Direct visualization of charge transport in suspended (or free-standing) DNA strands by low-energy electron microscopy.

Low-energy electrons offer a unique possibility for long exposure imaging of individual biomolecules without significant radiation damage. In addition, low-energy electrons exhibit high sensitivity to local potentials and thus can be employed for imaging charges as small as a fraction of one elementary charge. The combination of these properties makes low-energy electrons an exciting tool for imaging charge transport in individual biomolecules. Here we demonstrate the imaging of individual deoxyribonucleic acid (DNA) molecules at the resolution of about 1 nm with simultaneous imaging of the charging of the DNA molecules that is of the order of less than one elementary charge per nanometer. The cross-correlation analysis performed on different sections of the DNA network reveals that the charge redistribution between the two regions is correlated. Thus, low-energy electron microscopy is capable to provide simultaneous imaging of macromolecular structure and its charge distribution which can be beneficial for imaging and constructing nano-bio-sensors.

Moiré structures in twisted bilayer graphene studied by transmission electron microscopy.

We investigate imaging of moiré structures in free-standing twisted bilayer graphene (TBG) carried out by transmission electron microscopy (TEM) in diffraction and in-line Gabor holography modes. Electron diffraction patterns of TBG acquired at typical TEM electron energies of 80-300 keV exhibit the diffraction peaks caused by diffraction on individual layers. However, diffraction peaks at the scattering angles related to the periodicity of the moiré structure have not been observed in such diffraction patterns. We show that diffraction on moiré structure can create intense diffraction peaks if the energy of the probing electrons is very low, in the range of a few tens of eV. Experimental diffraction patterns of TBG acquired with low-energy electrons of 236 eV exhibiting peaks attributed to the moiré structure periodicity are shown. In holography mode, the intensity of the wave transmitted through the sample and measured in the far-field can be enhanced or decreased depending on the atomic arrangement, as for example AA or AB stacking. Thus, a decrease of intensity in the far-field must not necessarily be associated with some absorption inside the sample but can simply be a result of a particular atomic arrangement. We believe that our findings can be important for exploiting graphene as a support in electron imaging.

Metal Adsorption and Nucleation on Free-Standing Graphene by Low-Energy Electron Point Source Microscopy.

The interaction of metals with carbon materials (and specifically with graphene) is of importance for various technological applications. In particular, the intercalation of alkali metals is believed to provide a means for tuning the electronic properties of graphene for device applications. While the macroscopic effects of such intercalation events can readily be studied, following the related processes at an atomic scale in detail and under well-defined experimental conditions constitutes a challenge. Here, we investigate in situ the adsorption and nucleation of the alkali metals K, Cs, and Li on free-standing graphene by means of low-energy electron point source microscopy. We find that alkali metals readily intercalate between the layers of bilayer graphene. In fact, the equilibrium distribution of K and Cs favors a much-higher particle density between the layers than on the single-layer graphene. We obtain a quantitative value for the difference of the free energy of the binding between these two domains. Our study is completed with a control experiment introducing Pd as a representative of the nonalkali metals. Now, we observe cluster formation in equal measure on both single-layer and bilayer graphene; however, there was no intercalation. Our investigations thus constitute the first in situ study of metal-atom sorption of different specificity on free-standing graphene.

Imaging the potential distribution of individual charged impurities on graphene by low-energy electron holography.

While imaging individual atoms can routinely be achieved in high resolution transmission electron microscopy, visualizing the potential distribution of individually charged adsorbates leading to a phase shift of the probing electron wave is still a challenging task. Low-energy electrons (30 - 250 eV) are sensitive to localized potential gradients. We employed low-energy electron holography to acquire in-line holograms of individual charged impurities on free-standing graphene. By applying an iterative phase retrieval reconstruction routine we recover the potential distribution of the localized charged impurities present on free-standing graphene.

Imaging proteins at the single-molecule level.

Imaging single proteins has been a long-standing ambition for advancing various fields in natural science, as for instance structural biology, biophysics, and molecular nanotechnology. In particular, revealing the distinct conformations of an individual protein is of utmost importance. Here, we show the imaging of individual proteins and protein complexes by low-energy electron holography. Samples of individual proteins and protein complexes on ultraclean freestanding graphene were prepared by soft-landing electrospray ion beam deposition, which allows chemical- and conformational-specific selection and gentle deposition. Low-energy electrons do not induce radiation damage, which enables acquiring subnanometer resolution images of individual proteins (cytochrome C and BSA) as well as of protein complexes (hemoglobin), which are not the result of an averaging process.

Direct Observation of Individual Charges and Their Dynamics on Graphene by Low-Energy Electron Holography.

Visualizing individual charges confined to molecules and observing their dynamics with high spatial resolution is a challenge for advancing various fields in science, ranging from mesoscopic physics to electron transfer events in biological molecules. We show here that the high sensitivity of low-energy electrons to local electric fields can be employed to directly visualize individual charged adsorbates and to study their behavior in a quantitative way. This makes electron holography a unique probing tool for directly visualizing charge distributions with a sensitivity of a fraction of an elementary charge. Moreover, spatial resolution in the nanometer range and fast data acquisition inherent to lens-less low-energy electron holography allows for direct visual inspection of charge transfer processes.

Design and implementation of a micron-sized electron column fabricated by focused ion beam milling.

We have designed, fabricated and tested a micron-sized electron column with an overall length of about 700 microns comprising two electron lenses; a micro-lens with a minimal bore of 1 micron followed by a second lens with a bore of up to 50 microns in diameter to shape a coherent low-energy electron wave front. The design criteria follow the notion of scaling down source size, lens-dimensions and kinetic electron energy for minimizing spherical aberrations to ensure a parallel coherent electron wave front. All lens apertures have been milled employing a focused ion beam and could thus be precisely aligned within a tolerance of about 300 nm from the optical axis. Experimentally, the final column shapes a quasi-planar wave front with a minimal full divergence angle of 4 mrad and electron energies as low as 100 eV.

Holography and coherent diffraction with low-energy electrons: A route towards structural biology at the single molecule level.

The current state of the art in structural biology is led by NMR, X-ray crystallography and TEM investigations. These powerful tools however all rely on averaging over a large ensemble of molecules. Here, we present an alternative concept aiming at structural analysis at the single molecule level. We show that by combining electron holography and coherent diffraction imaging estimations concerning the phase of the scattered wave become needless as the phase information is extracted from the data directly and unambiguously. Performed with low-energy electrons the resolution of this lens-less microscope is just limited by the De Broglie wavelength of the electron wave and the numerical aperture, given by detector geometry. In imaging freestanding graphene, a resolution of 2Å has been achieved revealing the 660.000 unit cells of the graphene sheet from a single data set. Once applied to individual biomolecules the method shall ultimately allow for non-destructive imaging and imports the potential to distinguish between different conformations of proteins with atomic resolution.

Low-energy electron holographic imaging of gold nanorods supported by ultraclean graphene.

An ideal support for an electron microscopy should be as thin as possible and be able to interact as little as possible with the primary electrons. Since graphene is atomically thin and made up of carbon atoms arranged in a honeycomb lattice, the potential to use graphene as a substrate in electron microscopy is enormous. Until now graphene has hardly ever been used for this purpose because the cleanliness of freestanding graphene before or after deposition of the objects of interest was insufficient. We demonstrate here by means of low-energy electron holographic imaging that freestanding graphene prepared with a platinum-metal catalysis method remains ultraclean even after re-exposure to ambient conditions and deposition of gold nanorods from the liquid phase. In the holographic reconstruction of gold particles the organic shell surrounding the objects is apparent while it is not detectable in SEM images of the very same sample, demonstrating the tremendous potential of low-energy electron holography for imaging of graphene-supported single biomolecules.

On artefact-free reconstruction of low-energy (30-250eV) electron holograms.

Low-energy electrons (30-250eV) have been successfully employed for imaging individual biomolecules. The most simple and elegant design of a low-energy electron microscope for imaging biomolecules is a lensless setup that operates in the holographic mode. In this work we address the problem associated with the reconstruction from the recorded holograms. We discuss the twin image problem intrinsic to inline holography and the problem of the so-called biprism-like effect specific to low-energy electrons. We demonstrate how the presence of the biprism-like effect can be efficiently identified and circumvented. The presented sideband filtering reconstruction method eliminates the twin image and allows for reconstruction despite the biprism-like effect, which we demonstrate on both, simulated and experimental examples.

Graphene unit cell imaging by holographic coherent diffraction.

We have imaged a freestanding graphene sheet of 210 nm in diameter with 2 Å resolution by combining coherent diffraction and holography with low-energy electrons. The entire sheet is reconstructed from a single diffraction pattern displaying the arrangement of 660.000 individual graphene unit cells at once. Given the fact that electrons with kinetic energies of the order of 100 eV do not damage biological molecules, it will now be a matter of developing methods for depositing individual proteins onto such graphene sheets.

Nondestructive imaging of individual biomolecules.

Radiation damage is considered to be the major problem that still prevents imaging an individual biological molecule for structural analysis. So far, all known mapping techniques using sufficient short wavelength radiation, be it x rays or high energy electrons, circumvent this problem by averaging over many molecules. Averaging, however, leaves conformational details uncovered. Even the anticipated use of ultrashort but extremely bright x-ray bursts of a free electron laser shall afford averaging over 10{6} molecules to arrive at atomic resolution. Here, we present direct experimental evidence for nondestructive imaging of individual DNA molecules. In fact, we show that DNA withstands coherent low energy electron radiation with deBroglie wavelength in the Angstrom regime despite a vast dose of 10{8} electrons/nm{2} accumulated over more than one hour.

Direct evidence for conduction pathways in a solid electrolyte.

The emission of silver ions from the apex of an amorphous electrolyte tip has been investigated by field ion microscopy. The ion emission patterns show discrete nanometer-sized spots. We present evidence that they represent the termination of bulk ion conduction pathways at the solid-vacuum interface. The analysis of the signals from individual emission sites suggests the existence of a network of such pathways in the solid. Auto- and cross-correlation measurements of the currents from individual sites provide quantitative information on the microscopic dynamics of charge transport in solid electrolytes as well as on the lateral extent of the pathway network.