicularians in the north-western Mediterranean in late autumn-winter and shows that their distribution is mainly related to the main mesoscale hydrographic features characterising this basin.

Biological Communities at Marine Shallow-Water Vent and Seep Sites

The review compiles, for the first time, data on the communities at 62 shallow-water hydrothermal vent and cold seep sites. ‘Shallow sites’ are defined as sites no deeper than 200 m. The communities at these sites are also compared with communities in reducing sediments at similar depths. Below 200 m, vent and seep obligate species tend to dominate the fauna living in areas where reducing fluids are released from the seabed. At the shallow sites, vent and seep obligate species of fauna are rare, only eight having being reported from shallow vents. No definite seep obligates have been found. Shallow vents and seeps are colonized by communities that consist of a subset of the background fauna, especially those species that are less sensitive to hydrogen sulphide toxicity. Conversely the zones directly surrounding shallow vent and seeps sites with varied topography, substrate type and food supply, often have a higher species diversity than the background area. The reasons for these differences are discussed.

Drifter observations in the summer time Bay of Biscay slope current

During the summer of 2012, 20 surface drifters drogued at 50 m depth were deployed on the continental slope to the north of the Bay of Biscay. Initially after release the drifters all crossed the slope, with 14 continuing equatorward, parallel to the slope following an absolute dynamic topography feature and 6 returning to the slope, in an eddy, visible in chlorophyll-a maps. Lagrangian statistics show an anisotropic flow field that becomes less tied to the absolute dynamic topography and increasingly dominated by diffusion and eddy processes. A weaker tie to the absolute dynamic topography allowed for total of 8 of the drifters crossed from the deep water onto the shelf, showing pathways for flow across the slope. A combination of drifter trajectories, absolute dynamic topography and chlorophyll-a concentration maps have been used to show that small anticyclonic eddies, tied to the complex slope topography provide a mechanism for on shelf transport. During the summer, the presence of these eddies can be seen in surface chlorophyll-a maps.

Drifter observations in the summer time Bay of Biscay slope current

During the summer of 2012, 20 surface drifters drogued at 50 m depth were deployed on the continental slope to the north of the Bay of Biscay. Initially after release the drifters all crossed the slope, with 14 continuing equatorward, parallel to the slope following an absolute dynamic topography feature and 6 returning to the slope, in an eddy, visible in chlorophyll-a maps. Lagrangian statistics show an anisotropic flow field that becomes less tied to the absolute dynamic topography and increasingly dominated by diffusion and eddy processes. A weaker tie to the absolute dynamic topography allowed for total of 8 of the drifters crossed from the deep water onto the shelf, showing pathways for flow across the slope. A combination of drifter trajectories, absolute dynamic topography and chlorophyll-a concentration maps have been used to show that small anticyclonic eddies, tied to the complex slope topography provide a mechanism for on shelf transport. During the summer, the presence of these eddies can be seen in surface chlorophyll-a maps.

Coastal upwelling and downwelling forcing of circulation in a semi-enclosed bay: Ria de Vigo

Semi-enclosed bays in upwelling regions are exposed to forcing related to winds, currents and buoyancy over the shelf. The influence of this external forcing is moderated by factors such as connectivity to the open ocean, shelter by surrounding topography, dimensions of the bay, and freshwater outflows. Such bays, preferred locations for ports, mariculture, marine industry, recreational activities and coastal settlement, present a range of characteristics, understanding of which is necessary to their rational management. Observations in such a semi-enclosed bay, the Ria de Vigo in Spain, are used to characterize the influence of upwelling and downwelling pulses on its circulation. In this location, near the northern limit of the Iberian upwelling system, upwelling events dominate during a short summer season and downwelling events the rest of the year. The ria response to the external forcing is central to nutrient supply and resultant plankton productivity that supports its high level of cultured mussel production. Intensive field studies in September 2006 and June 2007 captured a downwelling event and an upwelling event, respectively. Data from eight current profiler moorings and boat-based MiniBat/ADCP surveys provided an unprecedented quasi-synoptic view of the distribution of water masses and circulation patterns in any ria. In the outer ria, circulation was dominated by the introduction of wind-driven alongshore flow from the external continental shelf through the ria entrances and its interaction with the topography. In the middle ria, circulation was primarily related to the upwelling/downwelling cycle, with a cool, salty and dense lower layer penetrating to the inner ria during upwelling over the shelf. A warmer, lower salinity and less dense surface layer of coastal waters flowed inward during downwelling. Without external forcing, the inner ria responded primarily to tides and buoyancy changes related to land runoff. Under both upwelling and downwelling conditions, the flushing of the ria involved shelf responses to wind pulses. Their persistence for a few days was sufficient to allow waters from the continental shelf to penetrate the innermost ria. Longer term observations supported by numerical modeling are required to confirm the generality of such flushing events in the ria and determine their typical frequency, while comparative studies should explore how these scenarios fit into the range of conditions experienced in other semi-enclosed bays.

Sentinel-3 Mission Performance Centre: Ensuring a high-quality altimetric dataset

Sentinel-3A is scheduled for launch in Oct. 2015, with Sentinel-3B to follow 18 months later. Together these missions are to take oceanographic remote-sensing into a new operational realm. To achieve this a large number of processing, calibration and validation tasks have to be applied to their data in order to assess for quality, absolute bias, short-term changes and long-term drifts. ESA has funded the Sentinel-3 Mission Performance Centre (S3MPC) to carry out this evaluation on behalf of ESA and EUMETSAT. The S3MPC is run by a consortium led by ACRI [1] and this paper describes the work on the calibration/validation (cal/val) of the Surface Topography Mission (STM), which is co-ordinated by CLS and PML.