Jean-Nicolas Longchamp

Low-energy electron transmission imaging of clusters on free-standing graphene

We investigated the utility of free-standing graphene as a transparent sample carrier for imaging nanometer-sized objects by means of low-energy electron holography. The sample preparation for obtaining contamination-free graphene as well as the experimental setup and findings are discussed. For incoming electrons with 66 eV kinetic energy, graphene exhibits 27% opacity per layer. Hence, electron holograms of nanometer-sized objects adsorbed on free-standing graphene can be recorded and numerically reconstructed to reveal the objects shapes and distribution. Furthermore, a Moire effect has been observed with free-standing graphene multi-layers.

Ultraclean freestanding graphene by platinum-metal catalysis

While freestanding clean graphene is essential for various applications, existing technologies for removing the polymer layer after transfer of graphene to the desired substrate still leave significant contaminations behind. The authors discovered a method for preparing ultraclean freestanding graphene utilizing the catalytic properties of platinum metals. Complete catalytic removal of polymer residues requires annealing in air at a temperature between 175 and 350 °C. Low-energy electron holography investigations prove that this method results in ultraclean freestanding graphene.

Imaging proteins at the single-molecule level

Significance We report a method to image and reveal structural details of proteins on a truly single-molecule level. Low-energy electron holography is used to image individual proteins electrospray deposited on freestanding graphene. In contrast to the current state of the art in structural biology, we do away with the need for averaging over many molecules. This is crucial because proteins are flexible objects that can assume distinct conformations often associated with different functions. Proteins are also the targets of almost all the currently known and available drugs. The design of new and more effective drugs relies on the knowledge of the targeted proteins structure in all its biologically significant conformations at the best possible resolution. 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.

Non-destructive imaging of an individual protein

Imaging a single biomolecule at atomic resolution without averaging over different conformations is the ultimate goal in structural biology. We report recordings of a protein at nanometer resolution obtained from one individual ferritin by means of low-energy electron holography. One single protein could be imaged for an extended period of time without any sign of radiation damage. Since the fragile protein shell encloses a robust iron cluster, the holographic reconstructions could also be cross-validated against transmission electron microscopy images of the very same molecule by imaging its iron core.

Low-energy electron holographic imaging of individual tobacco mosaic virions

Modern structural biology relies on NMR, X-ray crystallography and cryo-electron microscopy for gaining information on biomolecules at nanometer, sub-nanometer or atomic resolution. All these methods, however, require averaging over a vast ensemble of entities and hence knowledge on the conformational landscape of an individual particle is lost. Unfortunately, there are now strong indications that even X-ray free electron lasers will not be able to image individual molecules but will require nanocrystal samples. Here, we show that non-destructive structural biology of single particles has now become possible by means of low-energy electron holography. Individual tobacco mosaic virions deposited on ultraclean freestanding graphene are imaged at one nanometer resolution revealing structural details arising from the helical arrangement of the outer protein shell of the virus. Since low-energy electron holography is a lens-less technique and since electrons with a deBroglie wavelength of approximately 1 Angstrom do not impose radiation damage to biomolecules, the method has the potential for Angstrom resolution imaging of single biomolecules.Modern structural biology relies on Nuclear Magnetic Resonance (NMR), X-ray crystallography, and cryo-electron microscopy for gaining information on biomolecules at nanometer, sub-nanometer, or atomic resolution. All these methods, however, require averaging over a vast ensemble of entities, and hence knowledge on the conformational landscape of an individual particle is lost. Unfortunately, there are now strong indications that even X-ray free electron lasers will not be able to image individual molecules but will require nanocrystal samples. Here, we show that non-destructive structural biology of single particles has now become possible by means of low-energy electron holography. As an example, individual tobacco mosaic virions deposited on ultraclean freestanding graphene are imaged at 1 nm resolution revealing structural details arising from the helical arrangement of the outer protein shell of the virus. Since low-energy electron holography is a lens-less technique and since electrons with a deBrogl...

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.

Coherent low-energy electron diffraction on individual nanometer sized objects

Todays structural biology techniques require averaging over millions of molecules to obtain detailed structural information. Derivation of the molecular structure from a scattering experiment with just one single 3D-molecule imposes major challenges. Coherent and damage-free radiation is needed to ensure sufficient elastic scattering events before destroying the molecule and a means to solve the phase problem is wanted. We have devised such a scheme using coherent low-energy electrons shaped into a collimated beam by an electrostatic microlens. Initial experiments using a carbon nanotube sample demonstrate the feasibility of coherent low-energy electron diffraction on an individual nanometer-sized object.

Fabrication and characterization of low aberration micrometer-sized electron lenses

Intrinsic spherical aberrations of electron lenses have been the major resolution limiting factor in electron microscopes for several decades. While effective correctors have recently been implemented, an alternative to correct these aberrations is to circumvent them by scaling down lens dimensions by several orders of magnitude. We have fabricated electrostatic lenses exhibiting one micrometer diameter apertures and evaluated their beam forming properties against predictions from numerical ray tracing simulations. It turns out that it is routinely possible to shape a paraxial low-energy electron beam by such micron-sized lenses. Beam profiles have been measured both at a distant detector as well as in a plane close to the lens. It is shown that the lens can form a parallel beam extending no more than 800 nm from the optical axes at a distance of 200 microm beyond the lens exit. We believe that these findings constitute a prerequisite to derive novel tools for high resolution microscopy using low-energy electrons.

Mapping unoccupied electronic states of freestanding graphene by angle-resolved low-energy electron transmission

We report angle-resolved electron transmission measurements through freestanding graphene sheets in the energy range of 18 to 30 eV above the Fermi level. The measurements are carried out in a low-energy electron point source microscope, which allows simultaneously probing the transmission for a large angular range. The characteristics of low-energy electron transmission through graphene depend on its electronic structure above the vacuum level. The experimental technique described here allows mapping the unoccupied band structure of freestanding two-dimensional materials as a function of energy and probing angle, respectively in-plane momentum. Our experimental findings are consistent with theoretical predictions of a resonance in the band structure of graphene above the vacuum level [V. U. Nazarov, E. E. Krasovskii, and V. M. Silkin, Physical Review B 87, 041405 (2013)].