bluuebunny/crossref_metadata_2025 huggingface dataset:
Flow cytometry, and Transmission electron microscopy; two valuable tools used in an innovative approach to isolate and describe new microorganisms
Authors: Khalil Jacques Bou
Published: December 2016

Flow cytometry and transmission electron microscopy are two reliable techniques used for decades in microbiology. In parallel, amoebal pathogens like giant viruses, and chlamydiae are of a big interest for the scientific community. Their ubiquity and diversity made by them a hot spot in environmental microbiology. The isolation of these pathogens of protists is the key to understand their evolution, their respective biotopes, and their potential or hidden pathogenicity. The co‐culture is the routinely used tool to isolate these microorganisms. Here we report the association of flow cytometry and electron microscopy to the co‐culture in a high throughput method capable of describing new isolates. Flow cytometry is used to detect and sort lytic or non‐lytic amoebal pathogens and electron microscopy characterizes the developmental stages and phenotypic features of the new isolates. These improvements based on enrichment systems, targeted use of antibiotics and high‐throughput methods associating FACS to TEM which are highly sensible regarding the old techniques, brought extreme benefits to the giant viruses and chlamydiae study and research fields.
Characterisation of Trypanosoma spp. in
A
ustralian wildlife
Authors: Cooper Crystal , Thompson Andrew , Botero Adriana , Clode Peta
Published: December 2016

The genus
Trypanosoma
comprises numerous species of flagellated vector‐borne protozoa that parasitise the blood and tissues of vertebrates. They are ubiquitous in terms of their geographical distribution and host range. In mammals, most species of
Trypanosoma
have been described from wildlife yet apart from their taxonomy, we know very little about the host parasite relationship, particularly those species in Australia.

Most of what is known about their host parasite relationships, life history, and developmental biology has been obtained from studies on the two species that invade cells and infect humans ‐
T. brucei
and
T. cruzi
‐ species endemic in Africa and South America respectively, and which are major causes of disease and death in humans and domestic animals.
T. brucei
is the cause of sleeping sickness as a result of its association with the nervous system, whereas
T. cruzi
is the cause of Chagas disease that results in cardiac, neurological, and digestive disorders.

Importantly, an Australian species ‐
T. copemani
G2 ‐ has also been found to invade cells, thus demonstrating a pathogenic potential previously not associated with trypanosomes from Australia. Recently several new species of
Trypanosoma
in Australian marsupials (
T. copemani
(G1 & G2),
T. vergrandis
, and
T. noyesi
) have been characterised. These have varying affinities to
T. cruzi
, including surprisingly similar genetic relationships (
e.g
. close genetic link of
T. noyesi
and
T. cruzi
) and behavioural traits (
e.g
. cellular invasion by both
T. copemani
and
T. cruzi
). Such observations not only raise concerns about the impact of Australian trypanosomes on wildlife health and conservation but also in terms of biosecurity and human health given the potential for local transmission of imported cases of Chagas disease.

Here we present correlative data across a range of length scales demonstrating the ongoing characterisation of several
Trypanosoma
spp. from Australian wildlife. In particular we have used live cell imaging to show host cell‐pathogen interactions (Figure 1), scanning (SEM) (Figure 2) and transmission (TEM) (Figure 3) electron microscopy for structural analysis, and Slice and View
TM
focussed ion beam‐scanning electron microscopy (FIB‐SEM)(Figure 4) to begin to image key structural features (
e.g
. kinetoplast, flagellum) at high resolution in 3‐dimensions.

Together these data are i) providing a greater understanding of the pathogenic potential and host‐parasite relationships of trypanosomes in Australian marsupials; ii) allowing for identification of biosecurity issues relating to potential local hosts and transmission of exotic species; and iii) generating information about the role of trypanosomes as a potential cause of disease in threatened and endangered Australian marsupials.
A
NanoWorkshop
Toolkit for in situ Nanoassembly and Nanocharacterization
Authors: Jonathan Smith Andrew , Rummel Andreas , Schock Klaus , Kleindiek Stephan
Published: December 2016

Fitting a Focussed Ion Beam / Scanning Electron Microscope (FIB/SEM) with a micromanipulators as well as a range of plug‐in tools transforms the microscope from a device purely used for observation to a workstation where materials can be manipulated, assembled, characterized, etc. ‐ at the micron, sub‐micron, and nano scale.

Typical applications include harvesting, arranging, and mechanically testing nanowires, nano tubes, and CNTs. It is also often of great interest to characterize nanowires, nanotubes, CNTs, etc. electrically.

Electrical and mechanical tests as well as structural investigations on MEMS devices are also commonly performed tasks.

This work will present a number of different experiments performed inside SEM or FIB/SEM tools. Among these are pick and place operations on sub‐micron sized particles, mechanical testing of nanowires and CNTs as well as in situ thermal experiments.

One of the described experiments entails mounting a strand of CNTs to a force measurement cantilever inside an SEM and subsequently performing a tensile experiment on the strand of CNTs. The CNTs are mounted to the force measurement cantilever using a special vacuum compatible adhesive. The adhesive can be applied in situ using a fine tip on the end of a micromanipulator. The tip is dipped into a small droplet of the adhesive in order to wet it. Next, some adhesive is transferred to the force measurement cantilever. In an additional step, some adhesive is used to extract a strand of CNTs from a large bundle. The extracted CNTs are brought into contact to the wetted force measurement cantilever and the adhesive is cured using the electron beam. Finally, the a force measurement is performed revealing the CNTs tensile strength.
Precise atomic column position measurements using ISTEM
Authors: H W van den Bos Karel , F Krause Florian , Béché Armand , Verbeeck Johan , Rosenauer Andreas , Van Aert Sandra
Published: December 2016

Nowadays, high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) is one of the most popular materials' characterisation techniques because of its ability to provide direct structural images at the atomic resolution [1]. Recently, Rosenauer et al. proposed a new imaging technique called imaging STEM (ISTEM) combining the conventional TEM imaging with STEM illumination [2]. This new, spatially incoherent imaging mode is particularly interesting as it provides direct structural images and visualisation of light elements, while it is robust towards chromatic aberrations. In this work, the ISTEM and STEM precision with which atomic column positions can be measured in a quantitative manner are compared.

In HAADF STEM imaging, statistical parameter estimation theory is an excellent tool to quantitatively extract structure parameters [3]. Here, an empirical model is fitted to an experimental image by optimising a criterion of goodness of fit. In this model, the shape of an atomic column is described by a Gaussian peak. The parameters of this empirical model can be linked to the unknown structure parameters of the material under study. The precision with which those parameters can be measured is mainly determined by the presence of shot noise and scan noise in the images. While post‐processing techniques reduce both effects [4,5], scan noise errors and probe instabilities have no influence in the ISTEM imaging mode as images for all probe positions are integrated. Therefore, it is expected that without the use of post‐processing techniques, atomic column positions can be measured more precisely for ISTEM imaging as compared to STEM imaging.

This assumption is tested on experimental images of PbTiO
3
. The ISTEM image in Fig. 1a clearly resolves the light oxygen columns, while in the HAADF STEM image, Fig. 2a, these columns cannot be resolved. In order to extract the atomic column positions, Gaussian models are fitted to both images, shown in Figs. 1b and 2b. The precision with which individual column positions can be estimated is determined by calculating the standard deviation on the distance between the columns (Fig. 3). These results confirm that atomic column positions are measured more precise for ISTEM as compared to ADF STEM imaging. Furthermore, it demonstrates that the position of individual light atomic columns, like oxygen columns in the present example, can be estimated with a precision in the picometer range.

In conclusion, it is shown that ISTEM is a very promising technique for precise measurements of atomic column positions.
In situ observation of heat‐induced degradation of perovskite solar cells
Authors: Divitini Giorgio , Cacovich Stefania , Matteocci Fabio , Cina' Lucio , Carlo Aldo Di , Midgley Paul , Ducati Caterina
Published: December 2016

The use of perovskite materials (particularly methylammonium lead iodide) in solar cells has become very attractive due to the fast increase in reported power conversion efficiencies over the last few years, leading to values above 20%. While this value is competitive with established photovoltaic technologies, the stability of perovskite‐based solar cells is still insufficient for commercial applications. In particular, it is very well known that some components, including the perovskite layer and the hole transporter, can degrade when exposed to a combination of heat and moisture. In situ TEM is an ideal tool for investigating such degradation and understanding the phenomena underpinning it.

In this work [1,2] we prepare methylammonium lead iodide cells using different approaches from the literature (with the perovskite conversion carried out in single‐ and double‐step in glovebox, in air or in vacuum); we prepare TEM cross‐sectional samples using focused ion beam milling.

For each cell, we carry out scanning TEM imaging and EDX elemental mapping (shown in Figure 1) as they are heated in situ in the TEM. This is a procedure that requires careful control over the temperature and the electron dose. To that aim we exploit recent advances in TEM‐related technology, such as Silicon Drift Detectors (SDD) for EDX, which collect energy‐dispersed X‐ray spectra with a good yield, and stable MEMS heaters, enabling the temperature to be cycled quickly and reproducibly. Moreover, we employ multivariate analysis (principal component analysis, PCA) to increase the signal‐to‐noise ratio of the spectral maps.

Cross‐sectional views acquired after heating are reported in Figure 2. We do not observe changes in the morphology or the elemental distribution in the perovskite layer for heating up to 150°C for short times (employing a heating ramp with 30' steps every 25°C). Since the ex‐situ heating of the same samples above 90°C causes a significant decay in cell performance, we attribute such decay to the degradation of the charge transport properties of the hole transporter (spiro‐OMeTAD in this case). Increasing the temperature further, different decomposition patterns emerge for the perovskite layer. In samples that had not been exposed to air, elemental migration of lead and iodine results in the formation of aggregates, which EDX suggests might be PbI
2
, clustering on the FTO electrode. In the sample exposed to air, a different phenomenon occurs – instead of forming aggregates, the elemental species diffuse from the perovskite into the hole transporter. This is visible both as an increased contrast in the high‐angle annular dark field images (HAADF) and as features in the EDX spectra; we hypothesise that the trapped moisture within the cell might be hindering the formation of PbI
2
and make elemental diffusion more favourable.
Transmission electron microscopy of deformed Laves phase
NbFe2
Authors: Šlapáková Michaela , Voss Simon , Kumar Sharvan , Liebscher Christian , Stein Frank
Published: December 2016

Laves phases are the largest class of intermetallic phases, showing very high strength up to temperatures above 1000 °C, but being very brittle at room temperature. The mechanical behavior and the deformation mechanism of these phases is very much different from that of pure metals and is still not well understood [1, 2].

The unit layer of a Laves phase AB
2
does not consist of only one plane but a slab of four planes, each of which is composed of either only A‐ or B‐atoms. This package of four atomic planes can be sub‐divided in one single B‐atom layer and a triple layer of successive A‐, B‐ and A‐atom planes [3].

The present work summarizes results obtained from transmission electron microscopy on single‐phase NbFe
2
before and after compression tests at high temperatures. The material was produced by levitation melting and has hexagonal
C
14‐type structure (
hP
12,
P
6
3
mmc
). The compressive stress‐strain curves are characterized by a pronounced stress peak in the stress‐strain curves at lower temperatures (up to 1200 °C) and by steady state flow at higher temperatures (above 1200 °C) [4].

Undeformed NbFe
2
is almost free of dislocations. It is assumed that the lack of dislocations in the as‐cast condition leads to the pronounced yield stress maximum observed during compression testing at lower deformation temperatures.

After deformation at 1200 °C new dislocations are introduced into the material. Widely‐extended stacking faults on the basal plane dominate the microstructure. They are bounded either by partial dislocations or terminate at low angle grain boundaries (Fig. 1).

Material deformed at 1300 °C shows a high density of dislocations, which are split into pairs of partial dislocations that bound stacking faults on the basal plane (Fig. 2). The observation that most dislocations are split up into Shockley partial dislocations could indicate deformation by the synchroshear process [5], as the occurrence of such partial dislocations is essential for this mechanism. Synchroshear is based on the idea of a synchronous shear motion of two adjacent planes within the triple layer.

Dislocation networks (Fig. 3 and 4) indicate the activation of dislocation climb. The improvement of dislocation mobility at higher deformation temperatures leads to the absence of a pronounced stress maximum in the stress‐strain curves.

The observed perfect dislocations are of (0001) 1/3<11‐20> type, the dissociated Shockley partials are (0001) 1/3<10‐10> type. The main deformation mechanism in the NbFe
2
C
14 Laves phase is basal slip.
A Variable‐Temperature Continuous‐Flow Liquid‐Helium Cryostat Inside a (Scanning) Transmission Electron Microscope
Authors: Börrnert Felix , Horst Alexander , Krzyzowski Michael A. , Büchner Bernd
Published: December 2016

The progress in (scanning) transmission electron microscopy development had led to an unprecedented knowledge of the microscopic structure of functional materials at the atomic level. Additionally, although not widely used yet, electron holography is capable to map the electric and magnetic potential distributions at the sub‐nanometer scale. This opens a route to investigate the phase structures of electronic and magnetic phenomena in condensed matter at a microscopic level. Many of the most interesting solid state phenomena occur at low temperatures only. Nevertheless, low temperature studies inside a (scanning) transmission electron microscope ((S)TEM) are extremely challenging because of the much restricted size and accessibility of the sample space. Up to date, there are no cryo‐(S)TEMs or special sample holders that are capable to cool a sample controllably to any but its base temperature below room temperature.

Recently, we introduced a concept for a dedicated in‐situ (S)TEM for flexible multi‐stimuli experimental setups with the capabilities of holographic recording and scanning electron microscopy type imaging. A central part was a large sample chamber with multiple ports. With a prototype instrument, we demonstrated a maximum resolving power of about 1 nm in conventional imaging mode and substantially better than 5 nm in scanning mode while providing an effectively usable pole piece gap of 70 mm [1].

Here, we report about the state of the first major plug‐in fitted into the prototype in‐situ (S)TEM: A variable‐temperature liquid‐helium continuous‐flow cryostat for nanometer resolved imaging and diffraction at controlled temperatures between 10 K and 300 K. Arbitrary temperatures in the offered range can be installed and held stable by a heating in the sample mount with the help of a PID controller. The cryostat has two operation modes, one with two cooled radiation shields for temperatures below 10 K and one without the shields for free sample access from outside the cryostat at temperatures down to 20 K. Sample drift due to negative thermal expansion is reduced by a circular cooled sample mount and a flexible copper strand to the cold finger. The design of a continuous flow cryostat with a low consumption rate offers a long working time at low temperature while sucking helium from a 100 l vessel. Additionally, the cryostat offers four cooled terminals for fixed electrical contacts and is prepared for a future incorporation of two mobile electrical probes.

Examples of experiments now possible with this new setup are the mapping of the phase structure of different electronic and magnetic phenomena, like charge density waves and Skyrmions.
The measurement of off‐plane magnetic field through electron vortex beams
Authors: Grillo Vincenzo , Harvey Tyler , Pierce Jordan , Venturi Federico , Balboni Roberto , Gazzadi Gian Carlo , Frabboni Stefano , McMorran Benjamin , Boyd Robert , Karimi Ebrahim
Published: December 2016

It is well known that a magnetic field produces a phase change proportional to the in plane magnetic field B [1]. Alas the out of plane magnetic field is more elusive. The only measurement that so far seems to produce  any out of plane information is the magnetic dichroism [2] as it actually measures the out of plane magnetization.

Unfortunately magnetic dichroism experiment is still a complicated experiment that requires a considerable control of the condition and of the material; moreover the measures of the magnetization , often with the objective on, is not directly a measure of the magnetic field itself.

However while a plane wave along the optical axis has no significant interaction with a magnetic field along z , vortex beams  with winding number l do feel such field through a Larmor phase effect.

We will demonstrate here that through the use of large vortex beams [3][4][5] we are able, for the first time, to measure the out of plane magnetic field generated by a magnetic pillar of Co [6].

We placed the vortex around the pillar ensuring as much as possible an axial symmetry and observed the phase effect.  The result is in good agreement with the typical magnetization expected for the pillar with an average field at surface of about 2T.

Fig 1 illustrates the A and B field produced by a magnetic pillar. The actual magnetic pillar fabricated by EBID deposition is illustrated in fig 1b. Fig 2 depicts the vortex beam density with L=200 as imaged in its focal plane. Thanks to an interferometric approach we were able to measure the magnetic phase contribution and bind it to the magnetic field in proximity of the sample. The magnetic phase is visible in the inset of fig 2 showing approximate axial symmetry.

The technique has been carried on in Low Mag mode with the main objective  lens off but there are no principle limitation to the application in conventional (S) TEM mode.

Moreover this promises  to be one of the best recognition of the importance of vortex beams in microscopy for material science.
Ultra‐thin epitaxial selenide films: Structure and two‐dimensional properties
Authors: Bazioti Calliope , Dimitrakopulos George , Tsipas Polychronis , Xenogiannopoulou Evangelia , Dimoulas Athanasios , Komninou Philomela
Published: December 2016

Ultra‐thin films of selenide compound epilayers were deposited epitaxially by molecular beam epitaxy (MBE) on AlN(0001) / Si(111) templates and were studied structurally using high resolution transmission electron microscopy (HRTEM), image simulations, and geometrical phase analysis (GPA). The films comprised Bi
2
Se
3
, MoSe
2
, and HfSe
2
epilayers, as well as composite heterostructures of these materials. Wurtzite AlN is a wide band gap semiconductor that strongly favors an excellent interfacial quality with the selenides under the employed growth conditions contrary to direct deposition on silicon that leads to interfacial amorphization and extended defects in the film. The structural observations were combined with angle‐resolved photoelectron spectroscopy measurements.

Bi
2
Se
3
is a topological insulator (TI), and the deposited films exhibited a surface Dirac cone making them promising for novel spintronics and quantum computing applications. HRTEM, combined with GPA, showed an epitaxial well‐ordered interface with (0001) AlN. The interfacial periodicity was manifested by a 3:4 plane matching. No interdiffusion or chemical reaction was observed at the interface. High quality and large scale 2D films with thicknesses of 3 and 5 quintuple layers (QLs) were deposited [1]. The films contained only vertical and in‐plane 180° rotational domain boundaries, as shown in Fig. 1.

In addition, high quality films of a few monolayers (MLs) of MoSe
2
and HfSe
2
compound semiconductors were deposited in extended scale by MBE directly on AlN(0001), showing promise for nanoelectronic device applications mediated by the van der Waals bonding [2]. In an alternative approach, Bi
2
Se
3
was employed as buffer layer in order to maintain low growth temperatures that favor large scale manufacture. Furthermore, various combinations of alternating selenide layers were achieved, signifying a versatility towards advanced nanodevice possibilities and prospects for combined 2D semiconductor/TI applications. Cross sectional HRTEM, in conjunction with image simulations elucidated the interfaces between dissimilar materials. Variations in lattice spacings were obtained by GPA. Such a heterostructure is illustrated in Fig. 2.
HRSTEM
and
EELS
study of
NMC
aging mechanisms for renewable energy application
Authors: Davoisne Carine , Hassine Mohamed Ben , Tessier Cécile , Drazic Goran , Dupont Loic
Published: December 2016

With the constant development of renewable energy, the associated energy storage devices need to improve their lifetime (15‐20 years), security and be of low cost. Among the possible materials for such applications, Li[Ni
1‐x‐y
Mn
x
Co
y
]O
2
(NMC) is a good candidate due to its lower cost and better thermal stability than NCA for example. Obtaining a better understanding of the aging mechanisms of NMC is then critical to create reliable prototypes. In this aim, NMC materials have been investigated after drastic cycling.

To investigate the microstructural and chemical evolutions of the NMC before and after cycling, we performed combined SAED, HRSTEM‐HAADF, EDS and EELS study using a TECNAI F20‐STWIN and a JEOL ARM 200 CF Cs‐corrected both equipped with an EDX and EELS. By compiling the different information, we were able to highlight several modifications:

a preferential dissolution
1
,
2
of first manganese then nickel with the cycling,

A preferential dissolution of the material along the lithium diffusion path resulting in the creation of cracks,

Microstructural evolution from a layered structure to a spinel
3
and a rock salt one (see figure 1),

Changes in the oxidation state of the manganese and cobalt (see figure 2).

These evolutions show a progressive degradation of the NMC during cycling until the apparition of phase (rock salt type) with limited cycling performance. Knowing this extreme behavior will help to adapt new generation (electrode formulation, …) of prototype cells.
Radial distribution function imaging by STEM diffraction: a method development in resolving the mysteries of amorphous materials
Authors: Mu Xiaoke , Wang Di , Feng Tao , Kübel Christian
Published: December 2016

Interpreting atomic structure of amorphous materials have attracted attentions for a century and, in recent years, especially heterogeneous nanoglasses have fueled the interest because of their unusual structure and properties [1]. However, only few experimental means offer a way to characterize the disordered structures. Atomic radial distribution function (RDF) is one of the important tools for the goal, which was first obtained from X‐ray diffraction data of organic solids [2], and afterward extended to electron diffraction for metallic glasses [3]. RDF describes the probability to find certain atomic pairs as a function of the pair separation and consequently, provides structural information in the short‐ and medium‐range. However, the traditional diffraction experiments only average large sample areas and lack spatial resolution especially at nanometer scale. Plenty information is hidden in the averaged signal.

In this work, we demonstrate a newly developed method, RDF‐imaging, combining diffraction imaging in scanning transmission electron microscopy (STEM) mode [4] with RDF analysis and spectral‐imaging analysis (e.g. multiple linear least square (MLLS) fitting) to achieve structural mapping of heterogeneous amorphous materials with 1 nm resolution. Figure 1 schematically shows the procedure for data acquisition and RDF calculation: a 4‐dimensional (D) diffraction‐image is acquired by recording diffraction patterns in STEM mode with quasi parallel nano‐beam configuration (0.8 mrad convergence angle) and 1 nm spot size. RDFs are calculated from the diffraction patterns according to description in [3][5]. A 3D data cube of RDFs (RDF‐cube) is constructed by relating pixel positions of the initial diffraction‐image to the calculated RDFs. The RDF‐cube can then be analyzed by MLLS fitting of reference spectra taking experimentally from pure phase or numerically from matrix decomposition by multivariate statistical analysis.

The method is extremely sensitive to atomic packing variation. Figure 2 shows an application to amorphous ZrO
2
(a‐ZrO
2
) and a‐ZrFe multilayers. Not surprisingly, both the a‐ZrO
2
and the a‐ZrFe phases are unambiguously distinguished by RDF‐image (figure 2d,e), but also an interface layer between ZrO
2
and ZrFe (figure 2c) is detected, which could not be identified in STEM‐EELS and EDX maps. The atomic structure (figure 2a, red dashed‐line) of the interfacial layers possesses the same atomic packing as that of the a‐ZrO
2
phase (figure 2a, blue line) but with a 0.04 Å shrinkage of the average bonding distance.  The shift in bonding distance could be introduced by Fe replacing Zr atoms in the ZrO
2
clusters due to diffusion of Fe atoms from the a‐ZrFe into the ZrO
2
layers.
Characterizing the protein corona of polystyrene nanoparticles
Authors: Kokkinopoulou Maria , Simon Johanna , Mailaender Volker , Lieberwirth Ingo , Landfester Katharina
Published: December 2016

Nanoparticles are of great interest for their use in biomedical applications like imaging and drug delivery. They are used as delivery vesicles to carry drugs to cells, while protecting them from degradation and allowing a targeted delivery. It is important to understand the interaction of nanoparticles with the biological systems, in order to make these processes more efficient. Upon injection of nanoparticles inside the blood, there is a competition of different biological molecules to adsorb on the surface of the nanoparticles. Numerous proteins are present in the plasma at high concentrations and interact with the nanomaterial surface forming a cloud of proteins known as the “protein corona”. The protein corona alters the size and composition of a nanomaterial, giving it an identity which is not the same as its synthetic identity [1]. The physical parameters of nanoparticles could affect the composition of protein corona, which is dynamic as the proteins are exchanging and the composition is evolving. This interface can be divided into the “hard” and the “soft” corona, depending on the binding strength and exchange rates of the proteins associated with the nanomaterial surface. The proteins with high binding affinities that are tightly bound to the nanoparticles form the “hard” protein corona and the ones that are loosely bound, have high exchange rates and can be easily replaced, form the “soft” corona. The cell or organ sees the nanoparticle‐protein complexes when interacting with a nanoparticle dispersed in a biological medium. That means that the corona defines the biological identity of nanoparticles and influences the cytotoxicity and endocytosis. Due to the complexity of the protein corona, it has been challenging to characterize. Approaching the project with microscopy techniques will answer critical questions about the structure of nanoparticles and their interactions with the biological systems, which can be used to make biomedical applications more efficient.
Amniotic Membrane Scaffold Enriched with Bladder Fibroblasts Promotes Establishment of Highly Differentiated Urothelium
Authors: Jerman Urška Dragin , Veranič Peter , Cirman Tina , Kreft Mateja Erdani
Published: December 2016

Introduction:
Human amniotic membrane (AM) is the innermost layer of placenta and is composed of three distinct strata, namely single epithelial layer, a thick basement membrane and avascular stroma. AM has been widely adopted as a biomaterial in tissue engineering and surgical reconstructions, since it promotes wound healing and epithelialization, has low immunogenicity, reduces inflammation and scarring, and was also shown to possess anti‐cancer properties (1). We have shown that AM promotes formation of highly differentiated urothelium, in particular when urothelial cells are cultured on AM stromal side (2). Although AM stroma scaffold allows establishment of differentiated urothelium, its orientation probably does not favour AM integration into surrounding tissue, if applied
in vivo
. Therefore, we established an
in vitro
AM model which would allow incorporation of fibroblasts into the AM stroma as well as promote urothelial differentiation.

Materials and Methods:
Porcine bladder fibroblasts were seeded onto porous membranes and were labelled with green lipophilic dye when subconfluent. Cryopreserved human AMs mounted into Scaffadex holders were de‐epithelized and laid onto cultures of fibroblasts in such a manner that fibroblasts were in close contact with the AM stromal layer. Fibroblasts were left to grow into AM stroma in medium adapted for fibroblasts for up to a week, when porcine urothelial cells were seeded on the opposite side of the AM scaffold i.e. on the AM basement membrane. The constructs were maintained for three additional weeks in the medium adapted for urothelial cells. As a control, urothelial cells seeded on the de‐epithelized AM scaffold without integrated fibroblasts were used to evaluate contribution of fibroblasts on urothelial differentiation. Growth of urothelial cells was monitored daily. Integration of fibroblasts and differentiation of urothelial cells was determined by electron microscopy, histological and immunofluorescence techniques.

Results:
Fibroblasts cultured in close contact with AM stroma in the medium adapted for fibroblasts integrated into AM stroma and remained viable after three additional weeks of maintenance in the medium for urothelial cells. Proliferation and differentiation of urothelial cells depended largely on the presence of fibroblasts. In comparison to the control, urothelial cells on fibroblast enriched AM scaffolds reached confluency faster, and more importantly, reached higher differentiation stage (Figure 1).

Discussion and Conclusion:
Our data demonstrate that bladder fibroblast enriched AM scaffold promotes urothelial differentiation
in vitro
. Besides being suitable
in vitro
research tool to study epithelial‐mesenchymal interactions, this model also indicates that de‐epithelized human AM could integrate into bladder lamina propria and could be as such potentially used as a graft for urothelial reconstruction.
HRTEM
and
STEM‐EELS
study of thin films
Pt‐CeOx
nanocatalysts for on‐chip fuel cell technology
Authors: Potin Valérie , Lavkova Jaroslava , Dubau Martin , Matolinova Iva , Matolin Vladimir
Published: December 2016

Platinum (Pt) is a versatile element in catalysis that efficiently mediates a multitude of chemical reactions. Unfortunately, Pt is a rare noble metal and its high price exceeding that of gold, limits large‐scale applications. Therefore, not surprisingly, reducing amount of Pt is the major driving force in catalysis research. There are two strategies to tackle this challenge: to replace noble metal by others, less expensive materials; and to use platinum as efficiently as possible. In this study, we handle both of them by growing extremely porous Pt‐CeO
2
structures prepared It was shown that sputtered thin cerium oxide films containing Pt, which had been deposited on the anode side of a fuel cell, exhibited a higher specific power compared to a conventional Pt−Ru catalyst [1]. Besides the large scale fuel cells, there is also an increasing interest in miniature fuel cells fabricated on silicon, which could be used as an on‐chip power supply for portable electronic devices.

In this study, nanometric Pt‐ceria thin films were characterized by TEM after elaboration by physical vapor deposition on various substrates (silicon, carbon foils, intermediate carbon films). The deposited layers exhibited different morphologies linked to the different substrates. It is shown that the roughness of the layers is dependent on the deposition conditions, the amount of deposited material and the type of the carbon substrate. The change of these parameters results in growth of flat, mushroom‐like or noodle‐like structures (Fig. 1). By the optimization and the suitable combination of materials we can tune the morphology of the catalysts. In addition to the substrate type, many effects as the formation of carbides or silicates at the interface, an interaction of ceria with platinum and the presence of the porosity influenced also the structure and the chemistry of the deposited layers [2,3].

In all samples, crystallites corresponding mainly to CeO
2
and to a less extent to CeC
2
crystallographic structures were observed (Fig. 1). STEM‐EELS measurements have been carried out on layers grown on silicon with and without intermediate carbon layer. Data analysis of the M
4,5
white lines of cerium have pointed out a variation of cerium oxidation state from Ce
4+
to a mixture of Ce
3+
and Ce
4+
depending of the localization of the measurement (Fig. 2).
In situ
TEM
study of nanoalloys in solution
Authors: De Clercq Astrid , Margeat Olivier , Henry Claude R , Giorgio Suzanne
Published: December 2016

1. Introduction

Different Pt‐Pd nanoalloys were prepared from organic precursors in solution. Their nucleation and growth in the liquid was in situ studied in a graphene oxide liquid cell by the direct reduction in the electron beam [1‐3].

Then, the morphological evolution of the nanoalloys under gas pressure was in situ studied in an environmental sample holder [4] by standard TEM.

2. Pt@Pd core shell NPs

Increasing amounts of Pt could be deposited on Pd nanocubes cubes by sequential reduction, with a resulting concave shape as seen in fig. 1.

Fig.2 is a set of images during the Pt growth around the same Pd cube, during 20 mn. The operating conditions (magnification 800 K, electron density 3. 10
5
A/m
2
) corresponds to an increasing electron dose. During this time, the variation of liquid quantity in the drop was not visible. From the set of images, it seems that the Pt layer has an homogeneous thickness during the growth, so that the final concave shape results from other steps in the preparation process.

Then, the morphology of Pt@Pd nanocubes was in situ observed during oxido reduction cycles in a few mbar of pure O
2
and H
2
.

Fig. 3 clearly shows that the Pd@Pt nanocubes have concave shapes in pure H
2
. In pure O
2,
they are much rounded at the corners and the (110) facets are extended compared to the same samples in H
2
, as the anisotropy ratio between the surface free energies of (001) and (110) faces, increases from 0.7 in H
2
to 0.8 in O
2
.

3. Properties

A maximal reactivity in gas has been found for an equivalent thickness of 0.4 atomic Pt layers on the Pd nanocubes. For this thickness the core‐shell particles are more active than pure Pd cubes or similar Pt cubes reported in literature. This behavior is explained by a decrease of the adsorption energy of molecules, due a compressive strain, induced by the misfit between the two metal bulk lattices and by a ligand effect due to the modification of the electronic structure of Pt atoms in contact with Pd atoms. A similar qualitative evolution as a function of thickness of the Pt layer was already observed in electrocatalysis and was also explained by the decrease of the strength of adsorbed species.
Correlative nonlinear optical microscopy and infrared nanoscopy reveals collagen degradation in altered parchments
Authors: Latour Gael , Robinet Laurianne , Dazzi Alexandre , Portier François , Deniset‐Besseau Ariane , Schanne‐Klein Marie‐Claire
Published: December 2016

Non‐invasive investigation techniques are strongly needed to avoid sampling during the examination of heritage artefacts. This study aims at developing specific and non‐destructive mapping of the degradation of parchment, which is mainly composed of dermal fibrillar collagen. The main issue is to characterize gelatinization, its ultimate and irreversible alteration corresponding to collagen denaturation to gelatin, both from the morphological and chemical point of view. To that end, we implement correlative imaging of parchment using nonlinear optical (NLO) microscopy and nanoscale infrared spectroscopy (nanoIR).

NLO microscopy, also called multiphoton microscopy, advantageously provides non‐invasive three‐dimensional (3D) multimodal imaging of scattering samples with micrometer‐scale resolution [1]. Among the collected signals, SHG signals are specific for dense non‐centrosymmetric materials. These signals therefore provide a unique structural probe of fibrillar collagen at the micrometer scale since the small signals from the collagen triple helices at molecular scale are amplified by constructive interferences at macromolecular scale due to the tight alignment of the collagen molecules to form collagen fibrils [2]. Nanoscale infrared spectroscopy is carried out thanks to an Atomic Force Microscope (AFM) coupled with an IR pulsed tunable laser [3]. This IR nanoscopy allows acquiring chemical mapping and local IR spectra to characterize and image samples at nanoscale.

Using this multiscale approach, key information about collagen and gelatin signatures is obtained in parchments and assessed by characterizing the denaturation of pure collagen reference samples. A new absorbing band is observed near the amide I band in the IR spectra, colocalized with the onset of fluorescence signals in NLO images. Meanwhile, a strong decrease is observed in Second Harmonic signals (see figure 1).

NLO microscopy therefore appears as a powerful tool to reveal collagen degradation in a non‐invasive way. It should provide a relevant method to assess or monitor the condition of collagen‐based materials in museum and archival collections and opens avenues for a broad range of applications regarding this widespread biological material.
Ultrafast nano‐fabrication and analysis using Xe plasma‐
FIB
‐
SEM
microscope and its applications for Cu milling using the Rocking‐stage
Authors: Benkouider Abdelmalek , Sharang Sharang , Hrnčíř Tomaš , Oboňa Jozef Vincenc , Jiruše Jaroslav , Principe Edward
Published: December 2016

Conventional Ga FIB has a reasonable resolution (typically up to 2.5 nm). However, these instruments present some limitations, including Ga ion implantation and contamination, and slow sputtering rate. New liquid metal alloy ion sources (LMAIS) have been developed to overcome these limits[1]. However, none of the proposed LMAIS sources is suitable for rapid milling because they can only deliver probe current up to few tens of nA. Contrary, emerging Xe plasma FIB systems promise faster removal rates[2],[3].

Homogenous copper FIB milling arises from the need to perform various circuit edit operations below the dielectric layer following the copper layer.  If the layer beneath the dielectric is affected by inhomogeneous milling, it can lead to short‐circuit and eventual device breakdown. Failure analysis on an integrated circuit was performed using rocking stage with 6‐axes piezo movement capabilities together with the novel approach of the combined Xe‐plasma ion source FIB and SEM system (XEIA). The new Xe plasma FIB offers sputtering speed up to 50 times faster than the most powerful Ga FIBs. Compared to conventional Ga ion sources, the Xe plasma ion source reduces dramatically the time for cross‐sectioning from tens of hours or even days to a matter of hours[4],[5].

Site‐specific milling of copper with different milling strategies were tested to optimize time and homogeneity of the milling across the target surface and to overcome the channeling effect posed by polycrystalline copper. Only during the last few nanometers of copper layer the water vapor is used to protect the dielectric layer. The complete removal of copper was followed with XeF
2
assisted milling of the dielectric layer to observe the unharmed circuitry. Channeling effect was reduced by regulating the sputtering rates across different grains keeping the underlying dielectric layer safe. Ultra‐high‐resolution scanning electron microscopy (UHR‐SEM) imaging was used for constant monitoring of the removed material to help modulate the process for highest throughput in the least possible amount of time[6].
Nanoparticles quantification inside cells by near‐edge absorption soft X‐ray nanotomography
Authors: Conesa Jose Javier , Oton Joaquin , Chiappi Michele , Pereiro Eva , Carazo José María , Chichon Francisco Javier , Carrascosa Jose L.
Published: December 2016

Recent advances in superparamagnetic iron oxide nanoparticles (SPION) design have generated new possibilities for nano‐biotechnology and nano‐medicine. Achieving the full potential of SPION will nonetheless require precise quantitative analysis at sufficient resolution to model the interactions between nanoparticles and the cell environment. The results from this analysis will determine the dose for either drug‐delivery or hyperthermia treatments as well as its feasibility. To get an insight into this problem we incubated 15 nm SPION functionalized with dimercaptosuccinic acid with MCF‐7 breast cancer cells [1] as a model system to be analyzed exploiting the iron differential absorption contrast in the L2‐edge. Near‐edge absorption soft X‐ray nanotomography (NEASXT) combines whole‐cell 3D structure determination at 50 nm resolution, with 3D elemental mapping and high throughput. We solved three‐dimensional distribution of SPIONs within cells with sensitivity sufficient for detecting the density corresponding to a single nanoparticle (Fig. 1)[2].
Aluminum nanoflakes: relations between microstructure and reactivity
Authors: Madigou Véronique , Leroux Christine , Esposito Pierre‐Henri , Denoyel Renaud , Coulet Marie‐Vanessa
Published: December 2016

Aluminum powders are used as a component in propellant formulations, explosives and pyrotechnics [1]. Their reactive and propulsive properties are linked to the exothermic reaction of aluminum with an oxidant. Powders with a high specific surface area (higher than 10 m
2
/g) i.e. aluminum nanopowders (Al‐NPs) have a higher reactivity than micronic powders. Moreover, we have shown that shape and nanostructuration of the grains influence the reactivity [2]. The reactivity of the powders is characterized by thermogravimetric (TG) analyses. In this work, the powders which are obtained by high energy ball milling, show a flake like morphology (fig. 1). The influence of the milling parameters and additives on the morphology, structure and reactivity of the powder was investigated. Among the milling parameters, we have changed the size of the balls, the atmosphere of the process and the post‐treatment of the powder.

The nanoflakes are highly dispersed in lateral size and thickness; the biggest ones have micronic lateral sizes (1‐4 mm) and thickness less than 200 nm, and the smallest ones 100 nm in lateral size and 30 nm in thickness (fig. 2). The nanoflakes contain several crystallites with an orientation relationship; a [110] texture was observed (fig. 3). They are surrounded by an amorphous layer of aluminum oxide, whose thickness depends on the size of the crystallite. Small crystallites exhibit thicker amorphous layer at the edges than on basal planes (fig. 4), this could induce different oxidation rates of the powder. To remove the passivation agent used in the milling process, the powder is rinsed several times. This is a rather tedious process, so a simple annealing of the powder was tested as an alternative to remove paraffin. Heating the powder at 420°C led to an early crystallization of the Al
2
O
3
amorphous layer, which is an unwanted effect. In‐situ heating in a transmission electron microscope, under inert atmosphere, are in progress, in order to follow the crystallisation of the amorphous alumina layer and the different phase transitions, which are predicated to play an important role in the oxidation mechanism [3].
Direct mapping of Li‐enabled octahedral tilt ordering and associated strain in nanostructured perovskites
Authors: Zhu Ye , Withers Ray , Bourgeois Laure , Dwyer Christian , Etheridge Joanne
Published: December 2016

Self‐assembled nanostructures are promising for creating 2D and 3D superlattices with exceptional functionalities. Understanding the mechanisms driving the superlattice formation demands the underlying structural information. However, nanoscale structural modulations intrinsic to these superlattices are difficult to be characterized by conventional diffraction‐based structure determination. A real‐space, direct imaging method is necessary to probe the local structure characteristics, providing essential information for theoretical understanding and subsequent design of structure‐property correlations.

Using the aberration‐corrected scanning transmission electron microscopy (STEM), we developed an optimized atomic‐level bright‐field (BF) condition to image the oxygen octahedra in perovskite oxides. We used multislice calculations to determine detector collection angles that allow oxygen octahedra to be imaged sensitively and robustly over large specimen thicknesses. These calculations also provided a calibration by which the octahedral‐tilt angle can be measured quantitatively from the image of each octahedron.

Applying this real‐space octahedral‐tilt mapping on Li

0.5–3
x

Nd

0.5+
x

TiO
3
, a promising solid electrolyte in Li‐ion batteries, we directly revealed an unconventional superlattices with 2D modulated octahedral tilting. A mathematical description of the octahedral‐tilt modulation was derived based on the quantitative tilt maps, which explicitly identified the high‐order harmonic character of the modulation. Using simultaneous annular‐dark‐field (ADF) imaging, we also mapped the lattice parameters unit‐cell by unit‐cell, uncovering highly‐localized strain associated with the tilt modulation. Furthermore, we demonstrate the tunability of the tilt modulation by changing Li stoichiometry. Fascinatingly, we observe a reversible annihilation/reconstruction of the tilt modulation correlated with delithiation/lithiation process, suggesting the structural transformation that is associated with Li‐ion conduction in this promising Li‐ion conductor.
1

The above observations are largely inaccessible from conventional diffraction analysis,
2
and lead to an unprecedented mechanically‐coupled tilting competition model to explain the superlattice formation.
1
Our real‐space approach to quantify local octahedral structure and correlate it with strain can be applied to other advanced oxide systems.