bluuebunny/crossref_metadata_2025_split huggingface dataset:
Electron‐beam induced fluorescence superresolution with 100nm resolution in
CLEM
on labelled tissue sections
Authors: Voortman Lennard M. , Raja Aditi Srinivasa , Väkeväinen Aaro , de Boer Pascal , Giepmans Ben N.G. , Kruit Pieter , Hoogenboom Jacob P. 
Published: December 2016

We present a novel optical superresolution (SR) technique using integrated correlative light and electron microscopy. Recent advances in SR techniques has revolutionized the field of optical microscopy by achieving image resolutions well below the diffraction limit, the fundamental resolution limit of traditional optical microscopy. Current SR methods involve stochastic techniques, beam‐shaping in combination with confocal scanning, external control over excited state relaxation pathways, and/or structured illumination [1]. Correlation of SR data with ultrastructural images obtained with electron microscopy (EM) has been demonstrated [2], but requirements for SR microscopy are often in conflict with those for EM. Moreover, the optical localization accuracy in the correlation image may be severely compromised compared to the SR resolution by the additional error introduced in aligning the separate SR and EM images. Here, we demonstrate a novel approach for correlative SR‐EM using a focused electron beam to locally modify the fluorescence signal of fluorophores, and detecting the change in fluorescence intensity with a wide‐field epi‐fluorescence microscope.
We use an integrated light‐electron microscope [3] that is used for correlative light and electron microscopy (CLEM) [2]. The integrated light microscope allows us to record the fluorescence signal while scanning the electron beam through the optical field of view. By correlating changes in the fluorescence decay with the instantaneous electron beam position and the other EM signals, we obtain a SR fluorescence image (Fig.1). This SR fluorescence image is in perfect registry with the simultaneously acquired EM image.
In first experiments on rat pancreas tissue, immuno‐labelled for insulin and guanine quadruplexes using different Alexa Fluor dyes, we have achieved a lateral resolution below 100nm (Fig. 2). We will discuss further implementation of our technique towards higher resolution, paving the way towards precise localization, within the EM ultrastructure, of bio‐molecules labelled with standard fluorescent dyes.
Study on the robustness of electron ptychography for phase imaging in the
STEM
using fast pixelated detectors
Authors: Martinez Gerardo T , Yang Hao , Jones Lewys , Simson Martin , Huth Martin , Soltau Heike , Strüder Lothar , Sagawa Ryuusuke , Kondo Yukihito , Nellist Peter D 
Published: December 2016

The use of electron ptychography methods for phase imaging in the scanning transmission electron microscope (STEM) have gained renewed interest due to the recent developments of fast pixelated detectors. One of those being the pnCCD (S)TEM camera [1] developed by PNSensor and PNDetector, it allows the recording of a 4D‐dataset that consists of the full convergent beam electron diffraction pattern for each probe position that is scanned over the sample. This dataset contains all the scattering information generated by the electron sample interaction in the STEM experiment. The technique has allowed improved resolution beyond conventional limits [2] and phase contrast imaging at atomic resolution of different materials [3, 4]. In combination with other STEM techniques, it enables imaging of heavy and light elements of radiation‐sensitive materials at the atomic scale [4].
In this work, we present an analysis and discussion of two different phase reconstruction algorithms, such as the Single Side Band (SSB) [3, 5, 6] and the Wigner‐Distribution Deconvolution (WDD) [7] methods, in terms of their robustness to dynamical effects. The main difference of both methods is that the phase difference retrieved by the SSB method is still affected by the probe aberrations, meanwhile the WDD method allows to correct for this [4, 7]. However, in both cases a multiplicative interaction between the specimen function and the electron wave is assumed, which complicates the interpretation of the reconstructed wave when dynamical effects start to play a major role. Figures 1 and 2 show experimental results of retrieving the modulus (a) and the phase difference (b) of a gold nanoparticle measured with the pnCCD (S)TEM camera and using the discussed ptychography methods. It can be observed that by correcting the probe aberrations [3] while using the WDD method, the quality of the reconstructed image is dramatically improved. Moreover, a ring‐like shape of the reconstructed phase difference for some atomic columns is observed. This behavior is studied in detail by image simulations (c) and discussed in terms of how dynamical effects influence the reconstruction algorithm. Furthermore, we explore the comparison of this method with other phase imaging techniques.
Analytical electron tomographic investigations revealing self stabilization of core‐shell precipitates through opposing diffusion processes
Authors: Orthacker Angelina , Haberfehlner Georg , Taendl Johannes , Poletti Maria Cecilia , Sonderegger Bernhard , Kothleitner Gerald 
Published: December 2016

For a thorough understanding of a material, investigations at the nanoscale are often essential. Analytical techniques like electron energy loss spectroscopy (EELS) and energy dispersive X‐ray spectroscopy (EDXS) in scanning transmission electron microscopy (STEM) can reveal important chemical information necessary for the development and improvement of high‐tech materials. The integrative character of the signal acquired through transmission, however, might hide important morphological details of the material, relevant for its properties. Those details can be revealed through electron tomography, where the data is acquired at different tilt angles and, after alignment, reconstructed to form a full 3D model of the material under investigation. The combination of both techniques, analytical STEM and tomography, gives full insight into structure and composition of a material [1].
In this study an industrially cast aluminum alloy, containing scandium (Sc) and zirconium (Zr) rich nano‐precipitates, was investigated at different stages of ageing. High resolution STEM and analytical EELS and EDX tomographic investigations were carried out. The resulting 3D elemental reconstructions delivered otherwise inaccessible information on the samples' chemistry and structure (figure 1). Additionally, EDX‐spectra of a long aged and electron‐beam re‐solidified (EBRS) sample were reconstructed channel by channel, resulting in a data cube, in which each voxel (volume pixel) contained an entire spectrum. Via a high angle annular dark field (HAADF) reconstruction and 3D masking, the voxels of the core and the different shell regions of a nano‐precipitate were extracted and summed to obtain pure spectra of those volumes, thus overcoming the intrinsic limitations arising from the integrative character of analytical STEM. Comparisons of these results with data from high resolution STEM imaging, HAADF tomography and data from literature have led to the conclusion ‐opposed to previous believes [2]‐ that a self‐limiting diffusion process takes place within the precipitates stabilizing their core‐shell structure (figure 2).
Revealing the internal structure of microbial mats by
FIB
/
SEM
tomography
Authors: Zeitvogel Fabian , Obst Martin , Schroeppel Birgit , Burkhardt Claus J. 
Published: December 2016

Microbial mats are macroscopically visible communities of microorganisms, which play an important role for the interaction of microorganisms with their environment [1]. Important examples are mats dominated by Fe(II)‐oxidizing bacteria, which are typically found in wet, Fe(II)‐rich environments such as lake sediments and mine tunnels [2,3].
The microscopic constituents of microbial mats can be analyzed using 2D microscopy approaches such as SEM. Other properties such as the microbial community and elemental composition are accessible using bulk analytical methods [e.g. 3]. However, little is known about the 3‐dimensional structure of these mats.
Samples of two microbial mats from an abandoned mine tunnel were analyzed by FIB/SEM tomography using a Zeiss Auriga CrossBeam instrument. The samples were embedded in resin. Metal ions and minerals within the sample were responsible for contrast generation. The voxel size was 30 x 30 x 30 nm³. The resulting 3D reconstructions show the original arrangement of the microscopic constituents in great detail (Fig. 1,2). The two mats were dominated by different types of Fe(II)‐oxidizing bacteria, which formed characteristic structures that were visible in the two respective volumes. A large part of both volumes appeared void.
The results of this analysis provide explanations for a series of macroscopically observed effects, such as the physical stability of the mats, high water content, efficient interaction with the feeding water, and the creation of a habitat for a variety of different microbial species. In addition, the results represent a valuable reference for the detection of microfossils in sedimentary rocks.
Calcination of pd nanocatalysts on alumina: ex‐situ analysis versus in‐situ environmental
TEM
Authors: Koneti Siddardha , Roiban Lucian , Epicier Thierry , Gay Anne‐Sophie , Avenier Priscilla , Cabiac Amandine , Dalmas Florent 
Published: December 2016

Sustainable chemistry, reduction of pollution, greenhouse gases control, oil refinery, liquid and solid waste management, are essential societal topics related to what is called Environmental catalysis [1], a sector which is booming rapidly for the past 10 years. So, developing innovative catalysts is a very important aspect ahead of us. To develop more advanced catalysts, we have to understand the catalysts genesis in all the stages of preparation (impregnation, drying, calcination and reduction of the active phase). Nowadays Environmental Transmission Electron Microscopy (ETEM) enables dynamic studies of the catalysts down to the nanometer and even atomic scale., at high temperatures and under gaseous environment.
This contribution deals with the dynamic evolution of palladium nanoparticles (NP) supported on alumina during the catalyst preparation process. Here, the calcination step is studied in a FEI TITAN‐ETEM microscope operated at 300 kV. A Pd/delta alumina catalyst was investigated at different temperatures and under different gas pressures in order to follow the particles size evolution. Owing to the small size of the Pd NPs in the range of 1‐3 nm, we mainly used the STEM‐ADF imaging mode. Preliminary observations during heating under high vacuum up to 600°C show that particles as well as the supporting media remain stable and apparently not damaged by the electron beam under nominal and atomic resolution imaging STEM conditions (see Fig. 1). Some EELS (Electron Energy‐Loss Spectroscopy, Gatan Imaging Filter Tridiem Ers) analysis was performed in situ in order to ascertain the chemical nature of the observed particles. During this work it appeared essential to perform measurements systematically on the same areas at different temperatures. Fig. 2 is a typically illustration of the in situ evolution of palladium particles under oxygen partial pressure at different temperatures. From such micrographs, the NP size evolution was quantified and compared with post‐mortem TEM observations after ex‐situ experiments performed at same temperatures but atmospheric pressure in the course of the catalyst synthesis (Fig. 3).
This comparison shows that all measurements appear to be consistent except those performed in the bright field TEM imaging mode, where larger particles sizes are obtained, most probably due to irradiation effects which were further evidenced by a high‐induced mobility of NPs on their supporting media. This ETEM study brings direct in situ information on the transient stages that cannot be followed by post mortem experiments after ex‐situ treatments; in particular, we will also discuss about the crystallographic evolution of the Pd NPs during the calcination process [2,3].
EDX‐STEM phase mapping of semiconductor devices using multivariate statistical analysis tools
Authors: Saghi Zineb , Pimenta‐Barros Patricia , Goret Gael , Printemps Tony , Bernier Nicolas , Barraud Sylvain , Delaye Vincent 
Published: December 2016

With the increasing miniaturization of electronic devices, high resolution structural and analytical characterization tools are necessary for the optimization of fabrication processes. Scanning transmission electron microscopy energy dispersive X‐ray (EDX‐STEM) spectroscopy is a well‐established technique that has recently gained momentum thanks to the introduction of high‐brightness electron sources and the Super‐X EDX system (4 SDD detectors), allowing fast EDX mapping with high collection efficiency. While the traditional EDX data analysis consists in extracting the elemental map of each element present in the sample [1], it is often the case that the aim of the analysis is to investigate the spatial distribution, shape and thickness of the different chemical phases present in the sample.
Multivariate statistical analysis tools, such as non‐negative matrix factorization (NMF) and independent component analysis (ICA), were shown to yield simplified interpretation of spectral datasets by rapid identification of phases (e.g. [2,3]). In this work, we applied NMF to the EDX analysis of Si/SiGe multilayers to validate the Sidewall Image Transfer (SIT) process developed for their patterning [4]. A FIB‐prepared lamella was characterized in an FEI Titan Themis operating at 200kV and equipped with a probe corrector and 4 SDD EDX detectors. An EDX‐STEM map was acquired with TIA, using a pixel size of 1nm and a dwell time of 20ms/pixel, and exported to hyperspy, a python‐based software for hyperspectral data processing [5].
Spectral unmixing using NMF led to the identification of five chemical phases in the sample: Si, SiGe, SiO2, TiN and C (see the component spectra in Figure 1 and the corresponding loadings in Figure 2(a‐e)). More specifically, NMF succeeded in: (1) separating the Si signal emanating from pure Si, SiGe and SiO2 layers; and (2) deconvoluting the C, N and O peaks. This greatly simplified the compositional analysis (Figure 2(f)), and allowed a more straightforward estimation of the thickness of the different layers, as shown in Figure 2(g).
NMF combined to EDX‐STEM tomography was recently applied to superalloy systems for aerospace applications [6,7]. We will show that this approach has also the potential to address materials characterization challenges currently facing the semiconductor industry, such as the chemical analysis of dopants and impurities [8].
Single particle
EM
study of the E1 helicase from Papillomavirus in complex with
DNA
Authors: Coscia Francesca , Ryzhenkova Ksenia , Chaban Yurij , Sanders Cyril , Orlova Elena V 
Published: December 2016

The papillomaviruses (PV) are small dsDNA tumour viruses of significant medical importance and the prototype of the group is bovine PV (BPV‐1). PVs encode one highly conserved replication enzyme, E1, that acts as an initiator and a helicase. E1 forms hexamers and unwinds double stranded DNA (dsDNA) into single stranded DNA (ssDNA) products using the energy of ATP hydrolysis. However, how the helicase engages the replication fork at the molecular level is unclear. BPV‐1 E1 is comprised of four domains: a regulatory domain (residues 1‐158), a sequence specific
ori
DNA binding domain (OBD, residues 159‐299) and the C‐terminal half (residues 300‐605, E1HD) that has helicase activity and is further divided into the oligomerisation domain (OD, residues 308‐378) and the AAA+ ATP
ase
domain. A C‐terminal acidic tail domain (AT, residues 579‐605) is required for helicase processivity (Figure A). We have obtained structures of the full length E1 helicase (E1FL) in complex with a DNA replication fork with and without DNA labelling with protein tags. The 5’ end of dsDNA and the 5’ ssDNA end of the fork were labelled and helicase structures studied using single particle electron microscopy.

Negatively stained images (with 2% uranyl acetate) of E1FL/DNAfork/Fab /Streptavidin were taken and the structure obtained at a resolution of~ 20 Å (Figure B). We are currently working on the native structure by cryoEM (Figure C). The 3D reconstruction of E1FL (Figure D) has a three‐tier organisation with well‐defined domains (N‐terminal, DBD, OD, and AAA+). Domains were localised by docking of available atomic structures. The 3D structure of the E1/ labelled DNA fork confirmed the triple ring organisation with an internal small chamber above the helicase motor domain where DNA unwinding appears to take place. Interestingly, dsDNA enters into the chamber via a side tunnel above the helicase motor domain, with the 5́‐ssDNA strand leaving through a narrow tunnel located on the opposite side, while the 3́‐ssDNA is pulled through the hexamer's central tunnel (Figure D). Our findings are confirmed by DNA footprint experiments and FRET experiments. Recently we analysed a cryoEM structure of the E1FL in complex with a DNA fork at subnanometer resolution that reveals the same structural organization and provides more detail on the interaction with DNA.
Our structural studies demonstrate that the process of DNA separation takes place inside the E1FL complex rather than on the exterior surface of E1. In the light of our results we suggest that the current ‘steric exclusion’ model for strand separation should be revised. A molecular understanding of E1 function will be essential to shed light on the early phase of DNA replication and will assist in the understanding of E1 as a therapeutic target of viral DNA replication.
Defects and strain analysis of
GaAs/Si
nanostructures from high‐resolution
HAADF‐STEM
images
Authors: Kozak Roksolana , Prieto Ivan , Skibitzki Oliver , Dasilva Yadira Arroyo‐Rojas , Rossell Marta , Erni Rolf , Schroeder Thomas , von Känel Hans 
Published: December 2016

The increasing demand for high‐performance, ultra‐small size electronics and photonics requires the development of new nanostructured materials. Promising candidates for this purpose are monolithically integrated GaAs nanocrystals, selectively grown by metal organic vapor phase epitaxy (MOVPE), on top of Si nano‐tips and nano‐pillars (Fig. 1,2) with ~ 60 nm and ~ 40 nm diameter, respectively. Growth on substrate nanopatterns eliminates threading dislocations similar to “aspect ratio trapping” in submicron trenches and pits [1]. It may even prevent plastic strain relaxation by misfit dislocations in contrast to the growth of thin films on planar substrates by taking advantage of the compliant substrate effects [2]. Consequently, it is important to study the local atomic defects at the GaAs/Si interface and their evolution with changing shape/size of the substrate pattern, and to analyze the residual elastic strain of the grown crystals by means of the scanning transmission electron microscopy (STEM) technique in order to find the maximum crystal size below which elastic relaxation is favorable.
STEM investigation of the GaAs/Si nanostructures has been performed on lamellas prepared by focused ion beam (FIB). From the analysis of aberration‐corrected high‐resolution high‐angle annular dark‐field (HAADF) STEM images the presence of 60º misfit dislocations (MDs), as well as 90º and 30º Shockley misfit partial dislocations (MPDs) at the GaAs/(001)Si tip interface has been revealed (Fig. 3). The latter generate extrinsic and intrinsic stacking faults, respectively, which propagate through the entire crystal. Gliding of a group of 30º Shockley MPDs along parallel successive slip planes produces a nanotwin. In the GaAs crystals grown on (001) Si pillars two intrinsic stacking faults, lying in {111}‐type planes, meet at the interface and build a V‐shaped defect (Fig. 4). According to the literature [3], such V‐shaped configuration of stacking faults suggests their formation upon island coalescence. These stacking faults border at the interface a stair‐rod dislocation, which is formed by the interaction of two 30º Shockley MPDs located in the Si pillar. Among the observed defects, the most efficient ones for relaxing misfit strain are 60º MDs, whereas 30º MPDs release strain only partially. Geometrical phase analysis (GPA) [4] has been also applied to estimate the misfit strain in the GaAs crystals.
Optimizing electron channeling contrast imaging condition in scanning electron microscope
Authors: Han Luyang , Gamm Bjoern 
Published: December 2016

The electron channeling contrast imaging (ECCI) is a technique that makes use of the influence of back scattered electrons (BSE) by the relative orientation of crystalline lattice and incident electron beam [1]. The ECCI in scanning electron microscope (SEM) is especially useful to image the crystallographic defects such as dislocations, stacking faults and twin boundaries [2]. In this contribution the amount of channeling contrast in the total BSE signal is quantified and its dependence on different working conditions is explored.
An example of the ECCI on an epitaxial GaN thin film is shown in Figure 1. The sample is investigated using ZEISS GeminiSEM 500 FE‐SEM with a PIN‐diode BSE detector under the objective lens. By adjusting the strength of the scanning coil it is possible to move the pivot point of scanning electron down to the surface of the sample, forming the selected area channeling pattern (SACP) [1]. The intensity variation of BSE signal can be extracted by the line profile across the Kikuchi line, shown as the red dotted line in the figure. The relative variation of BSE signal in SACP with respect to average signal can thus be used as a quantification of channeling contrast.
The amount of channeling contrast is influenced by the properties of incident electron beam and the detector. In principle, the energy and convergence angle of the incident electron beam determines the channeling pattern itself, while the position and energy response of the detector influence the amount of channeling contrast. In this study a large parameter space is explored to optimize the channeling contrast, including electron beam energy, working distance, convergence angle and position of detectors. As an example, the influence of electron beam energy to channeling contrast is summarized in Figure 2. The SACPs are shown in the right part of the figure. The position of the lines as well as intensity variations within the pattern changes significantly between different energies. However, quantification of the channeling contrast shows a clear trend of increasing channeling contrast at lower electron beam energy.