Ute Kaiser
Materials Science Electron Microscopy,
Ulm University, Albert Einstein Allee 11, 89081 Ulm, Germany

Structural and electronic properties of different low-dimensional electron-beam-sensitive crystalline (graphene [1], ion-implanted graphene [2], MoS2 [3], MoSe2, SiO2 [4], CN [5], square ice [6], transition-metal clusters [7]) and amorphous (monolayer carbon, SiO2) [8] objects as well as a new structure of crystalline AuC [9] are obtained by analytical low-voltage aberration-corrected transmission electron microscopy following three main strategies: (1) Theory and image processing: For exact calculation of the contrast of dose-limited high-resolution TEM images for low-Z materials at low voltages, image theory and image processing needs to be improved taking into account elastic and inelastic scattering [10-11]. (2) Sample preparation: We demonstrate our method to clean graphene [12]. We show that sandwiching clean radiation-sensitive low-dimensional objects in-between two graphene layers [13] or embedding them into single-walled carbon nanotubes [14] allows to reduce electron-induced damage of the objects. (3) Low-voltage transmission electron microscope: We outline our unique voltage-tuneable low-voltage (20-80kV) spherical and chromatic aberration-corrected TEM and show first results obtained from its prototype [15].

Pavel Tomancak.
MPI-CBG, Dresden, Germany.

Ten years of technology development in light sheet microscopy have led to spectacular proof of principle demonstrations of this new imaging paradigm’s capabilities. The technology is now ready to assist biologists in tackling complex biological problems. However, are biologists ready for it? I will discuss the unique interdisciplinary challenges light sheet microscopy imposes on researchers in biological sciences and highlight the solutions and resources available to help them meet these challenges. In particular I will highlight the OpenSPIM open access hardware and Fiji open source software platforms and their applications for imaging early animal development.

Aleksandr Bagmut and Ivan Bagmut
National Technical University , Kharkiv, Ukraine

Hadas Sternlicht, Wolfgang Rheinheimer, Michael J. Hoffmann and Wayne D. Kaplan
Technion, Israel

Lilac Amirav
Schulich Faculty of Chemistry,
Technion – Israel Institute of Technology, Haifa, Israel

The solar-driven photocatalytic splitting of water into hydrogen and oxygen is a potential source of clean and renewable fuels. However, four decades of global research have proven this multi-step reaction to be highly challenging. The design of effective artificial photocatalytic systems will depend on our ability to correlate the photocatalyst structure, composition, and morphology with its activity.

Here, I will present our strategies, and most recent results, in taking photocatalyst production to new and unexplored frontiers. I will focus on unique design of innovative nano scale particles, which harness nano phenomena for improved activity, and methodologies for the construction of sophisticated heterostructures. I will demonstrate how vital is the ability to characterize our hybrid nanostructures on the atomic level, and how we can benefit from information on the structure-properties relationship for the future design of an efficient photocatalyst for solar-to-fuel energy conversion.

Amit Kohn
Department of Materials Engineering and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev

Holography in the transmission electron microscope (TEM) can achieve quantitative mapping of electrostatic and magnetic fields with nanometer scale spatial resolution.

I will describe holography methodologies in the TEM of ‘in-line’ and ‘off-axis’ and our experimental work to determine their quantitative capabilities.

For magnetic mapping, we examined an ‘in-line’ approach based on the ‘transport-of-intensity’ equation (TIE), to Lorentz TEM using Fresnel-contrast images. Our experimental study of sub-micrometer Permalloy sized elements tested the application of the TIE demonstrating quantitative mapping of magnetic fields in structures sized down to approximately 100nm wide and 5nm thick.

Using off-axis electron holography, we examined quantitative dopant mapping of silicon PN nanostructures in the form of implanted junctions. For the microelectronics industry, ongoing reduction of device dimensions, 3D device geometry, and failure analysis of specific devices require mapping of electrically active dopants in thin (<50nm) TEM samples prepared by focused ion beam.

I will demonstrate our current applications for off-axis electron holography, e.g. silicon nanowires with vertical doping, space charge zones in granular magnesium aluminate spinel, and heterojunctions of PbS/CdS core-arm nanostructures.

Laurie Palasse and Daniel Goran
Bruker Nano GmbH, Berlin, Germany

Shmuel Samuha, Benjamin Grushko and Louisa Meshi
Ben Gurion University of the Negev, Israel

An intermetallic phase with approximate stoichiometry of Al65Cu25Re10 was revealed in [1]. It was presumed that this phase belongs to a family of the Al-TM hexagonal phases, some of which are included in Table 1. These phases have very similar c lattice parameters and a lattice parameters are related by ~τ (golden mean, τ≈1.618). Similar to, mentioned in Table 1, κ and λ phases, the Al65Cu25Re10 phase exhibits pseudo-tenfold symmetry along the [250] orientation, indicating that it can be classified as an approximant of decagonal quasicrystals (D-QC). Fundamental building-blocks (structural subunits or clusters) of these phases are believed to approximate the local atomic arrangement of their related QCs.

Structure solution of the Al65Cu25Re10 phase was performed using Precession Electron Diffraction (PED) Tomography technique [4]. For this purpose, a sequence of off-axis PED patterns was collected manually with a constant angular separation of 1° at wide range of ±38°, using the Fischione tomography holder. For precession illumination, the Nanomegas "Spinning Star" precession unit was used (precession angle 1.5°). Following the data collection process, the PED frames were subsequently processed using the Analitex EDT-PROCESS package. Pattern merging resulted in the reconstruction of a 3D reciprocal space, providing 99.6% completeness up to the 0.76 Å diffraction resolution (employing the P63 symmetry proposed in [1] for the Al65Cu25Re10 phase). The structure was solved applying Direct Methods, utilized in the SIR2008 software [5], and refined using kinematical least squares refinement procedure in the Jana2006 program [6]. A final atomic model consisted of 92 atoms (62 Al, 20 Cu and 10 Re atoms) distributed along 18 unique atom sites. This is one of the most complex intermetallides solved exclusively from electron diffraction data. Moreover, it is the first structure measured and solved by 3D electron diffraction tomography in Israel.

Using the cluster-based approach, the structure of the Al65Cu25Re10 phase can be described by 3D chains constructed of interconnected complex icosahedra. Similar structural arrangement of clusters was reported also for the, mentioned earlier, λ and κ phases. Thus structural relationship among these and Al65Cu25Re10 structures was proposed. Comparing main zonal PED patterns taken from the studied Al65Cu25Re10 and κ-Al76Cr18Cu6 phases (see Figure 1); high degree of likelihood can be observed, in particular, similarity in the distribution of reflections with the highest intensity (i.e. strong reflections). Using the so-called 'strong reflection approach' [7], atomic positions of the Al65Cu25Re10 structure were extracted from a 3D Electron-Density Map [commercial, Analitex], they were derived using adopted structure-factor amplitudes and phases from the κ-Al76Cr18Cu6 phase. In this way – atomic structure of the Al65Cu25Re10 phase was predicted through a theoretical calculation, assuming it relates to the discussed family of approximants.

Since predicted and experimentally solved atomic structures of the Al65Cu25Re10 phase were practically identical, we introduce the Al65Cu25Re10 phase as a new member of the family of hexagonal approximants of the D-QC.

Haim Weissman
Weizmann Institute of Science, Israel

Supramolecular polymer systems are of primary importance for creating multifunctional adaptive materials as their structure and function can be reversibly controlled in situ. We will present our work on self-assembled nanostructures in aqueous media based on perylene diimide amphiphiles (PDI), whose structure was studied using cryogenic TEM and SEM. We used cryo-TEM and spectroscopic methods to study dynamic behavior of self-assembly of various systems thus gaining mechanistic insights about self-assembly and nano-crystallization. Some of our systems show photofunction and multiple stimuli-responsiveness, including reversible supramolecular depolymerization in situ through aromatic charging, which enables switching of mechanical properties and optical functions. Another system utilizes hierarchical supramolecular interaction for the formation of unique nanospirals (Figure 1) in aqueous solutions.

Yuval Golan
Department of Materials Engineering and the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Chemical bath deposition from solution offers a simple, inexpensive and scalable alternative for obtaining monocrystalline semiconductor thin films with well-defined orientation relations with the substrate, a phenomenon termed “chemical epitaxy”. This talk will highlight the chemical bath deposition pathway to chemical epitaxy, providing examples for well-defined orientation relationship between film and substrate in a variety of systems. The influence of the substrate on the incipient films, and the effect of deposition parameters such as solution composition, bath temperature and substrate pre-treatments on the film morphology and subsequent physical properties will be discussed. Finally, we will describe applications such as deposition of IR absorbing layers in a nanomaterials based short wave infrared night vision device.

Stavros Nicolopoulos
NanoMEGAS, Brussels, Belgium

Following the initial work by Vincent and Midgley in Bristol UK (1994) which developed the Precession Electron Diffraction (PED) technique in TEM, PED has become essential tool for several TEM applications. Today, more than 180 articles (that include PED technique) from various laboratories worldwide and dedicated issues of major scientific microscopy journals have been published the last decade.

Beam precession has been proved to enhance the reflections quality (quasi-kinematical, similar to X-ray intensities) ; one of the most important applications for electron crystallography , is the recently developed 3D PED diffraction tomography technique that allows from several PED patterns collection, a complete solution of various structures to atomic scale , from complex zeolites and minerals to metals and alloys.

Another important application including use of PED is the ASTAR technique where is possible to obtain TEM orientation and phase maps at 1-3 nm resolution (in case of FEGTEM) for a variety of materials (metals, semiconductors, oxides etc..) . The technique is becoming very popular and is similar to EBSD-SEM , but in ASTAR case , the technique is based on collection of several PED patterns which are compared via correlation template matching techniques with theoretically generated ED templates.

Precession diffraction has been also recently successfully applied to obtain Strain mapping analysis of several semiconductor materials at 1-4 nm resolution (in case of FEG-TEM, sensitivity 0.02%), based on comparison of NBD patterns from strained to reference unstrained areas. The technique is very easy to use at any TEM and provides very fast and accurate data (same order of magnitude as dark field holography) without any need to index diffraction patterns.

Another nice application of ED related techniques, is the study of amorphous materials. In case of amorphous routine crystallography fails to reliably characterize them. Alternatively, the Pair Distribution Function analysis from electron diffraction data (e-PDF) can be used for fingerprinting and characterize the crystalline order present in the compound. The advantage of using PDF analysis from electron diffraction data is the short data collection time (10 msec to several seconds) compared with long exposure times (15-24 hours) for PDF data acquired with laboratory X-Ray sources (Mo/Ag radiation).

Eyal Nir
Department of Chemistry and the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer Sheva, Israel

Natural molecular machines, made of proteins and RNA, play major roles in many biological processes, often with impressive operation yields and speeds. Artificial molecular machines, including machines made of DNA, however, are still slow and exhibits low operation yields which limits its applicability.

In the talk I will show how we use single-molecule total internal fluorescence microscopy (smTIRF) and microfluidics technology to develop and operate a DNA-based bipedal walker that stride on a DNA origami track and powered by sequential interactions with externally introduced fuels and anti-fuels DNA strands. Using the SMF technique, which enables structural dynamics analysis of the motor and the identification of unwanted side-reactions, and using computer controlled microfluidics device, which enables automatic introduction and removal of fuels and anti-fuels, we were able to rationally design and conveniently operate motors with superb operation yield and speed. With our recent design the motor walk 36 steps with 50% operational yield. This is equivalent to around 99% yield per chemical reaction (DNA binding and unbinding), and the stepping rate can be faster than several seconds. This is significantly better than any other DNA based motor introduce to date and opens the way for variety of future computer controlled DNA-based devices and applications.

Natan T. Shaked
Department of Biomedical Engineering, Faculty of Engineering,
Tel-Aviv University, Tel-Aviv, Israel

We propose new optical imaging methods for rapid non-invasive acquisition of the three-dimensional (3-D) refractive-index structure of live cells in suspension without using any labelling. The methods are based on the acquisition of off-axis interferograms of a single cell from different angles using external interferometric module, while fully rotating the cell using micro-manipulations. The interferometric projections are processed via computed tomographic phase microscopy reconstruction technique, which considers optical diffraction effects, into the 3-D refractive-index structure of the suspended cell. By inspection of the 3-D refractive index distribution of cells in suspension, the proposed methods can be useful for label-free morphological and contents characterization of biological processes and cell transformations from healthy to pathological conditions.

Einat Zelinger, Yael Heifetz, Leor Williams and Vlad Brumfeld
Hebrew University, faculty of agriculture, Rehovot, Israel

Michael Elbaum
Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, Israel

The realm of cryo-electron microscopy now extends from atomic resolution of macromolecules to 3D tomographic views of intact cells. Lacking heavy metal stains, the approach is almost synonymous with phase contrast. Recently we have been exploring scanning transmission EM (STEM) imaging for cryo-microscopy and tomography [1]. STEM contrast (dark field) is based on incoherent atomic scattering. Its quantitative nature is well recognized in mass measurement studies. STEM circumvents many of the pitfalls inherint in phase contrast. For example, depth of field and resolution can be tuned more flexibly so that images are formed in focus. Moreover, STEM is insensitive to inelastic scattering and there is no need for zero-loss energy filtering. These features make STEM particularly efficient for application to thick specimens. We demonstrate these advantages in tomography of both bacterial and mammalian tissue culture cells. Scattering profiles also depend on atomic number so STEM can be used for mapping of heavy elements on the organic background of carbon, nitrogen and oxygen, for example in bacterial poly-phosphate bodies. Using ferritin as a model system we explore the ultimate sensitivity for detection of trace metals in the biological context.

Anat Akiva, Karina Yaniv, Steve Weiner and Lia Addadi
Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel

Omer Ziv1, Assaf Zaritsky, Yakey Yaffe*, Reuven Edri and Yechiel Elkabetz
Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel

Yonatan Sivan, Yannick Sonnefraud, Matthew R. Foreman, Hugo G. Sinclair, Christopher Dunsby, Mark A. A. Neil, Paul M. French and Stefan A. Maier
Unit of Electro-Optic Engineering, Ben-Gurion University, Beer Sheva, Israel

Daniel Razansky
Institute for Biological and Medical Imaging (IBMI),
Technical University of Munich and Helmholtz Center Munich

Daniel Phifer, Brandon van Leer, David Wall, Anthony Burgess
FEI Company, Materials Science Business Unit, Eindhoven, The Netherlands

Since FEI’s DualBeam 620 became commercially available in 1993, focused ion beam (FIB) and SEM/FIB instrumentation has transformed scientists’ ability to investigate materials, to develop new sample preparation methods and become the “work-horse” for site-specific cross-section analysis, TEM/STEM sample preparation (cross-section or plan view), nanoscale patterning and prototyping applications. Increasingly, the demands for making sample with a particular quality are required over a variety of materials which lead to new DualBeam workflow solutions.

With demand for advanced TEM sample preparation at an all-time-high, electron microscopists and researchers require sample preparation to take much less than a day and to be suitable for a number of specific requirements. There are many factors that enable a system’s ease-of-use and flexibility including optimal beam parameters, system stability, software functionality, automation and novel approaches to sample preparation. Recent advances in all four have enabled SEM/FIB operators to generate high quality, site specific samples in 2 hours or less. For non-site specific materials the time can be reduced to much less than an hour.

Sample damage with high energy FIB is also well characterized among many materials. The use of low energy FIB to reduce the damage continues to generate interest in the scientific community. FIBs routinely operate in a low energy regime with imaging capabilities that allow for site specific pattern placement onto samples. Figure 1 reveals an example of 2 keV and 1 keV Ga+ focused ion beam imaging performance with resulting material damage.

Hitting a site-specific region of the target is also crucial for sample preparation. To that end, there have been many new end-pointing techniques, which allow the operator to watch the bulk milling and lamella formation process to maintain control of the milling process and provide visibility of the target structure. These techniques include simultaneous imaging while patterning, sequential image and mill, and FIB real time monitoring.

With the increasing use of FIBs, the requirement to work with ultra-thin (<45 nm thick) specimens increased significantly. Finishing a thin specimen now includes novel approaches such as inverting the sample prior to final polish. This allows successful milling of mixed composition interfaces by positioning a sample such that hard materials face the FIB to achieve ultra-thin samples in boundary regions which were previously much more difficult to prepare. Advances in the DualBeam workflows continues to lead to better control of sample thickness, uniformity and sample quality which can be directly seen in the latest atomic resolution (S)TEM analysis.

Figure 1. FIB damage in Silicon and FIB induced secondary-electron imaging performance for 2 keV and 1 keV Ga+ FIB beam

Andreas Schertel
Carl Zeiss Microscopy GmbH, Carl-Zeiss-Str. 22, D-73447 Oberkochen, Germany

Focused Ion Beam – Scanning Electron Microscopy (FIB-SEM) volume imaging of heavy-metal-stained biological specimens embedded in resin is a well-established technique to reconstruct and to analyse subcellular structures in all three dimensions, e.g. brain mapping [1,2]. Cellular ultrastructure is visualized by detecting the low loss backscattered electrons generated by the interaction of the primary electrons with the stained resin-embedded tissue. FIB-SEM block face imaging data is acquired in a complete automated process in contrast to the long-lasting and troublesome imaging and aligning of serial thin sections in transmission electron microscopy. The z-limitations given by conventional serial sectioning using an ultra-microtome are overcome by FIB milling which allows slice thicknesses down to several nm. Therefore, isotropic voxel sizes down to 5 nm and below can be achieved. For this reason, FIB-SEM 3D reconstruction closes the gap between electron and x-ray tomography.

The conventional resin-embedding preparation technique involves dehydration and impregnation with heavy metals by freeze substitution or chemical fixation followed by resin-embedding. In contrast, biologists aim to visualize cellular ultrastructure of specimens in their native or living state.

A new, very inspiring and exciting approach for FIB-SEM Microscopy is block face imaging of native biological samples in the high-pressure frozen state omitting any dehydration, chemical fixation or staining. In our recent work, we applied serial FIB milling and block face imaging to acquire 3D data cubes of high pressure frozen mouse optic nerves and bacillus subtilis spores at cryo conditions [3]. By using in-lens secondary electron detection, we succeeded to directly visualize the cellular ultrastructure in the freshly exposed serial FIB cross-sections. The observed contrast between lipid-rich membranes and water-rich areas allowed differentiating subcellular structures like Golgi apparatus, nuclear envelope, vesicles, endoplasmic reticulum and cristae within mitochondria. The new method is comparatively easy and extremely fast because cryo-immobilization is the only step of preparation to be ready for investigation in a SEM. Interesting potentials for the correlative light and electron microscopy workflow are provided by introducing this novel technique.

Recent cryo-FIB-SEM results on high pressure frozen HeLa cells, yeast cells and Algae Emiliania huxleyi (EHUX) are presented. The Cryo-FIB-SEM data cube of native frozen yeast and EHUX contains one complete cell.

References
This project was supported by the German Federal Ministry for Education and Research (grant No. FKZ: 13N11403).
[1] A. Merchán-Pérez et al., Frontiers in Neuroanatomy 3:18.10.3389/neuro.05.018.2009.
[2] M. Cantoni et al., Microscopy and Analysis 24(4): 13-16, 2010.
[3] A. Schertel et al., Journal of Structural Biology 184 (2013): 355-360

Andrey Denisyuk, Kateřina Klosová, Lukáš Hladík, Tomáš Hrnčíř
Tescan Orsay Holding, Brno, Czech Republic

In this talk we report on our recent developments in FIB/SEM technology. Our instruments combine the ability for precise FIB nanofabrication, high resolution SEM imaging and in situ analysis by means of fully integrated units.

Main application of FIB/SEM instrumentation is cross-sectioning and imaging. However FIB cross-sectioning of compositional species with differential milling yield can result in unpleasant curtaining artefacts. In order to overcome this effect and obtain smooth cuts we developed a special multi-tilt rocking stage that enables ion polishing from different directions (Fig. 1a). High resolution SEM imaging is achieved by means of immersion electron optics, which significantly minimize aberration of the electron beam and improves resolution particularly for low voltage SEM observation.

For the purpose of in situ analysis we implemented various novel techniques. One of them is Time of Flight Secondary Ion Mass Spectroscopy (TOF-SIMS) which is integrated to our FIB/SEM instruments. Such integration provides good lateral resolution and allows 3D chemical mapping, detection of light elements and isotope distinguishing (Fig. 1b). Another possibility for analysis is the integration of Raman Imaging and Scanning Electron microscopy (RISE). This technique provides high resolution SEM imaging with correlative Raman mapping and spectroscopy. Such combination is helpful for identification of different compounds and polymorphic phases, analysis of crystallinity, observation of internal stresses and damage (Fig. 1c).
Figure 1. Some examples of our recent developments in FIB-SEM technology. (a) Rocking stage: cross-sections without curtaining; (b) TOF-SIMS: Li distribution maps in a LiNbO3 sample; (c) RISE: stress observation in silicon by means of SEM imaging and Raman mapping.

Katya Rechav
Department of Chemical Research Support, Weizmann Institute of Science
Rehovot, Israel

Focused Ion Beam - Scanning Electron Microscope (FIB-SEM) instruments have been promoting a true revolution in biological research during the last few years. FIB-SEMs were designed for and primarily used in materials science and in the semiconductors industry. Their popularity in the field of biology is growing day by day, mostly thanks to the technique of serial surface view (SSV). Briefly - this method involves gradual removal of nanometer-thick sections from the sample surface, sequentially recording the images of the renewed surface and then reconstructing a three-dimensional model from the collected images.

Despite the considerable challenges associated with biological samples preparation for electron microscopy, FIB-SEM seems the most promising technology allowing large-volume 3D visualization with resolution at the nano-meter level. FIB-SEM also enables site-specific cross sectioning and TEM lamellae preparation of biomaterials, cells and their interfaces, possibly coupled with cryogenic capabilities.

Here we review some practical usages, advances and challenges of FIB-SEM techniques associated with biological research.

Prof. Gregor Hlawacek
Ion Beam Physics and Materials Research,
Helmholz-Zentrum Dresden – Rossendorf, Dresden, Germany

HIM is well known for its exceptional imaging and nanofabrication capabilities. After a brief introduction of the gas field ion source and the ion microscope, I will present a wide range of results obtained with either the Twente UHV Orion+ or the NanoFab at the HZDR in Dresden. Special emphasis will be given to the use of channeling and the role of defects created by the energetic ion beam. Ionoluminescence is used to obtain information on the latter. Helium Ion Microscopy has an unprecedented surface sensitivity. Recent results obtained on thin silver layers on Pt(111) demonstrate that work function differences as small as ~20 meV as well as surface reconstructions can be visualized. Finally, some preliminary results of Neon based materials modification and cross section preparation will be presented.

Yafit Fleger
Bar Ilan Institute of Nanotechnology & Advanced Materials,
Ramat Gan, Israel

Focused Ion Beam (FIB) was developed during the last two decades and became a central tool for micro and nano fabrication and analysis. The Processing and milling capabilities of FIB can be used for multiple applications in different research fields. Ga ion beam of the FIB system provides a micro to nano scale -milling capabilities of various samples with a resolution of ~15 nm while a He source can improve the milling resolution down to ~4 nm.

The most popular FIB is a dual beam system that contains SEM, Gas Injection System (GIS) and lift out probe. Altogether it provides better analysis system with the ability to image, lift out TEM samples and create local depositions of materials such as Pt and SiO. These three additives turns the FIB system into a powerful factory in the nano world.

In my talk I will describe a variety of FIB applications in physics, chemistry and material science for both academic and industrial purposes. These applications demonstrates the high-resolution milling and imaging, and includes electrochemistry micro patterning, fabrication of nano plasmonics devices, TEM lamella preparation for analyzing crystal defects and the study of carbon nano-tubes. FIB is also a powerful tool for failure analysis tests such as circuit editing and cross sections. This will be demonstrated for the limiting size of few tens of nm.Figure 1: a. cross section of an integrated circuit, b. TEM lamella of porous sample, c. Plasmonic device nano patterning.