Monday 29 August
08:00

"Monday 29 August"

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OC
08:00 - 09:00

Opening Ceremony

08:00 - 08:10 EMC 2016 President. Thierry EPICIER (LYON, France)
08:05 - 08:10 EMC 2016 Vice-President. Pascale BAYLE-GUILLEMAUD (GRENOBLE, France)
08:10 - 08:20 In the name of Laurent Wauquiez, President of Région Auvergne Rhône Alpes. Nora BERRA (France)
Former ministry
08:20 - 08:30 UDL President. Khaled BOUABDALLAH (France)
08:30 - 08:35 EMS President. Roger A. WEPF (Zürich, Switzerland)
08:35 - 08:40 Sfμ President. Guy SCHOEHN (Grenoble, France)
08:40 - 08:50 For the Institut Lumière. Philippe OUDOT (lyon, France)
08:50 - 09:00 Honorary Fellowship of the RMS Award. Peter NELLIST (Professor of Materials) (Oxford, United Kingdom)
RMS President
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PL2
09:00 - 10:00

Plenary Lecture 2

09:00 - 10:00 Plenary Lecture 2. Eric BETZIG (USA)
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10:30

"Monday 29 August"

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IM2-I
10:30 - 12:30

IM2: Micro-Nano Lab and dynamic microscopy
SLOT I

Chairpersons: Francisco José CADETE SANTOS AIRES (VILLEURBANNE CEDEX, France), Niels DE JONGE (Saarbrücken, Germany), Gerhard DEHM (Düsseldorf, Germany)
10:30 - 11:00 Following nanomaterial dynamics in their formation and application media with liquid-cell transmission electron microscopy. Damien ALLOYEAU (CNRS scientist) (LMPQ, Paris Diderot, France)
Invited - Last minute change
11:00 - 11:15 #4756 - IM02-OP054 Oxidation of Carbon Nanotubes Using Environmental TEM and the Influence of the Imaging Electron Beam.
Oxidation of Carbon Nanotubes Using Environmental TEM and the Influence of the Imaging Electron Beam.

Carbon nanotubes (CNTs) can be used as field emission electron sources in X-ray tubes for medical applications [1, 2]. In a laboratory setting, field emission measurements of CNTs are usually carried out in an ultra-high vacuum system with base pressure of about 1E-7 mbar or better. Under less stringent vacuum conditions, CNTs are found to exhibit lower emission currents and reduced lifetimes [3, 4].

 

Here, we report the direct study on the structural changes in CNTs as we heated and oxidized them in situ using an aberration-corrected environmental TEM [5].  We established a protocol whereby heating and oxidation were performed without an imaging beam and changes on identifiable nanotubes were documented after purging the gas from the chamber, to ensure that they were due to the effect of gaseous oxygen molecules on the nanotubes, rather than the ionized gas species [5].  Contrary to earlier reports that CNT oxidation initiates at the end of the tube and proceeds along its length, our findings show that only the outside graphene layer is being removed and, on occasion, the interior inner wall is oxidized, presumably due to oxygen infiltrating into the hollow nanotube through an open end or breaks in the tube [5].  The CNT caps are not observed to oxidize preferentially [5, 6].

 

In the environment of an ETEM, interaction between fast electrons and gas leads to ionization of gas molecules and increased reactivity. It is very important to evaluate the results to determine or ameliorate the influence of the imaging electron beam. We found that there is a two orders of magnitude difference in the cumulative electron doses required to damage carbon nanotubes from 80 keV electron beam irradiation in gas versus in high vacuum [7]. We anticipate that experimental conditions that delineate the influence of the imaging electron beam can be established, which will enable us to study the CNT field emission process in situ in an ETEM.

 

[1]. G. Cao et al., Med. Phys. 37 (2010), pp. 5306–5312.

[2] X. Qian et al., Med. Phys. 39 (2012), pp. 2090–2099.

[3] K. A. Dean and B. R. Chalamala, Appl. Phys. Lett. 75 (1999), pp. 3017–3019.

[4] J.-M. Bonard, et al., Ultramicroscopy 73 (1998), pp. 7–15.

[5] A. L. Koh et al., ACS Nano 7(3) (2013), pp. 2566–2572.

[6] R. Sinclair et al., Advanced Engineering Materials 16(5) (2014), pp. 476-481.

[7] A. L. Koh et al., Nano Lett. 16(2) (2016), pp. 856-863.

[8] The authors acknowledge funding from the National Cancer Institute grants CCNE U54CA-119343 (O.Z.), R01CA134598 (O.Z.), CCNE-T U54CA151459-02 (R.S.) and CCNE-TD #11U54CA199075. (R.S.)   Part of this work was performed at the Stanford Nano Shared Facilities.


Ai Leen KOH (Stanford, USA), Emily GIDCUMB, Otto ZHOU, Robert SINCLAIR
11:15 - 11:30 #5548 - IM02-OP057 Nucleation of Graphene and its Conversion to Single Walled Carbon Nanotube revealed.
Nucleation of Graphene and its Conversion to Single Walled Carbon Nanotube revealed.

During catalytic chemical vapor deposition, the chirality of single wall carbon nanotubes is determined when the growing graphene nucleus wraps around the catalyst and converts into a tubular structure. Elucidating this critical process is required to develop deterministic bottom-up strategies aiming at better chiral distribution control. Direct observations of carbon nanotube growth, and theoretical modeling and simulations of the nucleation have been published but experimental atomic-resolution evidence of single-walled carbon nanotube nucleation has, until now, eluded us.

The main obstacle is that nucleation involves a few atoms only and a short time scale, thus requiring a combination of high spatial and temporal resolution for direct observation. Here, we overcome the temporal resolution constraint by reducing the growth rate in order to match the temporal resolution of our recording medium. We employ an environmental scanning transmission electron (ESTEM), equipped with an image corrector and a digital video recording system, to follow SWCNT growth using Co-Mo/MgO catalyst and acetylene (C2H2) as a carbon source (see Methods). We present atomic-resolution movies that reveal the nucleation of graphene on cobalt carbide nanoparticles followed by its transformation to a single-walled carbon nanotube. We find that the surface termination of the faceted catalyst nanoparticles regulates the nucleation of the graphene sheet and its conversion into a nanotube. Additional density functional theory calculations show that the disparity in adhesion energies for graphene to different catalyst surfaces is critical for nanotube formation: strong work of adhesion provides anchoring planes for the tube rim to attach, while weak work of adhesion promotes the lift-off of the nanotube cap (Fig. 1). [1]

[1] Nucleation of Graphene and Its Conversion to Single-Walled Carbon Nanotubes. Nano Letters. 2014, 14, 6104−6108


Matthieu PICHER (Strasbourg), Ann Lin PIN, Jose L. Gomez BALLESTEROS, Perla BALBUENA, Renu SHARMA
11:30 - 11:45 #6776 - IM02-OP074 In-situ TEM growth of single-layer boron nitride dome-shaped nanostructures catalysed by iron clusters.
In-situ TEM growth of single-layer boron nitride dome-shaped nanostructures catalysed by iron clusters.

We report on the growth and formation of single-layer boron nitride dome-shaped nanostructures mediated by small iron clusters located on flakes of hexagonal boron nitride. The nanostructures were synthesized in situ at high temperature inside a transmission electron microscope while the e-beam was blanked (Figure 1). The formation process, typically originating at defective step-edges on the boron nitride support, was investigated using a combination of transmission electron microscopy, electron energy loss spectroscopy and computational modelling. The h-BN dome-shaped nanostructure of Figure 1 was used to simulate images of BN protrusions at various angles relative to the incident electron beam, by adjusting effectively the beam direction. Figures 2 presents simulated images for beam angles of 0° (2a), 30° (2b,c) and 50° (2d), respectively, relative to the h-BN plane normal, in comparison with experimentally observed features (Figures 2e-h). The image simulations are in striking agreement with the experimental images, consistent with the circular features being protrusions formed normal to the h-BN plane, whilst the hemispheres correspond to protrusions tilted with respect to the h-BN plane.  Computational modelling showed that the domes exhibit a nanotube-like structure with flat circular caps (Figure 3) and that their stability was comparable to that of a single boron nitride layer.

       Nanostructured carbon protrusions have been studied since 2001 [1-3], but the investigation of analogous BN structures has only just begun. In the present study, we have shown that even member rings are required for the formation of h-BN dome-shaped protrusions, but not in the form of active linear defects, containing B-B and N-N bonds, as observed recently in BN monolayers under electron beam irradiation [4]. Furthermore, according to our molecular simulations result the even members rings present in the half dome structure present B-B and N-N bonds (Figure 4). The BN dome-shaped nanostructures represent a new material that perhaps by hosting metal atoms may unveil new optical, magnetic, electronic or catalytic properties, emerging from confinement effects.

[1] Sharma, R.; et al. Journal of Electron Microscopy 2005, 54, 231-237.

[2] Chamberlain, T. W.; et al. Nature Chemistry 2011, 3, 732-737

[3] Nasibulin, A. G.; et al. Nature Nanotechnolgy2007, 2, 156-161.

[4] Cretu, O.; et al Nanoletters2014, 14, 1064-1068. 


A. LA TORRE (Nottingham, United Kingdom), E. H. ÅHLGREN, M. W. FAY, F. BEN ROMDHANE, S. T. SKOWRON, A. J. DAVIES, C. PARMENTER, J. JOUHANNAUD, A. N. KHLOBYSTOV, G. POURROY, E. BESLEY, P. D. BROWN, F. BANHART
11:45 - 12:00 #6199 - IM02-OP064 Structural changes of Au nanocones during in situ cold-field emission observed by high-resolution TEM.
Structural changes of Au nanocones during in situ cold-field emission observed by high-resolution TEM.

In situ transmission electron microscopy (TEM) allows imaging on the atomic scale of complex physical phenomena, which are induced by an externally applied stimuli. This provides a directly observed correlation between material structure and properties, which promotes the understanding of the material and the triggered phenomena. Here, a Nanofactory in situ TEM biasing holder with a nanomanipulator has been used to both manipulate Au nanostructures and also to enable the studies of electron cold-field emission (CFE).

The conical shaped Au nanostructures are produced by hole-mask colloidal lithography on an electron-transparent carbon film in macroscopic short-range-ordered arrays [1]. Individual nanocones typically feature a tip radius of around 5 nm and a height of around 180 nm. The entire macro-array is then transferred to a mechanically cut Au-wire that subsequently was inserted into the in situ TEM holder (Fig. 1).

The nanomanipulator of the TEM holder can move in 3D, with both coarse and fine motion. The coarse control utilizes a slip-stick mechanism for a mm-ranged motion. The piezo-driven fine control has a range of 10 μm and a resolution on the sub-Å level. The Au nanocones in Fig. 1 were transferred to the nanomanipulator, making the configuration seen in Fig. 2. The nanomanipulator was thereafter positioned opposite a nanocone that was in direct contact with the Au-wire (Fig. 2). During the experiment, the electrical potential between the two cones was increased until the electric field around the cathode nanocone was sufficiently high (several volts per nm) to initiate CFE.

Earlier work using a similar TEM holder reports about in situ CFE experiments using carbon-based nanotips over a distance of a few hundreds of nanometer [2-4]. Here, the distance between the two Au nanocones is around 20 nm, allowing for simultaneous imaging at high resolution of both cones during CFE. This allows a better understanding of the CFE process and the effects of electron bombardment. 

At 115 V applied voltage with a CFE current, ie, of 4 μA, structural changes of the anode Au nanocone were observed. The change in structure started with a faceting at the apex of the anode nanocone. At the same time, the anode nanocone material was redistributed forming an elongated structure, making the anode nanocone thinner over a region that stretched over more than 30 nm from the tip towards the base. The elongation was a multi-stage process, taking about 5 s to complete. See images in Figs. 3a and 3b, which are separated by 0.4 s.

The electron bombardment current was kept in the μA-range and resulted in an amorphization of the outmost atomic layers of the anode apex around 7 s after the structural changes of the nanocone had occurred.

During these events, no structural changes were observed on the cathode. This indicates that the structural changes to the anode Au nanocone is an effect of electron bombardment by the emitted and accelerated electrons.

[1]      H. Fredriksson, Y. Alaverdyan, A. Dmitriev, C. Langhammer, D. S. Sutherland, M. Zäch, and B. Kasemo, Adv. Mater. 19, 4297 (2007).

[2]      L. de Knoop, F. Houdellier, C. Gatel, A. Masseboeuf, M. Monthioux, and M. J. Hÿtch, Micron 63, 2 (2014).

[3]      L. de Knoop, C. Gatel, F. Houdellier, M. Monthioux, A. Masseboeuf, E. Snoeck, and M. J. Hÿtch, Applied Physics Letters 106, 263101 (2015).

[4]      F. Houdellier, L. de Knoop, C. Gatel, A. Masseboeuf, S. Mamishin, Y. Taniguchi, M. Delmas, M. Monthioux, M. J. Hÿtch, and E. Snoeck, Ultramicroscopy 151, 107 (2015).


Ludvig DE KNOOP (Gothenburg, Sweden), Norvik VOSKANIAN, Andrew YANKOVICH, Kristof LODEWIJKS, Alexandre DMITRIEV, Eva OLSSON
12:00 - 12:15 #6859 - IM02-OP077 Shape transformations during the growth of gold nanostructures.
Shape transformations during the growth of gold nanostructures.

Liquid cell transmission electron microscopy (LCTEM) has rapidly emerged as a potent tool for understanding the dynamical processes taking place at solid / liquid interfaces. Imaging colloidal solutions with the high temporal and spatial resolutions of TEM enables understanding the growth mechanisms that control the final size and morphology of nanoparticles. Nevertheless, conclusive LCTEM experiments require understanding the effects of electron-irradiation on the nanoscale phenomena under study. Radiolytic syntheses driven by the electron beam were performed in this work to study the effects of the dose history (including the instantaneous dose rate and the cumulative dose) and the solvent nature on the shape of gold nanoparticles. The straightforward control over the concentration of reducing agents (radiolytically-produced hydrated or solvated electrons) provides mechanistic insights on the growth of highly desired nanocrystal shapes for plasmonic applications.

 

Liquid cell experiments were carried out on an aberration corrected JEOL ARM 200F operated at 200KV, by using a commercial liquid-cell holder provided by Protochips Inc. 1mM HAuCl4 in water or methanol was analyzed in a 150 nm-spacer liquid cell. Growth experiments were conducted under two extreme regimes of dose rate (over 150 electrons/Å2s and below 1 electrons/Å2s) in both TEM and STEM modes.     

 

Under high dose rate we observed the growth of dendritic nanostructures (Fig. 1a). By comparing LCTEM observations with an extended diffusion-limited aggregation model (Fig. 1b), we explicitly reveal the molecular and atomic diffusion processes that impact the shape of these dendritic nanostructures.[1] Besides the well-established link between the dose rate and the growth speed of the nanostructures,[2,3] we have demonstrated that the cumulative dose in the irradiated area can also induce drastic transitions in the growth mode of the nanostructures. For instance, high dose rate observation severely affects the concentration of precursors in and around the irradiated area, resulting in the formation of anisotropic tree-like structures over spherical nanoparticles (Fig. 1a).

 

Under low dose rate, reaction-limited growth leads to the formation of highly facetted nanoclusters. The growth is then dominated by thermodynamic effects, because the lower adsorption rate of gold atoms provides enough time for the clusters to reach an equilibrium shape that depend on intrinsic and extrinsic parameters.[3] We show that crystal defects (intrinsic parameter) or the nature of the solvent that modulates the surface energies of crystal facets (extrinsic parameters) can both drive shape transformations during the growth of the nanoparticles. Remarkably, we reveal the formation mechanisms of highly symmetric 2D and 3D nanostars enclosed by high-index facets. . These in situ studies could help in designing new seed-mediated methods or capping strategies to fabricate metallic nanostars. 

      

[1] Ahmad et al. Advanced structural and chemical imaging, submitted (2016).

[2] Woehl et al. ACS nano, 10, 8599 (2012).

[3] Alloyeau et al. Nanoletters, 15, 2574 (2015).


Nabeel AHMAD (Paris), Christian RICOLLEAU, Yann LE BOUAR, Damien ALLOYEAU
12:15 - 12:30 #6667 - IM02-OP072 Analysis to reveal dynamical and correlated atomic displacements on gold surfaces depending on various environments.
Analysis to reveal dynamical and correlated atomic displacements on gold surfaces depending on various environments.

     Gold has a wide range of important applications, such as gold nanoparticles (AuNPs) for catalyst and various gold nanostructures for sensing technology. For the applications, it is necessary to understand the chemical reaction on gold surface in actual environments, at atomic resolution, and at high time resolution. Though gold is chemically stable, it is known that the supported AuNPs of the size smaller than about 5 nm exhibit higher catalytic activity. This partially originates from the small curvature of nanoparticle surfaces, so the gold surface structures such as facets, edges and corners could change in certain environments. Here, we analyze in-situ images of the surface of bulk gold with different curvatures that are acquired using spherical aberration (Cs)-corrected environmental transmission electron microscopy (ETEM) to derive dynamical and correlated atomic displacements in various environments.

     TEM characterization is carried out by Cs-corrected Titan ETEM G2 apparatus [1], where the accelerating voltage is 300 kV. Figure 1 shows the (E)TEM images of the gold surface with relatively small curvature in various environments (vacuum, oxygen, hydrogen, and nitrogen). In vacuum, the facets of {100} and {111} and the step edge are seen clearly. In contrast, the gold surface is rough in oxygen (oxygen partial pressure: PO2 = 100 Pa), where the surface gold atoms move continuously. In hydrogen and nitrogen (PH2, PN2 = 100 Pa), the surface is facetted as well as that in vacuum and the gold atoms hardly move on the surface.

     To shed light on the change of the gold surface in oxygen, we further investigated the dynamics of surface gold atoms in oxygen by high resolution in-situ ETEM observation with an advanced image acquiring system (Figure 2). By tracking the individual gold atoms in time-lapse images, we found that gold atoms at the step edge readily migrate on the surface compared to those of the terrace surface. We further found that as the oxygen partial pressure decreases, the gold surface becomes more stable structure. We also investigated the electron irradiation effect behind the dynamical changes of surface structures in gas environments, where the current density of the electron beam is varied from 25 A/cm2 to 0.1 A/cm2. As the current density of the electron beam decreases, the migration of the gold atoms in the surface moderates. The gold surface remains rough even in the very small current density of 0.1 A/cm2. Though the electron beam affects the structural changes of the gold surface in oxygen, the analysis result suggests that the surface of bulk gold could interact with oxygen gas molecules to some extent regardless of the electron beam.

     The dynamic surface structures of metals in actual environments most likely originate from the interaction between gas molecules and metal atoms on the surface. Full understanding of the dynamical behavior of metal surface in various environments crucially important for application to the nanomaterials and nanodevices. To this end, it is useful to detect the behavior of individual metal atoms at higher temporal resolution with high detection efficiency of electrons in atomic resolution ETEM. We have already successful in capturing the extraordinary atomic migration on gold surfaces by advanced in-situ Cs-ETEM. We will show some movies in our presentation that show the dynamics of individual surface gold atoms in various environments by time resolution better than 50 ms.


Reference

[1] S. Takeda, Y. Kuwauchi, H. Yoshida, Ultramicroscopy, 151 (2015) 178.


Ryotaro ASO (Ibaraki, Japan), Yohei OGAWA, Hideto YOSHIDA, Seiji TAKEDA
Amphithéâtre

"Monday 29 August"

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IM1-I
10:30 - 12:30

IM1: Tomography and Multidimensional microscopy
SLOT I

Chairpersons: Sara BALS (Antwerpen, Belgium), Wolfgang LUDWIG (Lyon, France), Sergio MARCO (Paris, France)
10:30 - 11:00 #8328 - IM01-S31 Determining atomic coordinates in 3D by atomic electron tomography.
Determining atomic coordinates in 3D by atomic electron tomography.

At a basic level, materials properties depend on the three-dimensional arrangement of atoms, and it is necessary to determine their coordinates to make correlative measurements of structure and functionality from basic principles. Traditional 3D reconstruction techniques (such as X-ray crystallography and single-particle Cryo-Em) continue to provide critical insights into structure/property relationships but average over many identical structures. This will blur out the defects inherent to inhomogeneous nanoengineered materials important to their functionality. Aberration-corrected HR-TEM and HAADF-STEM are now indispensable techniques in materials science to examine the atomic structure of materials systems with sub-Å resolution and single atom sensitivity. Combining these new tools with powerful iterative 3D reconstruction and peak finding algorithms for electron tomography is opening a new field with the ability to determine atomic coordinates of all atoms in a structure without the assumption of crystallinity. This talk will cover recent develops and future directions of Atomic Electron Tomography (AET), which will be critical to our understanding of the atomic structure of complex materials systems.
HAADF-STEM and the equally sloped tomography method were recently used to determine the atomic coordinates of 3,769 atoms in 9 atomic layers at the apex of an etched tungsten needle (Figure 1) [1]. A tungsten point defect was unambiguously located in the material for the first time in three-dimensions. Comparing the experimental positions to the ideal bcc tungsten lattice produces the atomic displacement field with ±19 pm precision. Kernel density estimation applied to the differentiation of the displacement field was used to calculate the 6 components of the strain tensor with ~1 nm 3D spatial resolution indicating expansion along the [011] axis (x-axis) and compression along the [100] axis (y-axis). It was determined by experiments, DFT simulations and MD simulations that the strain in the lattice was due to a surface layer of tungsten carbide and sub-surface carbon. This result shows the capabilities of AET to measure atomic coordinates of inhomogeneous objects without the assumption of crystallinity providing and the capability of directly measuring materials properties.
Measurements of material structure in their native environment are now being accomplished using in-situ TEM, but has been limited by the thickness of the SiN windows and the contained liquid volume. A recent advance in this field was the introduction of the graphene liquid cell (GLC) to minimize the combined window/liquid thickness allowing observation of the growth and coalescence of colloidal Pt nanoparticles at atomic resolution [2]. It was discovered that stable NPs in the GLC were randomly rotating thus providing many orientations that could be reconstructed using methods developed in single-particle Cryo-Em. A direct electron detector and aberration-corrected HR-TEM were combined with a GLC in a technique called 3D SINGLE (3D Structure Identification of Nanoparticles by Graphene Liquid Cell EM) to determine the atomic-scale facets, lattice plane orientations and multi-twinned grain structure of a Pt nanoparticle in liquid with 2.10 Å resolution (Figure 2) [3]. The particle is constructed of three distinct regions: a central disk region of well-ordered {111} atomic planes with conical protrusions attached on each side connected by screw dislocations.
[1] Xu, R. et al., Nat Mater, 14, 1099–1103 (2015).
[2] Yuk, J. M. et al., Science, 336, 61–64 (2012).
[3] Park, J. et al. Science, 349, 290–295 (2015).


Peter ERCIUS (Berkeley, USA), Rui XU, Chien-Chun CHEN, Li WU, Mary SCOTT, Wolfgang THEIS, Colin OPHUS, Jungwon PARK, Hans ELMLUND, Alex ZETTL, A. Paul ALIVISATOS, Jianwei MIAO
Invited
11:00 - 11:15 #6438 - IM01-OP047 Environmental Transmission Electron Tomography: fast 3D analysis of nano-materials.
Environmental Transmission Electron Tomography: fast 3D analysis of nano-materials.

Modern environmental Transmission Electron Microscopes (ETEM) enables chemical reactions to be directly observed with new perspectives in the operando characterization of nano-materials. However, morphological features are essentially missing in 2-dimensional observations, thus nano-tomography under environmental conditions is a new promising challenge. Obviously, the essential condition to achieve this goal is to run fast tilt series acquisitions as compared to the kinetics of the reactions which are followed in situ in the microscope. This contribution will show that such experiments are possible by comparing the volumes respectively obtained from a classic or a fast tilt series acquisition in the bright field mode.

Firstly, simulations were performed on ghost models in order to appreciate the influence of the goniometer rotation speed during image acquisition on quality of images (sharpness and blurring effects). A typical micrograph of a nano-object, e.g. metallic nanoparticles encaged into mesoporous silicalites, was used to reconstruct a 2D model. The 1D projections were calculated according to different conditions intending to reproduce the effects of a continuous tilt during the acquisition. Figure 1 a-c) show the models at zero tilt projected perpendicularly to the tilt axis marked by a cross (the vertical direction is the projection direction); in a), a fixed image is shown as obtained at a given rotation; it is compared to images simulated by integrating a blur effect to a rotation of 3° during the acquisition, either whit a centered (b) or not centered rotation axis (c). To give an order of magnitude, a 120° rotation performed in 1 minute with acquisition of images every second without interrupting the rotation leads to an angular blur of only 2° in each image. From the 1D projection series (not shown here), 2D reconstructions were calculated using the simple Weighted-Back Projection (WBP) algorithm. Results from fig. 1 d-e) show that, at least in the case of nanoparticles with strong absorption contrast as presented here, the tomograms obtained from the blurred series are not significantly different from the constructed volume obtained from the conventional step-by-step acquisition scheme.

In a second step, we performed experimental nano-tomography experiments on Pd/Al2O3 samples deposited on holey carbon grids. Volume reconstructions shown in Fig.2 were obtained from the same object using two bright field tilt series acquired in a FEI Titan-ETEM microscope operated at 300 kV and equipped with a dedicated Fischione high-tilt sample holder. The first one was acquired through a classical step by step tilt series acquisition from 74° to + 66° with a step of 2° in mode Saxton within 45 minutes. The second one was recorded by 'fast tomography' in 150 seconds. From these data, a quantitative analysis of the Pd nanoparticles (NP) distribution and size was performed and reported in Fig. 3. Although differences obviously exist (especially, the fast tomography approach misses some of the NPs smaller than nominally 2 nm and tends to overestimate the size of the largest ones), it can be concluded that acquisitions of tilting series in very short times of the order of one minute, or even less, represent a promising way to provide 3D information on samples studied under dynamic gas and temperature conditions such as typically nano-catalysts studied in an Environmental TEM. This fast tomography approach can also be of a great interest for beam sensitive samples where the material is generally not able to bear a long exposure to the electron beam without any specific and sometimes hazardous pre-treatment or preparation.

Acknowledgements

Thanks are due to CLYM (Consortium Lyon - St-Etienne de Microscopie, www.clym.fr) for the access to the microscope funded by the Region Rhône-Alpes, the CNRS and the 'GrandLyon'.

Acknowledgments are also due to BQR project SEE3D granted by Insa-Lyon, ANR project 3DClean, Labex iMUST and IFP Energies Nouvelles for the financial support.


Siddardha KONETI, Lucian ROIBAN (MATEIS / INSA, Lyon), Voichita MAXIM, Thomas GRENIER, Priscilla AVENIER, Amandine CABIAC, Anne-Sophie GAY, Florent DALMAS, Thierry EPICIER
11:15 - 11:30 #5519 - IM01-OP038 Investigating lattice strain in Au nanodecahedrons.
Investigating lattice strain in Au nanodecahedrons.

The three dimensional (3D) structural characterization of nanoparticles is crucial in materials science since many properties heavily depend on size, surface to volume ratio and morphology.  In addition, the ability to investigate the crystal structure is just as essential because the presence of defects and surface relaxation will directly affect plasmonic or catalytic properties. A well-known example of strained nanoparticles are the so-called “nanodecahedra” or “pentagonal bipyramids”. Such particles consist of five segments bound by {111} twin boundaries, yielding a crystallographic forbidden morphology. Therefore, measuring strain fields in nanodecahedra by transmission electron microscopy (TEM) has been the topic of several studies. However, it is important to note that such studies are based on 2D projections, hereby neglecting the 3D nature of the lattice strain. [1,2] Here, we will quantify the lattice strain in 3D based on high resolution electron tomography reconstructions. [3]

 

Therefore, a continuous tilt series of 2D projection images was acquired using high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) and a dedicated alignment procedure was applied. These projection images are then used as an input for a model based tomography reconstruction algorithm. A major disadvantage of conventional reconstruction techniques is that a continuous volume is reconstructed hampering the extraction of atom coordinates without the use of dedicated post-processing methods. [4] We could overcome this limitation by assuming that the 3D atomic potential can be modelled by 3D Gaussian functions. This hypothesis significantly simplifies the reconstruction problem to a sparse inverse problem, yielding the coordinates of the individual atoms as a direct outcome of the reconstruction.

 

Visualizations of the final 3D reconstruction, obtained for a Au nanodecahedron containing more than 90,000 atoms, are presented in Figure 1.a-c along different viewing directions. Since the coordinates of the atoms are a direct outcome of the reconstruction, it becomes straightforward to calculate the 3D displacement map. We computed derivatives of the displacement map in such a manner that 3D volumes were obtained corresponding to εxx and εzz. Slices through the resulting εxx and εzz volumes are presented in Figure 1.d and Figure 1.f. Furthermore, the variation of the lattice parameters was investigated along x and z based on the same slices (Figure 1.e and Figure 1.g). Both along the x and z direction a systematic outward expansion of the lattice can be observed. The expansion along z is limited to a few of the outer atomic layers and shows an asymmetry (Figure 1.f-g) that is likely related to the fact that the decahedron is deposited on a carbon support.

 

[1] C.L. Johnson, et al., Nat. Mater. 7 (2007) 120-124

[2] M.J. Walsh, et al., Nano Letters 12 (2012) 2027-2031

[3] B. Goris, et al., Nano Letters 15 (2015) 6996-7001

[4] R. Xu, et al., Nat. Mater. 14 (2015) 1099–1103

[5] The authors gratefully acknowledge funding from the Research Foundation Flanders (project numbers G.0369.15, G.0374.13  and a post-doctoral grant to B.G. and A.D.B.). S.B. and D.Z. acknowledge the European Research Council, ERC grant N°335078 – Colouratom. The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreements 312483 (ESTEEM2).  


Bart GORIS (Antwerp, Belgium), Jan DE BEENHOUWER, Annick DE BACKER, Daniele ZANAGA, Joost BATENBURG, Anna SANCHEZ-IGLESIAS, Luis LIZ-MARZAN, Sandra VAN AERT, Jan SIJBERS, Gustaaf VAN TENDELOO, Sara BALS
11:30 - 11:45 #6057 - IM01-OP042 Three dimensional confocal imaging using coherent elastically scattered electrons.
Three dimensional confocal imaging using coherent elastically scattered electrons.

To fully understand structure-property relationships in nanostructured materials, it is important to reveal the three dimensional (3D) structure at the nanometre scale.

Scanning electron confocal microscopy (SCEM) was introduced as an alternative approach to 3D imaging in 20031. The confocal method was originally developed in optical microscopy to image the 3D structure of biological samples2. The incident beam is focused at a certain depth in a thick sample, and the excited fluorescence signal is imaged onto the detector plane through the imaging system. A small pin hole before the detector only allows the signal from the confocal plane to reach the detector and blocks the out-of-focus signal. Critically, by using a fluorescent signal, the incident and outgoing waves lose their phase relationship and an incoherent 3D point spread function can be achieved.

The optical setup in SCEM is analogous to fluorescence confocal microscopy and also requires an incoherent signal to achieve the incoherent 3D point spread function. Several groups have developed different approaches to using inelastically scattered electrons to achieve an incoherent confocal condition in the TEM3,4,5, however, difficulties remain. Core-loss electrons have a suitably limited coherence length, however, the excitation probability is extremely low which leads to a poor signal to noise ratio (SNR). Low loss electrons give a much better SNR but still have a significant coherence length due to the collective nature of the  excitation.

In this work, we introduce a different approach to achieve 3D imaging in a confocal mode by using the elastically scattered, coherent electrons. This method exploits the depth sensitivity of electrons that have suffered a specific momentum change, rather than an intensity change.  According to Fourier optics, when a thin object is inserted at a distance z above the confocal plane, the new wave function at the confocal plane will be the original probe convoluted with the Fourier transform of the object function, together with a scale factor related to z (defined in fig.1). For crystalline specimens, the Fourier transform of the object function is a set of delta functions, so a diffraction-like pattern will be generated at the confocal plane. Importantly, the separation between the diffraction spots is proportional to the distance z (see fig.1), so that the resulting diffraction contrast is very sensitive to depth. This strong depth sensitivity is combined with the very strong SNR due to the use of the elastically scattered signal. Applications to the imaging of 3D engineered nanostructures are demonstrated (fig. 2 and 3).

1. S. P. Frigo, Z. H. Levine, and N. J. Zaluzec, Appl. Phys.Lett. 81, 2112 (2002 and N. J. Zaluzec, U.S. Patent No. 6,548,810 B2 (2003).

2. T. Wilson and C. Sheppard, Theory and practice of scanning optical microscopy (Academic Press, London ; Orlando, 1984)

3. Wang, P., Behan, G., Takeguchi, M., Hashimoto, A., Mitsuishi, K., Shimojo, M., & Nellist, P. D. (2010). Phys. Rev. Lett. 104(20), 200801.

4. Xin, H. L., Dwyer, C., Muller, D. A., Zheng, H., & Ercius, P. (2013). Microsc. Microanal., 19(04), 1036-1049.

5. C Zheng, Y Zhu, S Lazar, J Etheridge, Phys. Rev. Lett. (2014) 112 (16), 166101

Acknowledgement: The authors thanks staff at the Monash Centre for Electron Microscopy. The double-aberration Titan3 80-300 FEGTEM was funded by ARC grant LE0454166.


Changlin ZHENG (Melbourne, Australia), Ye ZHU, Sorin LAZAR, Joanne ETHERIDGE
11:45 - 12:00 #5183 - IM01-OP037 Multi-modal electron tomography for 3D spectroscopic analysis using limited projections.
Multi-modal electron tomography for 3D spectroscopic analysis using limited projections.

     Electron tomography applied to spectroscopic signals in the scanning transmission electron microscope (STEM) offers the possibility for quantitative determination of structure-chemistry relationships with nanometre spatial resolution. Electron energy loss spectroscopy (EELS) and X-ray energy dispersive spectroscopy (EDS), however, often require long exposure times or high beam currents for sufficient data quality for spectral tomography. Many materials samples are not sufficiently stable under the electron beam for the prolonged irradiation times necessary for conventional tilt-series acquisition and back-projection tomographic reconstruction schemes using STEM spectrum imaging signals. Reduced dose acquisition strategies will, in general, require the use of fewer projections for tilt-series electron tomography because signals with sufficient signal-to-noise must be recorded on the respective detectors for quantitative chemical reconstructions, establishing a limit on the minimum acquisition time for individual spectrum images using current detector technologies. While methods such as compressive sensing electron tomography (CS-ET) [1] show promise for reducing the number of projections required for successful tomographic reconstructions, combining information from multiple simultaneous imaging modes in the STEM provides a complementary strategy for further reducing electron dose in spectral tomography. Simultaneously acquired signals that offer structural contrast information (e.g. ADF STEM, low-loss EELS, qualitative EDS tomography) in many cases enable the spectral tomography problem to be re-cast as a recovery problem with reduced dimensionality. The 3D reconstruction of spectral data can then be recovered quantitatively from substantially fewer spectrum images. In the case of surface plasmon modes of silver particles, ADF STEM tomography has already been applied in conjunction with EELS spectrum imaging to reconstruct the surface charge distributions [2], a two-dimensional reconstruction problem (on a surface) defined in three-dimensional spatial coordinates.

     This approach has been extended to the recovery of voxel spectra from the cloudy zone, a spinodal decomposition of Fe-Ni in the Tazewell meteorite (Figure 1). Due to minimal ADF STEM contrast, qualitative EDS tomography using the Ni K-alpha signal was analysed for structural segmentation of the sample volume. Re-projections of the extracted binarized volumes for each of the two phases were then used as a thickness-series to re-cast the recovery problem as an overdetermined system of linear equations, assuming homogeneous composition within each phase. The spectral intensity at each energy channel was decomposed according to the thickness data for each phase available at each pixel in the two-dimensional spectrum images, allowing relative spectral intensities to be attributed to the voxels assigned to each of the two phases. The resulting tomographically unmixed spectra enabled improved EDS quantification of the relative Fe-Ni ratios in each phase, giving results within 2% of quantification by atom probe tomography of similar material from the cloudy zone of the Tazewell meteorite.

     Applications to core-loss STEM-EELS analyses will be presented, further extending this family of methods to cases involving plural-scattering corrections implemented in conjunction with the linear thickness unmixing approach. Comparisons of signal unmixing determined from multi-modal structural and spectral tomography and blind-source separation methods (e.g. non-negative matrix factorization or independent component analysis) of two-dimensional spectrum image data will also be discussed.

 

References: [1] Saghi, Z.; Holland, D.J.; Leary, R.K.; Falqui, A.; Bertoni, G.; Sederman, A.J.; Gladden, L.F.; Midgley, P.A. Nano Lett., 2011, 11, 4666-4673. [2] Collins, S.M.; Ringe, E.; Duchamp, M.; Saghi, Z.; Dunin-Borkowski, R.E.; Midgley, P.A. ACS Photonics, 2015, 2, 1628-1635.

Acknowledgements: The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Program (No. FP7/2007-2013)/ERC Grant Agreement No. 291522-3DIMAGE and (No. FP7/2007-2013)/ERC Grant Agreement No. 320750-Nanopaleomagnetism as well as the European Union’s Seventh Framework Program under a contract for an Integrated Infrastructure Initiative (Reference No. 312483-ESTEEM2).


Sean COLLINS (Cambridge, United Kingdom), Joshua EINSLE, Zineb SAGHI, Robert BLUKIS, Richard HARRISON, Paul MIDGLEY
12:00 - 12:15 #6946 - IM01-OP053 Random Beam Scanning Transmission Electron Microscopy and Compressive Sensing as Tools for Drastic Electron Dose Reduction in Electron Tomography.
Random Beam Scanning Transmission Electron Microscopy and Compressive Sensing as Tools for Drastic Electron Dose Reduction in Electron Tomography.

Electron tomography is a fantastic tool for deciphering the structural information of complex 3D samples. During the last years, several tools have been developped to improve the 3D reconstruction quality of thick specimens. The direct detector cameras have incredibly increased the SNR and resolution of thin samples 2D projections, bringing electron microscopy resolution at the level of the one of X-ray diffraction studies. However, the study of thick biological samples in tomography still suffers from the too important electron dose one has to use in order to retrieve high quality images and reconstructions. It has been shown that STEM tomography can generate more accurate reconstructions than TEM tomography while preserving better the sample integrity. Previous uses of compressive sensing enabled the reduction of tilt-angles in tomography studies, unveiling electron dose reduction. Here, we push further the electron dose reduction thanks to a more effective compressive sensing method which uses incomplete images as incoherent data. The generation of incomplete images being performed at the microscope during the acquisition process where the beam randomly scans the surface of the sample.


Sylvain TREPOUT (Institut Curie / INSERM U1196), Masih NILCHIAN, Cédric MESSAOUDI, Laurène DONATI, Michael UNSER, Sergio MARCO
12:15 - 12:30 #6266 - IM01-154 Comparison of propagation-based phase contrast tomography and full-field optical coherence tomography on bone tissue.
IM01-154 Comparison of propagation-based phase contrast tomography and full-field optical coherence tomography on bone tissue.

The current huge development of new 3D microscopic techniques (synchrotron microtomography, optical coherence tomography, light sheet microscopy, …) opens a large variety of new perspectives for life sciences. The contrasts of these new microscopies are mostly well understood on samples of known material content such as those used in physics or instrumentation studies. The situation is different when it comes to the interpretation of the contrasts observed with complex heterogeneous media found in biology. Therefore determining which 3D microscopy technique is suited for which biological question is a topic of current interest (see [1,2] for instance in our group).

In this communication, we propose a comparison of the contrast observed with full-field optical coherence tomography (OCT) and propagation-based phase contrast tomography (PCT) on bone tissue at similar spatial resolution. A first comparison of OCT with standard absorption microtomography was given in [3] for bones and we extend this comparison to PCT which is known to provide enhanced contrast on bones at multiple scales [4]. The contrast of both these techniques are a priori interesting to be compared since they both rely on discontinuities of refraction index. This produces phase shift in PCT which operates in the X-ray domain with a monochromatic beam (generated by a synchrotron) while this generates direct intensity reflexion with OCT which only resorts to white light in the visible domain.

As visible in Figure 1, we specifically focussed our attention on the contrast observed in both techniques around the same bone structural unit, a so-called osteon, at a microscopic scale with images of same spatial resolution (voxel size 3.5µm). It happens that the osteons are visible in PCT while they are not perceptible with conventional absorption micro computed tomography. Also, concentric lamellae, corresponding to the so-called Harvers system, appear clearly visible in OCT while they are not perceptible with PCT at this spatial resolution. The contrast between the osteon and the surrounding bone tissue, is found in terms of homogeneous regions in PCT. However, this less spatially resolved contrast in PCT is constant throughout the sample while it is spatially variable in OCT where a continuous degradation of the contrast is observed along the direction Z of the propagation of light. We found, as given in Figure 2, that a certain spatial average of some 30 µm along Z was able to improve optimally the contrast across the concentric lamellae when inspected at the surface (up to 500 µm depth) of the sample with OCT. This contributes to establish quantitatively the complementarity of OCT and PCT for the characterization of bones at the microscopic scale.

 

References:

 

[1] Rousseau, D., Widiez, T., Tommaso, S., Rositi, H., Adrien, J., Maire, E., Langer, M., Olivier, C., Peyrin, F. Rogowsky, P. (2015). Fast virtual histology using X-ray in-line phase tomography: application to the 3D anatomy of maize developing seeds. Plant methods, 11(1), 1.

 

[2] Rositi, H., Frindel, C., Wiart, M., Langer, M., Olivier, C., Peyrin, F., Rousseau, D. (2014). Computer vision tools to optimize reconstruction parameters in x-ray in-line phase tomography. Physics in medicine and biology, 59(24), 7767.

 

[3] Kasseck, C., Kratz, M., Torcasio, A., Gerhardt, N. C., van Lenthe, G. H., Gambichler, T., . Hofmann, M. R. (2010). Comparison of optical coherence tomography, microcomputed tomography, and histology at a three-dimensionally imaged trabecular bone sample. Journal of biomedical optics, 15(4), 046019-046019.

 

[4] Peyrin, F., Dong, P., Pacureanu, A., & Langer, M. (2014). Micro-and Nano-CT for the Study of Bone Ultrastructure. Current osteoporosis reports, 12(4), 465-474.

 

Acknowledgement : This work was supported by the European Synchrotron Research Facility (ESRF, project LS-2290) through the allocation of beam time.


Sylvaine DI TOMMASO, Hugo ROSITI, Max LANGER, Carole FRINDEL, Cécile OLIVIER, Françoise PEYRIN, David ROUSSEAU (VILLEURBANNE CEDEX)
Salle Bellecour 1,2,3

"Monday 29 August"

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MS4-I
10:30 - 12:30

MS4: Complex materials and nanocomposites
SLOT I

Chairpersons: Rick BRYDSON (Leeds, United Kingdom), Marc SCHMUTZ (CNRS-UNISTRA, Strasbourg, France)
10:30 - 11:00 Cryo MEB. Roger A. WEPF (Zürich, Switzerland)
Invited
11:00 - 11:15 #5792 - MS04-OP249 Application of Cryogenic Focused Ion Beam Scanning Electron Microcopy to Hydrogel Characterisation.
Application of Cryogenic Focused Ion Beam Scanning Electron Microcopy to Hydrogel Characterisation.

Hydrogels are an important material as support matrices for cells to promote growth. These systems have been characterized by electron microscopy, through the application of fixation or sucrose embedding followed by ultramicrotomy. In this way the porosity of the gels can be assessed by transmission electron microscopy (TEM) and/or scanning electron microscopy (SEM). While this approach does give a guide to porosity of samples the absence of water in a hydrogel will have a detrimental effect and its absence distorts the dimensions of the remaining gel. Focused ion beam scanning electron microscopy (FIB-SEM) has been used on dried hydrogels [1], however, we propose a new method of gel porosity characterization by the use of cryogenic-FIB-SEM (cryo-FIB-SEM).  

Cryo-SEM is a long established technique to preserve the water content of a sample and more recently it has been demonstrated that cryo- FIB-SEM can be used for biological and soft matter materials [2]. In this work, the authors have used cryo-FIB-SEM to investigate the porosity and structure of gels whilst in the presence of water. Gels were plunge frozen in slush nitrogen or using a metal mirror freezer and transferred under liquid nitrogen to the sample shuttle of a Cryo-SEM system (Quorum PPT 2000, Quorum Technologies). In the prep-chamber, the sample was coated for 60 seconds using a Pt sputter target. The samples were then loaded into the FIB-SEM (FEI Quanta 3D, FEI). Once in the SEM chamber, the gels were prepared for FIB by deposition (3-4 seconds) of a platinum precursor from the gas injector (set to 27 °C) of the microscope.

The hydrogel samples were milled using an initial current of 1-3 nA to make a rough cut and then by further cuts at lower milling currents (0.3 nA-50 pA), to remove the common milling artefact known as curtaining. SEM micrographs of the visible milled face showed dark patches with largely white areas in between. It was initially postulated that the darker areas were the pores of the gel. In order to test this, the temperature in the SEM chamber was raised to -90 °C, leading to slow sublimation of the water at the FIB milled face (Figure 1). Over approximately 20 minutes images of the slowly subliming gel were acquired. The resulting images show a transition from the black features amongst the majority of lighter contrast through to images with inverse contrast. The final sublimed gel images are clearly interpretable as a porous gel where now the lighter contrast features are identified as the gel strands and the pores are now darker and devoid of water. With a better understanding of the location of these components the original non-sublimed images can be re-examined and the black contrast correlated directly to the polymer and the white to the water.

By inverting the contrast of the original milled face image, it is possible to give an image equivalent to the dehydrated image, but which has all the water bound and is therefore a truer representation of the gel’s morphology (Figure 2). The major advantage to this is that the sample does not undergo shrinkage, and that the process of imaging the milled face hydrated saves time. Additional slices of the freshly milled face may then be acquired to yield a series of slices suitable for 3D rendering (figure 3).

 

References:

[1] A Al-Abboodi et al., Biotech and Bioengineering, 110 (2013), p. 328.

[2] M Marko et al., J Microsc. 222 (2006), p. 42


Chris PARMENTER (Nottingham, United Kingdom), Abdulraman BAKI, Kevin SHAKESHEFF
11:15 - 11:30 #5948 - MS04-OP252 Low dose analysis of nanoparticles suspended in vitreous ice for near native state imaging.
MS04-OP252 Low dose analysis of nanoparticles suspended in vitreous ice for near native state imaging.

Most formulated fine chemical products are complex systems that contain multiple components, with nanoparticles and any incorporated surface coatings interacting with other particles and the dispersant (which can be a liquid or solid). Assessing nanoparticles when suspended in a liquid can be challenging as the particles may disperse individually, agglomerate, aggregate, sediment, chemically-alter or even dissolve and re-precipitate.  With the appropriate sample preparation however, TEM can be used to measure the dispersion and any transformation of nanoparticles suspended in, for example, biological or environmental media [1,2].

Conventional transmission electron microscopy (TEM), with samples prepared by simply drop-casting suspensions onto a thin carbon film, enables imaging and analysis of individual nanoparticles but, because of the drying process, does not capture the particle agglomeration in the dispersion or the surface chemistry when hydrated [3]. To overcome this problem we have prepared thin sections of nanoparticle suspensions for TEM by plunge-freezing a blotted grid into liquid ethane to ensure the aqueous phase vitrifies with no significant redistribution of suspended material. We have used this technique to quantify the dispersion of polymer coated quantum dots, silica and zinc oxide nanoparticles in water and biological cell culture media, identifying the true form in which these nanoparticles are taken up into cells in vitro and thereby providing mechanistic insight to the cellular response at these exposures [3,4,5].

Low dose electron microscopy of nanoparticles suspended in vitreous ice provides opportunity for the analysis of the structure and chemistry of the dispersion, both vital characteristics to understand before any successful biomedical exploitation of nanoparticles. Here, dextran coated iron oxide nanoparticles agglomerated in aqueous suspension and captured in vitreous ice were imaged by bright field TEM and analysed by energy dispersive X-ray (EDX) spectroscopy. Careful control of the illumination conditions (electron dose) permit near native state imaging and confirmation of composition before inducing significant damage to the surrounding ice matrix and subsequent movement of the particles (Figure 1). HAADF STEM imaging was conducted using a 1.3 Å probe and 60 pA probe current, with a resulting EDX map collected in just over one minute showing an iron signal appropriately localised to the nanoparticles (Figure 2).

Going forward, we will use the recently installed FEI Titan Cubed Themis 300 G2 S/TEM at the University of Leeds which is equipped with FEI SuperX EDX spectrometers, a Gatan Quantum ER imaging filter and Gatan OneView CCD to explore the limits of nanoparticle structural analysis (incorporating diffraction and lattice imaging), as well as use of STEM-EDX and electron energy loss spectroscopy for detailed elemental analysis when encased in vitreous ice. In addition to examining the dispersion state of nanoparticles in different suspensions, our goal is to identify and analyse the surface coatings on nanoparticles in the frozen hydrated state, thereby extending the capability of near native state imaging and analysis of nanoparticle suspensions by TEM.

 

 

 

1. N. Hondow, A. Brown and R. Brydson (2015) Frontiers of Nanoscience, 8, 183 – 216.

2. R. Brydson, A. Brown, C. Hodges, P. Abellan and N. Hondow (2015) J. Microscopy, 260, 238 – 247.

3.  N. Hondow, R. Brydson, P. Wang, M.D. Holton, M.R. Brown, P. Rees, H.D. Summers and A. Brown (2012), J. Nanopart. Res., 14, 977.

4. Q. Mu, N.S. Hondow, L. Krzeminski, A.P. Brown, L.J.C. Jeuken and M.N. Routledge (2012) Particle Fibre Toxicol. 9, 1.

5. R. Wallace, A.P. Brown, R. Brydson, S.J. Milne, N. Hondow, P. Wang (2012) J. Phys. Conf. Ser. 371, 012080.

 

Acknowledgment: We thank FEI for the data shown in the figures which were collected as part of a demonstration at the FEI Nanoport, Eindhoven, and Steve Evans (Swansea University) for the dextran coated iron oxide nanoparticles.


Nicole HONDOW (Leeds, United Kingdom), Michael WARD, Rik BRYDSON, Andy BROWN
11:30 - 11:45 #6121 - MS04-OP254 Scanning electron diffraction of polyethylene.
Scanning electron diffraction of polyethylene.

Microstructural investigation of light elements and highly beam sensitive polymer materials using electron microscopy is attractive for elucidating nanostructure but presents numerous challenges. In particular, heavy element staining, often used to obtain image contrast, may obscure or degrade the structure of interest and the acquisition of detailed and spatially resolved information must be balanced with damage of the specimen. Here, scanning electron diffraction (SED) has been used to analyse the crystalline microstructure of unstained polyethylene, overcoming these challenges. SED involves scanning the electron beam across the specimen and recording a diffraction pattern at each position [1] at a high frame rate to enable the data to be acquired before severe degradation of the structure has occurred. In this way, electron diffraction patterns were obtained from an unstained polyethylene sample in 5 nm steps, over areas of a few microns squared and with a 10 ms dwell time. Radiation damage was further minimised by using a high electron acceleration voltage (300kV) to minimise radiolysis and cooling the sample with liquid nitrogen [2]. The diffraction patterns, acquired at every position in the scan, were indexed and analysed by plotting the intensity of a particular reflection as a function of electron probe position to form ‘virtual’ dark field (VDF) images. VDF imaging is much more effective than conventional imaging for visualizing the microstructure of polyethylene. Clear contrast is obtained without staining and the versatile post-facto nature of VDF image formation enables multiple complementary images to to be produced from a single acquisition.
Two novel observations from our SED experiments on polyethylene are highlighted here. The sample of polyethylene was extruded from a melt so as to form ‘shish-kebab’ structures confirmed through BF images of stained microtomed sections. For our experiments, again the samples were microtomed but now unstained to avoid any influence of the stain on the diffraction patterns. The first experiment highlights a lamella-like fragment of polyethylene crystal (likely to be a part of the ‘kebab’ structure). Fig 1 shows a ‘virtual’ BF image and a sample of diffraction patterns that can only be indexed assuming the orthorhombic crystal structure of polyethylene and that the lamella is twisting about a single axis almost parallel to the vertical axis of the image. Moreover, forming consecutive VDF images made it possible to visualize each region of the crystal having a particular orientation in the twisted lamella (Fig 2). In the second experiment, we found that the sample had several micron sized islands of hexagonal polyethylene first seen as a high pressure phase [3]. However, here the patterns reveal a √3 superstructure with weak spots at the 1/3[110]* position (Fig 3). VDF images (Fig 3(b-d)) formed by these supercell reflections revealed domains within which ‘striped’ contrast can be seen; these stripes run at an orientation of approximately 120° to one another. This work demonstrates the applicability of the SED technique to highly beam sensitive materials like polyethylene and the potential for new microstructural insights to be made in this way.


[1] Moeck P. et al., Cryst. Res. Technol., 2011, 46, 586-606
[2] Egerton R. F. et al., Micron, 2004, 35, 399-409
[3] Bassett D. C., et al., Journal of Applied Physics, 1974, 4146-415

   

PAM and SJK would like to acknowledge funding under ERC Advanced Grant 291522-3DIMAGE. DNJ receives a Vice Chancellor’s award from the University of Cambridge. HJ and HT thank Ms. Makiko Ito for her help in microtoming the polyethylene samples. The authors would like to thanks Anton Jan Bons (ExxonMobil) for initiating this research and stimulating discussions.


Sungjin KANG (Cambridge, United Kingdom), Duncan JOHNSTONE, Hiroshi JINNAI, Takeshi HIGUCHI, Hiroki MURASE, Paul MIDGLEY
11:45 - 12:00 #6743 - MS04-OP258 Non-rigid image registration of low-dose image series of zeolite materials.
Non-rigid image registration of low-dose image series of zeolite materials.

Zeolites are an important group of materials with a wide range of application in the catalysis industry. Many structural studies of zeolites rely on high resolution electron microscope imaging [1]. However, due to their high sensitivity to electron irradiation, zeolites deteriorate quickly under exposure to the electron beam. Low-dose imaging techniques use a reduced electron flux to slow the crystal degradation process, which gives more time for adjustment of the microscope configuration and better control over the progression of damage. However the disadvantage of low-dose imaging is poor signal to noise ratio which is often alleviated by averaging multiple image frames in a time series for improved image quality. Traditional rigid cross-correlation function (XCF) image registration methods work well for aligning high-dose time series of radiation-robust materials which experience little or no deformation during image acquisition. However, the deformation in radiation-sensitive materials, often manifest by sample shrinkage, means that the single translational shift vector from rigid image registration may not be sufficient for aligning time series and hence a non-rigid registration scheme is needed.

 

In this work, a low-dose time series of ZSM-5 zeolite consisting 60 image frames were recorded using an aberration-corrected JEOL2200MCO TEM (Figure 1). Two registration methods, a rigid XCF registration and a new non-rigid registration, were used to align the series respectively. The non-rigid registration method [2] is assisted by an IQ factor criterion, which evaluates the quality of the averaged image of the series as the registration proceeds and selects the best averaged image as the reference for future registration iterations.

 

The results show that the new non-rigid registration is helpful for alignment of low-dose TEM image series of radiation-sensitive materials that experience deformation during imaging, especially when the number of frames is small and when the sample is already damaged (Figure 2). This implies that, for TEM image series, the non-rigid registration approach is more effective in noise suppression and in avoiding the image components of a damaged sample compromising the final averaged image.

 
For further comparison, a low-dose STEM time series of zeolite Y, was registered by both rigid and non-rigid methods. A comparative analysis of IQ factor was carried out on the averaged images and showed that the non-rigid registration consistently outperforms the rigid XCF registration (Figure 3). The reason for this advantage is attributed to the fact that the STEM images often suffer from additional scan noise due to the pixel-by-pixel acquisition in STEM imaging. 
 
 
References
[1] M. Pan and P. A. Crozier, Ultramicroscopy. 1993 48(3):332–340.
[2] B. Berkels, P. Binev, D. A. Blom, W. Dahmen, and R. C. Sharpley, Ultramicroscopy. 2014 138:46-56. 

Chen HUANG (Oxford, United Kingdom), Benjamin BERKELS, Angus KIRKLAND
12:00 - 12:15 #6980 - MS04-OP259 Three dimesional nano- and interfacial structures in the Si rich SiC systems analysed by spectroscopic electron tomography.
Three dimesional nano- and interfacial structures in the Si rich SiC systems analysed by spectroscopic electron tomography.

Silicon (Si) nanopartcles (NPs) embedded in the insulating or semiconducting matrices has attracted much interest for the third generation of photovoltaics, so called “all-Si” tandem solar cells. In this work, the amorphous Si rich silicon carbide (SRSC) absorber layers with 30% carbon content were deposited using plasma enhanced chemical vapour deposition (PECVD) on quartz substrate at 500 ˚C, and then the SRSC films were annealed at 1100 ˚C in nitrogen for 1 hour 1. The thermal treatment leads to the SRSC films spinodally decomposed into a Si-SiC nanocomposite. The nanostructures of the phase separated Si and SiC presented in the 15 minutes and 1 hour annealed SRSC films were investigated by two dimensional (2D) energy-filtered transmission electron microscopy (EFTEM). After the thermal treatment, the coexistence of crystalline Si and SiC nanoparticles (NPs) were observed from the high resolution TEM (HRTEM) images and verified by the selected area diffraction (SAD) patterns. After 1 hour annealing, neither Si nor SiC phases are complelely crystallized, the detailed morphologies of Si and SiC nanostructures were studied by electron tomography. For the first time, we make use of EFTEM spectra-imaging (SI) dataset to reveal the three dimensional distributions of Si, a-SiC and c-SiC in sub-volumes. In particular, to obtain more detailed and quantitative information, we have fitted the plasmon spectra with reference plasmon peaks. This enables us to not only to get a quantitative 3D image of all components involved in the materials system in the final tomogram, but also to obtain information about hitherto undetected phases in this system. In such energy resolved plasmon tomograms, the 3D shape of a thin amorphous SiC layer (1) was observed at interface between the crystalline Si network like structure and crystalline SiC NPs. The appearance of the a-SiC interfacial layer is expected from nucleation theory.

Acknowledgements

The authors ackonwledge the support from the EU funded FP7 Project SNAPSUN, the Knut and Alice Wallenberg Foundation and the Swedish Science Council.

References

1.        Perraud, S. et al. Silicon nanocrystals: Novel synthesis routes for photovoltaic applications. Phys. Status Solidi a-Applications Mater. Sci. 210, 649–657 (2013).


Ling XIE, Karol JAROLIMEK, Rene VAN SWAAIJ, Klaus LEIFER (Uppsala, Sweden)
12:15 - 12:30 #5927 - MS04-OP251 Understanding the complex structures in nanoglasses.
MS04-OP251 Understanding the complex structures in nanoglasses.

In recent years nanoglass materials have attracted a lot of interests due to their special physical properties, which differ significantly from traditional bulk amorphous materials of the same composition [1,2]. For example, a Sc75Fe25 nanoglass exhibited a remarkable plasticity whereas the corresponding ribbon glass was brittle [3]. It has been suggested that these special properties originate from the interfacial regions between amorphous nano domains (analogous to grain boundaries changing the properties of nanocrystalline materials), which have either a different atomic configuration or chemical composition, or both compared to the domain core. Due to the amorphous structure of the materials, the structural variations between the core of a glassy grain and the interfacial region are difficult to distinguish, especially when the composition is similar. By STEM-EDX/EELS spectrum imaging and by EFTEM imaging, such composition variations were confirmed for a number of nanoglass systems synthesized using various methods [2], for example inert gas condensation (IGC), magnetron sputtering and ultra-high vacuum (UHV) cluster deposition. On the other hand, radial distribution function (RDF), which can be extracted from electron diffraction, has proven very sensitive to the small difference in atomic configurations [4], e.g., interatomic distances and coordination numbers. The newly developed STEM-RDF mapping has been shown to be capable of resolving the different amorphous structures at nanoglass core and at the interface, respectively.

In this presentation, we investigated the Sc75Fe25 nanoglass primary particles synthesized by IGC as well as the pellet pressed at 6 GPa. The primary particles were directly collected by a carbon coated TEM grid and the pellet sample was processed by FEI strata focused ion beam (FIB) for TEM observation. From the STEM-EDX mapping on the primary particles (Fig. 1), a Sc-rich shell is clearly resolved. Quantification of the integrated spectra from the out part and from the core of the particle reveals the Sc:Fe atomic ratio being around 4:1 and 2:1, respectively. After consolidation under high pressure, the inhomogeneity in primary particles remains and leads to Sc-rich interface between the areas originated from the particle cores. Additionally, by performing STEM-nanobeam diffraction on the pressed Sc75Fe25 sample, RDF mapping was obtained and two different types of RDFs are distinguished, indicating that there exist two major components, one with higher Fe-Sc coordination number (red curve in Fig.2), and the other one with higher Sc-Sc coordination number (green curve in Fig. 2). By multiple linear least square (MLLS) fitting, corresponding component maps are constructed and shown in Fig.2. In the color-mix map, the green areas represent the interface between particle cores, where Sc-Sc bonding is dominating and the red areas represent the particle cores, where Fe-Sc coordination is considerably higher.

In nanoglass systems synthesized by other method, e.g. NiTi-Cu by magnetron sputtering [5] and NiP by electrochemical deposition, composition and structure fluctuations were also observed. The different amorphous structures constrained locally can be successfully revealed by (S)TEM spectroscopic and nanobeam diffraction methods, which is an important step towards understanding the unique structure in nanoglasses compared to the conventional glasses. The correlation between synthesis and structure of nanoglasses makes it possible to design the amorphous nanomaterials with desired functionalities.

References

1. H. Gleiter, Acta mater. 48 (2000) 1.

2. Gleiter, Beilstein J. Nanotechnol. 4 (2013) 517.

3. J.X. Fang, U. Vainio, W. Puff, R. Würschum, X.L. Wang, D. Wang, M. Ghafari, F. Jiang, J. Sun, H. Hahn, H. Gleiter, Nano Lett. 12 (2012) 5058.

4. X. Mu, S. Neelamraju, W. Sigle, C.T. Koch, N. Totò, J.C. Schön, A. Bach, D. Fischer, M. Jansen, P. van Aken, J. Appl. Cryst. 12 (2013) 1105.

5. Z.Śniadecki, D.Wang, Yu.Ivanisenko, V.S.K.Chakravadhanula, C.Kübel, H.Hahn, H.Gleiter, Materials characterization 113 (2016) 26.


Di WANG, Xiaoke MU (Eggenstein-Leopoldshafen, Germany), Chaomin WANG, Tao FENG, Aaron KOBLER, Christian KÜBEL, Horst HAHN, Herbert GLEITER
Salle Prestige Gratte Ciel

"Monday 29 August"

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MS7-I
10:30 - 12:30

MS7: Materials for optics and nano-optics
SLOT I

Chairpersons: David MCCOMB (Columbus, USA), Jérôme PLAIN (Troyes, France)
10:30 - 11:00 #8634 - MS07-S84 Characterizing Localized Surface Plasmons using Electron Energy-Loss Spectroscopy.
Characterizing Localized Surface Plasmons using Electron Energy-Loss Spectroscopy.

Localized surface plasmon resonances (LSPRs) are the coherent and collective oscillations of conduction band electrons at the surface of metallic nanoparticles. LSPRs are known to localize far-field light to a sub-diffraction-limited length scale, yielding an intense electric field at the particle surface. This effect has been harnessed to dramatically enhance light-matter interactions, leading to a variety of applications such as surface-enhanced Raman spectroscopy (SERS), photothermal cancer therapy and solar energy harvesting. Though a variety of near- and far-field optical methods are used to probe LSPRs, the spatial resolution of these methods is on the order of tens of nanometers, limiting their effectiveness. In contrast, electron energy loss spectroscopy (EELS) performed in a scanning transmission electron microscope (STEM) combines sub-nanometer resolving power with the capability to excite both optical-accessible and –inaccessible plasmon modes and therefore has emerged as one of the leading techniques (Figure 1). In this presentation, I will briefly introduce the STEM/EELS technique and demonstrate the power of STEM/EELS in the characterization of LSPRs. In addition to the traditional use of STEM/EELS for LSPR imaging, we have recently demonstrated that STEM/EELS can also be used to spatially map LSP-semiconductor energy transfer at the nanoscale. The future of STEM/EELS as a window into the nanoscopic world is especially promising, and we expect continued advances in the molecular, optical, materials, information, and energy sciences as a result.


Guoliang LI (Notre Dame, USA), Charles CHERQUI, Yueying WU, Philip RACK, David MASIELLO, Jon CAMDEN
Invited
11:00 - 11:30 #7882 - MS07-S85 Optics at the nanoscale with fast electron spectroscopies.
Optics at the nanoscale with fast electron spectroscopies.

Electron microscopy techniques have been used to probe the optical properties of materials in the subwavelength scale. In particular, it has been shown that using cathodoluminesncence (CL) and electron energy loss spectroscopy (EELS), along with a propitious choice of electron beam energy and target material, resolution below 10 nm is attainable.

In this seminar we will describe recent advances electron spectroscopies in a STEM to probe individual quantum structures. Different experiments attempting to probe their optical properties will be presented (including their lifetimes). Typically, a 1-nm-wide 60 keV electron beam was used to excite the sample. Fundamentally, we have measured spectroscopic signals (CL and EELS) and the second order correlation function (g2(t)) of the emitted light of different materials using an optical spectrometer, an EEL spectrometer and a Hanbury-Brown and Twiss interferometer.

We will start by showing how monochromated EELS and CL experiments are well adapted to probe excitonic excitations in different materials. We will show how EELS is specially adapted to probe excitons in 2-dimensional materials, using MoS2 and MoSe2 monolayers as example. For thicker materials, CL is able to more easily produce meaningful information from excitoninc excitations. As examples, we will discuss the luminescence of GaN quantum wells in AlN nanowires (Figure 1) and excitons in hBN flakes.

However, only spectroscopic information does not give a complete picture of the behavior of a specific system. One example are single photon emitters (SPE). To characterize such systems, light intensity interferometry is necessary (a typical experiment in quantum optics). We will demonstrate how a similar experiment using CL is possible, using as example the neutral Nitrogen-Vacancy center in diamond nanoparticles (Figure 2). Also, we will show how this setup allowed the detection of new SPE in hBN.

Finally, we will discuss how a CL setup with a HBT interferometer in a STEM microscope can be used to measure the lifetime of individual quantum emitters with a high spatial resolution (< 15 nm). We name this technique spatially resolved time-correlated cathodoluminescence, SRTC-CL. As an example, we will show that the lifetimes of 8 GaN quantum wells separated by 15 nm in an AlN nanowire can be measured by SRTC-CL.


Luiz TIZEI (ORSAY)
Invited
11:30 - 11:45 #4605 - MS07-OP297 Tomography of particle plasmon fields by electron energy-loss spectroscopy.
Tomography of particle plasmon fields by electron energy-loss spectroscopy.

Tailoring shape of metallic nanoparticles and alignment of nanoparticle assemblies allows controlling the properties of localized surface plasmon resonances, such as peak positions or near field-coupling and enhancement [1]. Electron beam lithography is a versatile tool for nanoparticle manufacturing, but the technique usually suffers from imperfections, surface roughness, and limited spatial resolution, which leads to particle shapes that deviate from design objectives. Similar limitations apply to chemical synthesis, which leads to nanoparticle assemblies with size dispersion and geometry variations. Therefore, to exploit the full potential of plasmonics, full 3D characterization and simulation taking into account the imperfections of real structures become mandatory. Here we present two different tomography-based approaches to understanding complex plasmonic nanoparticles created by electron beam lithography.

In our first approach the precise 3D geometry of a particle dimer fabricated by means of electron beam lithography was reconstructed through electron tomography. This full 3D morphological information was used as an input for simulations of energy-loss spectra and plasmon resonance maps (Figure 1). Here excellent agreement between measured EELS data and theory was found, bringing the comparison between EELS imaging and simulations to a quantitative and correlative level [2].

In our second approach we directly reconstruct particle plasmon fields from a tomographic tilt series of EELS spectrum images. While first approaches and demonstrations of plasmon field tomography were limited to very small particles [3–5] – small enough to neglect retardation – we lift this limitation with our approach making plasmon field tomography generally applicable to nanoparticles of all sizes [6]. Formulation EELS tomography as an inverse problem allows reconstructing the complete dyadic Green tensor for plasmonic particles, which is linked to the photonic local density of states (LDOS). Using this approach we are able to reconstruct the full 3D LDOS for a silver metallic nanoparticle (Figure 2).

This work overcomes the need for geometrical assumptions or symmetry restrictions of the sample in simulations and generalizes plasmon field tomography to particles of all sizes, paving the way for detailed investigations of realistic and complex plasmonic nanostructures.

 

[1]          S.A. Maier, Springer US, Boston, MA, 2007.

[2]          G. Haberfehlner et al., Nano Lett. 15: 7726–7730 (2015).

[3]          A. Hörl et al., Phys. Rev. Lett. 111:076801 (2013).

[4]          O. Nicoletti et al., Nature. 502: 80–84 (2013).

[5]          S.M. Collins et al., ACS Photonics. 2: 1628–1635 (2015).

[6]          A. Hörl et al., ACS Photonics. 2: 1429–1435 (2015).

 

We thank Joachim Krenn and Harald Ditlbacher for access to and support with electron beam lithography and helpful discussion. This research has received funding from the European Union within the 7th Framework Program [FP7/2007-2013] under Grant Agreement no. 312483 (ESTEEM2). We acknowledge support by the Austrian Science Fund FWF under project P27299-N27, the SFB F49 NextLite, and NAWI Graz


Georg HABERFEHLNER (Graz, Austria), Anton HÖRL, Franz P. SCHMIDT, Andreas TRÜGLER, Ulrich HOHENESTER, Gerald KOTHLEITNER
11:45 - 12:00 #5857 - MS07-OP300 Investigating Surface Plasmon-Enhanced Local Electric Fields by EELS with tunable <60meV Energy Resolution.
MS07-OP300 Investigating Surface Plasmon-Enhanced Local Electric Fields by EELS with tunable <60meV Energy Resolution.

Recent improvements in energy resolution, enabled by the use of electron energy monochromators, have the potential to turn EELS into a tool able to provide quantitative information of localized surface plasmons (LSPs), such as damping effects in single particles and electron kinetics of single plasmon modes.[1] Crucial to the prospect of quantitative analysis of LSPs is the requirement that the experimental energy resolution must be better than the natural line width of the plasmon resonances, all the while retaining high enough signal-to-noise ratio to enable an accurate determination of the properties of interest.[1] The energy resolution of EELS is customarily determined by the full-width at half-maximum (FWHM) of the zero-loss (ZL) peak. The plasmon resonances, lying in the low-loss regime, often overlap with the broad tail of the ZL peak, blurring many spectral signatures of interest. Until now, several processing techniques had to be applied to overcome these issues, relying for instance on deconvolution algorithms [2] which can introduce artifacts [3] A new generation of electron monochromators now allows for high signal-to-noise ratios while varying the energy resolution controllably, down to the 10meV regime [4].

Here we present recent results aimed at spatially and spectrally resolving the plasmon resonances of individual plasmonic nanostructures and of functional plasmonic devices using a Cs-corrected and monochromated Nion UltraSTEM 100MC (‘Hermes’) microscope with a nominal energy resolution of 10meV. Figure 1 shows spatially resolved LSP modes of two individual Ag particles with different shapes and different number of crystalline domains for the energy ranges indicated in the figure. Both shape and crystallinity appear to affect the plasmonic response. We show how the energy resolution, which also affects the attainable signal-to-noise ratio and dictates the required integration (exposure) time, can be conveniently set and tuned, depending on the inherent properties of the system of interest. Figure 2A shows as-recorded EEL spectra taken from the centre of a Ag nanowire (inset) and showing a narrow bulk plasmon resonance at 3.85eV. We note that the FWHM of the peak does not decrease with decreasing energy resolution from 40meV to 16meV, meaning that 40meV must be below the natural line width of the resonance. In this context, we will discuss the prospect of not only characterizing bare metallic nanostructures, but of also interrogating chemically functionalized plasmonic nanostructures using EELS. We note that the accessible energy ranges (sub-40 meV) also allows us to probe molecules adsorbed onto metal nanostructures. In the raw spectra in Fig. 2B taken from different locations within a sample, spectroscopic signatures of aromatic thiols chemisorbed onto multiple Ag nanoparticles are shown.[5]

 

References:

[1] M. Bosman et al., Scientific reports, 2013, 3.

[2] R. F. Egerton in “Electron Energy-Loss Spectroscopy in the Electron Microscope“, Springer (New York), 3rd Ed., 2011.

[3] E. P. Bellido et al., Microsc. Microanal., 2014, 20, 767; V. Keast and M. Bosman, Microscopy research and technique, 2007, 70, 211; S. Lazar et al., Ultramicroscopy, 2006, 106, 1091.

[4] O. L. Krivanek et al., Nature, 2014, 514, 209-212

[5] SuperSTEM is the UK EPSRC National Facility for Aberration-Corrected STEM, supported by the Engineering and Physical Science Research Council. PZE acknowledges support from the Laboratory Directed Research and Development Program at Pacific Northwest National Laboratory. WPH is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences & Biosciences.


Patricia ABELLAN (Daresbury, United Kingdom), Patrick Z. EL-KHOURY, Fredrik S. HAGE, Josh COTTOM, Alan G. JOLY, Wayne P. HESS, Rik BRYDSON, Quentin M. RAMASSE
12:00 - 12:15 #5953 - MS07-OP302 How dark are dark plasmon modes - a correlative EELS and CL study on lithographed silver nanodisks.
How dark are dark plasmon modes - a correlative EELS and CL study on lithographed silver nanodisks.

Plasmonic nanostructures enable the concentration of light to the deep subwavelength regime and, thus, are the topic of intense fundamental and application oriented research. Nanoparticles acting as optical antennas are well known for their nanoscale mode volumes, due to the excitation of localized plasmon modes. In this context electron energy-loss spectroscopy (EELS) in a transmission electron microscope (TEM) became a powerful technique as it enables to map the full modal spectrum of plasmon eigenmodes with unprecedented high spatial resolution [1–3]. Beside EELS, Cathodoluminescence (CL) has also recently been used to gain information about the optical response taking advantage of the same high spatial resolution in a TEM [4]. While it is stated that EELS is linked to the full photonic local density of states (LDOS), the CL signal is related to the radiative LDOS [5].

In this work we present a combined EELS/CL study of plasmon eigenmodes on silver nanodisks, using fast electrons in a TEM. In particular we compare the differences of the EELS and CL response using experimental and simulated data. Precise variation of the disk size is achieved by means of electron beam lithography (Figure 1a), enabling a comprehensive study of plasmon excitations on silver nanodisks.

From theoretical considerations it is known, that for certain particle geometries (and therefore for specific surface charge distributions) there exist so called dark modes, which are “invisible” to photons but “visible” to electrons, and therefore can be measured with EELS but not with light [6]. Here we discuss how dark these dark modes are comparing EELS and CL (figure 1b+c). In particular, radial breathing modes (C in figure 1b+c) were predicted to be dark modes [6], although we will show how comparison between EELS and CL can mitigate this statement. Additionally, limitations for the theoretical predictions will be discussed, when the particle size is increased and therefore retardation effects become more important. In this case we show that dark modes are getting brighter. Furthermore symmetry breaking by the excitation source itself, a focused fast electron beam, will be discussed.

 

[1]          Nelayah et al., Nat. Phys. 2007, 3, 348−353.

[2]          Koh et al., Nano Lett. 2011, 11, 1323−1330.

[3]          Schmidt et al., Nat. Commun. 2014, 5, 4604.

[4]          Yamamoto et al., Phys. Rev. B 2001, 64, 205419.

[5]          Losquin et al., ACS Photonics 2015, 2, 1619–1627.

[6]          Schmidt et al., Nano Lett. 2012, 12, 5780−5783.

 

This research has received funding from the European Union within the 7th Framework Program [FP7/2007-2013] under Grant Agreement no. 312483 (ESTEEM2). We acknowledge support by the Austrian Science Fund FWF under project P21800-N20, the SFB F49 NextLite, and NAWI Graz.


Franz-Philipp SCHMIDT (Graz, Austria), Arthur LOSQUIN, Ferdinand HOFER, Joachim R. KRENN, Mathieu KOCIAK
12:15 - 12:30 #6630 - MS07-OP306 Systematic analysis of plasmon excitations and coupling by lithographic structure patterning and fast, monochromated STEM-EELS mapping.
MS07-OP306 Systematic analysis of plasmon excitations and coupling by lithographic structure patterning and fast, monochromated STEM-EELS mapping.

High energy resolution electron energy-loss spectroscopy (EELS) is now a recognised technique for mapping localized surface plasmon resonances and measuring their resonant energies with a nanometric spatial resolution [1–3]. In contrast to light-based techniques it can further excite and map high order harmonics that are optically forbidden. Here we move on from the study of isolated or tandem plasmonic structures randomly deposited on TEM grids, or more complex structures painstakingly patterned by FIB, to a fast, systematic EELS mapping of precisely patterned Au and Ag films on Si3N4 membranes. Following an electron lithography-based preparation, the spatial distributions and mode energies of plasmon resonances and coupling are studied in function of well-controlled variations in structure dimensions.

The measurements are made in STEM-EELS mode using a FEI Titan Themis 60-300 with Gatan Digiscan and GIF Quantum ERS spectrometer. The combination of the X-FEG gun and monochromator gives a sub-nm incident beam with 100–110 meV FWHM of the zero-loss peak and a current of up to 240–250 pA. A fundamental need of the work is that the plasmon excitation can be measured both in the pure Si3N4 membrane regions and in the metal film regions. To this end, a high tension of 300 kV is used because, by reducing the relative intensity of bulk plasmon scattering from the metal films, it improves surface plasmon excitation signal to noise. EELS data are normalized by the zero-loss intensity to give the true projected plasmon distribution, without “shadowing” by the metal film [4]. With the fast spectrum imaging mode and high beam current, dwell times are only 0.2–0.25 ms per pixel, allowing us to acquire maps with >105 pixels (e.g. 600 x 600 px) in < 10 minutes per map.

Applying this fast mapping with high spatial sampling to the lithographically-based structures of known layout and dimension gives a highly time-efficient method for studying plasmonic excitations in nanophotonic structures. Figure 1 shows example plasmon resonance low-loss EELS spectra and intensity maps. Parts (a–d) & (e–f) treat the well known plasmonic structures of a silver wire and nano-triangle. Owing to the energy resolution and good signal to noise ratio, there is no need to perform systematic deconvolution of the data to reveal plasmon excitations, even for modes at < 0.5 eV energy loss. High order multipoles are additionally well resolved for both non-penetrating and penetrating trajectories, such as the 2.8 eV breathing mode of the nano-triangle.

Figure 1 (g–h) shows data from more complex coupled gold heptamer apertures. We probe the effect of a nanoscale defect: a small 20 nm size gap in one of the heptamer arms. This feature induces the appearance of an additional low energy mode at 0.84 eV, and affects both the symmetry and intensity of higher order modes. This demonstrates how systematic study of varying geometries allows for in-depth analysis of plasmon resonances.

These experimental studies are combined with modeling of the excitations using a novel “in house” method which aids interpretation of multi-body interactions by simulating EELS spectra using the properties of plasmonic structures, and not the work done on electrons [5]. The simulations and experiments have a symbiotic relationship, with the modeling used to interpret experimental data, and the experimental data used to guide the modeling (e.g. for peak resonance values). Nevertheless, our experimental approach is significantly quicker than the modeling. While so far it is primarily applied to the systematic study of particles and apertures, in the future it will be used to explore the optical excitations of novel nanophotonic structures and materials.

 

References and Acknowledgements

[1] J. Nelayah et al., Nature Phys. 3 (2007) 348–353.

[2] M. Bosman et al., Nanotechnology 18 (2007) 165505.

[3] F.J. García de Abajo, Rev. Modern. Phys. 82 (2010) 209–275.

[4] N. Le Thomas et al., Phys. Rev. B 87 (2013) 155314.

[5] G.D. Bernasconi et al., J. Opt. Soc. Am. B 33 (2016) 768–779.

We thank the staff of the Center of Micro/Nanotechnology (CMI) of EPFL for support. This research was in part funded by the European Commission (FP7-ICT-2011-7, NANO-VISTA, under Grant Agreement No. 288263).


Valentin FLAURAUD, Gabriel BERNASCONI, Jérémy BUTET, Olivier MARTIN, Jürgen BRUGGER, Duncan ALEXANDER (Lausanne, Switzerland)
Salle Gratte Ciel 1&2

"Monday 29 August"

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IM5-I
10:30 - 12:30

IM5: Quantitative imaging and image processing
SLOT I

Chairpersons: Joanne ETHERIDGE (Director) (Melbourne, Australia), Jean-Christophe OLIVO-MARIN (Paris, France)
10:30 - 11:00 #6670 - IM05-S44 Retrieving atomic structure from dynamical rocking curve measurements in both real and reciprocal space.
Retrieving atomic structure from dynamical rocking curve measurements in both real and reciprocal space.

Transmission electron microscopy (TEM) data, in particular high-resolution TEM and conventional electron diffraction has the reputation of being not easily interpretable in a quantitative manner in terms of the object being probed by the fast electrons. The reason for this lies in the fact that multiple scattering makes the detected signal a non-linear function of the scattering potential. In cases where the structure is approximately known, refinement of structure factors from convergent beam electron diffraction (CBED) data [1] or atom positions from HRTEM images [2] is possible. But the ab-initio inversion of multiple scattering to recover the structure of an unknown object has not yet been shown to work in a routine manner for experimental data. Structure determination approaches thus typically revert to techniques which collect tilt-averaged data, such as precession electron diffraction (PED) and scanning transmission electron microscopy (STEM) with a convergent probe. Integrating the signal over a range of different relative orientations between object and electron beam wave vector averages over different multiple scattering conditions – however, a large amount of structural information encoded in the multiple scattering signal gets lost, including valuable information about the object’s 3D structure [3].  

Fig. 1 demonstrates that in the case of experimental large-angle rocking-beam electron diffraction (LARBED) data [4], i.e. diffraction data for a large range of beam tilts (about 20 times larger than possible in conventional CBED of silicon), the projected potential can be recovered by straight forward gradient optimization from a starting guess in which all structure factors were initialized to the same value, i.e. initially, all Ug = (0.01 + 0.01i) Å-2 [5]. Although rocking curves of only 121 diffraction spots were measured, 456 structure factors could be determined from this data, since, all possible difference vectors between reflections extracted from the diffraction pattern were also included in the dynamical diffraction calculation.

Although CBED and HRTEM are very different modes of operation of the microscope, the multiple scattering contribution to the signal in tilt series of HRTEM images allows us to retrieve the 3D scattering potential [6], where the position and height of peaks allows direct interpretation as atom species and positions (see Fig. 2). This inversion of multiple scattering is based on an interpretation of the multislice algorithm as an artificial neural network that is taught by feeding it TEM data recorded under different experimental conditions. This can be HRTEM tilt series, ptychography data sets, or scanning confocal electron microscopy (SCEM) data [7].[8]

[1] J.M. Zuo, M. Kim, M. O'Keeffe, and J C.H. Spence, Nature 401 (1999) p. 49.
[2] G. Möbus, M. Rühle, Ultramicroscopy 56 (1994) 54-70
[3] R.S. Pennington, W. Van den Broek, C.T. Koch, Phys. Rev. B 89 (2014) 205409
[4] C.T. Koch, Ultramicroscopy 111 (2011) 828.
[5] F. Wang, R.S. Pennington, C.T. Koch (2016) submitted.
[6] W. Van den Broek and C.T. Koch, Phys. Rev. Lett. 109 (2012) p. 245502.
[7] W. Van den Broek and C.T. Koch, Phys. Rev. B 87 (2013) 184108
[8] The authors acknowledge funding from the German Research Foundation (DFG) as well as the Carl-Zeiss Foundation.


Christoph KOCH (Berlin, Germany), Wouter VAN DEN BROEK, Feng WANG, Robert PENNINGTON
Invited
11:00 - 11:15 #6511 - IM05-OP112 Efficient and quantitative phase imaging in two- and three-dimensions using electron ptychography in STEM.
Efficient and quantitative phase imaging in two- and three-dimensions using electron ptychography in STEM.

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.

[1] P.D. Nellist, B.C. McCallum and J.M. Rodenburg, Nature 374 (1995) 630-632.

[2] T.J. Pennycook et al., Ultramicroscopy 151 (2015) 160-167.

[3] H. Yang et al., Ultramicroscopy 151 (2015) 232-239.

[4] J.M. Rodenburg and R.H.T. Bates, Philosophical Transactions of the Royal Society of London A, 339 (1992) 521-553.

[5] M.J. Humphry, B. Kraus, A.C. Hurst, A.M. Maiden, J.M. Rodenburg, Nat Commun, 3 (2012) 730.

[6] The authors acknowledge funding from the EPSRC through grant number EP/M010708/1.


Peter NELLIST (Oxford, United Kingdom), Hao YANG, Lewys JONES, Gerardo MARTINEZ, Reida RUTTE, Benjamin DAVIS, Timothy PENNYCOOK, Martin SIMSON, Martin HUTH, Heike SOLTAU, Lothar STRUEDER, Ryusuke SAGAWA, Yukihito KONDO, Martin HUMPHRY
11:15 - 11:30 #6213 - IM05-OP109 Atom-Resolved STEM Imaging Using a Segmented Detector.
Atom-Resolved STEM Imaging Using a Segmented Detector.

    In scanning transmission electron microscopy (STEM), differential phase contrast (DPC) imaging has been developed to visualize the local electromagnetic field distribution in materials at medium resolution [1, 2]. The electromagnetic field deflects the incident electron beam, and this deflection can be measured by taking the difference between signals detected in opposing detector segments. Recent rapid progress in high-sensitive segmented detectors has enabled DPC STEM imaging to be performed at atomic-resolution [3]. However, DPC STEM images are sensitive to thickness and defocus, because dynamical scattering strongly affects DPC imaging of crystals in zone axis orientations [4]. It thus remains a challenge to develop a practical imaging technique at atomic-resolution with the segmented detector.

    Fig. 1 shows images of SrTiO3 simultaneously obtained by different segments on a new segmented annular all field detector (SAAF2) installed in an aberration-corrected STEM (JEOL JEM-300F, 300kV). The relative orientation of the detector and the crystal structure is shown in Fig. 2. The images were at a defocus value of -3.7 nm relative to the defocus condition giving maximum contrast in annular dark field (ADF) imaging. These 512×512 pixel images were recorded with a dwell time of 38 µs per pixel, so the total imaging time is about 10 seconds. Each segment image can be qualitatively interpreted by electron beam deflection due to electric field from nuclei, including from the light oxygen atomic columns, though dynamical effects should be taken into account. According to image simulations, the DPC image appearance is largely unchanged with sample thickness if a defocus value is selected to obtain the highest contrast DPC image. This suggests that DPC STEM imaging at atomic resolution with a proper defocus value may be a new robust imaging mode that enables visualization of atomic column positions, including for light elements. Furthermore, this new imaging mode may contain information on charge redistribution due to charge transfer or orbital hybridization.

    In addition, we have found that the detector is sensitive enough to allow both segmented annular dark field imaging and DPC STEM imaging of single atoms to be performed. The details will be discussed in the presentation.

 

References

[1] J. N. Chapman et al., Ultramicroscopy 3, 203 (1978).

[2] M. Lohr et al., Ultramicroscopy 117, 7 (2012).

[3] N. Shibata et. al., Nat. Phys. 8, 611 (2012).

[4] R. Close et. al., Ultramicroscopy 159, 124 (2015).

[5] This work was supported by PRESTO and SENTAN, JST, and the JSPS KAKENHI Grant number 26289234. A part of this work was supported by Grant-in-Aid for Scientific Research on Innovative Areas (25106003). A part of this work was conducted in the Research Hub for Advanced Nano Characterization, The University of Tokyo, under the support of "Nanotechnology Platform" (Project No.12024046) by MEXT, Japan. This research was supported under the Discovery Projects funding scheme of the Australian Research Council (Project No. DP110101570).


Takehito SEKI (Tokyo, Japan), Gabriel SANCHEZ-SANTOLINO, Nathan LUGG, Ryo ISHIKAWA, Scott D. FINDLAY, Yuichi IKUHARA, Naoya SHIBATA
11:30 - 11:45 #5246 - IM05-OP104 ISTEM: A Realisation of Incoherent Imaging for Ultra-High Resolution TEM beyond the Classical Information Limit.
ISTEM: A Realisation of Incoherent Imaging for Ultra-High Resolution TEM beyond the Classical Information Limit.

The ISTEM (Imaging STEM) method [Phys. Rev Lett. 113, 096101(2014)] presented here constitutes a novel way for the realisation of TEM imaging with spatially incoherent illumination. It is well-known that such incoherent image formation allows for an increased resolution and higher robustness towards chromatic aberrations compared to coherent illumination as used in conventional TEM (CTEM). This has been realised in scanning TEM (STEM) via reciprocity, which however suffers from other resolution-limiting factors such as scan noise or the finite extent of the electron source.

The ISTEM mode circumvents these problems entirely. It combines STEM illumination with CTEM imaging as illustrated in Fig. 1: A camera is used to acquire images formed by the focused electron probe that is scanning over the specimen while the imaging system is in imaging mode. With an exposure time chosen equal to the STEM scan time, the resulting image corresponds to a sum over the images of all probe positions. Because different specimen positions are illuminated at different times, the corresponding intensities are summed up incoherently. ISTEM is therefore a spatially incoherent imaging mode and benefits from the associated improvement of resolution. Beyond this simple explanation, the equivalence of the ISTEM illumination and CTEM with an extended and incoherent electron source can be furthermore rigorously shown mathematically within the mutual intensity formalism. From this, the gain of resolution can be intuitively understood in the limit of total incoherence, in which case the transfer is given by the autocorrelation of the coherent transfer function. The theoretical considerations also show that neither scan noise nor source size have any influence on ISTEM-images. Aberrations and defocus of the condenser system cancel out completely as well. ISTEM imaging is therefore independent from probe correction.

In simulative studies the capability of ISTEM to extend the point resolution beyond the diffraction limit, its robustness towards temporal incoherence and the resulting possibility to overcome the information limit are demonstrated. These calculations are confirmed by experimental ISTEM-micrographs of GaN in [11-20] and [1-100] projection, as presented in Fig. 3, which are found in good agreement with simulations. For the [1-100] direction neighbouring gallium and nitrogen columns at a distance of only 63 pm are resolved despite an information limit of 83 pm of the image-corrected FEI TITAN 80/300 G1 microscope used for the acquisition. The classical information limit is thereby clearly overcome by 24%.

A further study was conducted that compared the results of strain-state analysis from simulated ISTEM images of a strained InGaAs-crystal with annular dark-field and bright-field STEM micrographs simulated for a probe-corrected microscope. The results are displayed in Fig. 2. They promise a significant increase in precision for ISTEM compared to STEM, due to the immunity to both scan noise and source size clearly recognisable by the smaller error bars. This was experimentally confirmed by an ISTEM study of PbTiO3 in which the heavy Pb and TiO atomic columns as well as the lighter oxygen columns are clearly resolved  and the evaluation based on a parametrically fitted model yields a significantly increased precision for position measurement compared to aberration corrected STEM images which were acquired from the same sample area.

With the help of the principle of reciprocity, ISTEM can finally be made equivalent to any STEM mode by appropriate choice of objective and condenser aperture, with the difference that ISTEM images will show no scan noise whatsoever. This will allow for the realisation of e.g. annular bright-field STEM, holding out the prospect of ultra-high resolution imaging of even lightest elements.


Florian F. KRAUSE (Bremen, Germany), Marco SCHOWALTER, Thorsten MEHRTENS, Knut MÜLLER-CASPARY, Armand BÉCHÉ, Karel W. H. VAN DEN BOS, Sandra VAN AERT, Johan VERBEECK, Andreas ROSENAUER
11:45 - 12:00 #6649 - IM05-OP113 Dark-Field Imaging with Electron Backscatter Diffraction Patterns.
Dark-Field Imaging with Electron Backscatter Diffraction Patterns.

Dark-field (DF) imaging can be performed by selecting a specific diffracted beam in the selected area diffraction pattern in conventional transmission electron microscope (CTEM) or in the convergent beam electron diffraction pattern in scanning transmission electron microscopy (STEM) mode [1]. The resultant micrograph provides high intensity of the objects in the probed volume that diffract in this particular direction. In contrast, dark-field micrographs can be obtained in STEM mode by capturing the signal from a specific range of scattering angles, with the most representative example being the high-angle annular dark-field imaging (HAADF) [2]. This leads to a contrast mostly based on atomic number differences between the different objects analysed [3].

These techniques were developed originally for CTEM and STEM. Because DF based on scattering angles is technically easy to obtain in a scanning electron microscope (SEM) by collecting the transmitted/diffracted signals with an electron detector below the thin specimen, it has been implemented in SEMs seriously since several years. This permitted taking advantage of the high contrast and low beam damage obtained at low accelerating voltages STEM in the SEM is now routinely achieved with a spatial resolution close to 1 nm in field-emission SEMs [4]. Despite these new possibilities, DF imaging only based on diffracted beams has not been achieved yet in a SEM.

The mostly used diffraction technique in the SEM has been, since the discovery of Venables [5], electron backscatter diffraction (EBSD) which has a spatial resolution of roughly 20-30 nm and which needs a limited bulk surface preparation compared to CTEM or STEM. EBSD is assumed to be related to the electron channeling pattern (ECP) diffraction technique by the reciprocity theorem [6], although its angular resolution is, at this time, limited by the pixel resolution of the acquisition equipment. Figure 1 is a comparison between an ECP and an EBSP acquired at 20 kV from a [001] (001) silicon wafer. In this work, pseudo-Kikuchi patterns (EBSP) recorded via EBSD were stored and reprocessed by reporting pixels or clusters of pixels intensities from a specific location in a reference EBSP to reconstruct the final image (EBSD map). A resulting micrograph (called EBSD-DF image) was produced with a direct link to the diffracted beams in the EBSP and hence, to the crystallography of the sample, i.e., a DF image. The origin of the contrast is then similar to that of electron channeling contrast image (ECCI) as shown in Figure 2, in which EBSD-DF micrographs of an indented compressed iron specimen with different reflections are displayed. However, the post-acquisition processing is an invaluable advantage over ECCI because it allows generating multiple micrographs at the same time with only one set of EBSPs recorded in a beam raster fashion. This opens new ways of extracting and using the information contained in each EBSP and the main applications, at this point, are understanding deformation behaviors and interpretation [7] of channeling contrast [8].

References:

[1] D.B. Williams and C.B. Carter, Transmission electron microscopy: a textbook for materials science. 2009: Springer.

[2] S. Pennycook, Ultramicroscopy, 30 (1989), pp. 58-69.

[3] O.L. Krivanek et al, Nature, 464 (2010), pp. 571-574.

[4] P.G.Merli et al, Microscopy and Microanalysis, 9 (2003), pp. 142-143.

[5] J. Venables and C. Harland, Philosophical Magazine, 27 (1973), pp. 1193-1200.

[6] O.C. Wells, Scanning, 21 (1999), pp. 368-371.

[7] N. Brodusch, H. Demers, and R. Gauvin, Ultramicroscopy, 148 (2015), pp. 123-131.

[8] S. Kaboli et al, Journal of Applied Crystallography, (2015), 48, pp. 776-785.


Raynald GAUVIN (Montreal, Canada), Hendrix DEMERS, Nicolas BRODUSCH
12:00 - 12:15 #6372 - IM05-OP110 Accurate and precise measurement of cluster sizes in localisation microscopy images using the Rényi divergence.
Accurate and precise measurement of cluster sizes in localisation microscopy images using the Rényi divergence.

Localisation microscopy is a super-resolution imaging technique based on detecting randomly activated single molecules in a sequence of images. A super-resolution image is then reconstructed as a collection of discrete points, using all of the localised single molecule positions. Clustering analysis of these points can provide quantitative information about sample structure, size of features and/or their number. The information obtained from clustering analysis allows characterisation of the functions or properties of biological systems, for example examining signalling pathways in T-cells antigen receptors. However, quantitative analysis of clusters in localisation microscopy images is challenging because the clusters are usually small and surrounded by relatively high noise.

The Rényi divergence quantifies differences between two distributions (in this case the observed data and a reference distribution). Its sensitivity to the degree to which one distribution differed from another can be tuned with a scaling parameter α, which allows us to adapt its robustness to noise. We approximated the data distribution by counting all points which were positioned closer to each other than a set threshold and the reference distribution as all the points concentrated in a single cluster. Ripley's K function, which is widely used for performing localisation microscopy analysis, is a special case of the Rényi divergence.

Here we present a comparison of the accuracy and precision of cluster size measurement performed using the Rényi divergence and Ripley's K function. Initial tests involved establishing noise adaptability of the two analysis methods using simulated data sets with characteristics similar to experimental images (with increasing noise levels), and optimising the tunable parameter of the Rényi divergence. We find that the adaptability of the Renyi divergence method is a particular advantage when dealing with localisation microscopy data, in which characteristics can vary a great deal between datasets. 


Adela STASZOWSKA (London, United Kingdom), Patrick FOX-ROBERTS, Susan COX
12:15 - 12:30 #5957 - IM05-OP106 STEM imaging of atom dynamics: novel methods for accurate particle tracking.
STEM imaging of atom dynamics: novel methods for accurate particle tracking.

Developments in scanning transmission electron microscopy (STEM) have opened up new possibilities for time-resolved imaging at the atomic scale. Recent examples include a study of the diffusion of dopant atoms in semiconductors [1] and, using environmental STEM, in situ studies of catalytic reactions [2]. Rapid imaging of single atom dynamics brings with it a new set of challenges. High frame rates and long total acquisition times mean novel methods are needed for handling and processing “big data” sets. Further, the need for short exposure times leads to severe problems with noise, but by exploiting the spatial and temporal correlations between frames, it is possible to considerably improve the signal-to-noise ratio using a method known as singular value thresholding [3]. Crucially, by employing robust procedures to automatically estimate the noise and motion characteristics, it is possible to optimize the process with little user input (Figure 1a,b). The identity and positions of individual atoms in the denoised data can then be determined using a newly-developed intensity-based classification algorithm. Building on the theme of automation, the classifier can be trained using simulated STEM images to robustly process long image sequences, where manual identification would be prohibitive.

As an example, we have applied these methods to investigate the diffusive behaviour of copper atoms on the (110) surface of silicon. The noise removal and atom identification steps are used along with particle tracking software [4] to extract a set of atomic trajectories from a series of annular dark-field STEM images (Figure 1c-e). The form of these trajectories can be related to the underlying silicon substrate, as in Figure 1f, which suggests the existence of preferred pinning sites for copper atoms. The interaction between adatoms and the substrate can be explored with unprecedented spatio-temporal resolution using rapid imaging, and interpreted by modelling with density functional theory (DFT) calculations (Figure 1g). This highlights the potential for combining time-resolved STEM with theory, forming a powerful approach to investigating and understanding the dynamic behaviour of materials at the atomic scale.

References

[1] Ishikawa R, et al. (2014). Phys. Rev. Lett. 113, 155501.

[2] Gai P, et al. (2014). Chemical Physics Letters. 592, 355-359.

[3] Furnival T, Leary R, Midgley PA. (2016). Manuscript submitted.

[4] Chenouard N, et al., (2013). IEEE Trans. Pattern Anal. Mach. Intell., 35, 2736-2750.

The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement 291522-3DIMAGE.


Tom FURNIVAL (Cambridge, United Kingdom), Eric SCHMIDT, Rowan LEARY, Daniel KNEZ, Ferdinand HOFER, Paul D BRISTOWE, Paul A MIDGLEY
Salle Tête d'or 1&2

"Monday 29 August"

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LS6-I
10:30 - 12:30

LS6: Interactions micro-organism-host
SLOT I

Chairpersons: Ilaria FERLENGHI (Structural Based Antigen Design) (Siena, Italy), Kay GRUNEWALD (Oxford, United Kingdom), Olivier LAMBERT (Bordeaux, France)
10:30 - 11:00 #8391 - LS06-S19 A huge but elusive macromolecular cage in enterobacterial stress response as seen by cryoEM and X-ray crystallography.
A huge but elusive macromolecular cage in enterobacterial stress response as seen by cryoEM and X-ray crystallography.

The E. coli inducible lysine decarboxylase LdcI is an important acid stress response enzyme, whereas the AAA+ ATPase RavA is involved in multiple stress response pathways. A complex between these two proteins is thought to counteract acid stress under starvation in E. coli. We solved the crystal structure of the RavA monomer and combined it with a negative stain EM reconstruction of the RavA-ADP hexamer (El Bakkouri et al., 2010). We also determined the crystal structure of the LdcI double pentamer (Kanjee et al., 2011). While these structures provided important insights into the functions of these individual proteins, the design principles of the LdcI-RavA complex appeared even more enigmatic because it was difficult to envision how a decamer can bind a hexamer.  CryoEM analysis allowed us to fit together the pieces of the jigsaw and revealed that the LdcI-RavA complex is a surprising macromolecular cage of the size of a ribosome formed by two LdcI decamers and five RavA hexamers (Malet et al., 2014). We identified molecular determinants of this interaction and specific elements essential for complex formation, as well as conformational rearrangements associated with the pH-dependent LdcI activation and with the RavA binding (Malet et al., 2014; Kandiah et al., 2016). Moreover, we uncovered differences between the LdcI and its close paralogue, the second E. coli lysine decarboxylase LdcC thought to play mainly a biosynthetic role, finally explaining why only the acid stress response enzyme is capable of binding RavA and forming the cage. Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions (Kandiah et al., 2016). Research on structure-functional relationships of this system by a combination of cryoEM, super-resolution fluorescence microscopy and other structural, biochemical, biophysical and cell biology techniques is on-going.

Kanjee, U. et al. EMBO J. 30, 931–944 (2011).

El Bakkouri, M. et al. Proc. Natl. Acad. Sci. U. S. A. 107, 22499–22504 (2010).

Malet, H. et al. eLife 3, e03653 (2014).

Kandiah, E. et al. Sci Rep. 6, 24601 (2016).

Acknowledgements

We thank Guy Schoehn for establishing and managing the cryo-electron microscopy platform and for providing training and support. We are grateful to all members of the Houry group and the Gutsche team who were or currently are involved in sample preparation and analysis for this study. For electron microscopy, this work used the platforms of the Grenoble Instruct center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The electron microscope facility (Polara electron microscope) is supported by the Rhône-Alpes Region (CIBLE and FEDER), the FRM, the CNRS, the University of Grenoble Alpes and the GIS-IBISA. DC is the recipient of the Grenoble Alliance for Integrated Structural and Cell Biology (GRAL) PhD fellowship. This work was supported by the ANR-12-JSV8-0002 and the ERC 647784 grants to IG, the CIHR MOP-130374 to WAH, the ANR-10-LABX-49-01 and the ANR-11-LABX-0003-01.


Eaazhisai KANDIAH, Hélène MALET, Diego CARRIEL, Julien PERARD, Maria BACIA, Walid A HOURY, Sandrine OLLAGNIER DE CHOUDENS, Sylvie ELSEN, Irina GUTSCHE (IBS, Grenoble)
Invited
11:00 - 11:30 #8370 - LS06-S20 Structure of the bacterial type 3 secretion system in action.
Structure of the bacterial type 3 secretion system in action.

Pathogenic bacteria commonly use the conserved type 3 secretion system (T3SS or Injectisome) to deliver virulence factors into the target eukaryotic cells in order to manipulate their functions. Injectisome is a multi-protein nanomachine assembled at the bacterial membranes containing the periplasmic basal body, cytoplasmic sorting platform and export apparatus1. A hollow needle extends from the periaplasmic part to the extracellular face serves as a channel for the secreted molecules.  The high resolution structure of the components of the injectisome is well understood: structure of the basal body at subnanometer resolution2 in combination with computational molecular analysis3 demonstrated the inter-subunit interaction interfaces and suggested a conserved oligomerization mechanism for the assembly. The cytoplasmic ATPase SctN (according to the general nomenclature) is structurally similar to the F- and V- type ATPases and is believed to be responsible for detachment of chaperones and unfolding the exported substrates4.

Recent fluorescent studies in Yersinia enterocolitica showed turnover of SctQ; the turnover was faster for secreting injectisomes therefore the injectisome was suggested to be a highly dynamic nanomachine5.  Additionally, upon activation of secretion newly formed injectisomes are established next to the existing ones6. Interestingly, trapping the exported substrate in the isolated needle complex (made of the basal body and the needle) results in only small conformational changes distant from the secretion path7. It is highly interesting to visualize the mechanics of substrate secretion happening inside the cell and understand the intermediate steps of this exciting process.

During my talk I will present the structural analysis of the injectisomes in Yersinia and Chlamydia performed by cryo electron tomography and subtomogram averaging.  Surprisingly, the basal bodies of injectsomes of Yersinia enterocolitica can vary in length up to 20% 8. This flexibility it attributed to the flexible domains of the proteins of the basal body. In Chlamydia the basal bodies of injectisomes strongly contract upon contact with the host cell membrane. This contraction is coupled to a structural stabilization of the cytoplasmic part of the injectisome9. This “pumping action” likely constitutes a general mechanism related to the injection of effectors. Higher resolution structural analysis in situ combined with mutagenesis will generate the clearer understanding of the mechanics of the T3SS action.

 

References: 

1.         Diepold, A. & Armitage, J. P. Type III secretion systems: the bacterial flagellum and the injectisome. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 370, (2015).

2.         Schraidt, O. & Marlovits, T. C. Three-dimensional model of Salmonella’s needle complex at subnanometer resolution. Science 331, 1192–1195 (2011).

3.         Bergeron, J. R. C. et al. A refined model of the prototypical Salmonella SPI-1 T3SS basal body reveals the molecular basis for its assembly. PLoS Pathog. 9, e1003307 (2013).

4.         Akeda, Y. & Galán, J. E. Chaperone release and unfolding of substrates in type III secretion. Nature 437, 911–915 (2005).

5.         Diepold, A., Kudryashev, M., Delalez, N. J., Berry, R. M. & Armitage, J. P. Composition, formation, and regulation of the cytosolic c-ring, a dynamic component of the type III secretion injectisome. PLoS Biol. 13, e1002039 (2015).

6.         Kudryashev, M. et al. Yersinia enterocolitica type III secretion injectisomes form regularly spaced clusters, which incorporate new machines upon activation. Mol. Microbiol. 95, 875–884 (2015).

7.         Radics, J., Königsmaier, L. & Marlovits, T. C. Structure of a pathogenic type 3 secretion system in action. Nat. Struct. Mol. Biol. 21, 82–87 (2014).

8.         Kudryashev, M. et al. In situ structural analysis of the Yersinia enterocolitica injectisome. eLife 2, e00792 (2013).

9.         Nans, A., Kudryashev, M., Saibil, H. R. & Hayward, R. D. Structure of a bacterial type III secretion system in contact with a host membrane in situ. Nat. Commun. 6, 10114 (2015).


Mikhail KUDRYASHEV (Frankfurt am Main, Germany)
Invited
11:30 - 11:45 #5273 - LS06-OP027 Characterisation of Trypanosoma spp. in Australian wildlife.
Characterisation of Trypanosoma spp. in Australian wildlife.

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 ViewTM 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.

Acknowledgements: The authors acknowledge use of the facilities at the Centre for Microscopy, Characterisation & Analysis, UWA, which is funded by State and Commonwealth governments; and funding from the West Australian Government's State NRM Program.


Crystal COOPER, Andrew THOMPSON, Adriana BOTERO, Peta CLODE (Crawley, Australia)
11:45 - 12:00 #5210 - LS06-OP026 Flow cytometry, and Transmission electron microscopy; two valuable tools used in an innovative approach to isolate and describe new microorganisms.
Flow cytometry, and Transmission electron microscopy; two valuable tools used in an innovative approach to isolate and describe new microorganisms.

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. 


Jacques BOU KHALIL (MARSEILLE)
12:00 - 12:30 #6220 - LS06-S21 Soft X-Ray cryo-tomography reveals ultrastructural alterations of the host cell during Hepatitis C infection.
Soft X-Ray cryo-tomography reveals ultrastructural alterations of the host cell during Hepatitis C infection.

Chronic hepatitis C virus (HCV) infection causes severe liver disease in millions of humans worldwide. Pathogenesis of HCV infection is strongly driven by a deficient immune response of the host, although intersection of different aspects of the virus life cycle with cellular homeostasis is emerging as an important player in the pathogenesis and progression of the disease.

Cryo soft X-ray tomography (cryo-SXT) was performed to investigate the ultrastructural alterations induced by the interference of hepatitis C virus (HCV) replication with cellular homeostasis. Native, whole cell, three-dimensional maps were obtained in HCV replicon-harboring cells and in a surrogate model of HCV infection at 40nm resolution. Tomograms from HCV-replicating cells show blind-ended endoplasmic reticulum (ER) tubules with pseudo spherical extrusions and marked alterations of mitochondrial morphology that correlated topologically with the presence of ER alterations, suggesting a short-range influence of the viral machinery on mitochondrial homeostasis.

Both mitochondrial and ER alterations could be reverted by a combination of sofosbuvir/daclatasvir, which are clinically approved direct-acting antivirals (DAAs) for the treatment of chronic HCV infection. In addition to providing structural insight into cellular aspects of HCV pathogenesis our study illustrates how cryo-SXT is a powerful three-dimensional wide-field imaging tool for the assessment and understanding of complex cellular processes in a setting of near native whole hydrated cells. Our results also constitute a proof of concept for the use of cryo-SXT as a platform that enables determining the potential impact of candidate compounds on the ultrastructure of the cell that may  assist drug development at a preclinical level.


Ana Joaquina PEREZ-BERNA (Barcelona, Spain), Maria Jose RODRÍGUEZ, Andrea SORRENTINO, Francisco Javier CHICHON, Martina FRIESLAND, Jose Lopez CARRASCOSA, P GASTAMINZA, Eva PEREIRO
Invited
Salon Tête d'Or

"Monday 29 August"

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LS4-I
10:30 - 12:30

LS4: Membrane Interaction
SLOT I

Chairpersons: Hans HEBERT (Huddinge, Sweden), Aurélien ROUX (Genève, Switzerland)
10:30 - 11:00 #8753 - LS04-S13 New insights into plasma membrane organization from super-resolution STED microscopy.
New insights into plasma membrane organization from super-resolution STED microscopy.

Plasma membrane interactions such as the transient protein-protein or protein-lipid complexes, the formation of lipid nanodomains (often denoted “rafts”), or diffusional restrictions by the cortical cytoskeleton are considered to play a functional part in a whole range of membrane-associated processes. However, the direct and non-invasive observation of such structures in living cells is impeded by the resolution limit of >200nm of a conventional far-field optical microscope. Here we present the use of the combination of super-resolution STED microscopy with fluorescence correlation spectroscopy (FCS) for the disclosure of complex nanoscopic dynamical processes. By performing FCS measurements in focal spots tuned to a diameter of down to 30 nm, we have obtained new details of molecular membrane dynamics, such as of transient lipid-protein interactions and of diffusional restrictions by the cortical cytoskeleton. Further insights will be given for molecular dynamics in the plasma membrane of immune cells, specifically during T-cell activation.


Christian EGGELING (Oxford, United Kingdom)
Invited
11:00 - 11:30 #8742 - LS04-S14 Characterization of the plasma membrane dynamics during cell signaling processes investigated by spot variation fluorescence correlation spectroscopy (svFCS).
Characterization of the plasma membrane dynamics during cell signaling processes investigated by spot variation fluorescence correlation spectroscopy (svFCS).

The plasma membrane organization is highly heterogeneous as a result of the intrinsic molecular Brownian agitation and the vast diversity of membrane components. Selective interactions take place in the formation of local complex multicomponent assemblies of lipids and proteins on different time scales. Still, deciphering this lateral organization on living cells and on the appropriate length and temporal scales has been challenging but is crucial to advance our knowledge on the biological function of the plasma membrane.

Among the methodological developments made during the last decade, the spot variation FCS (svFCS), a fluorescent correlation spectroscopy (FCS)-based method, has allowed the significant progress in the characterization of cell membrane lateral organization at the sub-optical level, including to providing compelling evidence for the in vivo existence of lipid-dependent nanodomains (see for review )1.This method provides particular insight to identify possible molecular confinement occurring at the plasma membrane 1, 2. The svFCS is performed by changing the spot of illumination underfilling the back aperture of the objective 3. Theoretical models have been developed to predict how geometrical constraints such as the presence of adjacent or isolated domains affect the svFCS observations 4, 5.

We will illustrate how the investigations based on svFCS have provided compelling evidence for the in vivo existence of lipid-dependent nanodomains 2, 6, 7 and have allowed significant progress in the characterization of cell membrane lateral organization for different kind of receptors 8-10.

 

 

1. He, H.T. & Marguet, D. Annu Rev Phys Chem 62, 417-436 (2011).

2. Lenne, P.F. et al. EMBO J 25, 3245-3256 (2006).

3. Wawrezinieck, L., Lenne, P.F., Marguet, D. & Rigneault, H. P Soc Photo-Opt Inst 5462, 92-102 (2004).

4. Wawrezinieck, L., Rigneault, H., Marguet, D. & Lenne, P.F. Biophys J 89, 4029-4042 (2005).

5. Ruprecht, V., Wieser, S., Marguet, D. & Schutz, G.J. Biophys J 100, 2839-2845 (2011).

6. Lasserre, R. et al. Nat Chem Biol 4, 538-547 (2008).

7. Wenger, J. et al. Biophys J 92, 913-919 (2007).

8. Blouin, C.M. et al. Cell in press (2016).

9. Chakrabandhu, K. et al. EMBO J 26, 209-220 (2007).

10. Guia, S. et al. Sci Signal 4, ra21 (2011).

 


Sébastien MAILFERT, Yannick HAMON, Hai-Tao HE, Didier MARGUET (MARSEILLE CEDEX 9)
Invited
11:30 - 12:00 #7863 - LS04-S15 Electron microscopy approaches to studying lipid–protein interactions.
Electron microscopy approaches to studying lipid–protein interactions.

Membrane proteins play crucial roles in many cellular processes such as signaling, nutrient uptake and cell adhesion.  Although the lipid bilayer influences many aspects of membrane protein function, our understanding of lipid–protein interactions is limited.  In the first part of my talk, I will describe how electron crystallography of the water channel aquaporin-0 reconstituted with lipids into two-dimensional crystals can be used to address very basic questions in membrane biology, such as the driving forces that define lipid–protein interactions and the effects of hydrophobic mismatch.  In the second part, I will discuss how we use membrane proteins reconstituted into nanodiscs to make it possible to study lipid–protein interactions by single-particle cryo-electron microscopy.  In particular, I will discuss how the mechanosensitive channel MscS adapts to different lipid bilayers.


Thomas WALZ (New York, USA)
Invited
12:00 - 12:15 #4615 - LS04-OP021 How nanoparticles disturb the lipid bilayer vesicles.
How nanoparticles disturb the lipid bilayer vesicles.

 

Meriem Er-Rafik*,(1),Khalid Ferji (2), Olivier Sandre (2), Carlos M. Marques (1), Jean-Francois Le Meins (2), Marc Schmutz (1)

1. Institut Charles Sadron, Université de Strasbourg-CNRS UPR 22, 67034 Strasbourg, France

2. Laboratoire de Chimie des Polymères Organiques (LCPO), Université de Bordeaux, CNRS,  33607 Pessac, France

 

         Innumerous molecules and particles, from simply water to complex proteins or self-assembled small liposomal carriers, can frequently interact with the cell membrane with possible modification on it. Despite a constant increase of the variety of new particles or molecular assemblies, due to rapid progress in nanotechnology, the molecular features determining how is the membrane behaviour with respect to a given molecule are not yet elucidated.

            Here, we will present a new mechanism of the lipid bilayer of liposomes behaviour in presence of nanoparticles investigated by cryo-electron tomography. Cryo-electron microscopy is a relevant technique allowing not only to inspect the structure of the membrane, by resolving for instance the two leaflets of the bilayer, but reveals also geometric features of nanoparticles such as size and shape that play an important role for the potential interaction with lipid bilayer (fig. 1). Cryo-electron tomography resolve in 3D space the relative positions of particles and membranes, providing insight into the interplay between particle-lipid interactions and the ensuing bilayer transformations.

We wish to thank for the financial support the research association ANR (Agence Nationale de Recherche) of the project ANR-12-BS08-0018-01 and the French society of microscopy (SFµ).


Meriem ER-RAFIK (STRASBOURG)
12:15 - 12:30 #6815 - LS04-OP022 In vitro/ex vivo behavior of a new optical imaging agent targeting αVβ3 integrin.
In vitro/ex vivo behavior of a new optical imaging agent targeting αVβ3 integrin.

Introduction Integrin αvβ3 is usually expressed at low or undetectable levels in most adult epithelia, but it is highly upregulated in tumors and correlates with disease progression. Moreover, unlike in quiescent endothelium, αvβ3 is highly expressed in tumor-associated vessels.

RGD (Arg-Gly-Asp) peptides carry the minimal integrin-binding sequence and are well-known to bind preferentially to αvβ3. Thus, RGD-based strategies have been widely adopted to design targeted molecules for cancer therapy and/or diagnosis.

We synthesized a new cyclic RGD-based peptidomimetic conjugated with a NIR fluorophore intended for intrasurgical use during tumor resection, to allow proper identification of tumor margins and sparse metastases by optical imaging. We present here the in vitro/ex vivo characterization of this molecule, meant to define its specificity for the target receptor, its ability to enter the cells and its in vivo behavior after administration to mice. Cellular models were used to preliminarily characterize the effects and the fate of the molecule as it contacts the cells. Mouse tumor models were used to investigate the distribution of the molecule in tumors after in vivo administration.

Methods Fluorescence microscopy, flow cytometry and immunofluorescence assays were used to define the features of the interaction of our molecule with cells expressing different αvβ3 levels.

Results The in vitro characterization of the molecule in contact with adherent cells reveals that it is internalized into the endosomal compartment and that it interferes with αvβ3-mediated cell adhesion. The affinity to both αvβ3 and HSA of our fluorescent RGD-based probe, demonstrated by flow cytometry and microscopy, is suitable for its intended use.

The ex vivo analyses of probe distribution in tumor tissues after in vivo administration highlight its ability to accumulate into the tumor mass and its specificity to delineate tumor margins.

Conclusions Our fluorescent RGD-based molecule is a promising optical imaging probe for fluorescence-guided surgical resection of tumors characterized by variable expression of αvβ3 integrin.


Chiara BRIOSCHI (colleretto giacosa (TO), Italy), Federica CHIANALE, Alessia CORDARO, Giovanni VALBUSA, Federico MAISANO, Fabio TEDOLDI
Salle Gratte Ciel 3
14:00

"Monday 29 August"

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IM2-II
14:00 - 16:00

IM2: Micro-Nano Lab and dynamic microscopy
SLOT II

Chairpersons: Francisco José CADETE SANTOS AIRES (VILLEURBANNE CEDEX, France), Niels DE JONGE (Saarbrücken, Germany), Gerhard DEHM (Düsseldorf, Germany)
14:00 - 14:30 #8381 - IM02-S35 In situ TEM for understanding electrical and thermal transport properties on nano and atomic scales.
In situ TEM for understanding electrical and thermal transport properties on nano and atomic scales.

In situ electron microscopy allows studies of transport of charges and matter in complex structures as well as thermal properties. We can study mechanically and thermally induced changes of charge transport properties using holders designed to enable different stimuli allowing the direct observation and correlation between material structure and properties. The direct correlation between structure and properties on the small scale involving individual interfaces, defects and atoms provides access to new information about which microstructural constituents that are active in determining the material properties on the macro, micro, nano and atomic scale. This talk addresses examples of in situ electrical, mechanical and thermal studies. A few examples are briefly described below.

 

The nanoscale dimensions of semiconducting nanowires (NWs) provide extended strain relaxation capability between lattice-mismatched materials and enable the fabrication of single-NW p-i-n junction solar cells on low-cost substrates. Due to its sub-wavelength dimension, semiconductor NWs can function as optical antennas and exhibit a “self-concentrating” effect that enhances optical absorption. The strain relaxation capability and the enhanced absorption cross section make NWs potential candidates as highly efficient and low-cost solar cells. Due to the effects of  elastic strain applied on the electronic band structure the strain can be used to achieve NW-based photovoltaic devices with new functionality. We have studied the effect of mechanical strain on the electrical resistance of nanowires. Electron energy loss spectroscopy was used to study the effect of strain on the electronic structure with emphasis on the low energy loss interval of 0 to 50 eV. Electron beam induced current measurements were also performed to study the effect of strain on the diffusion length of the charge carriers [1].

 

Heating of a transmission electron microscopy (TEM) specimen can be performed in several parallel modes and this talk will address three types of heating modes and show experimental results from nanostructured materials. One mode is by resistive heating of a ring shaped support in contact with the circumference of the entire TEM sample. An additional mode is by use of a heating wire patterned on the TEM sample where the wire is contacted by leads fed through the TEM sample holder. The third mode is by active Joule heating of the nanostructure of study, such as carbon nanotubes, graphene, or metal nanowires. The purpose of having several parallel modes of heating is to enable the separation of temperature dependence, effects of self-Joule heating, effects of radiative heating and thermal transport. It is also important to be able to extract the three dimensional information about the geometry of the investigated structures [2].

 

 

1.   L. Zeng, T.K. Nordkvist, P. Krogstrup, W. Jäger and E. Olsson, “Mechanical strain induced nonlinearity of the electrical transport properties of individual GaAs nanowires”, in manuscript.

2.   N. Voskanian and E. Olsson, “Heating holder for in situ three dimensional transmission electron microscopy studies”, in manuscript.


Eva OLSSON (Gothenburg, Sweden)
Invited
14:30 - 14:45 #6823 - IM02-OP075 In-Situ Hydration of MgO Nanocrystals to amorphous Mg(OH)2 using Liquid Cell Transmission Electron Microscopy.
In-Situ Hydration of MgO Nanocrystals to amorphous Mg(OH)2 using Liquid Cell Transmission Electron Microscopy.

The hydration reaction of MgO to amorphous Mg(OH)2 is a model hydration reaction and is important to diverse research fields, ranging from catalysis to Earth Sciences. Although the bulk thermodynamics and surface energies of these phases are well studied,[1,2] real time and real space analysis of the reaction at ambient pressure is lacking. In this study, the hydration of MgO nanocrystals is studied at the single particle level, both in real space and in diffraction space using in-situ Transmission Electron Microscopy (TEM) at near-ambient pressure and temperature. Upon exposure to water vapor and the electron beam, the MgO nanocrystals react with H2O and convert to amorphous Mg(OH)2.

 

Real-time recordings of the hydration reaction reveal that the reaction starts at the MgO nanocrystal surface and proceeds inwards at a constant rate while the Mg(OH)2 shell expands outwards. The growth rate is found to be constant throughout the reaction. Furthermore, as the applied dose rate is increased, the growth rate increases accordingly. Possible mechanisms for the beam-promoted transformation are discussed, including the role of defect formation and migration at the interior and at the surface of the MgO nanocrystals, H2O diffusion towards the MgO surface, and the possible influence of beam-generated H2O dissociation products. Assemblies of converting MgO/Mg(OH)2 nanocrystals exhibited a reorganization of the assembly framework due to the solid volume increase (~100%) of each individual nanocrystal.

References

[1] de Leeuw, N.H., Watson, G.W., and Parker, S.C., J. Phys. Chem., 1995, 99 (47), 17 219-17 225

[2] Geysermans, P., Finocchi, F., Goniakowski, J., Hacquart, R., und Jupille, J., Phys. Chem. Chem. Phys., 2009, 11 (13), 2228-2233


Wessel VLUG (Amsterdam, The Netherlands), Oliver PLÜMPER, Michael KANDIANIS, Alfons VAN BLAADEREN, Marijn VAN HUIS
14:45 - 15:00 #6563 - IM02-OP071 In-situ studies of the dendritic yttria precursor nanostructures growth dynamics at elevated temperatures using liquid-cell transmission electron microscopy.
In-situ studies of the dendritic yttria precursor nanostructures growth dynamics at elevated temperatures using liquid-cell transmission electron microscopy.

Yttria, a host for heavy rare earth elements, is an important up-conversion material, able to convert lower energy near-infrared light into higher energy visible light, opening the avenue for a wide spectrum of applications from laser technology, photovoltaics to theranostics [1,2]. The efficient use of yttria in the form of nanoparticles (NPs) is related to the understanding of the nucleation and early growth stage kinetics of yttria precursors, formed by the precipitation from the saturated solutions. In contrast to various analytical methods, where the kinetic data are deduced from large sampled volumes, in-situ transmission electron microscopy (TEM) combined with the specialized liquid cell offers the unique possibility to study the spatial and temporal evolution of NPs one-by-one, facilitating a complete reconstruction of early stage events that are vital for the formation of final products [3].

In-situ TEM experiments were performed by utilizing Jeol JEM 2100 LaB6 TEM operating at 200 kV and liquid cell TEM holder, Protochips Poseidon 300 with a heating capabilities up to 100 °C. The temperature controlled urea precipitation method was used for the synthesis of yttria precursors [4]. Namely, decomposition of urea at elevated temperatures releases precipitating agents (OH- and CO32-) homogeneously into the reaction system, avoiding localized distribution of the reactants, allowing precise control over the nucleation and growth of yttrium precursor, typically Y(OH)(CO3). The prepared solution was placed in a liquid sample enclosure contained in the liquid cell TEM holder. Water layer thickness during the observation was between 150 and 300 nm.

The initial solution was observed at a dose rate of 5000 e-/nm2/s, at room temperature (RT) for 30 minutes. Precipitation was not observed during that period, suggesting that additional chemical species that were created during the radiolysis of water by the incident electron beam did not have significant influence on the nucleation process at RT [5]. The formation rate of NPs increased drastically when the temperatures in the cell were raised above 90 °C. The resulting products were either faceted particles or dendritic nanostructures. While the faceted nanoparticles did not experience significant morphological changes during the observation, this was not true for dendritic nanostructures (Fig. 1). Dendrites first experience rapid growth by developing highly branched, hierarchical structure up to their final size of 50 nm in the first 45 s of the observation. In the second stage, in the period of about 45 s, dendrites undergo rapid fragmentation, resulting in the formation of several spherically shaped particles within the original dendrite volume that were dynamically changing either by the coalescence or Ostwald ripening. Finally, the spherical particles experience a complete dissolution within the observed area, accompanied by the appearance of faceted 150 nm sized NPs in the vicinity of the observation area.

We hypothesize that dendritic structure initially grew by the diffusion limited conditions to the stage when the depletion zone that developed around NPs hindered further growth, followed by coarsening as a result of surface area reduction. The dissolution and formation of NPs with faceted morphology is explained as a combined effect of water and urea decomposition at high temperatures, resulting in increase of [OH-] concentration, destabilizing initially formed particles and promoting a formation of more stable, plausibly Y(OH)3 hexagonal particles [6], as shown in Fig. 2.

 

References:

1 Feldmann, C., et al. (2003). Adv Funct Mater, 13 (7), 511-516.

2 Höppe, H. A. (2009). Angew Chem, Int. Ed., 48, 3572–3582.

3 Ross, F. M. (2015). Science, 350, 350 (6267), aaa9886-9.

4 Qin, H., et al. (2015). Ceramic International, 41, 11598-11604.

5 Schneider, N. M., et al. (2014). J Phys Chem, 118(38), 22373-22382.

6 Huang, S., et al. (2012). Mater. Chem., 22, 16136-16144.


Saso STURM (Ljubljana, Slovenia), Bojan AMBROZIC, Marjan BELE, Nina KOSTEVSEK, Kristina ZUZEK ROZMAN
15:00 - 15:15 #6492 - IM02-OP069 TEM compression of nano-particles in environmental mode and with atomic resolution observations.
TEM compression of nano-particles in environmental mode and with atomic resolution observations.

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 the in situ microstructural 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 performing in situ compression under gas pressure, with high resolution imaging. We will present in situ tests 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 CeO2 nanocubes 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 Ce2O3 under 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].

 

References

[1] Q. Yu, M. Legros, A.M. Minor, MRS Bulletin 40, 62-70 (2015).

[2] E. Calvié, L. Joly-Pottuz, C. Esnouf, P. Clément, V. Garnier, J. Chevalier, Y. Jorand, A. Malchère, T. Epicier, K. Masenelli-Varlot, J. Eur. Ceram. Soc. 32 2067-2071 (2012).

[3] S. Lee, J. Im, Y. Yoo, E. Bitzek, D. Kiener, G. Richter, B. Kim, S.H. Oh, Nature Communications 5:3033 (2014).

[4] I. Issa, J. Amodéo, J. Réthoré, L. Joly-Pottuz, C. Esnouf, J. Morthomas, M. Perez, J. Chevalier, K. Masenelli-Varlot, Acta Materialia 86, 295-304 (2015).

[5] This work is performed within the framework of the LABEX iMUST (ANR-10-LABX-0064) of Université de Lyon, within the program “Investissements d’Avenir” (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). The authors thank the CLYM (Consortium Lyon-Saint Etienne de Microscopie, www.clym.fr) for the access to the microscope and A.K.P. Mann, Z. Wu and S.H. Overbury (ORNL, USA) for having provided the samples.


Thierry EPICIER, Lucile JOLY-POTTUZ (MATEIS / INSA, Lyon), Istvan JENEI, Douglas STAUFFER, Fabrice DASSENOY, Karine MASENELLI-VARLOT
15:15 - 15:30 #6125 - IM02-OP063 Microsecond time- and subnanometer spatial-scale in situ observations of crystallization process in amorphous antimony nanoparticles by the UHVEM newly developed at Osaka University.
Microsecond time- and subnanometer spatial-scale in situ observations of crystallization process in amorphous antimony nanoparticles by the UHVEM newly developed at Osaka University.

Fast in situ observation by TEM is one of useful techniques in researches on phase transitions of nanoparticles. In our previous study, it was evident that amorphous antimony nanoparticles can be crystallized with ease by stimulation from the outside. For example, when lead atoms are vapour-deposited onto amorphous antimony nanoparticles kept at room temperature, crystallization of the amorphous antimony nanoparticles is abruptly induced by an interfacial strain between an antimony nanoparticle and crystalline lead nanoparticles attached. On the other hand, knock-on displacements by high energy electron irradiation also become one of the stimulations for the crystallization of the amorphous nanoparticles. In the present study, electron-irradiation-induced crystallization processes of amorphous antimony nanoparticles have been studied by microsecond time- and subnanometer spatial-scale in situ observations by ultra-high voltage electron microscope developed with JEOL Ltd. at Osaka University recently.

Amorphous antimony nanoparticles supported on thin amorphous carbon substrates were prepared by a vapour-deposition method. Electron irradiation experiments and the simultaneous in situ observations were carried out by JEM-1000EES UHVEM operating at an accelerating voltage of 1 MV and the electron flux of the order of approximately 1024 e m-2 s-1, which was equipped with Gatan K2-IS electron direct detection CMOS camera. The time for one frame was 625 μs.

The figure 1 shows a typical example of migration of interface between an amorphous and crystalline phase during crystallization in an approximately 60 nm-sized nanoparticle as indicated by arrows. As indicated in fig. 1(a), the nucleation site of the crystalline phase is located on the particle surface. At the early stage of the crystallization, the interface has a small curvature as shown in (b) ~ (f), but at the steady state of (g) ~ (j), the interface becomes flat. The velocity of the interface migration is estimated to be approximately 10 μm s-1.

Atomic scale observations by HREM were carried out. The figure 2 shows the snapshots during crystal growth in about 20 nm sized nanoparticle. In fig, 2(a), 2 nm-sized crystalline nucleus appears on the surface of the particle, and the FFT pattern from the particle is in set. Week four spots are recognized as indicated by four arrows in the FFT pattern, and correspond to nucleation of the small crystal. In fig. 2(b), the nucleus grows up to approximately 5 nm in diameter, after that the amorphous nanoparticle is crystallized in the whole nanoparticle. In the FFT pattern, the week four spots change to an obvious net pattern, which is indexed as the [2-21] zone axis pattern of an antimony crystal. In this case of the 20 nm-sized nanoparticle, the velocity of interface migration is estimated to be approximately 20 μm s-1. The velocity of the interface migration depends on the particle size, and it was confirmed that the smaller the particle size is, the faster the velocity is. From the observation, the critical particle size for crystallization all over the nanoparticle is estimated to be approximately 5 nm. A strain on the interface between this crystalline nucleus and the amorphous nanoparticle may induce the crystallization all over the nanoparticle. A schematic illustration of crystallization mechanism in amorphous antimony nanoparticles is shown in the bottom of figure 2. The amorphous nanoparticle has to jump beyond the activation energy for the crystallization. At the early stage of the crystallization, small nucleus fluctuates between an appearance and a disappearance. However, when the size of the nucleus is larger than the critical size for crystallization, the strain energy of interface between this crystalline nucleus and the amorphous nanoparticle will be larger than the activation energy. It is suggested that the strain energy is a trigger for crystallization in amorphous antimony nanoparticles.


Hidehiro YASUDA (Osaka, Japan)
15:30 - 15:45 #6831 - IM02-OP076 Studying the Formation Dynamics of VLS Silicon Nanowire Devices using in situ TEM.
Studying the Formation Dynamics of VLS Silicon Nanowire Devices using in situ TEM.

Control of the electrical properties of Si nanowires, and in particular their connection to the macroscale environment, is important when developing nanowire applications. We therefore use in situ TEM to create suspended Si nanowire devices so that we can correlate the structure and transport properties of the nanowires and their contacts. In an ultra high vacuum TEM, we grow Si nanowires by the vapor-liquid-solid process using AuSi eutectic droplet catalysts and disilane gas. The nanowires grow from one microfabricated heater [1,2] across to a second heater 2-3 micrometers away (Figure 1). Temperature can be controlled in the VLS growth range 450-600oC, and we can control the voltage across the nanowire at the moment of contact, and perform IV measurements on the final nanowire device [3] at room temperature. We have shown that novel nanowire contact geometries such as necked or bulged contacts can be formed [4] by tuning the balance between the Si growth rate and the migration of Au from the contact region. This is achieved by controlling the growth conditions during contact formation.

Here we examine an additional parameter that is even more effective in controlling the contact geometry. This is electromigration, induced by flowing current through the nanowire during contact formation. In Figure 2 we show the effect of current flow (as well as disilane pressure) on the deposition of Si and the volume of AuSi. In Fig 2(a) a TEM image series shows the formation of a 10nm nano-gap by a nanowire (Si NW) connecting to a Si cantilever side wall with an AuSi droplet, and removing the AuSi by using electromigration. In Fig 2 (b, c), the AuSi and deposited Si volumes is plotted along with (b) disilane pressure and (c) current through the wire. In (b) Si is incorporated only at high disilane pressure; when pressure is reduced, the morphology becomes constant. In (c), once a current is flowed through the nanowire, the AuSi starts to shrink at 5400nm3/s due to Au electromigration; as Au moves away, the Si is deposited at 1400nm3/s. The net decrease in volume creates the 10nm gap. Hence the current flow can cause rapid loss of Au from the contact site, forcing a rapid segregation of Si from the AuSi droplet. This we show can controls the contact formation dynamics to create bulged, straight, necked or nanogap contacts [4].

Once contact has been established, the nanowire device can be electrically characterized and further modified, for example by oxidation of the Si surface. We find that nanowires can sustain tens of volts before disconnecting, and exhibit fairly consistent IV characteristics at room temperature, Figure 2(d).

The ability to control the contact structure, and measure its transport properties directly after formation, is helpful in understanding the behaviour of nanowires in processed devices. Electromigration appears to be a useful parameter that allows novel nanowire contact geometries to be created and hence greater flexibility in nanowire device design.

[1] C. Kallesøe et al., Small, vol. 6, 2010, pp. 2058–2064.

[2] K. Molhave et al., Small, vol. 4, Oct. 2008, pp. 1741–1746.

[3] C. Kallesøe et al., Nano Letters, vol. 12, Jun. 2012, pp. 2965–2970.

[4] S.B. Alam et al., Nano Letters, vol. 15, Oct. 2015, pp. 6535–6541.


Sardar B. ALAM, Federico PANCIERA, Ole HANSEN, Frances M ROSS, Kristian MØLHAVE (Lyngby, Denmark)
15:45 - 16:00 #5916 - IM02-OP060 Quantitative measurement of doping and surface charge in a ZnO nanowire using in-situ biasing and off-axis electron holography.
Quantitative measurement of doping and surface charge in a ZnO nanowire using in-situ biasing and off-axis electron holography.

Semiconducting nanowires (NWs) are widely studied because the properties that stem from their three-dimensional, nanoscale nature open new opportunities for device design. In particular ZnO NWs are widely studied for their interesting piezoelectronic properties. Though NWs can be readily grown today with increased carrier concentration due to doping, the measurement of the doping concentration at the nm scale remains challenging.

    We demonstrate that state-of-the-art off-axis electron holography in combination with electrical in-situ biasing can be used to detect active dopants and surface charges quantitatively in ZnO nanowires. The outline of the contacted NW is described in Fig. 1. We have acquired series of holograms and averaged the phase to increase signal to noise but avoid blurring due to specimen drift. The 0V bias images were used to remove contrast not related to the varying electrostatic potential and to verify the nanowire was not electrically modified during the experiment. We analyzed the depletion width in the nanowire due to an applied reverse bias to a Schottky contact on the nanowire, using a fit to the data.

    Comparison of the experimental data with 3D simulations that were similarly treated indicates an n-type doping level of 1x1018 at. cm-3 and a negative surface charge around -2.5x1012 charges cm-2. Fig. 2a shows the experimental vacuum corrected phase profiles converted to potential, and a fit to the data. The extracted depletion width is indicated with a cross. The inset shows the location of the phase profile in the NW core and two symmetrically defined phase profiles obtained in vacuum on either side of the NW. The average signal in vacuum was subtracted from the NW signal and the remaining phase signal was converted to potential using a thickness of 75 nm, much smaller than the 150 nm NW diameter. In Fig. 2b the experimental and simulated depletion length is compared for 3D simulations including varying doping and surface charge quantities. We expect that the real doping is between 1 and 2x1018 at. cm-3 by comparison of experiment and simulation. The surface charge results in a surface depletion to a depth of 36 nm. We found an active/undepleted core thickness of 70-75 nm, providing excellent agreement between the simulated thickness of the undepleted core and the active thickness observed in the experimental data.

    Off-axis electron holography thus offers unique capabilities for quantitative analysis of active dopant concentrations and surface charges in nanostructures with nanometer-scale spatial resolution.


Martien DEN HERTOG (Institut Neel CNRS, Grenoble), Fabrice DONATINI, Robert MCLEOD, Eva MONROY, Julien PERNOT
Amphithéâtre

"Monday 29 August"

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MS3-I
14:00 - 16:00

MS3: Semiconductors and devices
SLOT I

Chairpersons: Catherine BOUGEROL (Grenoble, France), Vincenzo GRILLO (Modena, Italy)
14:00 - 14:30 #8413 - MS03-S72 Cathodoluminescence and EBIC study of widegap semiconductors and devices.
Cathodoluminescence and EBIC study of widegap semiconductors and devices.

Cathodoluminescence (CL) and electron-beam-induced current (EBIC) are versatile techniques to characterize semiconductor materials and devices. In this talk, we review our achievement on the study of widegap semiconductors, GaN and SiC.

The major defects in GaN are dislocations. GaN wafers on sapphire include dislocations of 109 to 107 cm-2 due to lattice mismatch. Fig. 1 shows the secondary electron (SE) and CL images of GaN wafer with different thickness. Such dislocations may agglomerate and form the hexagonal pits of micrometer size. These defects are detrimental for the device performance. Homoepitaxial GaN wafer take over the dislocations of 106 cm-2 from the seeds. We have performed CL study to distinguish dislocation characters and to clarify the effect of dislocations.

The major defects in SiC are threading screw dislocations (TSD) and stacking faults (SF). TSD act as the killer defects due to the surface roughness at the dislocation core region. SF may be generated and expanded due to e-beam irradiation. EBIC is very effective to characterize these defects in SiC. The e-beam enhanced defect generation of SF (Fig.2) will be reviewed.

At the end, we demonstrate 3D spectra imaging of CL, which is very promising to analyze the details of extended defects.

This work was supported from “GaN project”, Study of Future Semiconductors for Sustainable Society in MEXT, Japan.


Takashi SEKIGUCHI (, Japan)
Invited
14:30 - 14:45 #6432 - MS03-OP245 A novel way of measuring lifetime at the nanometer scale using specific fast electron-matter interactions.
A novel way of measuring lifetime at the nanometer scale using specific fast electron-matter interactions.

Charge carrier lifetime is a key parameter for understanding the physics of electronic or optical excitations. For example the excited state can unveil details of environmental influence, specifically the role of non-radiative transitions. From a practical point of view, lifetimes can largely determine the performances of devices, such as Light Emitting Devices (LEDs) or photovoltaic cells. These usually rely on nanometer scale structures for which small details, such as the presence of single point defects, have to be known with atomic precision. Despite the success of super resolution optical microscopies, they fail as general tools for lifetime measurement at the nanometer scale. In this presentation we will show how we can take advantage of the nanometer probe size formed in a Transmission Electron Microscope and a phenomenon that we recently discovered (referred hereafter as the bunching effect [1]), to study lifetimes of emitter at the nanometer scale without using a pulsed electron gun.

The effect takes its name from the fact that the autocorrelation function g(2)(τ) of the CL signal coming from quantum emitters (points defects or more generally single photon emitters –SPE-, quantum confined structures…) may exhibit a peak at zero delay – which is a fundamental difference with PL. To measure this effect, we use an intensity interferometry experiment that measures the CL g(2)(τ). Figure 1 shows that, at low incoming electron currents (I < 100 pA), the g(2)(τ) of the CL signal intensity I(t) displays a large nanosecond-range peak at zero delay (g(2)(0) > 35) (bunching), the amplitude of which depends on the incoming electron current. This behavior strongly departs from the PL g(2)(τ) function which is flat when multiple independent SPE are excited. In this presentation we will show that it occurs because an emitter, like a quantum well, will be excited multiple times by a single electron and will emit a bunch of photons on a time window close to its radiative lifetime. As it will be proved, by simply fitting the experimental curve of the g(2)(τ) function by an exponential we can retrieve the lifetime of the emitter.

Using this effect we were therefore able to measure very efficiently lifetimes of Gallium Nitride quantum wells (QWs) separated by less than 15 nm, together with their emission energy and atomic structure (Figure 2). Experiments on well separated individual quantum structures shows an excellent agreement with combined time-resolved μ-photoluminescence. We also demonstrate the possibility to measure the lifetimes of emitters of different kinds (defects, QWs, bulk) within a distance of a tenth of nanometers even for spectrally overlapping emissions. This technique is readily applicable to large ensembles of single photon sources and various emitters such as QWs, quantum dots, point defects and extended defects, such as stacking faults (SF).

 

[1] Meuret et al, PRL 114 197401 (2015)

[2] L. H. G. Tizei and M. Kociak, PRL 110  153604 (2013)


Sophie MEURET (CEMES, Toulouse), Luiz TIZEI, Thomas AUZELLE, Thibault CAZIMAJOU, Romain BOURRELLIER, Rudee SONGMUANG, Huan-Cheng CHANG, François TREUSSART, Bruno DAUDIN, Bruno GAYRAL, Mathieu KOCIAK
14:45 - 15:00 #6170 - MS03-OP242 Nanocathodoluminescence reveals the mitigation of the Stark shift in InGaN quantum wells by silicon doping.
Nanocathodoluminescence reveals the mitigation of the Stark shift in InGaN quantum wells by silicon doping.

InGaN quantum wells (QWs) show high internal quantum efficiencies over the ultraviolet to green spectrum and in white light emitting diodes (LEDs). However a persistent challenge to the development of higher efficiency devices is the strong polarisation field across the across the QWs along the polar axis. The polarisation induced internal electric fields cause the spatial separation of the electron and hole wavefunctions in the QWs, known as the quantum confined Stark effect (QCSE). It has been proposed that the internal electric field can be suppressed by silicon doping the quantum barriers (QBs) [1]. Moreover, Kim et al. have theoretically shown that the device efficiency may be improved by variations in the silicon dopant concentration through the QWs [2]. To confirm the simulated properties though, it is crucial to resolve the spectral properties of individual QWs.

In this study, nano-cathodoluminescence (nanoCL) reveals for the first time the spectral properties of individual InGaN QWs in high efficiency LEDs and the influence of silicon doping on the emission properties [3]. A silicon doped layer at 5×1018 cm-3 is included immediately prior to the growth of the 1st QW and the QBs between the QWs are subsequently doped to 1×1018 cm-3 (sample A). Two further multiple QW InGaN/GaN structures were also grown for reference with QB doping levels of 1×1018 cm-3 (sample B) and 1×1017 cm-3 or less (sample C). NanoCL reveals variations in the emission wavelength that directly correlate with individual QWs. With QB doping greater than 1×1018 cm-3, there is a continuous blue shift in the emission wavelength of each of the subsequently grown QWs. The inclusion of a higher doped layer immediately prior to the growth of the 1st QW in the LED structure leads to a blue shift unique to the 1st QW.

The experimental variations in the emission wavelengths were reproduced by Schrödinger-Poisson simulations. The blue shift in emission wavelength through the QWs due to QB doping is found to be caused by screening of the internal electric fields. The reduction in the emission wavelength of the first grown QW due to the higher doped layer is also found to be the result of screening of the internal electric field. The mitigation of the QCSE and consequently stronger overlap of the electron and hole wavefunction, thus should result in an increase in the radiative recombination. NanoCL thus may serve as an experimental approach to study and refine  the design of future optoelectronic nanostructures, including the effects from doping and lead to improvements in device efficiency and functionality.

[1] T. Deguchi, et al., Appl. Phys. Letts. 72, 3329 (1998)

[2] D. Y. Kim, et al., IEEE Photonics. 7, 1 (2015)

[3] J. T. Griffiths, et al., Nano Letts. 15, 7639 (2015)


James GRIFFITHS (Cambridge, United Kingdom), Siyuan ZHANG, Bertrand ROUET-LEDUC, Wai Yuen FU, Dandan ZHU, David WALLIS, Ashley HOWKINS, Ian BOYD, David STOWE, Colin HUMPHREYS, Rachel OLIVER
15:00 - 15:15 #5923 - MS03-OP240 Advanced characterization of colloidal semiconductor nanocrystals by 2D and 3D electron microscopy.
Advanced characterization of colloidal semiconductor nanocrystals by 2D and 3D electron microscopy.

Due to the specific size-dependent photoluminescence spectra of semiconductor nanocrystals (NCs), their use is promising as building blocks for new electronic and optical nanodevices such as light-emitting diodes, solar cells, lasers and biological sensors.1,2 In order to design these NCs with tailored properties for specific applications, a high level of control over their synthesis is of key importance. Therefore, it is of great importance to characterize both the shape as the composition of these systems. Here, a range of different colloidal semiconductor NCs are characterized using 2D and 3D electron microscopy techniques.

We will discuss, 2D semiconductor CdSe nanoplatelets (NPLs), both flat as helical shaped3, which are investigated using electron microscopy techniques. The aim is to retrieve structural information using high resolution imaging which enables us to study the growth mechanism of these NPLs. The flat NPLs have mainly {100} edges (Figure 1.A) and only a thickness of 4 to 5 atomic layers (Figure 1.B). The analysis of the helical NPLs shows that they are zinc blende and that the helices are folded uniquely around the ⟨110⟩ axis (Figure 1.D). In order to retrieve the helicity of the ultrathin helical shaped platelets, electron tomography is applied. The three-dimensional tomographic reconstructions confirm that the observed helices fully rotate over a diameter of ∼25 nm and that they are not preferentially left- or right-handed (Figure 1.C).

Furthermore, heteronanocrystals (HNCs) are studied as they improve the stability and, thereby, the surface passivation of the NCs when overgrown with a shell of a second semiconductor with a higher bandgap.  In this manner, the robustness of the system and the photoluminescence quantum yield of the core is increased.4  In order to understand the growth process of HNCs, both the 3D structure as the position of the core inside that structure is of key importance. We investigate two types of CdSe/CdS core/shell HNCs, with either a nanorod or bullet shape. High resolution HAADF-STEM microscopy enables us to investigate the crystal structure of the core-shell nanostructure (Figure 2.A,C). Advanced electron tomography based on novel reconstruction algorithms5 is used to investigate the 3D shape and to reveal the position of the CdSe core in the CdS shell (Figure 2.B,D). For the CdSe/CdS core/shell bullets, the presence of two types of morphologies was revealed (Figure 2.D). High resolution STEM imaging was used to characterize the surface facets of both morphologies, which enabled us to compare the surface energy of both morphologies. For the CdSe/CdS nanorods, a sequential topotactic cation exchange pathway that yields CuInSe2/CuInS2 nanorods with near-infrared luminescence is further investigated6.

[1]  Somers, R. C.; Bawendi, M. G.; Nocera, D. G. Chem. Soc. Rev. 2007, 36, 579–591.

[2]  Talapin, D. V.; Lee, J.-S.; Kovalenko, M. V.; Shevchenko, E. V. Chem. Rev. 2010, 110, 389–458.

[3]  Hutter, E. M.; Bladt, E.; Goris, B.; Pietra, F.; van der Bok, J. C.; Boneschanscher, M. P.; de Mello Donegá, C.; Bals, S.; Vanmaekelbergh, D. Nano Lett. 2014, 14, 6257–6262.

[4]  Dabbousi, B. O.; Rodriguez-Viejo, J.; Mikulec, F. V; Heine, J. R.; Mattoussi, H.; Ober, R.; Jensen, K. F.; Bawendi, M. G. J. Phys. Chem. B 1997, 101, 9463–9475.

[5]  Goris, B.; Van den Broek, W.; Batenburg, K. J.; Heidari Mezerji, H.; Bals, S. Ultramicroscopy 2012, 113, 120–130.

[6]  van der Stam, W.; Bladt, E.; Rabouw, F. T.; Bals, S.; de Mello Donega, C. ACS Nano 2015, 9, 11430–11438.

The authors acknowledge financial support from the Research Foundation - Flanders (FWO).


Eva BLADT (Antwerpen, Belgium), Bart GORIS, Eline HUTTER, Ward VAN DER STAM, Relinde MOES, Celso DE MELLO DONEGA, Daniël VANMAEKELBERGH, Sara BALS
15:15 - 15:30 #6244 - MS03-OP243 Picometre-precision atomic structure of inversion domain boundaries in GaN.
Picometre-precision atomic structure of inversion domain boundaries in GaN.

Here, we report on the precise analysis of the atomic structure of inversion domain boundaries (IDBs) in GaN by scanning transmission electron microscopy.  IDBs are a common defect in GaN that traps carriers and leads to a slightly modified luminescence wavelength [1,2].

Our analysis of IDBs in MOCVD grown nanowires confirms recent coherent Bragg imaging (CBI) results [3] stating that the atomic structure of this IDB is different or slightly different from the one determined in 1996 by first-principle calculations (IDB*) [4].  CBI experiments measured a 8 pm shift of the c-planes of the two domains [3], whereas  first-principle calculations predicted no shift. A previous study by STEM [5] found a shift of "ca. 0.6 Å" (60pm), corresponding roughly to the switch of the Ga and N positions without any additional shift. Here in addition to a shift along  c, we show that the interface configuration corresponds qualitatively to the IDB* model (cf. Fig. 1) and that there is a 10 pm dilatation perpendicular to the interface (shown in Fig. 3) in agreement with this model, while CBI did not find a dilatation.

To facilitate the measurement of atom positions across the IDB with picometre-precision, we use HAADF-STEM to avoid coherent effects leading to artefacts. Scanning and drift artefacts are being suppressed by acquiring series of rapid STEM images and aligning them using the newly developed Zorro code. This algorithm is based on calculating estimated drift positions by correlating every frame to multiple frames and minimizing the error of the overdetermined system to obtain a best estimate for the frame positions relative to each other. The sub-pixel aligned frames are then averaged and the peak positions are determined via TeMA (template-matching algorithm).

Our quantitative analysis of experimental and simulated STEM images shows that when atomic columns are very close to each other the measured distance can be slightly different from the real value.  For instance, when the distance between atomic columns becomes smaller than 0.1 nm, the difference between the measured and real values can account for several picometres. This effect can be well observed when an IDB kinks perpendicular to the observation direction leading to closely projected columns in the overlap region of the two domains as seen in Fig. 2. Electron scattering simulations show that the apparently wider distance between atoms is a channeling effect.

These results have provided elements to revisit previous theoretical models of IDBs in GaN.

  

[1] T. Auzelle et al., Appl. Phys. Lett. 107, 051904 (2015).

[2] R. Kirste et al., J. Appl. Phys., 110, 093503(2011).

[3] S. Labat et al., ACS Nano 9, 9210 (2015).

[4] J. E. Northrup et al., Phys. Rev. Lett. 77, 103 (1996).

[5] F. Liu et al., Adv. Mater. 20, 2162 (2008).


Benedikt HAAS (GRENOBLE CEDEX 9), Robert A. MCLEOD, Thomas AUZELLE, Bruno DAUDIN, Joël EYMERY, Frédéric LANÇON, Jian-Min ZUO, Jean-Luc ROUVIÈRE
15:30 - 15:45 #5980 - MS03-OP241 Si:B doping measurement by dark-field electron holography.
Si:B doping measurement by dark-field electron holography.

In modern MOS devices, sources and drains are of nanometric dimensions and highly doped (dopant concentration typically > 1020 at.cm-3). Measuring such dopant concentrations and visualizing their spatial extensions in silicon, although mandatory for the development of the technology, is elusive in practice. Several TEM techniques such as EELS and EDX seem suitable to map dopant concentrations with the required resolution but while they are accurate to measure impurities concentrations, they cannot assess whether these impurities are on interstitial or substitutional sites, what is essential to define doping levels. Moreover, the detection of boron suffers from other physical limitations. Finally, bright-field electron holography has been reported to be suited for such measurements but transforming the electrostatic fields which are measured into doping concentrations is far from straightforward.

In this work, we have explored the possibility to extract boron concentrations from the measurement of changes of the silicon lattice parameter induced by the substitution of boron atoms. For this we use dark-field electron holography (DFEH) on specifically designed samples.

In a first part, we will present the DFEH principle [1]. This is an interferometry technique able to map strain with a precision of the order of 10-4 and a few nanometers spatial resolution over micrometer fields of view. Two diffracted beams, one passing through an unstrained region of the lattice and acting as a reference, the other one passing through the region where strain has to be measured, are forced to interfere by using an electrostatic biprism and thus create an interference pattern (see figure 1). A phase map is extracted from the pattern by Fourier transform and converted into an atomic displacement field. By using two non-collinear diffraction vectors, all the components of the strain tensor in the observation plane can be obtained.

For our experiment, a sample consisting of five 50 nm-thick doped layers of increasing boron concentrations ranging from 3E18 at.cm-3 to 8.5E19 at.cm-3 was grown by RP-CVD, under conditions insuring both extremely low concentrations of impurities and the full activation of boron [2]. The sample was further checked by SIMS and ECVP measurements, demonstrating that 100 % of boron atoms are on substitutional sites in all the doped layers. DFEH was used to measure the deformation of the doped layers. We could thus deduce the silicon lattice expansion coefficient (β) resulting from the adding of boron atoms in the crystalline silicon network, from these measurements, as explained below.

The boron atoms being on substitutional sites, the Si:B doped layers can be seen as solid solutions as confirmed by the homogeneity of the deformations imaged by DFEH. These layers are pseudomorphic on the pure silicon lattice as confirmed by the mapping of the in-plane strain by DFEH. Thus, the change of the lattice parameter resulting from the incorporation of boron atoms is solely supported by the out-of-plane strain, through the Poisson’s reaction of the material (figure 2). From the modeling of this sample by FEM and taking into account the relaxation affecting the thin lamella used for DFEH, we are able to retrieve the values of the relaxed Si:B lattice parameter as a function of the substitutional boron concentration. As expected for a solid solution, we find a linear relation between these two parameters. Knowing the boron concentration and the Si:B lattice parameter profiles, we are able to deduce that β coefficient equals -6.5E-24 cm3 (figure 3). Figure 4 compares the results we have obtained with those found in the literature, often measured by XRD.

Finally, β, the coefficient relating the boron concentration to the lattice parameter, allows us to transform a strain map obtained by DFEH into a “substitutional boron concentration” map with a precision of 3E19 at.cm-3 and a spatial resolution of 5 nm. We will illustrate the DFEH effectiveness to measure and image dopant concentrations in “real samples” through few examples, and will discuss the complementarity of the information obtained by this method and by bright-field electron holography.

[1] M.J. Hÿtch, F. Houdellier, F. Hüe and E. Snoeck, Nature 453, pp. 1086-1090, 2008.

[2] F. Gonzatti, J.M. Hartmann, K. Yckache, ECS Transactions 16, pp. 485-493, 2008.


Victor BOUREAU (Toulouse), Daniel BENOIT, Jean-Michel HARTMANN, Martin HŸTCH, Alain CLAVERIE
15:45 - 16:00 #6633 - MS03-OP248 In situ tracking of the heat-induced replacement of GaAs by Au in nanowires.
In situ tracking of the heat-induced replacement of GaAs by Au in nanowires.

For devices, the junctions between the semiconductors and any metal contacts are crucial for the device performance. A heat treatment is commonly applied to improve the quality of the contacts. For GaAs nanowires with a Au-based contact, annealing can lead to a well-defined metal-GaAs junction within the nanowire [1]. Here, we report an in situ heating, high-angle annular dark-field scanning transmission electron microscopy (HAADF STEM) study on the formation and structural characteristics of such junctions. The nanowires are dispersed on a high-stability TEM heating chip, and local Au contacts are made by lithography before in situ heat treatment within the microscope (Figure 1) [2]. A replacement of GaAs by Au can take place and our study determined key aspects of the reaction mechanism and its kinetics such as the reaction rate and the activation energy. In general, the replacement proceeds one GaAs(111) bi-layer at a time, as demonstrated by lattice resolved HAADF STEM (Figure 2). Ga dissolves in Au and As desorbs, as was previously reported for planar GaAs-Au structures [3]. Using scanning precession electron diffraction (SPED) it was found that there is no fixed epitaxial relation between the newly formed 1D Au-phase and the original GaAs nanowire. The reaction rate and the activation energy for the exchange are accurately determined by tracking the interface between the two phases over relatively long (~0.5 μm) distances.

 

The morphology of the solid 1D Au-phase is the same as for the original GaAs nanowire. Within it, growth twins are observed. The morphology and growth twins do not alter upon cooling and reheating. For the case where the nanowire is attached to a relatively large Au contact (Au reservoir), the contact acts as a Ga diffusion sink, and only negligible amounts of Ga are found in the formed 1D Au-phase. The in situ STEM and electron diffraction results prove that the replacement reaction takes place in the solid state. For the case where the nanowire is attached to a limited Au supply, as would be the case for a small volume Au deposition onto the nanowire or a Au catalyst droplet used for the nanowire growth, the growing metal segment gradually becomes richer in Ga as the exchange reaction proceeds. The reaction rate is slowed down over time due to the Ga enrichment. Eventually and at sufficiently high temperatures, the Au-Ga segment becomes liquid. Upon cooling of such segments, different Au-Ga intermetallic phases form and the main phases could be identified using a combination of SPED and machine learning (Figure 3).

 

References:

[1]: M. Orrù, et al, Phys. Rev. Appl., 4, 044010, 2015. DOI: http://dx.doi.org/10.1103/PhysRevApplied.4.044010
[2]: V. T. Fauske, et al, submitted.
[3]: T. Sebestyen, Electronics Lett., 12, 96, 1976. DOI: 10.1049/el:19760075

 

The authors acknowledge: The Research Council of Norway for the support to the NorFab (197411/V30) and the NORTEM (197405) facilities, and the FRINATEK program (214235), NTNU for support of the initiative “Enabling Technologies” and the EU for support via ERC grant no. 259619 and grant no. 312483 ESTEEM2.


Vidar FAUSKE, Junghwan HUH, Giorgio DIVITINI, Mazid MUNSHI, Dasa Lakshmi DHEERAJ, Caterina DUCATI, Helge WEMAN, Bjørn-Ove FIMLAND, Antonius VAN HELVOORT (Trondheim, Norway)
Salle Bellecour 1,2,3

"Monday 29 August"

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IM5-II
14:00 - 16:00

IM5: Quantitative imaging and image processing
SLOT II

Chairpersons: Joanne ETHERIDGE (Director) (Melbourne, Australia), Jean-Christophe OLIVO-MARIN (Paris, France)
14:00 - 14:30 #8366 - IM05-S45 Exposing New Atomic-scale Information about Materials by Improving the Quality and Quantifiability of Aberration-corrected STEM Data.
Exposing New Atomic-scale Information about Materials by Improving the Quality and Quantifiability of Aberration-corrected STEM Data.

Aberration-corrected scanning transmission electron microscopy (STEM) is providing previously unattainable views of materials at the atomic scale. The quality of STEM data is now often limited by environmental and experimental factors instead of instrument factors (e.g. electron optics). Some of these environmental limitations can be overcome by collecting and processing STEM data using new data science techniques. These techniques expose new atomic-scale materials information by improving our ability to measure atomic column positions, 3D structure, single point defects, and atomic-scale composition.

The precision in locating atomic column positions in STEM images is fundamentally limited by the image signal to noise ratio (SNR), but typically practical limits including sample and microscope instabilities that produce distortions in STEM images are encountered before reaching the SNR limit. We have developed a non-rigid registration (NRR) technique that corrects image distortion of all length scales and enables averaging to enhance the SNR.[1-2] Sub-pm precision images of single crystal materials have been achieved by NRR and averaging high angle annular dark field (HAADF) STEM images. NRR has allowed measurements of pm-scale bond length variations of Pt nanocatalyst atoms that may help explain their catalytic activity. Figure 1 shows NRR and averaged HAADF STEM data of a Pt nanocatalyst on an alumina support from a side-view that exhibits ~2 pm precision. Displacement measurements of each atomic column position reveal moderate Pt surface bond length contraction and strong but localized strain of Pt atoms near nanoparticle-support interface. Some of the interface strain is transferred up the twin boundary.

Determining the three dimensional atomic structure of materials from two dimensional S/TEM images is a major hurdle. The standardless atom counting technique is one promising route to measure local sample thickness by quantitatively comparing experimental and simulated HAADF STEM images and can be used to deduce 3D structure.[3] Unlike previous examples, NRR and averaging STEM images have allowed standardless atom counting with the uncertainty no longer dominated by Poisson noise [1]. This should allow the unique determination of the number of atoms in atomic columns, although this has not yet been demonstrated due to other sample limitations.

Point defects are critical to the properties of a wide range of materials, but imaging single defects is challenging. Quantitative STEM has allowed imaging single substitutional and interstitial dopant impurity atoms[4], but experimentally imaging single vacancies has remained elusive. We have used HAADF STEM frozen phonon multislice simulations to predict the detectability of La vacancies in LaMnO3 by the reduced atomic column intensity and atomic column distortions around the vacancy. NRR and averaging HAADF STEM images of LaMnO3 improves the SNR and the image precision sufficiently to potentially detect single La vacancies. Experimental images contain candidate single La vacancies that have local atomic column distortions and intensity variations which match simulated predictions.

Atomic-resolution composition maps can be created using STEM energy dispersive x-ray spectroscopy (EDS) spectrum imaging (SI). However, long total dwell times that may introduce spatial distortions are required because of low x-ray production and collection efficiency. The most common approach to minimize distortions is to sum multiple SIs using online drift-correction software that discards the individual SIs and HAADF images. The quality of EDS SIs of a Nd2/3TiO3 sample was improved by saving the simultaneously acquired raw HAADF and EDS SI series, and applying post acquisition NRR and averaging. The resulting elemental maps show less spatial distortions and more atomic localization of x-rays. In addition, a novel non-local principle component analysis further enhances the quality of EDS SIs compared to conventional denoising methods.[5]

[1] Yankovich et al Nature Communications, 5 4155 (2014)

[2] Yankovich et al Advanced Structural and Chemical Imaging 1:2 (2015)

[3] LeBeau et al Nano Letters, 10 4405 (2010)

[4] Voyles et al Nature, 416 826 (2002)

[5] Yankovich et al Nanotechnology, submitted (2016)


Andrew YANKOVICH (Göteborg, Sweden), Torben PINGEL, Jie FENG, Alex KVIT, Thomas SLATER, Sarah HAIGH, Dane MORGAN, Paul VOYLES, Eva OLSSON
Invited
14:30 - 14:45 #6658 - IM05-OP114 Nanoparticle Structure from Genetic Algorithm Refinement Against Quantitative STEM Data.
Nanoparticle Structure from Genetic Algorithm Refinement Against Quantitative STEM Data.

We have developed a structure refinement method based on genetic algorithm optimization to create structural models of individual nanostructures based on quantitative scanning transmission electron microscopy (STEM) data [1].  We defined a cost function C for a structural model s, as C(s) = E(s) + αχ2[I(s), Iexp], where E is the simulated potential energy of s, χ2 is goodness-of-fit between the experimental STEM data Iexp and the simulated STEM data I(s), and α is a weighting parameter.  A genetic algorithm (GA) is used to minimize C over structures s, resulting in a structure that is both at a local minimum in the (simulated) energy and in good agreement with experimental data.  The advantage of combining the energy and goodness-of-fit to experiments over optimization on just one or the other is the ability to refine structures that are not at a global energy minimum (like most nanoparticles) from experimental data that does not completely constrain the three-dimensional structure (like a STEM image in one orientation).

We have validated the approach and implementation using simulated experimental data from a metastable, 309-atom Au inodecahedron, as shown in Figure 1.  Figure 1(a) is the test structure, and Figure 1(b) is the simulated STEM image from that structure.  The energy is calculated using an embedded atom method empirical potential for Au.  Figure 1(d) shows the evolution of the two terms in the cost function and the total cost function over the course of the optimization.  Neither term decreases monotonically for the entire optimization, but the entire C(s) does.  Figure 1(c) shows the STEM image of the refined structure after 2200 generations, which is an essentially perfect match for the input image in (b).  Figure 1(e) shows that the 3D structures are also a perfect match, with a maximum difference in atomic positions of 0.02 Å.

As a first test, we have refined the structure of a ~6000 atom colloidal Au nanoparticle, as shown in Figure 2.  Figure 2(a) is the experimental STEM image of the particle [2].  Figure 2(c) shows the evolution of the cost function as it converges over 4000 generations to reach the final structure in Figure 2(b).  In this case, the optimization was allowed to change the number of atoms in the structure as well as their position.  The result faithful reproduces the image of the sample, including the outline and the twin boundary.  Figure 2(d) shows the displacement of matching atomic columns in the two images.  The large displacements near 0.3 Å arise from surface atoms which are not well-imaged in the experiment due to surface atom mobility under the electron beam, but which are recovered in the refined model.  Additional applications to Pt and Pt-Mo catalysts will be discussed.

1. “Integrated Computational and Experimental Structure Determination for Nanoparticles” Min Yu, Andrew B. Yankovich, Amy Kaczmarowski, Dane Morgan, Paul M. Voyles (submitted)

2. “High-precision scanning transmission electron microscopy at coarse pixel sampling” Andrew B. Yankovich, Benjamin Berkels, W. Dahmen, P. Binev, and Paul M. Voyles Advanced Chemical and Structural Imaging 1, 2 (2015).  DOI: 10.1186/s40679-015-0003-9


Paul VOYLES (Madison, USA), Zhewen SONG, Dan ZHOU, Zhongnan XU, Andrew YANKOVICH, Dane MORGAN
14:45 - 15:00 #6196 - IM05-OP108 Non-destructive nanoparticle characterisation using a minimum electron dose in quantitative ADF STEM: how low can one go?
Non-destructive nanoparticle characterisation using a minimum electron dose in quantitative ADF STEM: how low can one go?

Aberration-corrected STEM has become a powerful technique for materials characterisation of complex nanostructures. Recent progress in the development of quantitative methods allows us to extract reliable structural and chemical information from experimental images in 2D as well as in 3D. In quantitative STEM, images are treated as datasets from which structure parameters are determined by comparison with image simulations or by using parameter estimation-based methods [1]. So-called scattering cross-sections, measuring the total scattered intensity for each atomic column, are useful values for quantification [2, 3]. Their high sensitivity and robustness for imaging parameters in combination with a statistical analysis enables us to count atoms with single-atom sensitivity [4].  An example is shown in Figure 1, where atom-counting from a single image combined with an energy minimisation approach [5] is used to reconstruct the 3D atomic structure of a Au nanorod. The close match with the 3D tomography reconstruction resulting from images recorded along 3 viewing directions [6] demonstrates the accuracy of the method.

    

Reducing the number of images by avoiding tilt tomography will be of great help when studying beam-sensitive nanostructures. However, the tolerable electron dose is often still orders of magnitude lower than what is typically used for atomic resolution imaging. Therefore, the question arises: how to optimise the experiment design in order to reduce the electron dose? To investigate this, we have developed a statistical framework that allows us to study the effect of electron shot noise, scan noise, and radiation damage on the atom-counting precision. Figure 2 shows that even for low-dose image acquisitions, statistical parameter estimation theory is a powerful tool to refine structure parameters of an incoherent imaging model and to measure scattering cross-sections. However, the presence of electron shot noise in this dose regime is limiting the atom-counting precision. Severe overlap in the distributions of scattering cross-sections related to columns of different thicknesses hampers one to achieve single-atom sensitivity. We will show how the precision improves with increasing electron dose until scan noise, followed by radiation damage, become the main limiting factors. This analysis allows one to balance atom-counting reliability and structural damage as a function of electron dose.

 

Finally, it will be shown how quantitative ADF STEM may greatly benefit from statistical detection theory in order to optimise the detector settings [7]. This is illustrated in Figure 3, where the number of atoms of a beam-sensitive Pt particle is determined from a STEM image acquired under the computed optimal detector settings. In addition, use is made of a novel hybrid method to count the number of Pt atoms, in which the benefits of a statistics-based and image simulations-based method are efficiently combined in one framework. In conclusion, new developments in the field of quantitative STEM will be presented enabling one to quantify atomic structures in their native state with the highest possible precision.

     

[1] S. Van Aert et al., IUCrJ 3 (2016) 71-83.

[2] S. Van Aert et al., Ultramicroscopy 109 (2009) 1236-1244.

[3] H. E et al., Ultramicroscopy 133 (2013) 109-119.

[4] S. Van Aert et al., Physical Review B 87 (2013) 064107.

[5] L. Jones et al., Nano Letters 14 (2014) 6336-6341.

[6] B. Goris et al., Nature Materials 11 (2012) 930-935.

[7] A. De Backer et al., Ultramicroscopy 151 (2015) 46-55.

  

The authors acknowledge financial support from the Research Foundation Flanders (FWO,Belgium) through project fundings (G.0374.13N, G.0369.15N and G.0368.15N) and postdoc grants to A.D.B. and B.G. S.B. and A.B. acknowledge funding from the European Research Council (Starting Grant No. COLOURATOMS 335078 and No. VORTEX 278510). The research leading to these results has also received funding from the European Union Seventh Framework Programme [FP7/2007- 2013] under Grant agreement no. 312483 (ESTEEM2).


Sandra VAN AERT (Antwerp, Belgium), Annick DE BACKER, Annelies DE WAEL, Lewys JONES, Gerardo T MARTINEZ, Bart GORIS, Thomas ALTANTZIS, Armand BÉCHÉ, Sara BALS, Peter D NELLIST
15:00 - 15:15 #5795 - IM05-OP105 Quantitative annular dark-field imaging at atomic resolution.
Quantitative annular dark-field imaging at atomic resolution.

Quantitative annular dark-filed (ADF) imaging in scanning transmission electron microscopy (STEM) enables us to identify the type and number of atoms of local crystal structures. A quantification procedure of ADF images was proposed by LeBeau and Stemmer, in which the signal at each pixel is placed on an absolute scale by normalizing the current reaching an ADF detector by the incident probe current [1]. Their procedure made possible a direct comparison between experimental and simulated ADF images without any arbitrary scaling parameters. In this study we acquired quantitative ADF images of a graphene and compared with simulated images to investigate how accurately the scattering intensities match between experiments and simulations.

We used a Titan3 microscope (FEI) equipped with spherical aberration correctors (DCOR and CETCOR, CEOS) operating at an acceleration voltage of 80 kV. An ADF detector (Model 3000, Fischione) and an analog-to-digital (A/D) converter (DigiScan II, Gatan) were used. We evaluated a nonlinear response of the ADF signal detection system, which had not been analyzed. Relationship between an ADF detector current (IADF [pA]) and an ADF image signal (SADF [count]) was measured as shown in Fig. 1. Quantitative contrasts QADF [%], i.e. IADF normalized by the incident probe current I0, were calculated from SADF using the nonlinear response. The quantification procedure was performed using an in-house DigitalMicrograph (Gatan) scripts.

The range of ADF detection angle was experimentally measured. The ADF inner angle (48.4 mrad) was measured by scanning an incident probe on the ADF detector. We found that the ADF outer angle (200 mrad) is limited by the aperture in the microscope column above the ADF detector, and the actual outer angle was measured by observing the shadow of the objective aperture [2]. The STEM image simulation was performed using a multislice program (xHREM with STEM Extension, HREM), in which defocus spread and residual aberrations (up to 5th order) were taken into account.

 

Figure 2 shows (a) a quantitative ADF image of graphene with 1–4 layers and (b) the histogram of the quantitative ADF image. The mean contrast, which was measured by averaging the value in areas including several unit cells, was 0.054% at a single-layer region. Since the mean value of a simulated image was 0.053%, the mean quantitative contrast exhibited excellent agreement between experimental and simulated images. We can instantly decide the number of graphene layers based on the quantitative ADF image.

Next we examined atomic-resolution ADF images of a single layer graphene, as shown in Fig. 3. To reproduce atomic ADF image profiles, an effective source distribution, which corresponds to a demagnified source image on the specimen, should be implemented in STEM simulation. Although a Gaussian function has been often utilized as the effective source distribution, we found that the linear combination between Gaussian and Lorentzian (G+L in Fig. 3c) well reproduces experimental results. We also found that there is a small systematic deviation, which is probably due to time-dependent aberrations (e.g., coma). Highly-stable microscope system and/or real-time aberration assessment are required for the advanced quantitative STEM imaging at atomic resolution.

References:

[1] J M LeBeau and S Stemmer, Ultramicroscopy 108 (2008), 1653.

[2] S Yamashita et al, Microscopy 64 (2015) 143 (doi: 10.1093/jmicro/dfu115).

[3] S Yamashita et al, Microscopy 64 (2015) 409 (doi: 10.1093/jmicro/dfv053).

This study was partly supported by the JST Research Acceleration Program and the Nano Platform Program of MEXT, Japan. The authors thank Dr. T. Nagai, Mr. K. Kurashima and Dr. J. Kikkawa for support in the STEM experiments.


Shunsuke YAMASHITA, Shogo KOSHIYA, Kazuo ISHIZUKA, Koji KIMOTO (Tsukuba, Japan)
15:15 - 15:30 #5050 - IM05-OP103 The atomic lensing model: extending HAADF STEM atom counting from homogeneous to heterogeneous nanostructures.
The atomic lensing model: extending HAADF STEM atom counting from homogeneous to heterogeneous nanostructures.

Counting the number of atoms in each atomic column from different viewing directions has proven to be a powerful technique to retrieve the 3D structure of homogeneous nanostructures [1]. In order to extend the atom counting technique to heterogeneous materials, this work presents a new atomic lensing model facilitating both atom counting and 3D compositional determination in such materials.

In the quantitative evaluation of high angle annular dark field scanning transmission electron microscopy (HAADF STEM) images the so-called scattering cross-section (SCS) has proven to be a successful performance measure [1-3]. Its monotonic increase with thickness can be used to count the number of atoms in homogeneous materials with single atom sensitivity [4]. However, for heterogeneous materials, small changes in atom ordering in the column can change the SCS (Fig. 1), significantly complicating atom counting. This depth dependency requires a quantitative method to predict SCSs of all possible 3D column configurations, already more than 2 million for a 20 atoms thick binary alloy. Image simulations can provide this information, but the amount of required simulations makes it an impossible task in terms of computing time. Therefore, a new atomic lensing model is developed based on the principles of the channelling theory [5], where each atom is considered to be an electrostatic lens resulting in an extra focussing effect on the probe. This model allows one to predict the SCSs of mixed columns based on the lensing factors of the individual atoms in monotonic atomic columns. As compared to a linear model neglecting channelling, this new approach leads to a significant improvement in the prediction of SCSs which is not restricted to the number of atom types (Fig. 2) and can be used for a wide range of detector angles (Fig. 3).

The power of the atomic lensing model to accurately predict SCSs enables one to extend the atom counting technique to heterogeneous materials. Here, simulated SCSs can be matched to the measured experimental SCSs. Next, the 3D structure can be determined by combining atom counts from different viewing directions. In this presentation, this technique will be demonstrated on experimentally recorded images of an Au@Ag nanocrystal. Another advantage of the atomic lensing model is its ability to accurately predict the atom ordering in the column from experimental SCSs (Fig. 1). Therefore, it opens up the possibility to extract 3D information from a single image. This ability will be presented on a simulated image of an Au@Ag nanorod.

In conclusion, a new atomic lensing model is developed which is of great importance for extending the atom counting technique from homogeneous to heterogeneous nanostructures.

 

References

[1] Van Aert et al., Nature 470 (2011), p. 374

[2] Van Aert et al., Ultramicroscopy 109 (2009), p. 1236

[3] E. et al., Ultramicroscopy 133 (2013), p. 109

[4] Van Aert et al., Physical Review B 87 (2013), 064107

[5] Van Dyck et al., Ultramicroscopy 64 (1996), p. 99

 

The authors acknowledge financial support from the Research Foundation Flanders (FWO,Belgium) through project fundings (G.0374.13N, G.0369.15N and G.0368.15N) and research grants to K.H.W. van den Bos and A. De Backer. S. Bals and N. Winckelmans acknowledge funding from the European Research Council (Starting Grant No. COLOURATOMS 335078). The research leading to these results has also received funding from the European Union Seventh Framework Programme [FP7/2007- 2013] under Grant agreement no. 312483 (ESTEEM2).


Karel H W VAN DEN BOS (Antwerp, Belgium), Annick DE BACKER, Gerardo T MARTINEZ, Naomi WINCKELMANS, Sara BALS, Peter D NELLIST, Sandra VAN AERT
15:30 - 15:45 #6452 - IM05-OP111 Mapping 2D strain components from STEM moiré fringes.
Mapping 2D strain components from STEM moiré fringes.

Artificial moirés are created in a STEM by deliberately choosing a low magnification where the scan step is close to the crystalline periodicity (see Figure 1a) [1]. A moiré contrast then results from the interference between the scan and the crystal lattice. The technique has been developed to analyse strain [2], and has been applied to the study of strained-silicon devices [3].

 

In reciprocal space, STEM moiré fringes can be understood as the convolution of the lattice created by the scan, characterized by the scan-step, s in real-space, or s* in reciprocal space, and the reciprocal lattice of the crystalline lattice, characterized by d* (Figure 1b). Interference between neighbouring periodicities gives rise to moiré fringes of periodicity qM. In general, only a few moiré fringe periodicities will be present in the image as the scan reciprocal lattice is in reality multiplied by the MTF of the probe: the size of the probe limits the possible interference terms (indicated by the yellow area in Figure 1b). The moiré peridocity qM is related vectorially to s*, which is usually 1 or 2 pixel-1, and the d* (or g-vector) for a particular set of lattice fringes (Figure 1c).

 

Here we show how the strain information can be extracted using the concept of geometric phase, previously used for the analysis of high-resolution TEM images [4]. The advantage of this formulation is that the moiré fringes do not need to be aligned exactly with the crystalline lattice, thus freeing up the experimental work. The periodicity of the moiré fringes (Figure 2a) is identified from the power spectrum of the image and the corresponding geometric phase determined (Figure 2b). The strain is in turn calculated from the geometric phase (Figure 2c). It is not necessary to know the exact calibration of the original image providing a reference region of known crystal parameter is present, as the vectorial relationship (Figure 1c) provides a strong constraint.

 

We have also developed a procedure to determine maps of the 2D strain tensor from two differently oriented SMFs [5] (Figure 3). The results from two separate moiré fringe images need to be aligned (Figure 3b) and combined to determine the full 2D in-plane strain tensor. Whilst the examples here are simulated, we anticipate presenting experimental results from devices and piezo-electric materials.

 

[1] D. Su and Y. Zhu, Ultramicroscopy 110, 229–233 (2010).

[2] S. Kim, Y. Kondo, K. Lee, G. Byun, J. J. Kim, et al., APL 102, 161604 (2013).

[3] S. Kim, Y. Kondo, K. Lee, G. Byun, J. J. Kim, et al., APL 103, 033523 (2013).

[4] M. J. Hÿtch, E. Snoeck, R. Kilaas, Ultramicroscopy 74, 131–146 (1998).

[5] STEM Moiré Analysis (HREM Research Inc.) a plugin for DigitalMicrograph (Gatan)

 

Acknowledgments

The authors greatly acknowledge to Yukihito Kondo (JEOL) for valuable advice during the development of the DM plug-in. This work was funded through the European Metrology Research Programme (EMRP) Project IND54 Nanostrain. The EMRP is jointly funded by the EMRP participating countries within EURAMET and the European Union. MJH and CG acknowledge the European Union under the Seventh Framework Programme under a contract for an Integrated Infrastructure Initiative Reference 312483-ESTEEM2.


Martin HYTCH (TOULOUSE CEDEX), Christophe GATEL, Akimitsu ISHIZUKA, Kazuo ISHIZUKA
15:45 - 16:00 #5993 - IM05-OP107 Precision and application of atom location in HAADF and ABF.
Precision and application of atom location in HAADF and ABF.

Precision and application of atom location in HAADF and ABF

Yi Wang1, Dan Zhou1*, Wilfried Sigle1, Y. E. Suyolcu1, Knut Müller-Caspary2, Florian F. Krause2, Andreas Rosenauer2, Peter A. van Aken1

1Stuttgart Center for Electron Microscopy, Max Planck Institute for Solid State Research, Heisenbergstraße 1, 70569, Stuttgart, Germany

*Current: Materials Science and Engineering, University of Wisconsin-Madison, 1509 University Avenue, Madison, WI 53706, USA

2Institut für Festkörperphysik, Universität Bremen, Otto-Hahn-Allee 1, 28359 Bremen, Germany

 

The multifaceted magnetic, electrical, and structural functionalities of perovskite oxides are underpinned by the distortions of the crystal lattice [1]. These distortions include the displacement of cations, deformation of oxygen octahedra (BO6, where B is a transition metal atom), and collective tilts of the octahedral network. Controlling and engineering these distortions in the constituent oxides are crucial in designing and fabricating heterostructures with novel functional properties that are absent in the bulk form. Atomistic understanding of these distortions and elucidation of their influence on the final properties requires imaging and measuring of atomic positions of both cations and oxygen. With the application of spherical aberration (Cs) correctors, sub-Angstrom atomic resolution is nowadays regularly achievable in both TEM and STEM. The recent application of the annular bright-field (ABF) imaging technique in perovskite oxides has become increasingly popular, as it enables simultaneous imaging of heavy and light elements and allows for simultaneous acquisition of other signals [2, 3].

Here, we report the development of a software tool, written in Digital Micrograph scripting language [4], to extract quantitative information of the crystal lattice and of oxygen octahedron distortions of perovskite oxides from high-angle annular dark-field (HAADF) and ABF STEM images. Center-of-mass and two-dimensional (2D) Gaussian fitting methods are implemented to locate positions of individual atom columns. As shown in Fig.1, under daily reproducible working conditions, e.g. sample drift and contamination present, the precision is in the range of 3–4 pm. Applications of this tool will be presented.

The accuracy of atom location by ABF can be significantly influenced by atom-column tilts introduced by inadequate alignment by the operator or by strain near crystal defects. The influence of such tilts was quantitatively analyzed using image simulations. Figure 2 shows exemplarily simulated HAADF and ABF images for 0 and 10 mrad tilt [5].

 

References:

 

[1] R H Mitchell “Perovskites: Modern and Ancient”, (Almaz, Thunder Bay)

[2] S D Findlay et al., Appl. Phys. Lett. 95 (2009), p.191913.

[3] E Okunishi et al., Microsc.Microanal.164 (2009), p.15.

[4] D R G Mitchell, B Schaffer, Ultramicroscopy 103 (2005), p.319.

[5] The research leading to these results has received funding from the European Union Seventh Framework Program under Grant Agreement 312483-ESTEEM2 (Integrated Infrastructure Initiative I3).


Yi WANG, Dan ZHOU, Wilfried SIGLE (Stuttgart, Germany), Y. Eren SUYOLCU, Knut MÜLLER-CASPARY, Florian F KRAUSE, Andreas ROSENAUER, Peter VAN AKEN
Salle Prestige Gratte Ciel

"Monday 29 August"

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MS7-II
14:00 - 16:00

MS7: Materials for optics and nano-optics
SLOT II

Chairpersons: David MCCOMB (Columbus, USA), Jérôme PLAIN (Troyes, France)
14:00 - 14:30 #6254 - MS07-S86 Excitonic Properties of Inorganic-Organic Hybrid Perovskites and Nanophotonic Devices.
Excitonic Properties of Inorganic-Organic Hybrid Perovskites and Nanophotonic Devices.

Excitonic Properties of Inorganic-Organic Hybrid Perovskites and Nanophotonic Devices

Qihua Xiong

School of Physical and Mathematic Sciences & School of Electrical and Electronic Engineering

Nanyang Technological University, Singapore, 637371 

Abstract: In this talk, we present investigations of vapor phase synthesis of purely inorganic or organic-inorganic perovskites nanoplatelets by a van der Waals epitaxy mechanism and their excitonic properties. Those crystals exhibit 2D well-faceted triangular, hexagonal or square geometry with thickness range of tens to hundreds of nanometers. Optical spectroscopy investigations suggest that the crystals have large exciton binding energy, high external quantum efficiency and long diffusion lengths. The naturally formed high-quality planar whispering-gallery mode cavities ensure adequate gain and efficient optical feedback for low-threshold optically pumped in-plane nanolasers ranging from ultraviolet and near-infrared, with an exceptionally high quality factor (>4000) in purely inorganic perovskite square-shaped crystals. Our findings open up a new class of wavelength tunable nanomaterials potentially suitable for on-chip integration and flexible optoelectronic devices. Progress in light-emitting diode and laser cooling will also be discussed.

 

References:

  1. S.T. Ha, C. Shen, J. Zhang and Q.H. Xiong*, “Laser Cooling of Organic-inorganic Lead Halide Perovskites”, Nature Photonics 10, 115-121 (2016)
  2. Q. Zhang, S.T. Ha, X.F. Liu, T.C. Sum* and Q.H. Xiong*, "Room-Temperature Near-Infrared High-Q Perovskite Whispering-Gallery Planar Nanolasers", Nano Lett. 14, 5995–6001 (2014)
  3. S.T. Ha, X.F. Liu, Q. Zhang, D. Giovanni, T. C. Sum and Q.H. Xiong*, "Synthesis of Organic–Inorganic Lead Halide Perovskite Nanoplatelets: Towards High-Performance Perovskite Solar Cells and Optoelectronic Devices”, Adv. Opt. Mater. 2, 838-844 (2014)
  4. J. Xing, X. F. Liu, Q. Zhang and Q. H. Xiong*, “Vapor Phase Synthesis of Organometal Halide Perovskite Nanowires for Tunable Room-Temperature Nanolasers”, Nano Lett. 15, 4571 - 4577 (2015).

Qihua XIONG (singapore, Singapore)
Invited
14:30 - 14:45 #5150 - MS07-OP298 Angle-resolved cathodoluminescence of plasmonic crystal waveguide.
Angle-resolved cathodoluminescence of plasmonic crystal waveguide.

Slow-light manipulation in a photonic crystal (PhC) waveguide is expected to improve future optical information processing and communication technologies such as optical buffering and light compression [T. Baba, Nat. Photon. 2, 465-473 (2008)]. Waveguiding using bandgap of plasmonic crystal (PlC) has also been demonstrated [S. I. Bozhevolnyi et al. Phys. Rev. Lett. 86, 3008-3011 (2001)]. However, the dispersion characteristics of the guided modes, which are essential to control surface plasmon polariton (SPP) pulses, have not yet been understood. Electron beam spectroscopies at high spatial resolution are powerful characterization tools to observe electromagnetic modes nowadays. Momentum-resolved spectroscopy in electron microscopy is especially useful to investigate detailed optical properties of locally-modified structures introduced into a PhC [R. Sapienza et al. Nat. Mater. 11, 781-787 (2012)] and a PlC [H. Saito and N. Yamamoto, Nano Lett. 15, 5764-5769 (2015)]. We have studied the dispersion characteristics of SPPs in a PlC waveguide by angle-resolved chatodoluminescence performed in a STEM. The guided SPP modes were found to have two unique features : i) energy dependence of the phase shift at the wall, and ii) waveguide bandgap (WBG) due to the periodicity originating from PlC structure, which resulted in small group velocity of the guided SPP modes over a wide energy range.

The investigated PlC waveguide is composed of a silver dot array with a triangular lattice and silver plane surface as shown in Fig. 1a, which was structured by electron beam lithography and physical deposition. A full bandgap is formed from 1.8 eV to 2.3 eV in the present PlC. The SPPs with the energies in the full bandgap are confined in the flat waveguide area and guided parallel to the Γ-K direction as illustrated in Fig. 1a. Figure 1b shows the dispersion pattern measured in the PlC waveguide area with the waveguide width W of 650 nm, angle-scanned parallel to the direction of the waveguide. The details of the experimental setup for angle-resolved chatodoluminescence measurements are explained elsewhere [K. Takeuchi and N. Yamamoto, Opt. Express 19, 12365-12374 (2011)]. The guided SPP mode is observed (indicated by green ellipse). We also find the small gap about 0.01 nm-1 along the curve. The guided SPP mode can be approximately modelled as the guided wave between two interfaces with total internal reflections considering an energy-dependent phase shift. The details of this model will be explained in the congress. The theoretical curves relatively well fits the experimental curves for various waveguide widths except for the gaps. The measured dispersions indicate that the SPPs in the PlC waveguide become much slower than light in vacuum. The guided SPP in the waveguide with W = 520 nm is 7.5 times slower than light in vacuum. The velocity is even more slowed as the energy approaches the gap about 0.01 nm-1.

To understand the origin of the gap, photon map imaging was performed for W = 1040 nm. Interestingly, the interference fringes appear in the direction of the waveguide with the period of 300 nm, indicating the dot row facing the waveguide causes Bragg reflection, resulting in the WBG. The antinode positions for lower band-edge energy and upper band-edge energy are different from each other as illustrated in Figs. 1c and 1d. The antinodes of the lower band-edge mode are extended between the dots facing the flat waveguide area (Fig. 1c) while the upper band-edge mode is more tightly confined within the flat waveguide area (Fig. 1d). This difference in the effective waveguide width generates the energy difference between the band-edge modes, i.e. WBG.

The present results indicated that the PlC waveguide has potential advantages in manipulation of ultrashort pulses since it follows the linear dispersion with small group velocity over a wide energy range. Although the dispersion mainly inside the light cone was measured in this fundamental study, it could be shifted outside the light cone for a practical use. One of the possible solutions is a use of a hybrid waveguide composed of a dielectric strip on a metal surface [T. Liu et al. Opt. Express 22, 8219-8225 (2014)].

This work was supported by Kazato Research Foundation, the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) Nanotechnology Platform 12025014.


Hikaru SAITO (Fukuoka, Japan), Naoki YAMAMOTO, Takumi SANNOMIYA
14:45 - 15:00 #5764 - MS07-OP299 Sub-nanosecond electron beam blanking for deep-subwavelength lifetime probing of nanophotonical devices and nanoparticles.
Sub-nanosecond electron beam blanking for deep-subwavelength lifetime probing of nanophotonical devices and nanoparticles.

Optical techniques are used to probe carrier or excited energy dynamics down to femtosecond time-scales, but they lack the resolution to address nanoscopic length scales [1]. Higher spatial resolution can be obtained using pulsed beams of electrons, but this typically requires dedicated microscopes with laser-triggered electron sources [2]. Here, we use a standard scanning electron microscope equipped with an electrostatic beam blanker operated in conjugate mode (Fig. 1). We show the generation of sub-nanosecond electron pulses (Fig.2). This system is used to probe nanophotonical devices and nanoparticles using time-resolved cathodoluminescence (CL). In these devices, optical emitters couple with the nanoscale environment leading to a position-dependent excited state lifetime. The pulsed electron beam excites the emitters, giving rise to CL. The CL emission is detected using an integrated light microscope [3]. Photon arrival histograms are obtained using time-correlated single photon counting, synchronized with the input signal of the electron beam blanker, thus measuring spatially resolved CL lifetime.

 

We demonstrate the ability to identify nanoparticles based on their lifetime as well as their emission wavelength, which provides an additional source of information in nanoparticle-based biological imaging. Moreover, we conduct a nanoscopic version of the seminal work performed in the 1960s and 1970s by Drexhage, who showed that the lifetime of emitters depends on their distance to a metallic mirror [4]. We locally excite Ce3+ emitters in YAG, which is partially covered with a thin aluminum film (Fig. 3). We then measure the CL lifetime as a function of distance d to the metal, with deep subwavelength (<λ/10) resolution (Fig. 4). Our results firmly establish time-resolved electron spectroscopy of nanophotonical devices as a powerful characterization tool for nanophotonics.

 

 

 

[1]     MacDonald, K. F.; Sámson, Z. L.; Stockman, M. I.; Zheludev, N. I., Ultrafast active plasmonics. Nat. Photonics 2008, 3 (1), 55-58.

[2]     Yang, D. S.; Mohammed, O. F.; Zewail, A. H., Scanning ultrafast electron microscopy. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (34), 14993-8.

[3]     Zonnevylle, A. C.; Van Tol, R. F.; Liv, N.; Narváez, A. C.; Effting, A. P.; Kruit, P.; Hoogenboom, J. P., Integration of a high-NA light microscope in a scanning electron microscope. J Microsc 2013, 252 (1), 58-70.

[4]     Drexhage, K. H., Influence of a dielectric interface on fluorescence decay time. J. Lumin. 1970, 1–2 (0), 693-701.


Robert MOERLAND, Mathijs GARMING (Delft, The Netherlands), Gerward WEPPELMAN, Pieter KRUIT, Jacob HOOGENBOOM
15:00 - 15:15 #5869 - MS07-OP301 Optically Coupled Plasmonic Nanopores Observed by Cathodoluminescence Scanning Transmission Electronmicroscopy.
Optically Coupled Plasmonic Nanopores Observed by Cathodoluminescence Scanning Transmission Electronmicroscopy.

Control of the optical properties of nano-plasmonic structures is essential for next-generation optical circuits and high-throughput biosensing platforms. Realization of such nano-optical devices requires optical couplings of various nanostructured elements and field confinement at the nanoscale. In particular, symmetric coupling modes, also referred to as “dark modes”, have recently received considerable attention because these modes can confine light energy to small spaces. Although the coupling behavior of plasmonic nanoparticles has been relatively well-studied, couplings of inverse structures, i.e., holes and pores, remain partially unexplored. Even for the most fundamental coupling system of two dipolar holes, comparison of the symmetric and anti-symmetric coupling modes has not been performed. Here, we present a systematic study of the symmetric and anti-symmetric coupling of nanopore pairs using cathodoluminescence by scanning transmission electron microscopy (CL-STEM) and electromagnetic simulation.

The nanopore samples were fabricated by colloidal lithography and film transfer by wet etching of the sacrificial layer. To achieve very close separation of nanopore pairs and to obtain high spatial resolution in STEM, we chose ultra-thin, free-standing film structures. For the measurement of single and coupled pairs of nanopores, 135 nm nanopores in an AlN(8 nm)/Au(16 nm)/AlN(8 nm) trilayer membrane were used (Figure 1b). With this sandwich layer structure, it is possible to obtain very thin and stable metal layers, even at high temperatures.

For CL-STEM measurement, 80 kV acceleration was used to avoid possible damage due to relatively high beam current. (5nA) Depending on the electron beam position it is possible to distinguish the symmetric and anti-symmetric dipolar coupling modes.(Fig. 1) The observed symmetric coupling mode, approximated as a pair of facing dipoles, appeared at a lower energy than that of the anti-symmetric coupling mode, indicating that the facing dipoles attract each other. The anti-symmetric coupling mode splits into the inner- and outer-edge localized modes as the coupling distance decreases. These coupling behaviors cannot be fully explained as simple inverses of coupled disks. Electromagnetic simulation by finite difference time domain (FDTD) also showed consistent coupling behaviors. Models of FDTD simulation showed that the inner- and outer-edge anti-symmetric modes become fully localized with minimal influence of the opposite edges as the coupling distance decreases. Symmetric and anti-symmetric coupling modes are also observed in a short-range ordered pore array (Fig. 2), where one pore supports multiple local resonance modes, depending on the distance to the neighboring pores.


Takumi SANNOMIYA (Yokohama, Kanagawa, Japan), Hikaru SAITO, Junesch JULIANE, Naoki YAMAMOTO
15:15 - 15:30 #6028 - MS07-OP303 Excitation and probing of hyperbolic phonon polaritons in hexagonal boron nitride structures by fast electrons.
Excitation and probing of hyperbolic phonon polaritons in hexagonal boron nitride structures by fast electrons.

Hexagonal boron nitride (hBN) is a representative material of a wide class of two-dimensional systems in which individual atomic layers are only weakly coupled by van der Waals interaction, resulting, among others, in extreme optical anisotropy. The latter gives rise to hBN’s hyperbolic phonon polaritons (h-PhPs), i.e. coupled excitations of optical phonons and light that exhibit hyperbolic dispersion [1, 2] at mid-infrared (mid-IR) energies, specifically in the range of 90-200 meV. Hyperbolic polaritons might be a key to many emerging photonic technologies that rely on nanoscale light confinement and manipulation, such as nanoscale imaging or sensing [3]. Thus, efficient design and utilization of hBN (nano)structures require spectroscopic studies with adequate spatial resolution and energy range.

Electron energy loss spectroscopy (EELS) performed in scanning transmission electron microscope (STEM) is a versatile technique that employs fast electrons as an effective localized electromagnetic probe for spectroscopy with nanoscale spatial resolution. Successfully employed at visible and near-IR energies, this technique has limited capabilities for mid-IR spectroscopy primarily due to lack of monochromaticity of the primary electron beam which typically masks low energy excitations under ~200  meV with the zero loss peak (ZLP) originating from the elastic electron scattering and also limits the spectral resolution to ~100 meV.

Here we demonstrate that by an optimization of microscope data acquistion and signal processing it is possible to significantly reduce the ZLP width down to 50 meV (with corresponding resolution enhancement), placing mid-IR spectroscopy within the reach of standard TEM instruments.

To this end, we perform experimental mapping of the spectral signature at an hBN edge.  As summarized in Fig. a, we clearly observe the variation in spectral peak position as a function of the electron impact parameter (position of the electron beam with respect to the edge). As revealed by our theoretical analysis, this behavior is the manifestation of polaritonic nature of the induced excitations. Indeed, our developed analytical and numerical models of EELS in structured hyperbolic materials show the existence of multiple EEL peaks depending on the impact parameter (see Fig. b). These peaks are due to the reflection of propagating h-PhPs from the hBN edge, which proves that fast electrons can and do couple to the hyperbolic polaritons. After mimicking the experimental spectral resolution (via convolution of the calculated spectra with a Gaussian of proper experimental width), we obtain a good agreement with the experimental data (see Fig. c).

Our work provides first steps in understanding polaritonic excitations produced by fast electrons in hyperbolic materials and sets grounds for the rigorous analysis of the observed low-energy EELS. With the ongoing improvements of STEM-EELS instrumentation [4], we expect further enhancement of the spectral resolution and an extension of the applicable energy ranges in near future, thus enabling EELS in STEM as a versatile technique for infrared spectroscopy of polaritons.

 

 

References:

[1] Dai S. et al. Science 343 (2014), 1125.

[2] Yoxall, E. et al. Nat. Photonics 9 (2015), 674.

[3] Li, P. et al. Nat. Commun. 6 (2015), 7507.

[4] Krivanek, O. L. et al. Nature 514 (2014), 209.


Andrea KONEČNÁ (San Sebastian, Spain), Alexander GOVYADINOV, Andrey CHUVILIN, Irene DOLADO, Saül VÉLEZ, Javier AIZPURUA, Rainer HILLENBRAND
15:30 - 15:45 #6598 - MS07-OP304 Spatiotemporal imaging of few-cycle nanoplasmonic fields using photoemission electron microscopy.
MS07-OP304 Spatiotemporal imaging of few-cycle nanoplasmonic fields using photoemission electron microscopy.

Surface plasmons are capable of concentrating light on both a nanometre spatial and femtosecond temporal scale, thus serving as a basis for nanotechnology at optical frequencies. However, the simultaneously small and fast nature of surface plasmons leads to new challenges for spatiotemporal characterization of the electric fields. An especially successful method for this purpose is photoemission electron microscopy (PEEM) in combination with ultrashort laser pulses. This method uses the high spatial resolution offered by electron microscopy together with the temporal resolution offered by femtosecond laser technology. By combining PEEM with state-of-the-art sources of ultrashort bursts of light, we have contributed to two pathways towards the ultimate goal: the full spatiotemporal reconstruction of the surface electric field at arbitrary nanostructures.

The first approach is based on extending interferometric time-resolved PEEM (ITR-PEEM) [1] to the few light cycle regime by using two synchronized pulses from an ultra-broadband oscillator. Because the photon energy (1.2-2.0 eV) is well below the material work function, photoemission occurs through a multiphoton process. The measurement is performed by scanning the delay between two identical, sub-6 fs pulses and measuring the local photoemission intensity (Fig. 1a). We have applied this method to a variety of nanostructures, including rice-shaped silver particles, nanocubes, and gold bow-tie nanoantennas. As an example, results from the rice-shaped silver nanoparticles are shown in Fig. 1. We excited multipolar surface plasmons at grazing incidence, and imaged the photoelectrons emitted from the two ends of the nanoparticle (Fig. 1b). Upon scanning the delay between the two pulses, the interference fringes measured from the two ends of the nanoparticle are shifted with respect to each other (Fig. 1c). We show that these shifts correspond to locally different instantaneous frequencies of the near-field within the same nanoparticle, and that these differences occur due to a combination of retardation effects and the excitation of multiple surface plasmon modes [2].

The second approach is based on using high-order harmonic generation (HHG) to produce attosecond pulses in the extreme ultraviolet (XUV) region. Attosecond XUV pulses have been proposed to enable a direct spatiotemporal measurement of nanoplasmonic fields with a temporal resolution down to 100 as [3]. However, PEEM imaging using HHG light sources has turned out to be a major challenge due to numerous issues such as space charge effects, chromatic aberration, and poor image contrast [4-6]. To address these issues, we perform HHG using a new optical parametric chirped pulse amplification system delivering 7 fs pulses at 200 kHz repetition rate. We show how the XUV pulses generated by this system allow for PEEM imaging with both higher resolution and shorter acquisition times. For comparison, Fig. 2 shows PEEM images of silver nanowires on a gold substrate, imaged using high-order harmonics at 1 kHz repetition rate (Fig. 2a, acquisition time is 400 s) and at 200 kHz repetition rate (Fig. 2b, acquisition time is 30 s). The image quality is clearly improved (Fig. 2c). We also show how the higher repetition rate allows for PEEM imaging using only primary (“true”) photoelectrons, whereas previous studies have acquired images using secondary electrons [4-6].

[1] A. Kubo et al., Nano Lett. 5, 1123 (2005).

[2] E. Mårsell et al., Nano Lett. 15, 6601 (2015).

[3] M. I. Stockman et al., Nat. Photon. 1, 539 (2007).

[4] A. Mikkelsen et al., Rev. Sci. Instrum. 80, 123703 (2009).

[5] S. H. Chew et al., Appl. Phys. Lett. 100, 051904 (2012).

[6] E. Mårsell et al., Ann. Phys. (Berlin) 525, 162 (2013).


Erik MÅRSELL, Arthur LOSQUIN (Lund, Sweden), Chen GUO, Anne HARTH, Eleonora LOREK, Miguel MIRANDA, Cord ARNOLD, Hongxing XU, Johan MAURITSSON, Anne L'HUILLIER, Anders MIKKELSEN
15:45 - 16:00 #6624 - MS07-OP305 Toroidal dipole plasmon resonance modes in upright split ring resonators.
Toroidal dipole plasmon resonance modes in upright split ring resonators.

Nanoscale split ring resonators (SRRs) have been a popular topic of study due to their surface plasmon resonance (SPR) modes and their many interesting interactions with light. They can be used as components in metamaterials exhibiting, among other properties, a negative refractive index. The surface plasmon properties of these structures are strongly dependent on their size and spatial arrangement. Most studies so far have focussed on the horizontal SRR due to the ease of fabrication. However, there are some advantages to be gained in the design of materials using upright SRRs. We are studying a structure composed of four upright SRRs as shown in Figure 1. The coupling of these four upright SRRs produces a magnetic dipole moment and a toroidal dipole moment.

The toroidal dipole moment, when compared to electric and magnetic dipole moments, shows a higher quality factor and lower gain threshold for a nanoscale laser analogue, the spaser (surface plasmon amplification by stimulated emission of radiation) [1]. The presence of a strong toroidal dipole moment isolated from magnetic and electric dipole moments makes the structure under study a promising candidate for a spaser for use in on-chip telecommunications.

A similar structure was first realized experimentally in the microwave regime of the electromagnetic spectrum [2]. Scaling the geometry down to nanoscale dimensions has been shown by simulation to shift the toroidal dipole energies into the near infra-red regime [1]. In this work we demonstrate the experimental fabrication (Figure 2) and characterization of this structure using electron energy loss spectroscopy (EELS), with confirmation of the modes provided by finite element method (FEM) simulations.

We have fabricated this structure using a double patterning process in electron beam lithography, with precise alignment of the second lithography layer to the first. The structures are made from gold deposited on a 50 nm thick silicon nitride membrane. We probe the plasmon modes using EELS on a monochromated scanning transmission electron microscope, collecting spectrum images with nanometer spatial resolution and 60 meV energy resolution. We extract site-specific spectra (Figure 3a) and energy-resolved maps of the SPR modes (Figure 3b, c). We apply the Richardson-Lucy algorithm to further increase the effective energy resolution and identify the magnetic and toroidal dipole modes at energies of 0.52 eV and 0.72 eV, with SPR maps as shown in Figure 3b and 3c, respectively.

We are able to correlate our EELS results with COMSOL Multiphysics FEM simulations. The simulated SPR response is given in Figure 3a, d, and e, showing close agreement in the peaks with our experimental data. Simulations confirm the low energy magnetic dipole mode (0.56 eV) and reveal two closely spaced toroidal dipole modes (0.61 eV, 0.66 eV) which are not perfectly resolved in the EELS data. We are able to tune the energy and strength of the toroidal dipole moment through tuning of the fabrication parameters; with careful design this structure is a promising spaser design for a range of applications near telecommunications frequencies.

References

[1] Y.-W. Huang, et al., Sci. Rep., vol. 3, Feb. 2013.

[2] T. Kaelberer, et al., Science, vol. 330, no. 6010, pp. 1510–1512, Dec. 2010.

Acknowledgements: We gratefully acknowledge the financial support of NSERC and the province of Ontario.


Isobel BICKET (Hamilton, Canada), Edson BELLIDO, Ahmed ELSHARABASY, Mohamed BAKR, Gianluigi BOTTON
Salle Gratte Ciel 1&2

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LS1-I
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LS1: Macromolecular assemblies, supra molecular assemblies
SLOT I

Chairpersons: Bettina BOETTCHER (Edinburgh, United Kingdom), Karen DAVIES (Staff Scientist) (Berkeley, USA), Guy SCHOEHN (Grenoble, France)
14:00 - 14:30 #8323 - LS01-S01 Native machinery of membrane-associated protein synthesis.
Native machinery of membrane-associated protein synthesis.

A large fraction of ribosomal synthesis occurs at organellar membranes. At the endoplasmic reticulum (ER), the inner mitochondrial, and the thylakoid membrane, nascent proteins are co-translationally inserted or transported into other compartments. Here, we structurally study membrane-bound ribosomes and their associated machineries in their native settings using cryo-electron tomography (cryo-ET) in combination with subtomogram analysis (Fig. 1).

Studies of ribosomes associated to isolated rough ER vesicles reveal the structure of the native ER translocon, as well as its compositional variability. Core components of the ER translocon are the protein-conducting channel Sec61, the translocon associated protein complex (TRAP), and the sub-stoichiometric oligosaccharyl transferase complex (OST), which all bind to the ribosome. Subnanometer resolution subtomogram averages indicate that the ribosome alone, even without a nascent chain, is sufficient for lateral opening of Sec61, contrary to recent mechanistic models. To elucidate the structures and functions of TRAP and OST in detail, we make use of mutations involved in congenital disorders, as well as their evolutionary diversity across different organisms. Analysis of cryo-tomograms from focused-ion-beam-milled whole cells allows studying the compositional variability of the ER-translocon and the relative arrangement of ER-associated ribosomes in vivo, which reveals a highly characteristic polysome organization.

Mitochondrial ribosomes specialize on the synthesis of few, very hydrophobic membrane proteins. Cryo-electron tomographic analysis of mitochondria isolated from Saccharomyces cerevisiae reveals the binding mode of mitoribosomes to the inner mitochondrial membrane, as well as their molecular organization into polysomes. The structures of mammalian mitoribosomes differ dramatically from their fungal counterparts and we study the consequences of these differences on membrane association and polysome organization. State-of-the-art phase plate imaging helps to overcome the contrast limitations set by the extremely dense and optically barely electron transparent mammalian mitochondria.

Chloroplast ribosomes constitute the third realm of eukaryotic ribosomes. We analyzed the in situ structure and intracellular distribution in green algae. The interaction mode of ribosomes with the thylakoid membrane appears to be much less defined than those of their cytoplasmic and mitochondrial counterparts.

In summary, in situ studies using cryoelectron tomography put atomic-level structural information of ribosomal complexes into context with their associated organellar membranes and their respective co-translational processing machineries, revealing high evolutionary diversity for organelles and organisms.


Stefan PFEFFER, Robert ENGLMEIER, Friedrich FOERSTER (martinsried, Germany)
Invited
14:30 - 15:00 #8743 - LS01-S02 Structure of hibernating ribosomal complexes from Gram-positive pathogenic bacteria Staphylococcus aureus, solved by single particle cryo-EM.
Structure of hibernating ribosomal complexes from Gram-positive pathogenic bacteria Staphylococcus aureus, solved by single particle cryo-EM.

Protein synthesis is a universally conserved process that is assured by a macromolecule called the ribosome (3.4 – 4.5 Mda). In spite of the conservation of the ribosome among all orders of life, its structure presents significant differences between eukaryotes and bacteria. Bacterial ribosome, smaller than its eukaryotic counterpart, presents specific particularities to which we owe the efficiency of numerous commonly used antibiotics that target the latter without hindering protein synthesis in the host. Such structural differences are found in all steps of protein translation.

Here, we attempt to explore by cryo-electron microscopy the ribosomal hibernation, one of the most mysterious regulation processes of protein translation in bacteria. Hibernation is a vital process that can be triggered as a response to stress and aims at shutting down translation in a reversible manner so that translation can recover quickly after the alleviation of stress. Here, we show several 3D reconstructions of hibernation ribosomal complexes from the Gram-positive pathogenic bacteria Staphylococcus aureus. Our structures display the formation of a disome (ribosomal dimer) mediated by a peculiar hibernation factor (HPF), thus setting up the disome in a unique fashion, different than other dimers of known bacterial species indicating the uniqueness of this process in S. aureus and perhaps in gram-positive bacteria more generally. In spite of the size of the imaged asymmetric complexes (~7 Mda, ~550Å diameter), our data-processing yielded several structures after particle sorting presenting an average resolution of ~3.7Å, thus enabling the modelling ab initio of S. aureus HPF, so far of unknown structure.

Our results represent a significant advance and pinpoint a unique process that can be targeted for designing drugs of improved specificity and efficiency against this dangerous pathogen and Gram-positive bacteria more generally.


Iskander KHUSAINOV, Quentin VICENS, Anthony BOCHLER, Alexander MYASINKOV, Srefano MARZI, Pascale ROMBY, Gulnara YUSUPOVA, Marat YUSUPOV, Yaser HASHEM (STRASBOURG CEDEX)
Invited
15:00 - 15:15 #5775 - LS01-OP001 Cryo-electron microscopy structure of La Crosse orthobunyavirus polymerase in presence or absence of viral RNA.
Cryo-electron microscopy structure of La Crosse orthobunyavirus polymerase in presence or absence of viral RNA.

Bunyaviridae is the largest family of segmented negative strand viruses (sNSV) which also include Orthomyxoviridae and Arenaviridae. Central to their viral cycle is the RNA-dependent RNA polymerase which replicates and transcribes the genome segments within circular ribonucleoprotein particles (RNPs). Here we describe a cryo-electron microscopy reconstruction of the full length La Crosse polymerase in complex with viral RNA (Figure 1), together with a reconstruction of its apo truncated form (Δ-Cterminal construct, Figure 2). Combined with the X-ray structure determined in the group, we provide a partial pseudo-atomic model of La Crosse polymerase (Figures 1 and 2). Identification of distinct template and product exit tunnels (Figure 3) and structural analysis of RNP (Figure 4) allows proposal of a detailed model for template-directed replication with minimal disruption to the circularised RNP. The similar overall architecture and vRNA binding of monomeric LACV to heterotrimeric influenza polymerase, despite high sequence divergence, suggests that all sNSV polymerases have a common evolutionary origin and mechanism of RNA synthesis.

Reference: Structural Insights into Bunyavirus Replication and Its Regulation by the vRNA Promoter. Gerlach P*, Malet H*, Cusack S¦, Reguera J¦. Cell. 2015 Jun 4;161:1267-79.


Helene MALET (Grenoble), Piotr GERLACH, Juan REGUERA, Stephen CUSACK
15:15 - 15:30 #6610 - LS01-OP006 Structure-function insights reveal the human ribosome as a cancer target for antibiotics.
Structure-function insights reveal the human ribosome as a cancer target for antibiotics.

Abstract

Many antibiotics in clinical use target the bacterial ribosome by interfering with several mechanistic steps of the protein synthesis machinery. However, targeting the human ribosome in the case of protein synthesis deregulations such as in highly proliferating cancer cells has not been much considered up to now. Here we report the first structure of the human 80S ribosome with a eukaryote-specific antibiotic and show its anti-proliferative effect on cancer cell lines. Structural sorting of the cryo electron microscopy data shows that the cycloheximide ligand induces an equilibrium shift of ribosome subpopulations in different states, revealing that the mechanism of action relies on an active release of the tRNA from the exit site. The structure provides unprecedented insights into the detailed interactions in a ligand-binding pocket of the human ribosome that are required for structure-assisted drug design. Furthermore, anti-proliferative dose response in leukemic cells and interference with synthesis of c-myc and mcl-1 short-lived protein markers reveals specificity of a series of antibiotics towards cytosolic rather than mitochondrial ribosomes, establishing the human ribosome as a promising cancer target. In addition, we present a protocol that primarily uses the crystallographic tools for atomic model building and refinement into cryo-EM maps, as exemplified by our recent human ribosome structure.


Alexander MYASNIKOV, Kundhavai NATCHIAR (Illkirch), Marielle NEBOUT, Isabelle ISABELLE HAZEMANN, Véronique IMBERT, Heena KHATTER, Jean-François PEYRON, Bruno KLAHOLZ
15:30 - 15:45 #6802 - LS01-OP007 Tripartite assembly of RND multidrug efflux pumps in nanodisc and amphipol.
Tripartite assembly of RND multidrug efflux pumps in nanodisc and amphipol.

 

*Two first authors equally contributed to this work

 

Tripartite multidrug efflux systems of Gram-negative bacteria export a large variety of antimicrobial compounds at the expense of ATP or the proton motive force, thereby conferring resistance to a wide variety of antibiotics. Pseudomonas aeruginosa MexAB-OprM and Escherichia coli AcrAB-TolC, are prototypic proton motive force-driven efflux systems from Resistance Nodulation and cell Division (RND) family. These efflux systems, composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein, are assumed to form ducts inside the periplasm, facilitating drug exit across the outer membrane. Using lipid nanodisc system, we recently reported the reconstitution of native MexAB-OprM tripartite system. Single particle analysis by electron microscopy revealed the lipid nanodisc-embedded inner and outer membrane protein components linked together via the MexA periplasmic adaptor protein [1].

Recently the replacement of detergent by specially designed amphiphilic molecules such as amphipol or styrene–maleic acid copolymers is an alternative option to stabilize the membrane proteins in a free-lipid environment. To further investigate key parameters controlling the formation of the tripartite system, we focus our study on MexA that in its mature form is anchored to the inner membrane via its palmitoyl moiety. To assess whether the lipid anchor is required for the reconstitution, we report here on the reconstitution of OprM and MexB in amphipol and compare with our previous results obtained with nanodiscs.

 

 

[1] Daury L, Orange F, Taveau JC, Verchère A, Monlezun L, Gounou C, Marreddy RK, Picard M, Broutin I, Pos KM, Lambert O (2016). Tripartite assembly of RND multidrug efflux pumps. Nat Commun. 7:10731.


Dimitri SALVADOR, Marie GLAVIER, Cyril GARNIER, Martin PICARD, Isabelle BROUTIN, Jean-Christophe TAVEAU, Laetitia DAURY, Olivier LAMBERT (Bordeaux)
15:45 - 16:00 #6546 - LS01-OP004 The supramolecular packing of the gel-forming MUC5B and MUC2 mucins and its importance for cystic fibrosis.
The supramolecular packing of the gel-forming MUC5B and MUC2 mucins and its importance for cystic fibrosis.

Text:

Introduction: The genetically related gel forming mucins, MUC2 (intestine), MUC5B (airways), MUC5AC (airways, stomach) and MUC6 (stomach) have large sizes with heavily glycosylated mucin domains in the central part. The C-termini form intermolecular dimers. The N-terminal regions are evolutionarily similar with identical domain organization important for the oligomerization. MUC5B is vital for normal mucociliary clearance of the lungs whereas MUC2 in colon forms an inner dense and attached stratified layer impermeable to bacteria, and an outer loose and unattached layer habituating commensal bacteria. The MUC2 N-terminus (D1-D2-D′D3 domains) was shown to form concatenated polygone-structures under low pH- and high calcium conditions (1).
The Aim is to understand the cellular packing of the MUC5B and MUC2 mucins and how this influences their secretion.
Method: The N-terminal and D´D3 domains of MUC2 and MUC5B were expressed, purified and then analyzed by subsequent gel filtration, transmission electron microscopy and single particle image processing.
Results: MUC5B multimerizes by disulfide bonds between the D3-domains giving the MUC5B polymer a linear structure (2). Analysis of the MUC5B N-terminus at lower pH and higher calcium concentration revealed a tight dimer+dimer packing where the second dimer is turned upside down by 180 degrees and then slightly rotated. This way of packing the MUC5B in the granulae will allow a slow unwinding of a linear molecule.
Conclusion: The MUC5B and MUC2 mucins are packed in the mucin granulae in a way allowing the formation of linear strands or net-like structures, respectively.

References:
1. Ambort, D., Johansson, M. E. V., Gustafsson, J. K., Nilsson, H., Ermund, A., Johansson, B. R., Koeck, P. J. B., Hebert, H., and Hansson, G. C. (2012) Proc. Natl. Acad. Sci. U. S. A. 109, 5645-5650
2. Ridley, C., Kouvatsos, N., Raynal, B. D., Howard, M., Collins, R. F., Desseyn, J. L., Jowitt, T. A., Baldock, C., Davis, C. W., Hardingham, T. E., and Thornton, D. J. (2014) J. Biol. Chem. 289, 16409-16420

 


Harriet E. NILSSON (Huddinge, Sweden), Sergio MUYO TRILLO, Anna ERMUND, Malin BÄCKSTRÖM, Elisabeth THOMSSON, Daniel AMBORT, Philip J. B. KOECK, David J. THORNTON, Gunnar C. HANSSON, Hans HEBERT
Salle Tête d'or 1&2

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LS7-I
14:00 - 16:00

LS7: Organism development and imaging
SLOT I

Chairpersons: Grégoire MICHAUX (Rennes, France), Cristina PUJADES (co-chair) (Barcelona, Spain)
14:00 - 14:30 #8649 - LS07-S22 Eavesdropping on the molecular signatures of embryonic development.
Eavesdropping on the molecular signatures of embryonic development.

The study of embryonic development has been dramatically advanced by the wealth of high-throughput molecular studies that have defined the genes and proteins involved.  This wealth of data now presents the challenge of integrating a working knowledge of how these molecular components, often present at vanishingly small concentrations, generate reliable patterns of cell migration and cell differentiation.  In typical cell biology approaches, cultures of isolated cells have been used reveal mechanism.  What is needed to understand development is to carry out studies on cells in their normal context interacting with other cells and signals in the intact embryo.  Key events of embryonic development take place over dimensions of less than 500um in less than 5 hours, making it tractable for light imaging tools to be used to answer this challenge.   

 Imaging techniques are challenged by major tradeoffs between spatial resolution, temporal resolution, and the limited photon budget.  We are attempting to advance this tradeoff by constructing light sheet microscopes that maintain subcellular resolution in thick and scattering specimens.  Our two-photon light-sheet microscope, combines the deep penetration of two-photon microscopy and the speed of light sheet microscopy to generate images with more than ten-fold improved imaging speed and sensitivity.  As with other light sheet technologies, the collection of an entire 2-D optical section in parallel offers dramatically speeds acquisition rates.  By adopting two-photon SPIM is far less subject to light scattering, permitting subcellular resolution to be maintained far better than conventional light sheet microscopes.  The combination of attributes permits cell and molecular imaging with sufficient speed and resolution to generate unambiguous tracing of cells and signals in intact systems, which presents a major challenge in data management, processing and analysis. 

Multispectral imaging offers the chance of asking multiple questions of the same embodied cells.  Multiplex analyses permit the variance and the “noise” in a system to be exploited by asking about the analytes that co-vary with a selected gene product.  Transforming fluorescent spectra to a point on a 2D-plot with sinusoidal functions (phasors) provides a powerful tool for the cumbersome tasks of visualizing and analyzing hyper-spectral imaging data.  This technique offers excellent performance in the face of biological and instrumental uncertainties. Our results on live zebrafish embryos document the accuracy of hyper-spectral phasors and demonstrates their capability to distinguish multiple spectrally overlapping fluorophores in low signal-to-noise and fast analysis time.

Combined, these tools offer the multi-dimensional imaging required to follow key events in embryos as they take place, and allow us to use variance as an experimental tool rather than a limitation. 


Scott FRASER (Los Angeles, USA)
Invited
14:30 - 14:45 #4890 - LS07-OP028 Germ-line cysts organisation in clitellate annelids – from electron microscopy to live cell imaging.
Germ-line cysts organisation in clitellate annelids – from electron microscopy to live cell imaging.

 

At the onset of gametogenesis in animals, the germ-line cells usually form syncytial cysts (clusters). The germ cells in such cysts are interconnected by wide cytoplasmic channels known as intercellular (cytoplasmic) bridges that are modified contractile rings that do not close during late cytokinesis. These bridges ensure the cytoplasmic continuity between the interconnected cells and, as a result, the cytoplasm of the sister cells is common. It is believed that the interconnections of germ cells into syncytial clusters regulate and synchronise germ cell development. However, it seems that the actual role of the cell clustering is sex dependent.

                The germ-line cyst architecture (spatial organization) varies between taxa. Currently, we know several different modes of cysts organisation. The best characterised examples are: linear cysts in which the interconnected cells form chains (e.g. mammal spermatogenesis); branched cysts in which some cells may have more than two intercellular bridges and, as a result, “branchings” occur in these sites (e.g. male and female cysts in Drosophila); and cysts with germ cells that are clustered around a central anuclear mass of cytoplasm (e.g. in C. elegans).

              Clitellate annelids (Clitellata) is a monophyletic taxon of segmented worms, which comprises earthworms and their allies and leeches. At the early stages of spermatogenesis and oogenesis in Clitellata, germ-line cysts are also formed (with one known exception – female cells in Capilloventer australis). In both lines, male and female, the cysts have the same basal plan of organization – each germ cell is connected to the central and anuclear cytoplasmic mass, the cytophore via one cytoplasmic bridge (Fig. 1 and 2). However, the cyst morphology may differ substantially between taxa and even between sexes in the same specimen. These differences may be caused by: a different number of interconnected germ cells (from 16 in the pot worm Enchytraeus albidus (Fig. 2) to over 2 000 in the sludge worm Tubifex tubifex (Fig. 3)); a different shape and ratio of cytophore development (the cytophore in male cysts is usually spherical and well developed, while in female cysts the cytophore usually has the form of long cytoplasmic strands and is often poorly developed). Additionally, whereas male cysts are not associated with somatic cells, female clusters are usually enveloped by somatic cells and together form characteristic structures such as egg follicles or ovary cords.

              During our long-term studies, we have analysed the formation, architecture and functions in both male and female germ-line clusters in several representatives of clitellate annelids (e.g. the earthworm Dendrobaena veneta, the pot worm E. albidus, a sludge worm T. tubifex and the European medicinal leech Hirudo medicinalis). Using classical (light and fluorescent microscopy, electron microscopy) and modern tools (live cell imaging see Fig. 4, 3D reconstructions in scanning electron microscopy – SBEM method), we have revealed, among others, the great plasticity in cyst architecture and sex-dependent differences in their functions.

Acknowledgements This work was partially funded by National Science Centre, Poland. Contract grant number: DEC-2012/05/B/NZ4/02417.


Piotr ŚWIĄTEK, Anna Z. URBISZ, Karol MAŁOTA, Szymon GORGOŃ, Natalia JAROSZ, Piotr ŚWIĄTEK (Katowice, Poland)
14:45 - 15:15 #8757 - LS07-S23 Optical investigation of a novel sensory interface linking spinal fluid to motor circuits in vivo.
Optical investigation of a novel sensory interface linking spinal fluid to motor circuits in vivo.

The cerebrospinal fluid (CSF) is a complex solution circulating around the brain and spinal cord. Behavior has long been known to be influenced by the content and flow of the CSF, but the underlying mechanisms are completely unknown. CSF-contacting neurons by their location at the interface with the CSF are in ideal position to sense CSF cues and to relay information to the nervous system. By combining electrophysiology, optogenetics, bioluminescence monitoring with calcium imaging in vivo, we demonstrate that neurons contacting the CSF in the spinal cord detect local bending and in turn feed back GABAergic inhibition to multiple interneurons driving locomotion in the ventral spinal cord. Behaviour analysis of animals deprived of this mechano-sensory pathway reveals its contribution in modulating key parameters of locomotion. Altogether our approach developed in a transparent animal model shed light on a novel pathway enabling sensory motor integration between the CSF and motor circuits in the spinal cord.

 

References

Djenoune et al., Frontiers in Neuroanatomy 2014.

Fidelin et al., Current Biology 2015.

Bohm et al., Nature Communications 2016.

Hernandez et al., Nature Communications 2016.

Sternberg et al., Current Biology, in press.

Hubbard et al., Current Biology, in revision.

Knafo et al., submitted.


Claire WYART (Paris)
Invited
15:15 - 15:30 #6574 - LS07-OP029 The posterior neural plate in axolotl can form mesoderm or neuroectoderm.
The posterior neural plate in axolotl can form mesoderm or neuroectoderm.

Gastrulation is the developmental process where germ layers are formed. It was generally assumed that germ layer specification is finished by the end of gastrulation. However, previous studies in amphibians revealed that posterior neural plate and fold, although traditionally regarded as neural, have a mesodermal bias and give rise to tail and posterior trunk muscles whereas only more anterior regions give rise to spinal chord [1-4]. In addition, recent studies in zebrafish, chick, and mouse revealed bipotential stem cell populations at the posterior ends of these embryos. The stem cells can give rise to neural and mesodermal tissues depending on local signaling in the tail bud [5-7]. Here, we reinvestigated morphogenesis and fate of the posterior neural plate in an amphibian model, the axolotl (Ambystoma mexicanum), using GFP-labeled grafts of the posterior third of the neural plate (reg. 3; stage 15; Figure 1) for detailed lineage analysis. In situ hybridisation with riboprobes against mesodermal (brachyury, bra) and neuronal/stem cell (sox2) markers revealed an ambiguous determinative state of reg. 3 at the time of transplantation. While its central and posterior part is bra-positive, small left and right anterior regions expressed sox2. The more anterior plate regions 2 and 1 are sox2-positive and bra-negative. The border of the two markers is ill defined and shows some overlap. Histological analysis of reg. 3 grafts at early tailbud stages revealed that the cells of the most posterior part of reg. 3 start moving from dorsal to ventral forming the posterior wall. Then they turn anteriorly, become connected with paraxial presomitic mesoderm and form somites on either side of the embryo (Figure 1). As a result of this movement, the posterior half of reg. 3 gives rise to posterior trunk and anterior tail somites whereas the anterior half forms posterior tail somites and tail spinal cord. Only cells that conduct this anterior turn and pass the Wnt/b-catenin positive posterior wall will contribute to paraxial mesoderm. Cells that remain in the dorsal part of the tail-bud eventually form lateral and dorsal parts of the tail spinal chord. The floor plate of the posterior spinal chord and the axial mesoderm do not contain any reg. 3 cells which indicates that they may be formed from the chordoneural hinge, a connection between the posteriormost reg. 2 cells and the tip of the notochord [8]. Additional grafting experiments showed that the notochord is formed from axial mesoderm that was already involuted during gastrulation and underwent massive elongation, presumably by convergence and extension. Therefore, axial and paraxial mesoderm of the tail are formed by different mechanisms and are probably specified at different time points, i.e. during gastrulation and tail bud development.

Taken together, these data show that germ layer specification is not complete after gastrulation, but that part of the putative neural plate is specified during morphogenetic movements of tail bud stages in order to become paraxial mesoderm.

 

[1]        JH Bijtel, Roux Arch. EntwMech. Organ. 125 (1931) 448-486

[2]        AS Tucker and JMW Slack, Curr. Biol. 5 (1995) 807-813

[3]        CW Beck, WIREs Dev Biol 4 (2014) 33-44

[4]        Y Taniguchi, T Kurth, et al., Sci. Rep. 5 (2015) 11428

[5]        BL Martin and D Kimelman, Dev. Cell 22 (2012) 223-232

[6]        CM Bouldin, AJ Manning, et al., Development 142, 2499-2507

[7]        H Kondoh and T Takemoto, Curr. Opin. Genet. Dev. 22 (2012) 374-380

[8]        LK Gont, H Steinbeisser, et al., Development 119 (1993) 991-1004

[9]        Financial support by DFG (EP 8/11-1) is gratefully acknowledged.


Yuka TANIGUCHI, Thomas KURTH (Dresden, Germany), Verena KAPPERT, Saskia REICHELT, Susanne WEICHE, Akira TAZAKI, Cora RÖHLECKE, Hans-Henning EPPERLEIN
15:30 - 16:00 #7505 - LS07-S24 Tracking tumor metastasis in vivo at high-resolution.
Tracking tumor metastasis in vivo at high-resolution.

Three reasons explain why most of the critical events driving normal and pathological scenarios had been less investigated: they occur rarely in space and time, they are highly dynamic, they differ when studied in situ in an entire living organism. Metastasis is the primary cause for cancer-related mortality, but its mechanisms remain to be elucidated. Intravital imaging has opened the door to in vivo functional imaging in animal models of cancer, however it is limited in resolution. Ultrastructural analysis of tumor metastasis in vivo has so far been hindered by the limited field of view of the electron microscope, making it difficult to retrieve volumes of interest in complex tissues. We recently developed a multimodal correlative approach allowing us to rapidly and accurately combine functional in vivo imaging with high-resolution ultrastructural analysis of tumor cells in a relevant pathological context. The multimodal correlative approach that we propose here combines two-photon excitation microscopy (2PEM), microscopic X-ray computed tomography (microCT) and three-dimensional electron microscopy (3DEM). It enables a rapid and accurate correlation of functional imaging to high-resolution ultrastructural analysis of tumor cells in a relevant pathological context. As an example, we are now capable of providing high- and isotropic (8nm) resolution imaging of single metastasizing tumor cells previously imaged in the process of extravasation in the living mouse brain. This reliable and versatile workflow offers access to ultrastructural details of metastatic cells with an unprecedented throughput opening to crucial and unparalleled insights into the mechanisms of tumor invasion, extravasation and metastasis in vivo.


Jacky GOETZ (Strasbourg)
Invited
Salon Tête d'Or

"Monday 29 August"

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SCUR - I
14:00 - 16:00

The Skin Imaging Society meeting
SLOT I

14:00 - 14:10 Opening of the SCUR meeting.
14:10 - 15:10 Session 1. Oral communications.
15:10 - 16:00 Invited lecture: Recent advances in cryomethods for skin studies. Roger A. WEPF (Zürich, Switzerland)
Salle Gratte Ciel 3
16:30

"Monday 29 August"

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SCUR - II
16:30 - 19:00

The Skin Imaging Society meeting
SLOT II

16:30 - 17:30 Session 2. Oral communications.
17:30 - 19:00 Annual General Assembly of SCUR.
Salle Gratte Ciel 3