ISM 2015 - The 49th annual meeting of the Israel society for microscopy
Dear ISM Members and Friends,
Thank you all for participating and contributing to a very inspiring meeting.
Special thanks to all our plenary and invited speakers !
We also wish to thank our partners from the industry and from the academia for their generous financial support, the dedicated team of the Institute for Nanotechnology and Advanced Materials (BINA) at Bar-Ilan university.
- Anat Akiva, Department of Structural Biology, Weizmann Institute of Science, recipient of the Lev Margulis Prize for her work titled: “Boning Up on Bone Formation: Tracking the Bone Formation Pathways in the Larval Zebrafish Tail”.
- Shmuel Samuha, Department of Materials Engineering and NRCN, Ben-Gurion University of the Negev, recipient of the SIG4 Prize for his work titled: “Structure Solution of the Al65Cu25Re10 phase by 3D Electron Diffraction Tomography”.
- Mahdi Halabi, Department of Materials Engineering and the Ilse Katz Institute for Nanoscale Science & Technology, Ben-Gurion University of the Negev, the recipient of the Best poster Prize in Materials Sciences, for his poster titled: “Measuring the Space Charge Potential in Granular Magnesium Aluminate Spinel using Off-Axis Electron Holography”.
- Gal Mor Khalifa, Department of Structural Biology, Weizmann Institute of Science, the recipient of the Best poster Prize in Life Sciences, for her poster titled: “Biomineralization Pathways in Foraminifera: A Cryo Correlative Approach”.
- Olga Kleinerman, Department of Chemical Engineering, Technion Israel Institute of Technology, the recipient of the Best Micrograph Prize for her micrograph “Nanocigar”.
We hope to meet you again at ISM2016 – the Golden Jubilee Annual Meeting of ISM !
Eyal Shimoni, Yaron Kauffmann
Chair, ISM Secretary, ISM
|08:30 - 09:30||Registration|
|PLENARY SESSION (Auditorium)|
|Session Chair:||Eyal Shimoni, ISM Chairperson|
|09:30 - 09:50||Eyal Shimoni - Greetings & Presentation of the Lev Margulis & SIG4 Prizes
|09:50 - 10:35||Plenary Lecture: Pavel Tomancak, MPI-CBG, Dresden, Germany.||Plenary|
|10:35 - 10:55||Coffee Break|
|10:55 - 11:40||Plenary Lecture: Ute Kaiser, Ulm university, Germany||Plenary|
|SOUND BITE SESSION (Auditorium)|
|Session Chair:||Amit Kohn, Ben Gurion University of the Negev, Israel|
|11:50 - 12:20||Posters Sound Bites|
|12:20 - 13:15||LIFE SCIENCES POSTERS |
MATERIALS SCIENCE POSTERS
|ISM GENERAL ASSEMBLY|
|13:15 - 14:15||Lunch|
|14:15 - 16:05||LIFE SCIENCES SESSION
MATERIALS SCIENCE SESSION
|16:05 - 16:20||Coffee Break|
|16:20 - 17:55||LIFE SCIENCES SESSION
MATERIALS SCIENCE SESSION
|18:00 - 18:30||Beer & Snacks + best posters & best micrograph nominations|
Materials Science Session:
|Session Chair:||Yossi Lereah, Tel-Aviv university, Israel|
|14:15 - 14:40||Stavros Nicolopoulos, Nanomegas, Belgium.||invited|
|14:40 – 15:05||Yuval Golan, Ben Gurion University of the Negev, Israel||invited|
|15:05 - 15:25||Haim Weissman, Weizmann Institute of Science, Israel|
|15:25 - 15:45||Shmuel Samuha, Ben Gurion University of the Negev, Israel||SIG 4|
|15:45 - 16:05||Laurie Palasse, Bruker Nano GmbH, Berlin, Germany|
|16:05 - 16:20||Coffee Break|
|Session Chair:||Louisa Meshi, Ben-Gurion university, Israel|
|16:20 - 16:45||Amit Kohn, Ben Gurion University of the Negev, Israel||invited|
|16:45 – 17:10||Lilac Amirav, Technion, Israel.||invited|
|17:10 - 17:30||Hadas Sternlicht , Technion, Israel|
|17:30 - 17:50||Aleksandr Bagmut, National Technical University , Kharkiv, Ukraine|
|18:00 - 18:30||Beer & Snacks + best posters & best micrograph nominations|
Life Science Session:
|Session Chair:||Edith Suss-Toby, Technion, Israel|
|14:15 - 14:40||Daniel Razansky, Technical University of Munich, Germany||invited|
|14:40 - 15:00||Yonatan Sivan, Ben Gurion University of the Negev, Israel|
|15:00 - 15:20||Yechiel Elkabetz,Tel Aviv University, Israel|
|15:20 - 15:40||Anat Akiva, Weizmann Institute of Science, Israel||Margulis|
|15:40 - 16:05||Karina Yaniv, Weizmann Institute of Science, Israel||invited|
|16:05 - 16:20||Coffee Break|
|Session Chair:||Dror Fixler, Bar-Ilan university, Israel|
|16:20 - 16:45||Michael Elbaum, Weizmann Institute of Science, Israel||invited|
|16:45 - 17:05||Einat Zelinger, Hebrew University, Israel|
|17:05 - 17:30||Natan T. Shaked, Tel-Aviv University, Israel||invited|
|17:30 - 17:55||Eyal Nir, Ben Gurion University of the Negev, Israel||invited|
|18:00 - 18:30||Beer & Snacks + best posters & best micrograph nominations|
- Anat Akiva, Department of Structural Biology, Weizmann Institute of Science, for her work titled: “Boning Up on Bone Formation: Tracking the Bone Formation Pathways in the Larval Zebrafish Tail”.
The ISM board wishes to thank the jury of the Lev Margulis Prize for the year 2015.
- Shmuel Samuha, Department of Materials Engineering and NRCN, Ben-Gurion University of the Negev, for his work titled: “Structure Solution of the Al65Cu25Re10 phase by 3D Electron Diffraction Tomography”.
|Eyal Shimoni||Chairperson||Weizmann Institute of Science|
|Yaron Kauffmann||Secretary||Technion – Israel Institute of Technology|
|Zahava Barkay||Treasurer||Tel-Aviv University|
|Yuval Garini||Head of local organizing committee||Bar Ilan University|
|Efrat Bodner||Secretary of local organizing committee||Bar Ilan University|
|Dror Fixler||Companies coordinator||Bar Ilan University|
|Yael Goldfinger||Local organizing committee||Bar Ilan University|
|Aryeh Weiss||Local organizing committee||Bar Ilan University|
|Louisa Meshi||Ben-Gurion University|
|Edith Suss-Toby||Technion – Israel Institute of Technology|
|Dganit Danino||Technion – Israel Institute of Technology|
|Maya Bar-Sadan||Ben-Gurion University|
|Amit Kohn||Ben-Gurion University|
|Inna Popov||Hebrew University|
Life science committee:
|Edith Suss-Toby||Chair||Technion – Israel Institute of Technology|
|Aryeh Weiss||Bar Ilan University|
|Dganit Danino||Technion – Israel Institute of Technology|
|Eyal Shimoni||Weizmann Institute of Science|
|Yuval Garini||Bar-Ilan University|
|Ilan Tsarfaty||Tel-Aviv University|
Materials science committee:
|Louisa Meshi||Chair||Ben-Gurion University|
|Inna Popov||Hebrew University|
|Yaron Kauffmann||Technion – Israel Institute of Technology|
Lev Margulis prize committee:
|Prof. Clare Waterman||NIH, Bethesda Maryland, USA|
|Prof. Benny Geiger||Weizmann Institute of Science, Israel|
SIG4 prize committee:
|Dr. Ronit Popovitz||Weizmann Institute of Science, Israel|
|Prof. Misha Talianker||Ben Gurion University of the Negev, Israel|
Best Poster prize committee:
|Dr. Luba Burlaka||Bar Ilan university, Israel|
|Dr. Inna Popov||Hebrew University, Israel|
|Dr. Vladimir Ezersky||Ben Gurion University of the Negev, Israel|
|Dr. Elena Kartvelishvili||Weizmann Institute of Science, Israel|
|Dr. Nitsan Dahan||Technion - Israel Institute of Technlogy, Israel|
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 , ion-implanted graphene , MoS2 , MoSe2, SiO2 , CN , square ice , transition-metal clusters ) and amorphous (monolayer carbon, SiO2)  objects as well as a new structure of crystalline AuC  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 . We show that sandwiching clean radiation-sensitive low-dimensional objects in-between two graphene layers  or embedding them into single-walled carbon nanotubes  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 .
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
Phase transformations in solid state accompanied with the changes of density of matter. The review and analyses of author's electron-microscopic investigations, concerning relative density changes η at phase transition from amorphous to crystalline state (fig. 1) and from hexagonal close packed (hcp) to face centred cube (fcc) structures (fig 2) is the aim of this work. Films were prepared by pulsed laser sputtering of rotating targets in vacuum ore in oxygen medium. The substances of laser erosion were deposited on the (001) surface of KCl crystal. The films, transparent for an electron beam, were investigated. Crystallization was initiated by thermal ore electron beam influence on amorphous film in a column of electron microscope. The film structure was investigated by electron diffraction and transmission electron microscopy methods. We used electron microscope PEM-100-01 (image resolution 0.4 nm) and EMV-100 LM (image resolution 0.5 nm) of JSC "SELMI" (Sumy, Ukraine). Investigations were carried out at an accelerating voltage of 100 kV.
Laser sputtering provided the formation of micro-babbles of sputtered substance in condensed film. These micro-babbles we used as bench marks. The distance X1 between them was changed and after phase transformation it was X2. So, performing the measurements of X1 and X2, the relative density changes can be calculated as η = (X1/X2)3–1. The results are collected in table.
It was established, that in the case of hcp - fcc structure transition in Ni η is equal to 18.5 2.9% (row 6 in the table). It agrees with the data of the JCPDS tables. According to it the density of hcp Ni lattice ρ1=7.372 g/сm3. For fcc Ni lattice ρ2=8.911 g/сm3. So, the value of η is equal to 20.9%. This value is in the confidence interval from 15.6% to 21.4%, that we have defined in our work. After annealing, that initiates the hcp - fcc structure transformation in Ni, the magnetic state of films changes noticeably . Namely, the magnetic moment increases essentially and hysteresis is observed at magnetization reversal (fig. 3).
Hadas Sternlicht, Wolfgang Rheinheimer, Michael J. Hoffmann and Wayne D. Kaplan
General GBs in polycrystalline SrTiO3 and GBs between a single crystal diffusion bonded to polycrystalline SrTiO3 were characterized using aberration corrected transmission electron microscopy (TEM). TEM was used to identify steps, which are hypothesized to be the active mechanism for grain boundary motion following the terrace ledge kink (TLK) model. The combined GB chemical excess and structure (complexions) were characterized and correlated to experimentally measured changes in mobility.
The GBs in SrTiO3 were found to be non-stoichiometric. Steps were found along the boundaries, which are assumed to be active in GB migration and to reduce strain caused by the change in local atomistic order. Specific types of steps were found regardless of the annealing temperature or atmosphere. The steps at different conditions lie along the same crystallographic planes even if the GB mobility is drastically different. This suggests that the step free energy in SrTiO3 is anisotropic, and that the rate of step-motion defines the GB mobility.
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.
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
As an alternative, the recently introduced Transmission Kikuchi Diffraction (TKD) technique is a SEM based method capable of delivering the same type of results as EBSD but with a spatial resolution improved by up to one order of magnitude [1, 2]. Such analysis is conducted on an electron transparent sample using a commercial EBSD system. The high spatial resolution (<10 nm) compared to conventional EBSD will be demonstrated through TKD application examples.
We will also address the challenges of this technique. Indeed, due to the non-optimum sample-detector geometry, the transmitted Kikuchi patterns exhibit much stronger gnomonic projection induced distortions as compared with normal EBSD patterns. In addition, most of the transmitted signal does not reach the detector screen which results in a loss of signal and can have implication in the measurement quality. We will review these limitations and propose new alternative.
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 . 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  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 . 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  for the Al65Cu25Re10 phase). The structure was solved applying Direct Methods, utilized in the SIR2008 software , and refined using kinematical least squares refinement procedure in the Jana2006 program . 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' , 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.
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.
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.
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).
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
We have used tannic acid in combination with iodide to significantly improve the contrast of challenging tissues for high resolution images. We used the reproductive apparatus of female Drosophila and Arabidopsis plant meristem as model systems.
Another challenge was to visualize the stored sperm inside the Drosophila female storage organs that had no contrast with all stains tested. To overcome this problem we used a correlative approach. The fluorescent sperm (DJ-GFP) was imaged in situ using a confocal microscope and the same sample was subsequently fixed, stained with our technique and scanned in the X-Ray tomographic microscope. A complete 3D mapping of the sperm was obtained by manual registration of the images.
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 . 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
We combined two spectroscopic techniques in order to characterize the transient mineral phases involved in the bone formation process: Raman spectroscopy, and x-ray diffraction, which is coupled with an x-ray fluorescence detector. Together with our colleagues from the MPI in Golm, we developed a new setup for fluorescence-Raman confocal microscopy . This unique micro-Raman setup allows for real time spectroscopy of stained mineral aggregates. In this set-up the living fish is stained beforehand with caclein. The spectroscopic signature originating from the fluorescent mineral aggregates is detected directly by the micro-Raman spectrometer. This setup assures that the detected mineral phase is indeed the phase that is present in vivo. X-ray diffraction (XRD) and X-ray fluorescence (XRF) are used as complementary techniques to detect the mineral particles and calcium distribution in a wider area of the caudal fin, which cannot be done by Raman spectroscopy alone.
Our results show the presence of mineral aggregates in close proximity to the blood vessels, between and within the intra-ray region, and relatively far from the bone. Some of the intracellular mineral vesicles were observed in cells which also carry an endothelial fluorescent marker (figure 1). This might imply the existence of a common lineage for endothelial and osteoblast cells. Raman spectroscopy coupled with fluorescence imaging performed on a living fish, shows the presence of a disordered calcium phosphate phase with characteristic features of an octacalcium–like (OCP-like) phase in mineral aggregates located between bones . XRD combined with XRF and FIB shows that the transient mineral phase is homogenously distributed at the edges of the bones as nano-particles.
The observation that mineral particles form at diverse locations, and involve different cell types including cells which express an endothelial reporter entails a conceptual change in our understanding of the comprehensive mechanism of bone formation in vertebrates, from the blood to the bone. The achievement of in vivo correlative confocal imaging and spectroscopy of cells and mineral in a living animal advances our understanding of bone mineralization pathways in vertebrates, including humans.
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
Here we describe a newly developed quantitative approach to analyze dynamic features in human neural stem cells derived from pluripotent stem cells. We used neural rosettes – an in vitro structure resembling the cortex cytoarchitecture. We have previously shown that cells within neural rosettes contain the information to reiterate in culture the process of cortical lamination – the orchestrated and timely ordered generation of cortical neurons. We specifically imaged and analyzed the apical to basal nuclei migration – a typical feature of neuroepithelial cells of the cortex that is also considered to be critical for maintaining NSC numbers during cortical development.
Using these measures we first quantitatively confirmed the repetitive radial apical to basal polarized cellular motion in rosette stem cells. We found that this apical basal radial nuclei movement becomes more precise as rosette size increases, suggesting that preciseness of motion is enforced via mechanical constraints of the confining structure. This also implies that also during real development, as embryonic cortical tissue expanses, radial movements of nuclei are more robust, consequently further contributing to NSC maintenance.
We further used these measures to characterize cellular dynamics within rosette structures derived from two developmentally and functionally distinct rosette stages - early emerging rosettes that are rich for NSCs with broad developmental potency and hence correspond to founder NSCs of the cortex, and their later derived and more differentiated stage rosettes that are characterized by reduced NSC capacity and lower developmental potential.
We show that early rosettes maintain faster and more precise radial cellular motions compared to the later stage rosettes. This is accompanied by increased ratios of basal motions, i.e. towards rosette periphery, compared to apical motions. Moreover, these basal motions were also found to be more radial (more precise motion) compared to apical motions. The increase in the ratios of basal motions was evident for all early rosettes but not the late rosettes, reflecting that our quantitative measures can distinguish early from late NSC stages of development. Furthermore, this increase in basal motion ratios was not dependent on rosette size, suggesting an additional, rosette size-independent mechanism for enhancing radial motion preciseness. Third, we found that late rosettes are characterized by dynamic temporal instability, again reflecting that progressive change in NSC number and function are reflected in cellular dynamics.
We conclude that our measures can serve as new readout for early versus late rosettes, which respectively correspond to early and late stages in cortical development: the higher scores rosette get, the earlier NSC stage is, greater NSC numbers are, and the broader potential prevails.
We propose that our quantitative kinetic measures have the potential to serve as readout for functional molecular studies, drug screening and diagnosis and may be implicated to gain novel insights into pathology of NSCs in health and disease.
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
One of the prominent super-resolution technique is stimulated-emission-depletion (STED) nanoscopy, which offers superb resolution along with fast acquisition times. The STED nanoscope, however, suffers from the need for high intensities required for efficient depletion.
We show that metal nanoparticles (NPs) can be used to improve the performance of STED nanoscopes. Compared with a standard STED nanoscope, we show theoretically a resolution improvement by more than an order of magnitude, or equivalently, depletion intensity reductions by more than 2 orders of magnitude; these come along with a strong photostabilization due reduction of photobleaching. We also show that such performance improvement can be attained without excessive heating, making it useful for live-cell studies.
We demonstrate the technique experimentally by comparing the resolution attained by imaging dye-doped silica nanoparticles with or without a thin shell of gold. We observe that an optimum resolution, limited by the particle sizes, can be reached for the NPs for a power of the STED beam 4 times smaller than for the bare cores, see Figure below, in good agreement with the theoretical calculations. We show that the experimental obstacles encountered all have very satisfactory solutions. Accordingly, we expect to reach the theoretical predictions once the measurements will be repeated with particles of optimal geometries. Our experimental demonstration opens the way to improvement of existing STED nanoscopes and assisting the development of low-power, low-cost nanoscopes. This has the potential to increase the availability of STED nanoscopes and lead to an expansion of our understanding of nanoscale biological phenomena.
Institute for Biological and Medical Imaging (IBMI),
Technical University of Munich and Helmholtz Center Munich
The talk will further deal with the new realm of 5-dimensional (5D) optoacoustic imaging , which enables simultaneous acquisition of information across all the 3 spatial dimensions, the time and the spectral (optical wavelength) dimension. The 5D imaging capability makes it possible to visualize diverse endogenous contrast and administered contrast agents on multiple scales, namely, from cell to whole organ on the spatial scale and from fast heart beat to longitudinal tumor development on the temporal scale. Applications are explored in the areas of in-vivo cell tracking, imaging of agent kinetics and biodistribution, targeted molecular imaging studies, as well as functional imaging of the brain and heart. Clinical translation activities are further discussed.
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
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 . 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.
This project was supported by the German Federal Ministry for Education and Research (grant No. FKZ: 13N11403).
 A. Merchán-Pérez et al., Frontiers in Neuroanatomy 3:18.10.3389/neuro.05.018.2009.
 M. Cantoni et al., Microscopy and Analysis 24(4): 13-16, 2010.
 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.
Department of Chemical Research Support, Weizmann Institute of Science
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.
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.