Radiation damage is the fundamental limitation for the attainable specimen resolution of electron micrographs of radiation-sensitive objects. To avoid atom displacement, the accelerating voltage must be lower than the knock-on threshold. At higher voltages, the low intrinsic contrast and the high susceptibility of low-Z materials to knock-on damage prevents for example defect analysis of graphene, carbon nanotubes, and functionalized fullerenes. In order to achieve atomic resolution and high contrast at low acceleration voltages, the correction of both chromatic aberration and spherical aberration is mandatory. The novel Cs/Cc-corrector of the SALVE (Sub-Angstrom Low-Voltage Electron) microscope compensates for these aberrations. In addition, the corrector eliminates the off-axial coma providing a large field of view with more than 2000 equally-well-resolved image points per diameter. The corrector consists of two identical units, each composed of a magnetic quadrupole doublet, a mixed electric and magnetic quadrupole, and two octopoles. The mixed quadrupoles compensate for the chromatic aberration and the octopoles eliminate the spherical aberration The Cc/Cs-corrector has recently been installed into the SALVE microscope and is tested at present. So far, the SALVE microscope was equipped with a monochromator, the imaging omega filter, and the hexapole corrector which only compensates for the spherical aberration and the isotropic off-axial coma. Apart from high-resolution imaging the microscope enables angle-resolved electron energy-loss spectroscopy with high S/N. In addition, we obtain basic information on the atomic structure of defects, their dynamical properties of carbonaceous materials and amorphous silica. Moreover, we shall demonstrate the feasibility of functionalized grapheme as substrate for in-situ experiments and outline the prospects and advantages of the fully (Cc and Cs) corrected SALVE microscope operating in the range between 20 and 80kV.

The new electron optical capabilities owing to the successful development of correctors of the spherical the chromatic aberration have opened a new field for electron microscopy: the low-voltage TEM. For further investigations in this field of microscopy a dedicated low-voltage TEM has been developed. The key component of this instrument is a new quadrupole-octupole corrector for the compensation of the chromatic and spherical aberration of the objective lens. Besides this off-axial aberrations must be compensated or, at least, taken into account during the optical design. Thereby, a large field of view can be imaged at high resolution even at low beam energies. The novel instrument has been designed and constructed for the German SALVE project at Ulm University. It will be available for materials science and life science applications from summer 2013 onwards. Before, however, the instrument development has to be fully completed and the theoretical specifications have to be proven. The optical specifications are related to the energy and we should be able to achieve from 20 kV - 80 kV always an acceptance angle of 50 mrad with respect to the specimen. This would lead to parameters as shown in Tab.1. The prototype corrector is ready for incorporation into a Zeiss LIBRA TEM. Beam-down is scheduled for July, 2012. At the meeting we will report about our first experience and give some impressions about low-voltage TEM. Also first results from the instrument should already be available and will be demonstrated

The roles of transmission electron microscopes (TEMs) and scanning electron microscopes (SEMs) with low acceleration voltages (in the range of several tens of kilovolts) have been converging. Owing to the growth of demand for light-element materials (i.e., radiation-sensitive materials) in green-innovation industries, the importance of analysis in this acceleration-voltage range has recently been increasing. Some projects have been developing aberration correctors for low-voltage TEMs and scanning transmission electron microscopes (STEMs) [1-4] and have reached atomic resolution. On the other hand, diffractive imaging, which provides a structural image of a specimen from a diffraction pattern with iterative phase retrieval, has started to open up the possibility of atomic resolution with an SEM-based microscope at low voltage [5, 6]. This imaging method has an advantage in terms of high resolution because resolution in this case is defined by recorded diffraction angle and lens aberration is not a critical factor. The developed microscope, which was used to verify atomic-resolution imaging of single-wall carbon nanotubes (SWCNTs) at 30 kV, is based on a conventional in-lens-type SEM (S-5500, Hitachi High-Technologies Corp.). The appearance of CNTs grown from a matrix is evaluated from SEM image obtained with this microscope, and certain parameters concerning a CNT (i.e., diameter, chirality) are evaluated from the obtained diffraction pattern. The results of these evaluations are used to characterize the crystallizability of the CNTs in low-damage range of acceleration voltage. An SEM with this diffractive-imaging function is presently being applied to SWCNTs, graphene, and nanoparticles. SEM has various functions for obtaining information concerning, for example, wide-area surface morphology, voltage contrast, and difference between elements and that between orientations of polycrystalline grains. Combining the functions for crystalline characterization from diffraction patterns and atomic-resolution structural analysis by diffractive imaging (collectively called “diffraction microscopy”) with the inherent functions of SEM will increase the applications of electron microscopy in developing materials and devices (especially film, fiber, and particles). We would like to thank Prof. Shinohara&’s group at Nagoya University for growing the SWCNTs. Part of this work was supported by the Japan Science and Technology Agency (JST). [1] O. L. Krivanek et al., Nature, 464 571 (2010). [2] K. Suenaga et al., Nat. Chem., 1 415 (2009). [3] U. Kaiser et al., Ultramicroscopy, 111 1239 (2011). [4] J. C. Meyer et al., Nano. Lett., 8 3582 (2008). [5] O. Kamimura et al., Ultramicroscopy, 110 130 (2010). [6] O. Kamimura et al., Appl. Phys. Lett., 98 174103 (2011).

Monochromatic, aberration-corrected, dual-beam low energy electron microscopy (MAD-LEEM) is a novel technique aimed at high resolution imaging of organic materials, nanoparticles and surfaces that utilizes electrons with landing energies in the range of 0 to a few hundred eV for imaging. MAD-LEEM combines a monochromator, a mirror aberration corrector and dual electron beam illumination in a single instrument. The monochromator reduces the energy spread of the illuminating electron beam, which significantly improves spectroscopic and spatial resolution. The aberration corrector is needed to improve the spatial resolution in order to achieve sub-nm resolution at landing energies of a few hundred eV. The dual flood illumination approach eliminates charging effects generated when a conventional low voltage electron microscope is used to image insulating specimens. The low landing energy of electrons is critical for avoiding electron beam damage, as high energy electrons with keV kinetic energies cause irreversible damage to many specimens, in particular biological materials. The electron-optical properties of the objective lens combined with an electron mirror aberration corrector have been analyzed up to 5th order for electron energies ranging from 1 to 1000 eV. The spherical and chromatic aberration coefficients of the electron mirror are fine-tuned iteratively to cancel the spherical and chromatic aberration of the objective lens for a range of electron energies, thus providing a path for sub-nanometer spatial resolution. A potential application for MAD-LEEM is in DNA sequencing. Image contrast simulations of the detectability of individual nucleotides in a DNA strand have been developed in order to refine the LEEM optics blur and nucleotide contrast requirements. Experimental images of DNA structures immobilized on a gold substrate obtained in a LEEM demonstrate that high contrast is achievable at low electron energies in the range of 1-10eV. Electron reflectivity measurements derived from these LEEM images over a range of landing energies show that small changes in landing energy have a strong impact on the DNA contrast and thus hold promise for distinguishing individual nucleotides without heavy atom labels. The MAD-LEEM approach promises to significantly improve the performance of a LEEM for a wide range of applications in the biosciences, material sciences and nanotechnology where nanometer scale resolution and analytical capabilities are required.

It has recently been demonstrated that electrons with kinetic energies in the range of 50-250 eV do not cause radiation damage to biomolecules, enabling the investigation of an individual molecule for an extended period of time. Since the electron wavelength associated with this kinetic energy range is between 0.77 Å (for 250 eV) and 1.7 Å (for 50 eV), low-energy electrons have the potential for non-destructive imaging of single biomolecules and in particular individual proteins at atomic resolution. We have developed and implemented an experimental scheme for low-energy electron coherent diffraction imaging. A sharp tungsten tip acts as source of a divergent beam of coherent low-energy electrons, which is collimated by a dedicated electrostatic microlens of only 2.5 micron in diameter. The resulting parallel beam is directed towards the sample and at a distant detector its diffraction pattern is recorded. For our investigations objects of interest can be deposited onto an ideally transparent support such as graphene. Graphene has already been employed as a support for imaging objects such as hydrogen atoms, gold nanoparticles, CoCl2 nanocrystals and biological molecules in TEM. When graphene is visualized with low-energy electrons, it is sufficiently transparent and does not get damaged during continuous exposure to the radiation, which makes graphene an ideal substrate for coherent diffraction imaging of biomolecules using low-energy electrons. Here, we report coherent diffraction patterns of freestanding ultra-clean graphene obtained with electrons of 150-250 eV kinetic energies. We show the first reconstruction of low-energy electron coherent diffraction pattern at 2Å resolution. Finally, we will discuss the next steps towards atomic resolution imaging of biomolecules with coherent low-energy electron diffraction.

Approaches to study free-standing graphene and its adsorbates at high temperatures (300 K - 2000 K) could be already realized in a transmission electron microscope (TEM) with atomic resolution at low acceleration voltage (80 kV). We demonstrated the effect of in-situ Joule heating on graphene membranes during HRTEM studies at temperatures until 2000 K [1] observing the transformation of physisorbed hydrocarbon adsorbates via amorphous carbon monolayers (at 1000 K) into polycrystalline graphene (at 2000 K) [2]. However, the electronic properties could not be studied yet. Here we investigate the electronic properties of graphene by 4-terminal electrical measurements whereas 4 other contacts remain for heating and temperature sensing. The magnetic field of the microscope's objective lens was determined at 80 kV applying a custom-made Hall probe as TEM specimen. This one-time-only calibration procedure allows us to carry out Hall measurements at different temperatures. Characteristics of free-standing graphene membranes were measured in-situ during TEM observation at temperatures up to 500 K before and after in-situ Joule heating. The reliability of the obtained carrier concentration and mobility values could be assured by their temperature dependence. The dependence of the carrier mobility on the pre-annealing temperature will be discussed. These first measurements happened strictly without exposing the sample to the electron beam. Later, TEM images and electrical measurements have been recorded simultaneously in order to reveal the remaining amount of hydrocarbon residues and their influence on the mobility. We observed a continuous drop of the mobility during TEM imaging which might be a consequence of contaminants burnt into the surface by the electron beam. Our results demonstrate the importance of considering the effect of electron irradiation on adsorbates, especially when the task is a clean surface in order to obtain high mobilities. References [1] B Westenfelder, J C Meyer, J Biskupek, G Algara-Siller, L G Lechner, J Kusterer, U Kaiser, C E Krill III, E Kohn and F Scholz, J Phys D: Appl. Phys. 44 (2011), 055502. [2] B Westenfelder, J C Meyer, J Biskupek, S Kurasch, F Scholz, C E Krill III and U Kaiser; Nano Lett. 11 (2011), p. 5123.

Chromatic aberration correction in the transmission electron microscope (TEM) allows large energy windows and large objective aperture sizes to be used without compromising the spatial resolution of energy-loss images. Background-subtracted chemical maps can then be recorded on the atomic scale with a signal-to-noise ratio that is suitable for quantitative high-resolution energy-filtered TEM (EFTEM), even at accelerating voltages of 80 kV or below. We have acquired preliminary experimental atomic-scale EFTEM images of perovskite oxides and thin layered materials such as BN and MoS2 at accelerating voltages of 80 - 300 kV using the chromatic and spherical aberration-corrected ‘PICO&’ TEM installed in Juelich. Our experimental results show that, in order to obtain reliable atomic-scale EFTEM images, a higher degree of aberration control and microscope stability is required compared to experiments with conventional, non energy-filtered HRTEM images. The strengthened requirements are essentially due to the preservation of elastic scattering and phase contrast in background and core-loss images in combination with acquisition times on the order of several seconds. Furthermore, the calculation of core-loss intensities necessitates alignment of the background and core-loss images to sub-Ångstrom accuracy. In confirmation of theoretical predictions, our results show that a knowledge of specimen thickness and defocus for a given accelerating voltage is important for the reliable interpretation of exit-plane intensities and therefore elemental distributions on the atomic scale.

Functionalized QDs are of emerging interest nowadays in the broad field of ultrastable contrast agents for bioimaging applications [1]. Irradiation damage and diminishing contrast are the major problems for conventional TEM investigations. Low energy electrons show significant different scattering behaviour compared to commonly used faster electrons with an energy between 80keV and 300keV as: (1) the inelastic (as well as the elastic) scattering cross section increases, and therefore an significant increase in image contrast of EFTEM (as well as TEM) images can be expected; (2) the elastic (electron-nucleus) damage mechanisms decrease significantly and the inelastic (electron-electron) damage mechanisms increase. Here we report the 20kV EFTEM investigation of core shell CdSe/CdZnS QDs with an albumin polymere surface coating [2] on single-layer graphene, the thinnest substrate possible. The investigations were performed with the monochromized and imaging aberration-corrected SALVE1 microscope [2] operating at 20 kV equipped with an corrected incolumn Omega energy filter and a 4kx4k CMOS Tietz camera (F416) with an energy slit width of 1.1 eV, an energy resolution of 0.18 eV (FWHM of ZLP). A series of energy filtered images was taken up to an energy loss of 40 eV with an energy delta of 0.5 eV between each image and an illumination time of 5s per image. This datacube was used to obtain EEL spectra by selecting specific sample positions and integrating over 20x20 pixel. The extacted spectra reveal significant changes in the peak shape and position for the different sample positions like single and double layered graphene, the protein and the core and shell of the QDs. One leftover question is now the influence of electron irradiation on the light efficiency of the QDs. Therfore dose rate and accelerating voltage depend studies in the SEM were started. [1] Liu, Y.; Solomon, M.; Achilefu, S. Med Res Rev. 2010, 31. [2] U.Kaiser et al Ultramicroscopy 111 (2011) 1239 [3] This work was supported by the DFG (German Research Foundation) and the Ministry of Science, Research and the Arts (MWK) of Baden-Wurttemberg in the frame of the SALVE (Sub Angstrom Low-Voltage Electron microscopy and spectroscopy project.

Grain boundary migration in polycrystalline solids is a great interest for materials scientists because grain boundaries (GBs) are strongly influencing the mechanical, electronic, thermal and chemical properties. For 3D materials however investigation of the underlaying mechanisms of GB migration has been reserved to computer simulations as experimental techniques are lacking the temporal and spatial resolution to capture the dynamics of individual atoms in the core region of a GB. For 2D materials, however these technical limitations can be overcome by bypassing the projection problem of transmission techniques. Here we use aberration-corrected TEM to study grain boundaries in polycrystalline graphene on atomic scale. We lowered the primary beam energy to 80kV to minimize knock-on damage [1] but the high energy electrons still offer enough energy to induce bond rotations [2] allowing the system to undergo structural relaxations (similar to local melting and recrystallization). For straight grain boundaries we do not observe a preferred movement, instead the atomic structure alters between more or less curved shapes. However for small grains (in the nm range) we find a distinct driving force towards reducing the grain diameter that can result in total annealing of the grain as we have observed for the flower defect [3]. [1] J. Meyer et al., PRL 108, 196102 (2012) [2] J. Kotakoski et al., PRB 83, 245420 (2011) [3] S. Kurasch et al., Nano Lett., 2012, 12 (6)

Single-walled carbon nanotubes (SWNTs) recently attract great attention for their aptitude as nano-test tubes and have proven to be ideal containers for atomically resolved imaging of interactions and dynamics of sub-nanometer sized molecules. In our experiments SWNTs do not only offer a necessary confinement and protection against undesired beam damage effects as a result of ionization or chemical etching: SWNT for instance provide a unique environment for the investigation of carbon-based reactions on the atomic scale.[1] Furthermore SWNT represent a precious test-system for a detailed study of interaction mechanisms between the specimen and the energetic electron-beam by nature omnipresent in transmission electron microscopy (TEM) observations. In this work we study sub-nanometer sized d-element metal-nanoclusters (W, Re, Os, Ru, Fe) enclosed in SWNTs by means of low voltage aberration-corrected high-resolution transmission electron microscopy (AC-HRTEM).[2,3] This particular technique combines imaging tool and irradiation source in one integral experiment while further benefiting from the SWNTs low and regular contrast combined with minimized susceptibility to head-on collisions of the electrons with the carbon atoms nuclei (knock-on damage) while operating with electron energies less or equal 80 keV.[4] In our experiments we pursue the methodology of varying both, the metal type and the electron acceleration voltage (between 20 kV, 40 kV and 80 kV). Thus we are able to separate the influence of the electron beam from that of the specimen which is a necessary condition for the detailed and atomically resolved study of sample or irradiation induced structural SWNT-modifications. Finally it is our aim to explicitly describe the interaction mechanisms e.g. those of different "beam-damage" processes. While irradiating, in order to answer the demand of comparability, time series of interactions are recorded until reaching the same total electron dose of 1010 e-/nm2. Experiments are conducted using a Cs-corrected FEI Titan 80-300 operated at 80 kV and a monochromated/Cs-corrected SALVE (Sub-Ångstroslash;m Low-Voltage Electron Microscopy) Zeiss LIBRA prototype microscope operated at 20, 40 and 80 kV. [1] T. Zoberbier, T. W. Chamberlain, J. Biskupek, N. Kuganathan, S. Eyhusen, E. Bichoutskaia, U. Kaiser, A. N. Khlobystov, J. Am. Chem. Soc. (2012) [2] A. Chuvilin, A.N. Khlobystov, D. Obergfell, M. Haluska, S. Yang, S. Roth, U. Kaiser, Angew. Chem. Int. (2010) [3] U. Kaiser, J. Biskupek, J. C. Meyer, J. Leschner, L. Lechner, H. H. Rose, M. Stöger-Pollach, A. N. Khlobystov, P. Hartel, H. Müller, M. Haider, S. Eyhusen, G. Benner, UM (2011) [4] J. C. Meyer, F. Eder, S. Kurasch, J. Kotakoski, H. J. Park, S. Roth, A. Chuvilin, S. Eyhusen, G. Benner, A. V. Krasheninnikov, Ute Kaiser, Phys. Rev. Lett. (2012)

Tranmission Electron Microscopy (TEM) is a widely used materials characterization method. Due to the fact that organic materials are mostly composed of low atomic number elements, various techniques are often needed to increase the contrast in TEM images. Heavy metal stains can be environmentally hazardous and may cause changes in microstructure. A tabletop sized, low-voltage (~5 kV) electron microscope that operates in scanning, transmission, scanning transmission and electron diffraction modes has been employed to investigate organic materials at high contrast without staining. Electron beam damage at this voltage is discussed and compared with normal high accelerating voltage electron microscopes. Ultrathin carbon, silicon nitride, graphene and graphene oxide supporting films have been studied and compared. Various samples including organic crystals, block copolymers, peptides have been imaged and show enhanced contrast as compared with normal high voltage electron microscopy.

Fundamental concepts for surface science are well-established and their applications are straightforward for metals and semiconductors, but not for insulators materials. Bombardment of insulator surfaces by charged particles such as electron or ion beams can be induced a charge on their surfaces. One can see the charging/discharging dynamics of the insulating material during this ion-beam bombardment. Sometimes, an electron shower using a tungsten filament placed nearby a sample is used to reduce the sample charging. However, electron-shower failure can cause sample damage. Therefore, we have developed a low-energy atom scattering spectroscopy system for the analysis of these insulator surfaces. The primary beams were 3 keV-4He neutral particles which were pulsed with 100 kHz by chopping plates. Actually, flight times were measured using MCP (micro channel plates) on this detection system. MgO is an exceptionally important material, which used in catalyst, toxic-waste remediation agent, or as an additive in refractory, paint as well as for fundamental and application studies. The 111 surface gives a hexagonal arrangement of atoms. We have obtained the image pictures which consists of Mg atoms and Oxygen atoms on outermost layers using this technique.

The correction of TEM-lens aberrations has made atomic resolution possible even at low accelerating voltages. However, the image resolution for most organic and some inorganic specimens is limited by radiation damage, and can be a factor of 10 or 100 worse than the electron-optical resolution. Radiolysis, electrostatic charging and thermal effects are the main damage mechanisms in insulating materials, whereas knock-on displacement predominates in metals and semiconductors. The influence of radiolysis can be reduced by lowering the specimen temperature and by coating the specimen or increasing its thickness. Charging and thermal effects can be controlled by reducing the incident-beam current, at the expense of data-recording time. Knock-on displacement can sometimes be avoided by reducing the incident-electron energy below some threshold value, which is typically exceeds 200kV for atom displacement within a crystal but is considerably lower for displacement from the surface (electron-induced sputtering) or along the surface (beam-induced atom motion). The dose-limited resolution also depends on the contrast and electron-collection efficiency of the imaging or spectroscopy mode employed in the TEM or STEM. It is potentially better for phase-contrast imaging, compared to bright-field or dark-field scattering-contrast methods. Resolution also depends on the detective quantum efficiency (DQE) of the electron detector, and an improvement by a factor of two is possible by using an electron-counting detector rather than regular CCD recording.

Structural DNA as a templating technology for energy, sensing and other applications has much promise because of its ability to self-assemble complex architectures with nanoscale precision. These architectures can vary in form from linear DNA, to dendrimers, to custom DNA origami or tiles, and they can by tagged site-specifically with fluorophores or nanoparticles to achieve specific functions such as fluoresence resonance energy transfer (FRET). In order to assess yield and to examine stuctural characteristics, high-resolution characterization methods are essential. Heretofore such characterization has been performed primarily with atomic-force microscopy, a method that is often limited in its lateral resolution and that can disturb the structure under examination through its contact. Given its extraordinary resolution, TEM would seem to have great potential. Since, however, most such work with biological structures has involved high-energy beams and negative staining, templates, or other techniques that can compromise resolution. Moreover, that the DNA materials of interest are mostly composed of low-Z atoms means that contrast is paramount and ultrathin substrates a neccesity. In this study, we report on a method for TEM imaging of low-contrast biomolecules using suspended graphene supports. A sacrificial silicon membrane beneath the graphene provides crucial mechanical support during the aqueous sample deposition but is then eliminated in a final step using a XeF2 dry etch. Taking advantage of these supports, we perform direct unstained imaging of DNA origami and other DNA nanostructures in TEM (JEOL 2200 FS, 200 kV) mode for non-aberration phase contrast imaging and in low-voltage STEM mode (aberration-corrected, FEI Titan 80keV, Nion UltraSTEM, 60keV) for lowered damage to both the DNA structure and the graphene support. STEM characterization targets both tagged and untagged DNA origami and dendrimer structures. We find that the high substrate quality of the fluorinated graphene opens a new way to probe DNA structures and measure the efficacy of the self-assembly. In general, we were able to directly characterize DNA structures and found variations in deliberate shape design of up to 15% in the origami, which may be due to flexibility within the molecule, as well as issues of adhesion to the graphene.

The ultimate goal of developing novel microscopy techniques is associated with the visualization of the atomic arrangement of individual molecules, in particular the complex structures of proteins. This goal can be realized when using radiation with very short wavelengths of the order of one Ångstrom or less, such as X-rays or electrons. However, when imaging biological specimens, the obtainable resolution is mainly determined by the radiation damage threshold. Low-energy electrons (50-250 eV) have been proven to be the best known radiation for imaging individual biological molecules at high resolution, as they have a sufficiently short wavelength (0.7-1.7 Å) and inflict the least radiation damage. Two dedicated low-energy electron microscopes for imaging individual biomolecules are operated in our group: using either holographic or coherent diffractive imaging (CDI). In both microscopes, a coherent divergent spherical electron wave is generated by field emission from a sharp tungsten tip. In the holographic microscope, part of the divergent wave is scattered by an individual molecule placed about 1um distant from the tip. The scattered and unscattered wave form an interference pattern - the hologram. In the CDI microscope, the divergent electron wave is collimated by a microlens and the parallel plane wave incidents onto the individual molecule. The far-field diffraction pattern is recorded at a 68 mm distant detector. Holograms and diffraction patterns of individual biomolecules are then subject to numerical reconstruction. Images of individual molecules reconstructed from their holograms, such as DNA and bacteriophages at sub-nanometer resolution will be presented. The intrinsic properties of CDI and holography, in particular, the achievable resolution with low-energy electrons will be addressed. We will also show how holography and CDI can be merged into one superior technique: holographic coherent diffraction imaging (HCDI). In HCDI, two images of the same sample, a hologram and a diffraction pattern, are used. In the reconstruction, HCDI employs an iterative phase retrieval algorithm where the initial phase distribution is not random as in conventional methods, but directly obtained from the hologram. Such well-defined initial phase distribution provides a stable convergence of the iterative procedure towards a unique solution. Thus, reconstructions obtained by HCDI combine the highest possible resolution and uniqueness of the solution. Reconstructions of experimental low-energy electron diffraction patterns of carbon nanotubes and of free-standing graphene at 2.13 Å resolution will also be presented.

In recent years the TEAM Project (Transmission Electron Aberration-corrected Microscope) was concluded that was sponsored by the US Department of Energy. As a result a next generation electron microscopes of extraordinary performance is now operated at the National Center for Electron Microscopy (NCEM) with great success. These new instruments allow for dynamic studies (<1 kHz) with single atom sensitivity across the Periodic Table of Elements at a resolution limit around 0.5 Å [1-3]. Their unprecedented abilities also reveal that the image formation process is now limited at a fundamental level by the Coulomb scattering process itself and by beam-sample interactions. As a result, efforts to push for higher resolution are coming to an end and there is room for assessing the future of the field. This talk describes progress beyond resolution improvements that was recently made by highlighting new capabilities and concepts, which enable new scientific investigations addressing functionality at a single atom level. Instrumental advancements include the implementation of atomic-resolution, low-dose rate techniques at variable voltage (20 kV - 300 kV) [4] and atomic-resolution imaging at elevated temperature and pressure. Thus, it becomes feasible to study the functionality of materials and possibly of single molecules in chemically meaningful environments with atomic resolution and single atom sensitivity. Such capabilities are of general interest to material, chemical, and biological sciences. In particular, they can help to improve our understanding of artificial photosynthetic systems that will produce transportation fuels from sunlight.

Gas-mediated electron beam induced etching (EBIE) is used to analyze damage generation in ultra nano-crystalline diamond (UNCD) irradiated by a low-energy electron beam. Specifically, we use H₂O-mediated EBIE to quantify the volatilization rate of UNCD, and show that it is rate-limited by an electron stimulated carbon restructuring process. The observed behavior contradicts existing EBIE models which predict a volatilization rate that is proportional to the precursor (H₂O) dissociation rate. The models are modified to reproduce the measured etch kinetics, and can now be used to characterize low-energy electron beam damage kinetics in UNCD. EBIE is a nano-scale, direct-write technique analogous to gas-assisted focused ion beam (FIB) milling. However, low-voltage EBIE is a chemical process that does not involve sputtering or knock-on damage. H₂O-mediated EBIE of carbon proceeds through electron induced dissociation of surface adsorbed H₂O molecules generating fragments (e.g., O and OH) that react with the substrate. Volatile species (e.g., CO and CO₂) produced in these reactions can desorb, thus giving rise to localized chemical dry etching under an electron beam. EBIE of diamond, carbon nanotubes and amorphous carbon has been demonstrated previously. However, the etch mechanisms have not been investigated in detail, and quantitative EBIE has previously not been used to compare volatilization rates and detect electron restructuring effects in different types of carbon. We show that as-grown UNCD and highly ordered pyrolytic graphite (HOPG) both exhibit negligible etching ascribed to a low volatilization rate of sp² and sp³ rich carbon by reactive fragments produced by electron induced dissociation of H₂O adsorbates. Etching of UNCD accelerates significantly upon irradiation by low-energy (<~ 20 keV) electrons, and the rate scales inversely with electron beam energy and directly with energy density deposited into the solid. EBIE of UNCD is shown to proceed through an electron restructuring pathway that generates defect rich carbon that is susceptible to volatilization, a behavior that is not observed in HOPG, which etches slower than electron irradiated UNCD. Electron restructuring of a substrate has previously not been shown to rate limit EBIE. Most detailed prior work on restructuring of diamond and graphitic carbon by charged particles is limited to electrons with energies above ~ 100 keV, and energetic ions. Our results demonstrate the utility of EBIE for characterizing damage produced by low-energy electrons, where atom displacements caused by momentum transfer to carbon nuclei are negligible.

We have been investigating the use of low dose and low voltage transmission electron microscoy techniques for examining defect structures in ordered polymer and organic molecular thin films. Recent results will be presented, with particular focus on conjugated semiconducting materials such as functionalized thiophenes, acenes, and anilines. We have been particularly interested in the nature and extent of structural relaxations near grain boundaries, dislocations, and vacancies.

Electron microscopy is becoming one of the most important experimental tools to characterize thin films of organic semiconductors. One of the most widespread techniques is transmission electron microscopy which continues to be the method of choice in unraveling the nanomorphology formed by the interpenetrating networks of conjugated polymers and fullerene in films for organic photovoltaics [1]. From a general perspective optoelectronic devices based on organic semiconductors are seeing large improvements in their performances with the use of dopants mixed at low weight-percentages (<10%) with the semiconductor [2, 3]. Clustering of dopant molecules, changes to the semiconductor morphology and in general dopant distribution inside the film are all crucial aspects for the development and design of this technology. However, such aspects have been poorly addressed, likely because of the lacking of suitable experimental techniques. In this communication we present experiments demonstrating the possibility to map dopant distributions with nm resolution in organic semiconductor thin films. The technique which is based on low-voltage scanning electron microscopy (SEM) provides information of the semiconductor morphology as well and promises to be a method of choice for these functional soft materials. We have investigated two sets of samples obtained by spin coating the technologically relevant polymers poly(3-hexylthiophene) (P3HT) and poly[2,1,3-benzothiadiazole-4,7-diyl[4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b:3,4-b']dithiophene-2,6-diyl] ] (PCPDTBT) on silicon. The polymers had 2,3,5,6-tetrafluoro-7,7,8,8-tetracyano-quinodimethane (F4-TCNQ) added as dopant inducing an excess of holes in the semiconductors. We have observed that a low acceleration voltage, below 1 KV, is critical in obtaining images of the polymer morphology and identifying areas in which dopant molecules are clustering. The two samples show different characteristics; PCPDTBT exhibit a lamellar morphology as pristine, which is slightly disrupted by the presence of F4-TCNQ at 5% ratio, without seeing clustering of F4TCNQ molecules on a tens of nm scale. On the other hand, P3HT exhibits clustering of F4-TCNQ with a patchy pattern on a hundreds of nm scale for concentrations above 5%. By performing imaging at different scan rates and simulating the electron back-scattering yields for the materials constituting the films we show that imbalanced charging between the doped and undoped regions is the origin of the observed contrast. The results show that low-voltage SEM is a powerful tool in investigating the morphology of doped organic semiconductors. [1] M. Hallermann, I. Kriegel, E. Da Como et al. Adv. Funct. Mater. 2009, 19, 3662. [2] F. Deschler, E. Da Como, T. Limmer et al. Phys. Rev. Lett. 2011, 107, 127402. [3] A. Tunc, A. De Sio, D. Riedel, F. Deschler, E. Da Como et al. Org. Electr. 2012, 13, 290.

Calcit (CaCO3) is one of the most common carbonates of the earth crust. The most important use for calcite is for the manufacture of cements and lime for mortars. Limestone is the chief raw material, which when heated about 900° C forms quicklime (CaO) by the reaction: CaCO3→CaO+CO2uarr;. Apparently calcite undergoes the same phase transformation to lime (CaO) by electron-irradiation damage. Therefore the new challenge arises to search for conditions and instrumental settings to study in situ the phase transformation on an atomic scale. The irradiation damage is caused predominantely by ionisation, thermal heating or by knock on damage. The influence of knock on damage is studied by reducing the accelerating voltage from 300 kV down to 20 kV by using a CM20 operating at 200 kV, equipped with a LaB6 cathode, a TITAN operating at 300 kV and 80 kV equipped with a field emission gun and an imaging-side Cs-corrector and the newly developed SALVE prototype microscope equipped with a field emission gun, an image-side Cs-corrector, corrected Omega-Filter and a monochromator [1] operating at 20, 40 and 80 kV. At high accelerating voltages the irradiation damage of calcite is starting immediatly with the electron-irradiation and can be divided up in three stages: first the amorphisation of the crystalline structure, second hole production and third recrystalisation in a polycrystalline structure with significant volume and mass loss during the reaction time. We found that the reduction of the accelerating voltages slows down the irradiation damage in calcite and gives the freedom to align the instrument to obtain information on the atomic scale before significant beam damage effects appear. This enables dose rate depend quantitative studies of the volume and mass losses during the phase transformation. [1] U.Kaiser et al Ultramicroscopy 111, 8,( 2011), p.1239 [2] This work was supported by the DFG (German Research Foundation) and the Ministry of Science, Research and the Arts (MWK) of Baden-Wuerttemberg in the frame of the SALVE (Sub Angstrom Low-Voltage Electron microscopy and spectroscopy project. We thank the Institut of Mineralogy Münster (P. Schmid-Beurmann) for the samples, S. Groezinger for the sample preparation.

The aim of the SALVE (Sub-Angstrom Low-Voltage Electron microscope) project is to visualize low-Z materials with atomic resolution and minimum radiation damage by employing voltages in the range between 20 and 80kV. To understand the image contrast, it is necessary to perform image calculations. The conventional image simulations consider only elastic scattering, which suffices at medium voltages for most objects. However, because at 20kV all atoms are strong scatters, we must incorporate the effect of inelastic scattering in the image calculation. For inelastic scattering, the sample states and the incident wave are coupled. Therefore, we cannot ignore anymore excitations of the object as in the elastic case. Our approach is based on the mixed dynamic form factor and the light-optical concept of mutual coherence. However, this procedure still poses a big challenge for the computer since it involves 4D Fourier transforms. In order to reduce the computing time, it is necessary to look for suitable approximations to transform the 4D Fourier transform into 2D Fourier transforms. In this work we will present a new approximation which has the maximum similarity with the original function; and calculated EFTEM images based on it for voltages as low as 20kV and 40kV. We employed the experimental EELS spectra for the simulation of the EFTEM images. For mono-atomic layered structures, our approach takes one elastic scattering and one inelastic scattering into account; for multi-layered structures, our method can take two inelastic scattering and multiple elastic scattering into account. Our calculation shows convincingly the effect of inelastic and elastic double scattering within a mono-layered structure at 20kV, exemplified by graphene, as well as two inelastic scattering and multiple elastic scattering within multi-layered structure at 40kV, exemplified by Si<110> with the thickness of 54nm. The smaller the energy loss, the more delocalized is the inelastic process and the lower is the blurring of the image formed by the doubly (elastically and inelastic) scattered electrons. Energy filtered inelastic images are only visible when Cc is corrected. However, in order to obtain the same S/N ratio as for the elastic image, the dose must be 10^6 times higher.

Electrons have large scattering cross-sections and can be focused into small probes that are ideal for single molecule diffraction and imaging. However, radiation damage is the main limiting factor that prevents its realization. At medium high electron energies (10 to 60 keV), the elastic scattering cross section increases with decreasing electron energies but damage per elastic scattering event is constant[1], except for the knock-on damage. Knock-on damage associated with atomic bonding can be avoided by using medium electron energies below the damage threshold. However, the threshold is significantly lowered not for molecular bonding with weak Van der Waals forces. To better understand this, we used C60 molecules confined inside single-walled carbon nanotubes (C60s@SWCNT or peapod) as a model system to study [2]. A 25 nm diameter electron beam from a field emission gun source is used to record diffraction patterns from individual peapods using imaging plates. The electron beam illuminates about 25 C60 molecules. Experimentally, we found that the molecules diffract inside ~15% of the host nanotubes. With the help of simulations, we calculated the knock-on damage threshold for elastic scattering at large diffraction angles and the limits this places on electron molecular diffraction. Furthermore, we examined the electron diffraction sensitivity to the molecular configurations. We show that the combination of molecular confinement, electron diffraction and electron direct imaging provides the best approach to single molecule imaging. [1] Henderson, R., 1995, Quarterly Review of Biophysics, 28, 171-193. [2] Ran, K., Zuo, J.M., Chen, Q., Shi, Z.J., 2011. Electron Beam Stimulated Molecular Motions. Acs Nano 5, 3367-3372.

Modern characterization techniques, such as focused ion beam serial sectioning (FIBSS) and electron tomography, provide a relatively direct route to a full 3D quantification of a microstructure. Multi-modal data acquisition (for instance, simultaneous or asynchronous acquisition of secondary and backscattered electron images, electron backscatter diffraction maps, and energy dispersive x-ray signals) can generate a large amount of information from which a rather complete microstructure model can be obtained. However, merging/fusing of these data streams and segmentation of the microstructure continue to pose significant barriers to the routine application of these 3D techniques. In this contribution we will review a couple of promising approaches that employ forward modeling, i.e., the use of the proper interaction physics, to extract the microstructure from the data. Forward modeling (also known as model-based reconstruction) starts from a model of the microstructure (as obtained, for instance, from a preliminary reconstruction in the serial sectioning case, or a simple filtered backprojection in the tomography case) and computes what the images would look like if this primary result were the correct microstructure. Consideration of the differences between the predicted images/diffraction patterns and the experimental ones then allows one to construct an iterative algorithm to extract the best possible microstructure model, given an experimental data set and prior knowledge about the sample and the imaging modalities. We will illustrate this approach by means of two examples: FIBSS reconstructions using a simple physics-based model for the electron beam interaction volume, and high angle annular dark field electron tomography, employing a Rutherford scattering forward model that includes the direct beam and elastic diffraction contributions. Results from each approach as well as the underlying physics-based forward models will be discussed in detail.

The creation of high spatial resolution scanning electron microscope (SEM) images has been a major goal of instrument developers since the first commericial SEM's were introduced in the mid-1960's. The major strategy to achieve this goal has been the production of electron beams of smaller and smaller size that would have sufficient probe current to generate a statistically significant signal above the inherent noise in a reasonable time. Over time, it was recognized that conventional tungsten and lanthanum hexaboride sources were limited by both source brightness concerns and chromatic aberrations and the field moved to cold field emission and Shottky sources since they have high brightness and low chromatic aberrations. As the gun potentials used to extract the electrons are high, methods were developed to de-accelerate the electrons prior to striking the sample to acheive low voltage operation and a small spot size. In the research described here it will be shown that conventional sources can be used to obtain high resolution low voltage images by using relatively large probes typical of conventional sources at low voltage . This is accomplished by using a series of images combined with a knowledge of the point spread function of the electron beam. The combination of these factors with a new image processing algorithm makes it possible, in principle, to determine intensity data from regions considerably smaller than the electron beam size. This is particularly interesting when used with conventional sources since they are capable of producing much larger probe currents at the sample than field emission sources. Thus, a lot of data can be collected quickly and processed. Examples of this new method as well as the underlying theory will be presented.

Electron microscopy has offered valuable insights into the shape distribution, interactions and other properties of engineered nanomaterials. Yet, polydisperse materials properties have been extremely difficult to quantify. This is a particularly difficult problem for soft (polymers) and hybrid materials, as recently reviewed [1]. Low voltage instruments offer several advantages over traditional ones for the characterization of soft engineered nanomaterials, including their ability to image the materials without using staining agents due to their inherently higher contrast. Some of the advantages are, however, less evident and are the consequence of the peculiar design of the instrument, like the possibility of retracing the location of a region of interest using combined modes of observation of the sample; an otherwise inherently difficult task. On the other hand, the optical path in LVEM5 instruments [2] offer opportunities as well as challenges for the proper processing of the images obtained with them. In this presentation, we will discuss new strategies for the rapid processing of large datasets obtained using LVEM5 instruments and their implementation in GPGPU clusters for the rapid characterization of engineered nanomaterials. This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. References 1. Sweeting the small stuff. Lauren K Wolf, Chemical and Engineering News, pages 48-50, May 28, 2012 2. http://en.wikipedia.org/wiki/Low-voltage_electron_microscope

Hybrid materials have been the focus of significant recent attention due to their potential for exhibiting novel material properties by synergistically combining constituents. Because of their high interfacial area to volume ratio, core-shell hybrid nanoparticles have shown property enhancements in several applications including energy storage and sensing. A limiting factor for the performance of core-shell nanoparticles in these applications is synthetic reproducibility, and direct methods for characterization are needed in aiding reaction optimization and purification. Low Voltage Transmission Electron Microscopy (LVTEM) has traditionally provided high imaging contrast for materials composed of light elements such polymers, biomolecules and oxides. However, the density differences in core-shell materials are often small, and the internal interface between the core and the shell often occurs on top of a sloping background (such as the projected thickness of a sphere), making the boundary extremely difficult to visualize. In this work LVTEM imaging is combined with a geometrical model to calculate the expected intensity profiles of core-shell nanoparticles based on their known material scattering cross sections. Shell thickness in assemblies of polystyrene functionalized silica nanoparticles was varied by controlling the molecular weight and grafting density of the polymer on the nanoparticle surface. Core-shell calculated intensity profiles were fit to 2D projection TEM images of the nanoparticles, thus producing an effective 3D model for the structure. Segmentations were produced via hybrid median noise filtering and quantitative measurements of particle core size and shell thickness distributions were made. For model validation, the mass-thickness parameter of the microscope was calibrated using layered nanocrystals of known composition and thickness. As a cross-calibration with an independent characterization technique, small angle X-ray scattering provided global material structural parameters such as average interparticle spacing in 3D assemblies. This work is expected to provide a basis for quantitative microscopy experiments where the measured intensity distribution is directly related to a structural and chemical model.

Due to their excellent electrical and optical properties, carbon nanotubes have been regarded as extremely promising candidates for high-performance electronic and optoelectronic applications. However effective and efficient distinction and separation of metallic and semiconducting single-walled carbon nanotubes are always challenges for their practical applications. In our experiment, horizontally-aligned and high-density SWCNT arrays synthesized via chemical vapor deposition (CVD) on a stable temperature-cut (ST-cut) quartz substrate were used. And we show that these metallic and semiconducting single-walled carbon nanotubes on SiO₂ can have obviously different contrast in scanning electron microscope, and thus can be effectively and efficiently identified. We have demonstrated that semiconducting and metallic single-walled carbon nanotubes on SiO₂, probably a variety of insulator substrates which were positively or negatively charging in low-voltage SEM, can have obviously different contrast in SEM, and thus can be effectively and efficiently identified. The correlation between conductivity and contrast difference has been confirmed by using voltage-contrast scanning electron microcopy, Peakforce Tunneling Atom Force Microscopy, and field effect transistor testing. This phenomenon can be understood via a proposed mechanism involving the e-beam induced surface potential of insulators and the conductivity difference between metallic and semiconducting SWCNTs. This method demonstrates great promise to achieve rapid and large-scale distinguishing between metallic and semiconducting single-walled carbon nanotubes, adding a new function to conventional SEM.

We study the catalyst state during chemical vapor deposition (CVD) of carbon nanostructures by complementary in-situ grazing-incidence X-ray diffraction, in-situ X-ray reflectivity, in-situ X-ray photoelectron spectroscopy and environmental transmission electron microscopy. For near-atmospheric pressure carbon nanotube forest CVD, we find that typical oxide supported Fe catalyst films form mixtures of bcc and fcc phased Fe nanoparticles upon reduction and that depending on this phase composition different growth modes occur. For fcc-rich Fe nanoparticle distributions, we find metallic Fe is the active catalyst phase, implying that carbide formation is not a prerequisite for nanotube growth. For bcc-rich catalyst mixtures, Fe₃C formation more readily occurs and constitutes part of the CNT growth process. Our data indicates that metastable catalyst phases dominate the CNT growth. We propose that this behavior can be rationalized in terms of kinetically accessible pathways, which we discuss in the context of the bulk iron-carbon phase diagram with the inclusion of phase equilibrium lines for metastable Fe₃C. In contrast, for low pressure CVD of single-wall nanotubes from Co catalysts we find that the catalyst state is purely metallic but that catalyst-support interactions (silicide formation) account for a remarkably narrow chiral distribution of the resulting tubes. Finally, we use the complementary in-situ metrology to compare the structural and chemical evolution of such nanoparticulate metals (1-d templates) to flat metal films (2-d templates) to elucidate similarities and differences between nanotube and graphene nucleation in CVD.

Carbon nanotubes (CNTs) are considered as prototypical “new” materials in nanoscience and nanotechnology. Indeed, CNTs provide two particularly exciting prospects. Firstly, the versatility of their properties according to their structure and their dimensions makes them objects of fundamental interest. Secondly, their outstanding mechanical strength, electrical and thermal conductivity and opto-electronic properties offer many opportunities for industrial applications. The combination of these exceptional and versatile properties might lead to the development of electronic systems where both active devices and interconnects are based on the same material. However, in spite of longstanding efforts, no major electronics application involving CNTs is available in the market yet. The main obstacle in the development of a CNT-based technology is that many aspects of their growth mechanisms remain obscure. In particular, the relationship between the nucleating/growing nanotube and the catalyst nanoparticle is not well understood. Despite some remarkable improvements in the control of CNTs features at the synthesis stage during the last decade [1-4], the optimization of CNT growth conditions remain mostly empirical and nanotube samples are frequently a mixture of different structures (number of walls, length, diameter and chiral angle) and morphologies (straight, bundled or entangled). Here, we have employed an in situ approach combining high resolution imaging and Electron Energy Loss Spectroscopy using an environmental scanning transmission electron microscope (ESTEM) in order to probe the relevant instants of a nanotube life at the atomic scale. The composition and the crystal structure of the catalytic nanoparticles were investigated as a function of the growth condition (temperature, pressure) and the nanoparticle size for various catalyst/carbon precursor combinations. The nature of the grown species (number of walls, diameter, crystallinity) were also systematically examined and correlated. Ex situ Raman measurements were used to characterize the in situ synthesized samples at a large scale. [1]. Harutyunyan AR, Chen G, Paronyan TM, Pigos EM, Kuznetsov OA, Hewaparakrama K, et al. Preferential growth of single-walled carbon nanotubes with metallic conductivity. Science. 2009;326(5949):116. [2]. Chiang W, Sankaran R. Linking catalyst composition to chirality distributions of as-grown single-walled carbon nanotubes by tuning NixFe1minus; x nanoparticles. Nat Mater. 2009;8(11):882-6. [3]. Bachilo S, Balzano L, Herrera J, Pompeo F, Resasco D, Weisman R. Narrow (n, m) Distribution of Single-Walled Carbon Nanotubes Grown Using a Solid Supported Catalyst. J Am Chem Soc. 2003;125(37):11186-7. [4]. Zheng L, O'connell M, Doorn S, Liao X, Zhao Y, Akhadov E, et al. Ultralong single-wall carbon nanotubes. Nat Mater. 2004;3(10):673-6.

We present a systematic study of the optical response of bilayer graphene as a function of misorientation angle using a combination of electron energy-loss spectroscopy in an aberration-corrected scanning transmission electron microscope. We find that an additional absorption peak (~4.40 eV), which has not been reported before, emerges in the ultraviolet region of the energy loss function of bilayer graphene for large misorientation angles (~30 degrees). The additional absorption peak is below the nominal π peak of bilayer graphene (~5.10 eV) and also at a different energy position of the π peak for monolayer graphene (4.95 eV). The analysis of the data also reveals that π peak as well as the π+σ peak (~15 eV) do not shift in energy as function of misorientation angle. The observations will be explained using total-energy first-principles calculations based on density functional theory within the random phase approximation. The results obtained in this study indicate that the misorientation angle between graphene layers can affect the optical properties, therefore suggesting that the misorientation angle between layers could also be used as a variable in the designing of nobel optoelectronic devices based on hybrid two-dimensional materials. This research was supported by Oak Ridge National Laboratory's Shared Research Equipment (ShaRE) User Facility (JCI), which is sponsored by the Office of Basic Energy Sciences, U.S. Department of Energy, and by the National Science Foundation grant No. DMR-0938330 (WZ).

One strategy to covalently functionalize single-walled carbon nanotubes (SWCNTs) is to oxidize the side-walls by an acidic treatment step to create COOH carboxylic functions and then convert these functions to COCl groups by reacting with SOCl2. The acid chloride functions can subsequently be reacted with many different grafting groups and offer great flexibility in terms of chemistry on tubes. However, the control of the functionalization process, i.e. the control of the number of covalent defects and the efficiency of the overall grafting process without destroying the intrinsic electronic and mechanical properties of CNTs, requires estimating the number of defects created at each step. In the present work, different oxidative conditions have been used to create SWCNTs with different kinds and densities of oxidized functions. A HiPco sample from NanoIntegris (SuperpureTM grade, highly purified sample), was used as one of the best starting materials commercially available on the market. The use of such a clean sample, with less than 2% of metallic impurities and less than 5% of carbonaceous impurities, is absolutely essential in order to obtain reliable results for quantitative analysis. This allowed us to quantify the number of created defects by spectroscopic, thermal and microscopic techniques, especially X-Ray photoelectron spectroscopy (XPS), thermogravimetric analysis coupled with mass spectrometry (TGA-MS) and high-resolution transmission electron microscopy (HR-TEM) associated with energy-dispersive X-ray spectroscopy (EDS). After the chlorination step with SOCl2, these techniques quantified the number of chlorine atoms grafted on the CNT side-walls. Finally, a grafting reaction with ferrocene derivatives and other electro-active groups was also successfully done on these samples. These functionalized CNTs were deposited on a glassy carbon electrode and cyclic voltammetry was used to evaluate the electrochemical activity of this modified electrode toward electron shuttle of biological interest such as nicotinamide adenine dinucleotide (NADH). The combination of techniques used in this study enabled estimating the level of functionalization at each step of the process, which gives a strong rationale for the optimization of such CNT treatment.

Ultra-thin materials such as graphene are an important class of function materials. They also provide atomically simple and clean systems that are well-suited for transmission electron microscopy (TEM). With the latest improvement made by aberration correction, reliable interpretation of TEM images become more practical and can be well combined with image simulation and first-principles simulations to understand the configuration and dynamics of various atomic structures such as intrinsic ripples, development of defects, and chemistry at the edges. We demonstrate the usefulness of such combined study with results on various nanostructures in ultra-thin materials.

Experimental studies of soft materials in the electron microscope are constrained by the damage caused by inelastic scattering processes. Typically, most soft materials can tolerate incident electron doses on the order of about 10 - 10,000 e/nm^^2, depending on the material, before exhibiting significant changes to their structure and/or chemistry. There has recently been substantial interest in using lower-energy electrons for soft-matter characterization. However, while lower-energy incident electrons can reduce knock-on damage, ionization damage remains the limiting factor, and, like the scattering cross section for inelastic scattering, it increases with decreasing electron energy. We have used a combination of scanned-probe microscopy, scanning electron microscopy (SEM), and confocal immunofluorescence imaging to study the effects of focused electron beams with energies ranging from 2 - 30 keV and point doses ranging from 10 - 1000 fC on thin films of poly(ethylene glycol) [PEG] ~100 nm thick on silicon substrates. STEM EELS studies at 200 keV show that PEG evolves hydrogen when irradiated and suggests that it undergoes free-radical radiolytic polymerization. We have observed at 2-30 keV that electron irradiation ultimately crosslinks the PEG and grafts it to the underlying substrate to create surfaces patterned with PEG microgels of interest for biomaterials applications. Consistent with the experimental data, Monte Carlo simulation of electron energy deposition identifies three types of structure within each microgel: a highly crosslinked core near the point of electron incidence; a lightly crosslinked near-corona surrounding the core; and a far-corona localized at the PEG-Si substrate created by electrons backscattered from the silicon substrate. The nature and relative sizes of these three regions and, hence, the protein-interactive and cell-interactive character of the microgels depend strongly on the incident electron energy and electron dose as well as on the molecular weight and thickness of the precursor PEG thin film.

Herein we discuss the capabilities and potentials of a newly acquired Zeiss Libra 120 Energy Filtered TEM (60kV-120kV) at the Center for Nanophase Materials Sciences (CNMS). As part of a user facility offered by the Department of Energy for the scientific community, the Libra 120 at CNMS is optimized for soft materials, including but not limited to block copolymers, organic semiconductors, surfactant covered nanoparticles, biological and bioinspired structures, soft and hybrid assemblies. We will elaborate on the application of analytical TEM and low electron dose techniques in some recent projects, which encompass three-window method for elemental mapping of low atomic-number elements such as sulfur, oxygen and nitrogen, low energy loss imaging, electron energy loss spectroscopy, energy dispersive spectroscopy, low-dose high resolution imaging, and low-dose selected area electron diffraction. We are actively developing cryo-TEM and 3-D tomography capabilities, and are making efforts to combine analytical and low-dose techniques with cryo-TEM in the near future.

Here we first are concerned with the dispersion of high-energy plasmons (>3eV), known as pi and pi-sigma plasmon in free-standing single- and multi-layer graphene. We have applied angle-resolved electron energy-loss spectroscopy in a low-voltage transmission electron microscope to measure the momentum-dependent energy-loss function of suspended single- and multi-layer graphene as well as mono-layer MoS2 for the two reciprocal symmetry direction Gamma-M and Gamma-K. Samples were prepared using mechanical exfoliation. Experiments were done on a Libra-based TEM prototype (ZEISS) operated at 20kV and 40kV. We determined the energy and momentum resolution to be 0.2eV and 0.1-0.2Å-1. Our achieved spatial resolution was around 100-200nm. For graphene we find the two plasmon peaks at small q-values to be around 5eV and 15eV for both symmetry directions. At smaller q-values the spectra for Gamma-M and Gamma-K are similar. We find significant differences at q-values larger than 0.5Å-1. In Gamma-M direction and above a value of 0.8Å-1 the pi-plasmon splits into two peaks with a shoulder at around 5eV similar to pi-plasmons observed in carbon nano-tubes and graphite. We see no shoulder for the Gamma-K direction. Comparison to density functional theory calculations in random phase approximation (RPA) shows, that in both cases the behavior of the pi-plasmon is well reproduced. In contrast RPA does not correctly describe the behavior of the pi-sigma-plasmon. Furthermore, for single-layer, free-standing graphene we report a quasi-linear dispersion of the pi-plasmon for both symmetry directions. In addition we present measurements, illustrating the changes of the energy-loss function with increasing number of graphene layers (up to 6). Here the behavior can be understood in terms of a simple layered electron gas model. Additionally we present the layer dependent plasmon dispersion behavior and compare it to graphite, showing that at small q-values 6-layered graphene is still significantly different from graphite but appears graphite-like at large q-values. In addition we will present experimental results of the energy-loss function of free-standing mono-layer MoS2, where we find significant differences to the bulk response of MoS2.

When operating at voltages low enough to avoid knock-on radiation damage, aberration-corrected scanning transmission electron microscopes (STEMs) allow volumes of matter as small as single atoms to be imaged and analyzed spectroscopically. The instrumental requirement for this type of work is that the STEM needs to be able to produce, at a low operating voltage, an electron probe not much bigger than 1 Å that contains a probe current of 0.1 nA and higher, and preferably has an energy spread of less than 0.4 eV. Such performance is now readily attainable. It has given us clear annular dark field (ADF) images of single atoms as light as boron, and it has identified the chemical type of single atoms in three different ways, sometimes simultaneously1: 1) by the atom&’s ADF intensity, 2) by its electron energy loss spectrum (EELS) and 3) by its energy-dispersive X-ray spectrum (EDXS). The EELS data typically shows fine structures in spectra from the single atoms, and the structures are often substantially different than when the same kind of atom is bonded in other materials. The fine structures can be used to answer questions relating to charge transfer and atomic coordination numbers, atom-by-atom. Acquiring EELS and EDXS data simultaneously from single atoms also allows theoretical cross-sections to be tested with unprecedented accuracy. Experimental results from materials systems such as single atom impurities in and on graphene and monolayer BN will be shown, and likely future progress will be discussed. Our more recent quest to develop a low to medium kV electron beam analytical instrument that may ultimately be able to identify single atoms of hydrogen by their low energy vibrational spectra will also be described. The new instrument is designed to reach EELS energy resolution down to 10 meV with an atom-sized probe. It uses a monochromator of a new design2 that acts on full-energy electrons and thus minimizes stochastic Coulomb effects that typically limit monochromator performance, and at the same time allows the monochromator and the energy loss spectrometer to be linked together in such a way that both short- and long-term drifts of spectrum energy are largely avoided. References: 1. See for instance O.L. Krivanek, W. Zhou, M.F. Chisholm, N. Dellby, T.C. Lovejoy, Q.M. Ramasse and J.C. Idrobo, “Gentle STEM of Single Atoms: Low keV Imaging and Analysis at Ultimate Detection Limits”, in: Low Voltage Electron Microscopy: Principles and Applications (D.C. Bell and N. Erdman, editors, RMS-Wiley) in press. 2. O.L. Krivanek, T.C. Lovejoy, G.J. Corbin, N. Dellby, M.F. Murfitt, N. Kurz, P.E. Batson and R.W. Carpenter “Monochromated STEM with high energy and spatial resolutions”, Microscopy and Microanalysis (proceedings 70th annual MSA meeting, Cambridge University Press, 2012), in press.

The potential application of graphene in future nanoelectronics and optoelectronics calls for understanding of the correlation between defect structures and local properties at the atomic scale [1]. Here, we investigate the link between atomic structure, bonding, electronic and optical properties of structural defects using aberration-corrected low-energy, low-dose, scanning transmission electron microscopy (STEM), also known as gentle STEM [2], in combination with density-functional quantum-mechanical calculations. Using a combination of quantitative annular dark-field (ADF) imaging and fast-scan sequential acquisition (FSSA), we show that single dopant atoms can be directly visualized and identified in graphene, and defect dynamics can be studied at the single-atom level. We observe that a Si dopant in graphene can oscillate between three- and four-fold coordinated configurations. The precise atomic configuration determines the local bonding and electronic structure, which are directly identified via atomic resolution electron energy-loss spectroscopy (EELS). Furthermore, with FSSA, the reversible oscillatory motion of a Si6 magic cluster trapped in a graphene nanopore can be directly imaged, following the calculated atomic-scale energy landscape, which provides a promising technique for quantitative study of molecular dynamics at the atomic scale. Atom-by-atom observation of both reversible and irreversible grain boundary migration in graphene will also be presented. The local optical response at various defect sites in graphene is studied by EEL spectrum imaging at the low-loss regime. We observe that a point defect complex acts as an atomic-scale antenna in the petaHertz (10^15 Hz) frequency range, inducing a localized surface plasmon enhancement at the sub-nanometer scale [3]. In addition, a new one-dimensional plasmon enhancement is observed at the open edge of monolayer graphene with a spatial extent of ~ 0.6 nm [4]. Finally, we show that highly localized plasmon modes are generated at a graphene quantum disk (GQD) due to the confinement from the edge of the GQD at all directions [5]. Our results open new possibilities for designing nanoscale optoelectronic devices based on monolayer graphene. References: [1] A. K. Geim, Science 234, 1530 (2009). [2] O. L. Krivanek et al. Ultramicroscopy, 110, 935 (2010). [3] W. Zhou et al. Nature Nanotechnology, 7, 161-165 (2012). [4] W. Zhou et al. Ultramicroscopy, in press (2012). [5] W. Zhou et al. J. Phys.: Condens. Matter, in press (2012). This research was supported by NSF grant No. DMR-0938330 (WZ), DOE grant DE- F002-09ER46554 (MK, STP), Oak Ridge National Laboratory's Shared Research Equipment (ShaRE) User Facility (JCI), which is sponsored by the Office of Basic Energy Sciences, U.S. Department of Energy; and the Office of Basic Energy Sciences, Materials Sciences and Engineering Division, U.S. Department of Energy (JL, ARL, SJP).

Lowering the accelerating voltage of TEM/STEM is becoming essential when one aims to image any beam sensitive objects. Especially, observation of small molecules made of light elements does require the reduced accelerating voltage, in order not to destroy the molecular structures by the knock-on effect and to enhance the image/EELS contrast. In order to compensate the poorer spatial resolution, more sophisticated electron optics is definitively required for the low voltage TEM/STEM to reduce the residual geometric/chromatic aberrations. I will summarize the low-voltage TEM/STEM developments under the triple C project and show exmaples for single atom spectroscopy (1-4). (1) K. Suenaga et al., Nature Chem. 1 (2009) 415-418. (2) K. Suenaga and M. Koshino, Nature, 468 (2010) 1088-1090. (3) Z. Liu et al., Nature Communications, 2 (2011) 213. (4) K. Suenaga, H. Kobayashi and M. Koshino, Phys. Rev. Lett., 108 (2012) 075501

Low dose imaging and the use of low accelerating voltage are effective techniques to reduce beam sample interaction in transmission electron microscopy. In some sensitive materials, especially those with mostly light elements, beam interaction produces a well-known decay of the sample during observation. Semiconductors are very sensitive to the beam and some of them develop planar and linear defects as wells as clear signs of knock on damage at accelerating voltages above 200 KeV. Normally the sample is heavily damaged after only a short time under the beam (minutes). This complicates acquisition of data for procedures such as exit wave reconstruction. Experiments with graphene show that low dose imaging can be used to reduce beam interaction with the sample and thus this investigation has been undertaken by using high dose and low dose conditions at 80 and 50 KeV to characterize quantum wells in the system CdSe-ZnSe. The samples have been prepared by atomic layer epitaxy on semi-insulating GaAs(001) substrates. These QWs are of interest for the fabrication of light emitting devices. CdSe and ZnSe have similar crystalline structures (fcc, zinc blende) but the strong lattice mismatch leads to strained interfaces when the QW is sufficiently thin (1 to 3 ML) and generation of defects above 4 ML of thickness. Thus the knowledge of their true structure and composition is of prime importance. The main result in this investigation is to reach atomic resolution under low dose and low voltage conditions without altering visibly the sensitive sample. Quantum wells are imaged under low dose conditions (around 100 e/A2s) at 50 and 80 KeV after exit wave reconstruction.

Electron tomography is a widely spread technique for recovering the three dimensional (3D) shape of nanostructured materials. Using a spectroscopic signal to achieve a reconstruction adds a fourth chemical dimension to the 3D structure. Up to date, energy filtering of the images in the transmission electron microscope (EFTEM) is the usual spectroscopic method even if most of the information in the spectrum is lost. Unlike EFTEM tomography, the use of electron energy-loss spectroscopy (EELS) spectrum-images (SI) for tomographic reconstruction retains all chemical information, and the possibilities of this new approach still remain to be fully exploited. In this work we prove the feasibility of EELS tomography at low voltages (80kV) and short acquisition times thanks to the recent advances in TEM and the use of Multivariate Analysis (MVA), applied to FexCo(3-x)O4@Co3O4 mesoporous materials. This approach provides a new scope into materials: the recovery of full EELS signal in 3D. Data acquisition was carried out on a probe Cs corrected FEI Titan operated at 80 kV acceleration voltage. Afterwards, MVA methods were applied, namely principal component analysis (PCA) and independent component analysis (ICA). From the noisy raw spectra, enhanced O (K), Fe (L3,2) and Co (L3,2) edges were retrieved after PCA analysis. ICA successfully retrieved the Fe oxide and Co oxide signals of the sample as well as the background signal before the oxygen K edge. Reconstruction of those signals was achieved, leading to volumes not only containing structural information, but also chemical information. Regarding chemical information, an interesting result was revealed: the comparison between iron and cobalt signals showed that some of the iron which was intended to penetrate into the structure remains instead on the outer surface. The particles are richer in iron at the border and therefore, iron related chemical signals give a sharp interface between the particle and the background, where HAADF signal is very low and has fallen to background levels due to the small thickness. These results show that iron signals reconstruct more precisely the edge of the particles than HAADF. On the other hand, the thickness signal has the drawback of underestimating the border more than the HAADF signal. However, the most interesting feature of this signal is that it is insensitive to the chemistry of our sample and independent of multiple scattering, a characteristic not found in any other signal used for electron tomography. As a conclusion, EELS SI tomography is shown to be able to reconstruct chemical information of a sample in three dimensions. Moreover, the application of MVA to the data opens a new range of applications, reducing the limitations due to beam sensitive materials or samples with components with overlapping edges, where core-loss extraction using background estimation fails.