bluuebunny/crossref_metadata_2025 huggingface dataset:
Formation of bacterial nanostructures: the impact of growth dead phase and antibiotics
Authors: Hovnanyan Margarita , Pepoyan Astghik , Hovnanyan Karlen
Published: December 2016

Background.
The existence of nanobacteria as a separate systematic unit is still under the doubt (1, 2). Nanobacteria are characterized as mycoplasmas and as L‐transformed forms, too (3).

The aim of current investigations was to evaluate the possibilities of formation of “nanostructure ‐ nanobacteria” in overgrowth bacterial cultures (deathphase of growth) and in the presence of antibiotics.

Material and methods.
E. coli
1257 and
S. flexnerii
130 were used in these investigations to follow the changes in bacterial membrane structures in death phase of growth and after the treatment of antibiotics. Bacteria were grown anaerobically in LB medium (48 hours, 37C°), solidified with 1.8% agar when necessary. The following antibiotics were used: tetracycline (15 µg/ml), doxycycline (15 µg/ml), amoxicillin (25 µg/ml), ampicillin (35 µg/ml), cefoxitin (50 µg/ml), kanamycin (50 µg/ml), gentamicin (50 µg/ml), chloramphenicol (30 µg/ml), and streptomycin (50µg/ml).

Transmission (TEM) electron microscopy technique was used to evaluate the changes in bacterial membrane structures.

Results.
The formation of spherical granules in 40‐100nm surrounding by membrane structures of S
. flexnerii
in growth death phase (Fig. 1) and formation of outgrowths on
E. coli
cell walls with different length during the ampicillin‐treatment (Fig. 2) were detected in these studies. These formations could be discussed as a basis for the development of nanobacteria under the specific conditions of environment.
Dynamical Holographic Moirés : Time average holographic interferometry
Authors: Gatel Christophe , Masseboeuf Aurélien , Houdellier Florent , Snoeck Etienne
Published: December 2016

Electron holography (EH) enable a sensitivity to electromagnetic fields at the nanoscale in a Transmission Electron Microscope (TEM) through the Aharonov‐Bohm effect (which was by far experimentally demonstrated with EH [1]). Beside EH we consider here the Moirés pattern than can arise with the fringe network of a hologram. Moirés originate from a superposition of two networks and their use in electron microscopy enable to reveal hidden details within crystal stacks (from stacking faults in crystals to graphene misalignments).

The technique presented in this contribution propose to reach one step beyond the sensitivity that is actually achieved in EH by combining it with Moirés. The basic idea of what we call Dynamical Holographic Moirés (DHM) is to superpose various and controlled states of the sample under observation within the same out‐of‐axis hologram giving rise to the apparition of Moirés in a holographic interferogram. Holographic interferometry was initially designed in optics [2] and then implemented in its double exposure at the beginning of electron holography development. Our approach can be described as the implementation of time‐average holographic interferometry by an application of
in situ
experiment with EH.

The system in use here is a Hard Disk Drive writing head that has been mounted to be characterized in operando by EH [3]. We can easily switch the writing pole state between two different states and thus reverting the magnetic flux sense. The DHM method [4] was thus used to fully characterize the magnetic flux escaping from such a magnetic pole in a quantitative way. By far, the obtained interferogram are directly quantized in term of magnetic flux quantum () linked to phase shift via the Aharonov‐Bohm effect. A typical phase image of the system along with an interferogram are displayed in Fig 2. The main advantages of this new TEM technique, namely frequential analysis, electron optic distorsion and disturbed reference problem or direct quantification, will be discussed.
Great advantages of using low voltage
HR‐SEM
in spatial resolution and sensitivity for low energy
X
‐ray analyses
Authors: Shunsuke Asahina , Hideyuki Takahashi , Masaru Takakura , Schüth Ferdi , Osamu Terasaki
Published: December 2016

Understanding surface fine features, such as topological and compositional information, is essential for controlling synthesis conditions of these materials and for designing novel materials and utilizing their functions. The materials are becoming complicated composites composed of Nano crystals in order to have functions with better performance. Therefore, higher spatial resolution, sensitivity and capability are required for characterization techniques are required now. The recent developments in compound type objective lens, electron beam deceleration method, high solid angle multi EDS system and Soft X‐ray Emission Spectrometry (SXES) of high‐resolution scanning electron microscopy (HR‐SEM) show great advances for the study of structures, chemical information, and electron state of Nano structured materials (1). Here, we will discuss newly developed spectroscopy approaches in EDS and SXES, and show some of their examples using low voltage (LV) HR‐SEM.

The electron beam deceleration method brings a lot benefits. Because it is improving size of electron beam diameter due to smaller aberration even at LV condition, high probe current condition and long working distance (2). Fig. 1 shows high spatial resolution EDS map from Au@TiO
2
Yolk‐shell type sample where a gold nanoparticle with 15 nm in diameter is encapsulated in a TiO
2
hollow sphere. The gold nanoparticles are well resolved within 3 min even at low electron landing‐energy (see observation conditions in the Fig caption).

The low energy SXES spectra obtained from Al‐B nano composites are shown in Fig. 2. SXES technique requires high probe current to acquire enough X‐ray signals as it is based on wavelength dispersive spectrometry (WDS). The spectra from small area of aluminum alloy (a) and aluminum boride (b) show different peak top energy and shape for aluminum L‐line at around 70 eV. In addition, the boron peak was also detected at around 67 eV. The spectra at this energy range, which corresponds to K‐line from light elements including Lithium, was hard to observe by EDS and WDS before. So the SXES detector has high‐energy resolution and can also detect extremely low energy X‐ray.

The electron beam deceleration method has advantages in SXES analysis. We demonstrated SXES line profiles in between metal Aluminum and Aluminum boride in Fig. 3. Actually, there is deference between with beam deceleration method or not even at 5 keV with 30 nA. Compares to Line (b) and (c), Electron beam deceleration method of line (c) shows better sharpness and details of spectrum than line (b). Probably it is due to smaller probe size was created by electron beam deceleration method. Here we have also tired low voltage condition that is 1 keV with ‐5 keV sample bias in line (a). The line (a) shows much higher spatial resolution line profile due to smaller interaction volume in sold than 5 keV.
Modified University of Wisconsin Solution With Melatonin and the Its Efficacy of Kidney Preservation Time
Authors: Coskun Abdurrahman , Yegen Cumhur , Arbak Serap , Gunal Omer , Attaallah Wafi , Seckin Ismail , Cetiner Banu , Yildirim Zeynep , Erdemgil Yigit , Elmas Merve Acikel , Demirci Sibel , Can Ozge , Serteser Mustafa , Ozpınar Aysel , Basdemir Gulcin , Belce Ahmet , Unsal Ibrahim
Published: December 2016

Organ transplantation solutions are used for protection of organ functions for a long time. In order to provide long lasting time period for organ preservation, donor organs are kept in special preservation solutions such as University of Wisconsin (UW), Histidine‐Triptophane‐Ketogluterate (HTK) and Celcior solutions. Although UW is the most preferred solution, preservation time even for the kidney tissue, has not exceeded over 36 hours. The aim of this study is to extend the preservation time and also provide more effective protection. In order to provide better preservation, the ingredients of the UW solution were changed (Mod UW), as well as melatonin was included (Mod UW+M) in the preservation medium. Time‐related morphological changes of rat kidneys in UW (group I), Modified UW (group II) and Modified UW+Melatonin (Mod UW+M) (group III) were comparatively investigated within this study. Totally perfused kidneys were removed and placed in UW, Mod UW and Mod UW +M solutions and kept during 2, 10, 24 and 72 h at 4° C. Kidney tissue samples were taken at all given time intervals and these samples were prepared for paraffin sections. Kidney tissue samples, taken at 72nd hour, were fixed in 2.5% glutaraldehyde and prepared for epoxy resin embedding. Histopathological scoring system were performed for light microscopical evaluation by using a scale ranging from 0 to 3 (0: none; 1: mild; 2: moderate; and 3:severe) for each criterion. Histopathological parameters were renal injury, based on tubular and glomerular degeneration, inflammatory cell infiltration and vasocongestion. Liquid samples, taken at 2, 10, 24 and 72 h at 4° C from the storage media, investigated for lactate dehydrogenase (LDH) activity which was measured using standard chemistry method (Siemens ADVIA 1800 chemistry System). Comparative findings at light and transmission electron microscopical levels revealed that preservation in Mod UW+M solution was statistically much more prominent in all time intervals, significantly at 72nd hour of preservation. In all groups, LDH levels were consistent with morphological results. We could conclude that Mod UW+M was the most effective solution among the experimental groups, especially suitable for preservation up to 72 h. In this study, the important efficacies of UW solution, a better protectant and melatonin, an effective agent to be used in such systems, were highlighted.
Laser induced yellowing of stonework: a combined
TEM
imaging and
STEM
‐
EELS
study on model samples
Authors: Godet Marie , Gauquelin Nicolas , Vergès‐Belmin Véronique , Andraud Christine , Saheb Mandana , Monnier Judith , Leroy Eric , Bourgon Julie , Verbeeck Johan , Tendeloo Gustaaf Van
Published: December 2016

Nd‐YAG Q‐Switched laser devices operating at 1064 nm have been considered in the 1990s as the most promising tool for cleaning stone sculptures, and more particularly eliminating indurated black gypsum crusts. However, the spreading of this laser technology has been undermined because of the yellow hue it occasionally conveys to the cleaned surfaces as seen on Figure 1. Especially in France, this yellowing effect is considered as a major esthetic issue by conservators and the laser technique has gradually disappeared from the restoration sites. This discoloration issue remains partly unexplained: a currently admitted hypothesis states that the iron containing compounds present in the black crusts would transform, upon laser irradiation, into yellow iron‐rich nanophase(s) that would re‐deposit on the cleaned substrate. To verify this hypothesis, model black crusts have been elaborated by mixing hematite α‐Fe
2
O
3
and gypsum CaSO
4
.2H
2
O in different proportions. The model crusts were irradiated using a Nd‐YAG QS laser resulting in ablation of numerous particles in a visible smoke and the color of the samples shifted instantaneously from red to a bright yellow. Transmission electron microscopy (TEM) has been used to characterize the morphology of the nanostructures generated by the laser, both in the smoke and on the surface of the samples as observed in Figure 2. In addition, the chemical composition of the neo‐formed nanophases was determined by aberration corrected scanning transmission electron microscopy in combination with electron energy loss spectroscopy (STEM‐EELS) and high angle annular dark field imaging (HAADF) as seen on Figure 3. It was found that both the surface of the samples and the ablated micro‐materials are covered by an irregular nano‐film and by dispersed spherical nanoparticles, all containing iron and oxygen. These results ascertain the link between the yellowing effect and the presence of iron containing nanophases after irradiation.
Combined stem‐eels and stem‐cl analysis of plasmonic coupling between chemically grown silver nanocubes
Authors: Lourenço Martins Hugo , Lee Yih Hong , Liu Yejing , Lee Hiang Kwee , Kociak Mathieu , Ling Xing Yi
Published: December 2016

These recent years, study of surface plasmon resonances in transmission electron microscope has undergone an increasing interest. Indeed, low‐loss EELS has proven its remarkable efficiency to probe plasmonic resonances at the nanometer scale while recent developments in cathodoluminescence (CL) spectroscopy brought a new insight into the coupling of plasmons and far field [1]. Several types of particles have already been numerically and experimentally studied ranging from nano‐disk to nano‐prism which plasmonic behaviors are now fairly understood [2,3]. However, despite this systematic effort to characterize the widest variety of plasmonic nano‐particles, some structures still remain challenging e.g. the nano‐cube. In the latest case, the difficulties encountered are plural. First, this structure being highly symmetric, plasmonic modes exhibit a significant number of natural degeneracies leading to superposition or hybridization of modes, which dramatically harden their understanding. Second, cube's plasmonic modes turned out to be very sensitive to the geometry of the underlying nano‐structure (e.g. edge rounding [4]) and to be strongly affected by the presence of substrate [5]. Third, particularly because of the degeneracies mentioned above, the coupling between two nanocubes brings unexpected difficulties, which are enhanced when the inter‐particle gap goes below 1nm. Although this coupling has been widely tackled recently, no definitive theory has been given and this question remains controversial [6,7].

In the present work, we experimentally and numerically investigated plasmonic silver nanocubes and their coupling using STEM‐EEL and STEM‐CL spectroscopies (see figures 1 and 2) aiming at giving a clear and complete understanding of these modes. The cube samples have been prepared by chemical growth, which provides them a high degree of crystallinity. This property dramatically enhance the CL signal and thus enable us to obtain remarkably relevant CL‐maps (see figure 2). For studying the coupling, in order to get rid of the quantum charge transfer problem arising at very small gap regime (<0.5nm), we restricted ourselves to inter‐particle separations larger than 5nm. Complementary BEM simulations [8] have been carried out to understand on a deeper level the observed plasmonic modes by computing the corresponding charge distributions. We observed a good agreement between computations and experiments, which strengthen our conclusion. In addition to be consistent with the earlier studies, our work bring an overall understanding of the nanocubes coupling.
Theoretical and numerical investigation of the interaction between phase‐shaped electron probes and plasmonic modes
Authors: Martins Hugo Lourenço , Guzzinati Giulio , Verbeeck Jo , Kociak Mathieu
Published: December 2016

Optical vortices ‐ i.e. electromagnetic fields with phase singularity ‐ are well known objects and have been used for a decade in a wide range of applications in e.g. optics or astrophysics. Recently, it has been demonstrated that such vortices can be created in an electron microscope by tailoring the phase of the beam [1] and these so‐called electron‐vortex‐beams have already proven their efficiency detecting magnetic state in a material or to probe chirality in a crystal [2].

Simultaneously, EEL spectroscopy in the low‐loss region has attracted a tremendous interest due to its efficiency in resolving plasmonic resonance at the nanometer scale [3] and the underlying formalism is now firmly established [4]. However, because of the invariance of the electron probe along the propagation axis, low‐loss EELS remained unable to detect plasmonic optical activity. However, electron‐vortex‐beams constitute a perfect candidate to overcome this limitation and measure the dichroic behavior of plasmons in an electron microscope ‐ as recently pointed out through simulations by Asenjo‐Garcia and García de Abajo [5].

In the present work, we developed a semiclassical formalism describing the interaction between an electron probe with an arbitrary phase profile and a plasmonic mode. Following [6], we used a quasi‐static and classical description of the plasmon resonances while the electron probe is described in a fully quantum way. We showed that the equation ruling this interaction takes the elegant form of a transition matrix ‐ between two electron states mediated by the eigenpotentials of the plasmon modes. Starting from this formalism, we built an analytical model describing the interaction between point charges and a vortex beam, which gave us a good insight into the physics of plasmonic dichroism. Important experimental inputs, such as convergence and collection angles, were considered. We also implemented a Matlab script within MNPBEM [7] in order to compute our equation and investigate the dichroic behavior of arbitrary plasmonic nano‐structures (see Figure 1).

In the conference, we will present the theoretical formalism and a wide variety of numerical studies of interactions between different nano‐structures (e.g. helix, rod) and phase shaped electron probes (e.g. vortex beams, HG‐like beams…), with a special emphasis on the experimental feasibility of the proposed geometries.
Structural dynamics of copper and nickel substrates during redox reactions studied by in‐situ
SEM
Authors: Cao Jing , Cao Jing , Rinaldi Ali , Wang Zhu‐Jun , Weinberg Gisela , Willinger Marc , Schlögl Robert
Published: December 2016

Many efforts have been made in order to investigate catalyst under relevant working conditions. Indeed, the action of a chemical potential has to be considered when describing the state of a catalyst and recent studies have demonstrated that the catalyst surface evolves dynamically under reaction conditions.
1,2
In order to complement
in‐situ
spectroscopic tools, such as near‐ambient X‐ray photoelectron spectroscopy (NAP‐XPS), with visual information about the active surface structure, we have implemented
in‐situ
scanning electron microscopy (SEM) for the observation of dynamic processes at the µm to nm scale.
3

By coupling
in‐situ
SEM with mass spectroscopy measurements we are able to relate structural dynamics at the surface of metal catalysts to changes in the gas phase composition. Under specific conditions, oscillatory behavior of the catalyzed chemical reactions was observed for the case of polycrystalline nickel and copper foils. These self‐sustained oscillations can be utilized to assist the identification of kinetic mechanisms
4
and provide insight in the active and inactive state of the catalyst. Figure 1. demonstrates the dynamic behavior of the Ni foil surface during hydrogen oxidation reaction at 600°C. The active state in the oscillatory reaction is dominated by the presence of metallic Ni, while the formation of NiO coincides with a decrease in the catalytic activity. Overall, it will be outlined how the implementation of complementary
in‐situ
SEM and mass spectroscopy techniques can enrich our understanding of the dynamic behavior of active catalyst and how it complements spatially integrated spectroscopic data that is recorded under similar conditions.

Figure 1. Self‐sustained oscillations during hydrogen oxidation over a Ni foil at 0.3 mbar with a hydrogen/oxygen ratio of 7:1. The SEM images correspond to the different points in the oscillation as indicated in the mass spectrum profile. During the active state of an oscillation (A), the Ni foil exhibits a smooth metallic surface. In the low‐active state (B), the surface of the Ni foil is covered by an oxide layer.
OKA
‐induced tau hyperphosphorylation decrease
BDNF
protein levels in primary cortical neuron cultures
Authors: Atasoy İrem L. , Yilmazer Selma , Dursun Erdİnç , Armağan Derya Metİn , Öztürk Melek , Gezen‐ak Duygu
Published: December 2016

Intracellular aggregation of abnormally phosphorylated tau in neurofibrillary tangles (NFTs) is a major neuropathological hallmark of Tauopathies such as Alzheimer's disease. Tau phosphorylation is controlled by the homeostasis of glycogen synthase kinase‐3β (GSK‐3β) and protein phosphatase‐2A (PP2A). Okadaic acid (OKA) is a potent inhibitor of PP2A, leading to abnormal tau phosphorylation. Brain‐derived neurotrophic factor (BDNF) is a member of the neurotrophin family and is selectively downregulated in AD brain. In this study, we investigated the effects of tau phosphorylation on secreted and cellular BDNF levels in primary cortical neurons. Primary cortical neurons were prepared from embryonic day 16 (E16) Sprague‐Dawley rat embryos. After 7 days in vitro, neurons were treated with 25 nM OKA for inducing tau hyperphosphorylation. Tau phosphorylation was assessed by Western blot using antibody against phospho‐Thr231 and non‐phosphorylated tau protein was detected with the Tau‐1 antibody. Levels of BDNF secreted to the culture medium were determined by ELISA at the 4th, 8th and 24th hours of treatment. Cellular localization and the protein expression of BNDF were determined by immunofluorescent labeling and fluorescent intensity measurements.

Our results show that after 8 hours of OKA treatment, tau phosphorylation at Thr231 increased, whereas Tau‐1 signal decreased (p<0.0001) Compared with the control groups, BDNF levels in the OKA treated group were significantly lower after 4 and 24 hours of treatment (p<0.0001) but were not significantly different at 8 hours of treatment (p>0.05). While prominent BDNF immunoreactivity was seen in cytoplasm and neurites of the neurons in control groups, BDNF immunoreactivity significantly decreased in the OKA treated group (p<0.0001) and this attenuation was significant especially at neurites.

Our results suggested that decreased BDNF protein levels might depend on the defects in axonal transport as a result of disrupted microtubule structure caused by tau hyperphosphorylation.
Identifying and Mapping the Polytypes and Orientation Relationships in
ZnO / CdSe
Core Shell Nanowire Arrays
Authors: Rapenne Laetitia , Consonni Vincent , Renou Gilles , Roussel Hervé , Gerard Lionel , Rauch Edgar
Published: December 2016

Core shell ZnO nanowire (NWs) heterostructures have emerged, over the past decade, as a potential building block for a large variety of nanoscale optoelectronic devices, including self‐powered UV photodetectors, dye‐sensitized solar cells (DSSCs), and extremely thin absorber (ETA) solar cells. These heterostructures benefit from a high absorption over the UV and visible parts of the electromagnetic spectrum through sophisticated optical processes (
i.e
., optically guided and radiated modes). In this work, we focused on ZnO / CdSe core shell NW heterostructures. The ZnO NWs are typically grown by chemical bath deposition on top of a ZnO seed layer deposited by sol‐gel process and strongly oriented along the polar c‐axis, but with no in‐plane orientation. Then, the growth of the CdSe shell was performed by molecular beam epitaxy.

CdSe is known to crystallize into the two following polytypes: cubic zinc blende (ZB) and hexagonal wurtzite (WZ). Identifying the different polytypes of CdSe by standard characterization techniques, such as x‐ray diffraction (XRD) and selected area electron diffraction (SAED) using transmission electron microscopy (TEM), as well as Raman scattering, is complicated in the present case. Both the ZB and WZ crystalline phases have very similar properties: most of the diffraction peaks and phonon modes of the ZB crystalline phase are located very closely to those of the WZ crystalline phase; similarly, the bang gap energy of both crystalline phases is almost identical. In addition to the identification of the different polytypes and potential epitaxial relationships, the spatial information on the local scale through mapping is of great interest, but requires the use and development of advanced TEM‐based experiments.

In order to address these issues, the morphology and structural properties of ZnO / CdSe core shell NW heterostructures are thoroughly investigated by field‐emission gun scanning electron microscopy (FEG‐SEM), XRD, Raman spectroscopy, TEM‐HRTEM (Figure 1) and ASTAR (Automated crystal phase and orientation mapping in TEM). We show the strong interest in ASTAR for identifying and mapping the different polytypes of the CdSe shell , but also for revealing the occurrence of orientation relationships with the ZnO NWs, as shown in figure 2.
The Determining Role of Solution Chemistry in Radiation‐Induced Nanoparticles Synthesis in the
STEM
Authors: Abellan Patricia , Arslan Ilke , Hasan Naila Al , Browning Nigel D. , Evans James E. , Grate Jay W. , Karim Ayman M. , Lucas Ivan T. , Moser Trevor H. , Parent Lucas R. , Park Chiwoo , Woehl Taylor J.
Published: December 2016

In the last decades, radiolytic synthesis routes have exploited the chemical effects of the absorption of high‐energy radiation on precursor solutions, to form nanostructures by reproducing a selective reducing/oxidizing environment. Radiation chemical synthesis provides a powerful means to form nuclei which are homogeneously distributed in the whole volume and where the growth rate can be easily controlled.[1] The latter has recently been achieved using liquid cell electron microscopy, with examples of formation kinetics of particles in polar (water)[2] and non‐polar (toluene)[3] solvents following a linear relation with applied dose rate. Fine control of particles size with conventional radiation sources typically requires increasing the production of radicals (thus applying “high doses”). On the contrary, the challenge in the scanning transmission electron microscope (STEM), where incident doses are inherently higher by orders of magnitude, is to reduce drastically such production. To illustrate this, we recently proposed the use of non‐polar systems (toluene), which are not typically used for radiolytic synthesis outside the STEM, as a solvent that produces much lower amount of radiolytic ionic species upon electron irradiation, as compared to water or other polar solvents such as alcohols (Figure 1 shows an example of

Thus far, most experiments in the liquid cell involved the use of water as a solvent, which explains the large amount of work dedicated to understanding the effects of the electron beam in aqueous conditions. Radiation chemical yields in water are large due to the relatively low bond energies in water molecules, meaning highly reactive oxidizing and reducing radicals and species are created in about an equal amount.[4] Reproducing net reducing conditions for nanoparticle growth can be achieved with the addition of substances that convert primary radicals into free reducing radicals (using OH · scavengers, for instance). The use of organic solvents in the liquid cell allows for tuning the polarity of the medium, giving access to a broader range of synthesis conditions but producing more complex radiolytic products. Here I will discuss, revisiting a number of examples from the literature and presenting our most recent work, general methods for finding more suitable synthesis environments for controlled nanoparticles formation in the liquid cell. Much of the presentation will focus on the solvent radiolysis which is what predominantly dictates the species and yields involved in the chemical processes leading to nanostructure synthesis.[4]
3‐D
EM
exploration of the hepatic microarchitecture ‐ lessons learned for large‐volume in situ serial sectioning
Authors: Shami Gerald , Cheng Delfine , Huynh Minh , Vreuls Celien , Wisse Eddie , Braet Filip
Published: December 2016

To‐date serial block‐face scanning electron microscopy (SBF‐SEM) dominates as the premier technique for generating three‐dimensional (3‐D) data of resin‐embedded biological samples at an unprecedented resolution and depth volume. Given the relative infancy of the technique, limited literature is currently available regarding the applicability of SBF‐SEM for the ultrastructural investigation of tissues that are inherently low in contrast, including hepatic tissue. Such tissues, relative to neural tissue which has been extensively investigated by means of SBF‐SEM require dedicated SBF‐SEM specimen preparation protocols combined with optimised imaging conditions in order to collect large‐volume 3‐D image data, at appropriate resolution which is devoid of imaging artefacts that are inherent to SEM investigations of uncoated samples.

Therefore, in this study, we provide a comprehensive and rigorous appraisal of different tissue preparation protocols for the large‐volume exploration of hepatic architecture at an unparalleled XY and Z resolution. In so doing, we qualitatively and quantitatively validate the use of a novel and time‐saving SBF‐SEM tissue preparation protocol, which involves the concomitant application of heavy metal fixatives, stains and mordanting agents for the subsequent imaging of contrast‐rich SBF‐SEM data, 3‐D reconstruction and modelling of the hepatic architecture. We further extrapolate large‐volume morphometric data relating to key tissue features of the murine liver, including hepatocytes, the hepatic sinusoids and bile canaliculi network (Fig. 1).

Furthermore, we combine our validated SBF‐SEM specimen preparation protocol with a correlative light and electron microscopy approach, to combine the respective advantages of confocal scanning laser microscopy and SBF‐SEM in order to selectively picture particular subcellular hepatic details in a novel manner. We propose that this correlated multidimensional light and electron imaging approach will allow the study of different liver diseases under relevant experimental tissue models.
Combined multidimensional microscopy as a histopathology imaging tool for the holistic evaluation of the hepatic microvascular and associated tissue cells
Authors: Shami Gerald , Cheng Delfine , Braet Filip
Published: December 2016

Background

Histological variations in the microanatomical organisation of the functional liver units or hepatic lobules adversely affect general body function. Typically, assessment of liver pathology is based on the examination of paraffin‐embedded histological slides counterstained with dyes using bright‐field light microscopy (BFLM). In some instances, further clinical electron microscopy is recommended to assess the extent of ultrastructural damage to the microvasculature, to determine the nature of hepatic inclusions, or to stage fibrosis, hepatitis, and malignancy in more detail.

Methods

Unfortunately, the above histopathology practice lacks the ability to cross‐correlate observations on the same tissue sample and within a large tissue volume. With the recent advent of smart tissue preparation methodologies and three‐dimensional (3‐D) imaging practices, this can be circumvented in a relatively swift manner, allowing the same tissue to be examined across different microscopy platforms. Herein, we outline the combined application of X‐ray micro‐computed tomography (Micro‐CT), BFLM and 3‐D backscattered electron microscopy (BSEM) on liver tissue as an alternative good practice multimodal imaging approach.

Results

A workflow is presented facilitating the collection of combined structure‐function 3‐D data (i.e., X, Y & Z) on liver architecture from the micron down to the nanometre scale using the same tissue preparation protocol. We illustrated the strength of this combined microscopy methodology to characterise various aspects of the hepatic vasculature, ranging from such large vessels as branches of the hepatic portal vein and hepatic artery, down to the smallest sinusoidal capillaries. Moreover, we were able to further characterise the subcellular features of a range of hepatic sinusoidal cells including, liver sinusoidal endothelial cells, liver‐associated natural liver cells and Kupffer cells. Above all, we demonstrate the capabilities of this specimen manipulation and microscopy workflow to generate quality microscopic detail and subsequently extract relevant quantitative 3‐D information.

Conclusions

This contribution illustrates the capability that advanced histology imaging can bring to the gastroenterologist and/or pathologist in the fine structure‐function assessment of the liver unit, its associated microvasculature, and the individual make‐up of parenchymal and non‐parenchymal tissue cells. This good practice further allows the instant generation of combined quantitative data about size, shape and volume changes of key microanatomical structures across multiple length scales.
Quantitative Electron Tomography Study of Metal Catalysts Supported on Heavy Oxides Combining Image De‐noising and Compressed Sensing Techniques
Authors: Lopez‐Haro Miguel , Tinoco Miguel , Hungria Ana Belen , Calvino Jose Juan
Published: December 2016

3D quantitative characterization of metal catalysts supported on heavy oxides by High Angle Annular Dark Field (HAADF) STEM electron tomography (ET) is a very challenging task. Noble metal nanoparticles (Au, Ru) supported on ceria or ceria mixed oxides illustrates the case. The difference, very small in some cases, between the atomic number (Z) of metal and support (Z
Ru
=44, Z
Au
=79, Z
Ce
=58) complicates the discrimination of the small particles. This makes that each step, acquisition, alignment, reconstruction and segmentation, has to be carefully optimized not only to improve the visualization of the nanoparticles but also to extract relevant quantitative results,
e.g
. metal loading, specific surface area, or particle size distribution. In this work, we have combined advanced image processing algorithms, based on undecimated wavelets transform (UWT) [1], to improve the contrast and denoising the tilt series projections, with new reconstruction algorithms, based on compressed sensing (CS) [2], to study a series of catalysts with a high potential in different processes related to the production of hydrogen, as CeO
2
Nanorods, Au/CeO
2
Nanocubes or Ru/Ce
2
Z
2
O
8
. The results obtained by ET have been compared to those determined by macroscopic characterization techniques as Inductively Coupled Plasma (ICP) or Brunauer, Emmett and Teller (BET) isotherm method.

HAADF‐STEM ET has been carried out using a FEI TITAN
3
THEMIS 60‐300 operated at 200kV recently installed at Cadiz University. Data collections were obtained by tilting the specimen about a single axis perpendicular to the electron beam. Series of projections were acquired between ‐70º and +70° either every 2° or 5º. The images series were aligned using Inspect3D and TomoJ and reconstructed using the ASTRA Toolbox implemented in Matlab [3]. In the particular case of CS, Total Variation Minimization (TVM) was carried by using the TVAL3 solver [2].

Figure 1 shows the 3D‐rendered voxel of a catalyst consisting of Au nanoparticles supported on CeO
2
Nanocubes after reconstructing the raw tilt series by SIRT (Figure 1a) and TVM (Figure 1b) and after denoising the tilt series projections by UWT and reconstructed by SIRT (Figure 1c) and TVM (Figure1d). Note how in the case of UWT‐TVM reconstruction an important improvement in terms of morphology of the support and visualization of nanoparticles is obtained. The 3D rendered surface of a similar sample and the 3D quantifications of relevant properties as metal loading, specific surface area and average particle size are shown in Figure 2. It is important to point out how the values are quite similar to those determined by macroscopic characterization techniques. These results indicate that this combination of techniques allows determining nanostructural features representative of the catalysts at macroscopic level.
TEMcompression of nano‐particles in environmental mode and with atomic resolution observations
Authors: Epicier Thierry , Joly‐Pottuz Lucile , Jenei Istvan , Stauffer Douglas , Dassenoy Fabrice , Masenelli‐Varlot Karine
Published: December 2016

Characterization of nanomaterials or materials at the nanoscale has drastically increased during the last decades. This increase can be explained by (i) the necessity to obtain materials with nanometer‐size grains, for instance nanocomposites, and by (ii) the use of nanoparticles in different fields, for instance lubrication applications. A challenge lies in thein situmicrostructural characterization of such materials as it can give access to valuable pieces of information regarding the microstructural changes induced by their use.

The availability of dedicated TEM (Transmission Electron Microscopy) holders equipped with force and displacement sensors is of a very high interest to test,in situ,the size‐dependent mechanical properties of nanometer‐sized objects [1,2]. On crystalline nano‐objects, Molecular Dynamics simulations have shown that dislocations nucleate at the surface [3,4]. Therefore, the surface state is of utmost importance in determining the nucleation stresses and types of dislocations. For materials which undergo surface reconstruction or changes in the surface chemistry under vacuum, it is necessary to perform experiments in a controlled environment (i.e. under gas pressure) which reproduces the real one.

Recently a Hysitron PI 95 Picoindenter has been installed on a Cs‐corrected FEI TITAN ETEM (Environmental TEM) microscope. It opens the possibility of performingin situcompression under gas pressure, with high resolution imaging. We will presentin situtests of cubic CeO2, a multifunctional oxide widely used in catalysis. Nanocubes are compressed along either under vacuum or under air pressure. Introducing oxygen inside the chamber limits or avoids the reduction of CeO2nanocubes induced more or less rapidly by the electron beam. A comparison of slopes of load‐displacement curves obtained under vacuum at different electron doses and under air pressure (see Figure 1) strongly suggests that ceria reduced as Ce2O3under the effect of an intense electron flux has a smaller Young modulus than unreduced or 'oxidized' ceria. Atomic resolution observations performed during the compression tests reveal the formation of dislocations and stacking faults (see Figure 2). Simulations are planned to further understand the deformation mechanisms as a function of the oxidation state (native, unreduced or oxidized states), as well as their reversibility [5].
Quantitative measurement of mean inner potential and specimen thickness from high‐resolution off‐axis electron holograms of ultra‐thin layered
WSe2
Authors: Winkler Florian , Tavabi Amir H. , Barthel Juri , Duchamp Martial , Yucelen Emrah , Borghardt Sven , Kardynal Beata E. , Dunin‐Borkowski Rafal E.
Published: December 2016

Off‐axis electron holography is a powerful tool to measure electrostatic and magnetic fields at the nanoscale inside a transmission electron microscope. The electron wave that has passed through a thin specimen can be recovered from an electron hologram and the phase can be related to the specimen thickness or the electrostatic potential in and around the specimen. However, dynamical diffraction may cause a deviation from the expected linear relationship between phase and specimen thickness, which emphasizes the need for comparisons with corresponding computer simulations.

Here, we study few‐layer‐thick two‐dimensional WSe
2
flakes by off‐axis electron holography. Voronoi tessellation is used to spatially average the phase and amplitude of the electron wavefunction within regions of unit‐cell size (see Fig. 1). A determination of specimen thickness of the WSe
2
is not possible from either the phase or the amplitude alone. Instead, we show that the combined analysis of phase and amplitude from experimental measurements and simulations allows an accurate determination of the local specimen thickness. Extremely thin specimens that are tilted slightly away from the [001] zone axis show an approximately linear relationship between phase and projected potential. If the specimen thickness is known, the electrostatic potential can be determined from the measured phase.

We used this combined approach to determine a value for the mean inner potential of 18.9 ± 0.8 V for WSe
2
, which is approximately 10% lower than the value calculated from neutral atom scattering factors. In this way, a comparison of high‐resolution electron holography data with simulations has been achieved on a quantitative level, enabling an assessment of the experimental conditions under which electrostatic potentials can be extracted directly from the phases of measured wavefunctions.

The authors are grateful to L. Houben, M. Lentzen, A. Thust, J. Caron and C. B. Boothroyd for discussions, as well as A. Chaturvedi and C. Kloc from the School of Materials Science and Engineering, Nanyang Technological University, Singapore for providing the WSe
2
crystals. We are also grateful to the European Research Council for an Advanced Grant and for funding by the German Science Foundation (DFG) grant MA 1280/40‐1.
EELS
analysis of
He
bubbles in
ODS
Steel and vanadium
Authors: Walls Michael , Brun Nathalie , Badjeck Vincent
Published: December 2016

The understanding and the assessment of neutron irradiation in nuclear materials is critical in the design of the next‐generation nuclear fission reactors. Here, one of the most promising structural and fuel cladding materials, an oxide‐dispersion‐strengthened (ODS) steel was implanted with He and Fe ion in order to simulate the transmutant He and the damage (He/dpa) caused by neutron irradiation. The fine distribution of Y‐Ti‐O nanoparticles (NPs, 1‐20 nm) in the Fe‐Cr ferritic matrix is expected to improve thermal and mechanical properties. STEM‐EELS was used to investigate the structure and chemistry of these NPs and the He bubbles generated. The ODS material (Fe‐14Cr‐1W‐0.3TiH
2
‐ 0.3Y
2
O
3
, wt.%) was prepared by mechanical alloying of Fe‐Cr‐W‐TiH2 and Y
2
O
3
powders followed by hot extrusion. Ion irradiation was carried out at 500°C, producing 5 dpa damage (Fe) with 1000 appm/dpa He implantation. Core loss spectrum‐images were denoised using principal component analysis [1]. Implanted He is shown to be trapped in some Y‐Ti‐O NPs (fig. 1) although bubbles also exist outside the NPs. The ion irradiation has also changed the Cr distribution, removing the Cr‐shell observed around the NPs in non‐irradiated ODS samples (fig. 1) [2,3] and rendering the Cr distribution in the metallic phase generally more heterogeneous.

The He‐K line (21.218 eV for free atoms) shifts to higher energy in the bubbles (ΔE = 0.5 to 4 eV); this is shown to be correlated with the He density. He quantification has been carried out with three different methods: spatial difference, curve‐fitting and Multivariate analysis (MVA) methods (see below), as well as hybrid approaches combining the latter two methods which are proving promising for the elimination of the problems associated with each method on its own. The density and pressure values are found to reach 105 nm
‐3
and 8 GPa respectively, although the pressure measurement is only semi‐quantitative given that the error bars can reach 30%. The curve‐fitting method allows us to map the He‐K energy position and intensity, yielding the density, in individual bubbles (fig. 2). The spectral response of individual bubbles can be separated in an SI containing many bubbles at different densities using independent component analysis (ICA: example shown in figure 3) or vertex component analysis (VCA). Bubbles larger than 4 nm are shown to be under‐pressurized or at equilibrium with the Fe‐Cr matrix. Below 3.5 nm, the He pressure is shown to increase markedly, passing into the over‐pressurised regime.

In the presentation we will also describe experiments and analysis on similar bubbles implanted in vanadium at 700 and 1000°C. Here the size/pressure relation is much less clear, but the MVA approach is similarly promising.
Efficient and quantitative phase imaging in two‐ and three‐dimensions using electron ptychography in
STEM
Authors: Nellist Peter , Yang Hao , Jones Lewys , Martinez Gerardo , Rutte Reida , Davis Benjamin , Pennycook Timothy , Simson Martin , Huth Martin , Soltau Heike , Strueder Lothar , Sagawa Ryusuke , Kondo Yukihito , Humphry Martin
Published: December 2016

Historically, the scanning transmission electron microscope (STEM) has not been widely used for phase contrast imaging because the small bright‐field detector required makes use of only a small fraction of the incident electrons and is therefore inefficient with respect to dose. This limitation has hindered the efficient imaging of light elements in STEM. Alternative modes also have limitations. For example, annular dark‐field (ADF) imaging of graphene only makes use of a few percent of the incident electrons, and annular bright‐field imaging (ABF) requires lens aberrations to form an effective phase plate to get contrast from weakly scattering objects.

Electron ptychography in the STEM was first demonstrated more than 20 years ago in the context of improving image resolution [1]. At that time, the image field of view was restricted by the limitations of the camera technology and data handling technology. Here we make use of the pnCCD (S)TEM camera, a direct electron pixelated detector from PNDetector, mounted on the JEOL ARM200‐CF aberration corrected microscope. The detector has a grid of 264x264 pixels and operates at a speed of 1000 frames‐per‐second (fps). The detector can achieve a speed of up to 20,000 fps through binning/windowing. ADF images can be recorded simultaneously, as shown by the schematic in Fig. 1.

The resulting 4D data set is formed of a series of coherent convergent beam diffraction patterns recorded as a function of illuminating probe position. Here we explore how the bright‐field and dark‐field regions of scattering can be used to enhance the capabilities of STEM. We compare a range of methods that can be used to form the phase image from this data set, including single side‐band [2,3], Wigner distribution deconvolution [4] (used to produce Fig. 2) and ePIE [5]. Phase imaging using ptychography has a relatively simple transfer function [3] and also provides an inherent filter of image noise without reducing the signal strength to form high quality phase images (Fig. 2). Furthermore, the four‐dimensional data set is highly redundant and it is possible to detect and correct for residual aberrations in the image.

The ability to deconvolve lens aberrations can further be used to extract three‐dimensional information from a single STEM image acquisition scan. This is achieved by reconstructing the phase image at a specific depth in the sample, which can be performed even though the microscope may not have been focused at that depth (Fig. 3). Finally, we explore the potential for using information outside the bright‐field disc to enhance STEM imaging.