Low-energy Electron Holography Research Articles

Coherent imaging with low-energy electrons, quantitative analysis

Low-energy electrons (20–300eV) hold the promise for low-dose, non-destructive, high-resolution imaging, but at the price of challenging data analysis. This study provides theoretical considerations and models for the quantitative analysis of experimental data observed in low-energy electron transmission microscopy and in-line holography. The scattering of low-energy electrons and the imaging parameters, such as the inelastic mean free path, point spread function, depth of focus, and resolution, are quantitatively described. It is shown that unlike high-energy electrons (20–300 keV), low-energy electrons (20–300eV) introduce a large phase shift into the probing electron waves. Using the projected potentials formalism, the maximal phase shift acquired by a 120eV electron wave scattered by a carbon atom is theoretically estimated to be 5.03 radian and experimentally measured to be between 3 and 7.5 radian. The point spread function evaluated for low-energy electrons shows that they diffract much stronger than high-energy electrons, and that only very thin objects of up to 3Å in thickness can be imaged in focus. Thus, when imaging an object of finite thickness, such as a macromolecule, the obtained image will always be blurred due to the out-of-focus signal. This can provide an explanation for a long-standing problem of limited resolution in low-energy electron holography of macromolecules. As for imaging of a macromolecule’s structure, it is shown that the amplitude of the wavefront reconstructed from the sample’s hologram provides the best match to the projected potential distribution of the macromolecule. To evaluate the absorption properties, the inelastic mean free path (IMFP) is considered. The IMFP values calculated from theoretical models agree with the measured values. The IMFP of about 5Å was measured by transmission through graphene of 50–200eV electrons. This result implies that the internal structure of only very thin samples can be imaged in transmission mode. A simple method to quantitatively evaluate the absorption of a specimen from its in-line hologram without the need to reconstruct the hologram is presented.

Imaging conformations of holo- and apo-transferrin on the single-molecule level by low-energy electron holography

Conformational changes play a key role in the biological function of many proteins, thereby sustaining a multitude of processes essential to life. Thus, the imaging of the conformational space of proteins exhibiting such conformational changes is of great interest. Low-energy electron holography (LEEH) in combination with native electrospray ion beam deposition (ES-IBD) has recently been demonstrated to be capable of exploring the conformational space of conformationally highly variable proteins on the single-molecule level. While the previously studied conformations were induced by changes in environment, it is of relevance to assess the performance of this imaging method when applied to protein conformations inherently tied to a function-related conformational change. We show that LEEH imaging can distinguish different conformations of transferrin, the major iron transport protein in many organisms, by resolving a nanometer-scale cleft in the structure of the iron-free molecule (apo-transferrin) resulting from the conformational change associated with the iron binding/release process. This, along with a statistical analysis of the data, which evidences a degree of flexibility of the molecules, indicates that LEEH is a viable technique for imaging function-related conformational changes in individual proteins.

Electrospray ion beam deposition plus low-energy electron holography as a tool for imaging individual biomolecules.

Inline low-energy electron holography (LEEH) in conjunction with sample preparation by electrospray ion beam deposition (ES-IBD) has recently emerged as a promising method for the sub-nanometre-scale single-molecule imaging of biomolecules. The single-molecule nature of the LEEH measurement allows for the mapping of the molecules' conformational space and thus for the imaging of structurally variable biomolecules, thereby providing valuable complementary information to well-established biomolecular structure determination methods. Here, after briefly tracing the development of inline LEEH in bioimaging, we present the state-of-the-art of native ES-IBD + LEEH as a method of single-protein imaging, discuss its applications, specifically regarding the imaging of structurally flexible protein systems and the amplitude and phase information encoded in a low-energy electron hologram, and provide an outlook regarding the considerable possibilities for the future advancement of the approach.

Phase Reconstruction of Low-Energy Electron Holograms of Individual Proteins.

Low-energy electron holography (LEEH) is one of the few techniques capable of imaging large and complex three-dimensional molecules, such as proteins, on the single-molecule level at subnanometer resolution. During the imaging process, the structural information about the object is recorded both in the amplitude and in the phase of the hologram. In low-energy electron holography imaging of proteins, the object's amplitude distribution, which directly reveals molecular size and shape on the single-molecule level, can be retrieved via a one-step reconstruction process. However, such a one-step reconstruction routine cannot directly recover the phase information encoded in the hologram. In order to extract the full information about the imaged molecules, we thus implemented an iterative phase retrieval algorithm and applied it to experimentally acquired low-energy electron holograms, reconstructing the phase shift induced by the protein along with the amplitude data. We show that phase imaging can map the projected atomic density of the molecule given by the number of atoms in the electron path. This directly implies a correlation between reconstructed phase shift and projected mean inner potential of the molecule, and thus a sensitivity to local changes in potential, an interpretation that is further substantiated by the strong phase signatures induced by localized charges.

Low-energy electron holography imaging of conformational variability of single-antibody molecules from electrospray ion beam deposition

Imaging of proteins at the single-molecule level can reveal conformational variability, which is essential for the understanding of biomolecules. To this end, a biologically relevant state of the sample must be retained during both sample preparation and imaging. Native electrospray ionization (ESI) can transfer even the largest protein complexes into the gas phase while preserving their stoichiometry and overall shape. High-resolution imaging of protein structures following native ESI is thus of fundamental interest for establishing the relation between gas phase and solution structure. Taking advantage of low-energy electron holography's (LEEH) unique capability of imaging individual proteins with subnanometer resolution, we investigate the conformational flexibility of Herceptin, a monoclonal IgG antibody, deposited by native electrospray mass-selected ion beam deposition (ES-IBD) on graphene. Images reconstructed from holograms reveal a large variety of conformers. Some of these conformations can be mapped to the crystallographic structure of IgG, while others suggest that a compact, gas-phase-related conformation, adopted by the molecules during ES-IBD, is retained. We can steer the ratio of those two types of conformations by changing the landing energy of the protein on the single-layer graphene surface. Overall, we show that LEEH can elucidate the conformational heterogeneity of inherently flexible proteins, exemplified here by IgG antibodies, and thereby distinguish gas-phase collapse from rearrangement on surfaces.

Wave-Mechanical Electron-Optical Modeling of Field-Emission Electron Sources

Electron source coherence has a very important influence on the imaging capabilities of modern electron microscopes. However, conventional electron source models that are based on geometrical electron optics implicitly assume that the emission from the source surface is fully incoherent, which can complicate the treatment of highly coherent field-emission sources. In an attempt to treat the wave-optical properties of electron sources, models inspired by light optics treatments of (partially) coherent sources, which assume a planar source and free wave propagation, have been developed. In this case the underlying assumptions are problematic, because the source surface of a field emitter can have a radius of curvature on the nanometer scale, and the emitted electrons are accelerated by a strong, inhomogeneous electrostatic field following emission. We introduce a model based on wave-mechanical electron optics that draws on a quantum mechanical description of electron emission and propagation to obtain a physically consistent treatment of the wave-mechanical properties of electron sources. We apply the model to investigate spatial resolution limits in low-energy electron holography and microscopy, where it is shown that aberrations and coherence properties of the electron source are crucial and interrelated. The wave-mechanical electron-optical model can, furthermore, be readily generalized to assess and improve electron source performance in other scenarios and techniques where spatial and temporal coherence, and electron-optical aberrations, are relevant.

Low-energy in-line electron holographic imaging of vitreous ice-embedded small biomolecules using a modified scanning electron microscope

Cryo-electron microscopy (cryo-EM) has become the method of choice in the field of structural biology, owing to its unique ability to deduce structures of vitreous ice-embedded, hydrated biomolecules over a wide range of structural resolutions. As cryo-transmission electron microscopes (cryo-TEM) become increasingly specialised for high, near-atomic resolution studies, operational complexity and associated costs serve as significant barriers to widespread usability and adoptability. To facilitate the expansion and accessibility of the cryo-EM method, an efficient, user-friendly means of imaging vitreous ice-embedded biomolecules has been called for. In this study, we present a solution to this issue by integrating cryo-EM capabilities into a commercial scanning electron microscope (SEM). Utilising the principle of low-energy in-line electron holography, our newly developed hybrid microscope permits low-to-moderate resolution imaging of vitreous ice-embedded biomolecules without the need for any form of sample staining or chemical fixation. Operating at 20 kV, the microscope takes advantage of the ease-of-use of SEM-based imaging and phase contrast imaging of low-energy electron holography. This study represents the first reported successful application of low-energy in-line electron holographic imaging to vitreous ice-embedded small biomolecules, the effectiveness of which is demonstrated here with three morphologically distinct specimens.

Development of a SEM-based low-energy in-line electron holography microscope for individual particle imaging

A new SEM-based in-line electron holography microscope has been under development. The microscope utilizes conventional SEM and BF-STEM functionality to allow for rapid searching of the specimen of interest, seamless interchange between SEM, BF-STEM and holographic imaging modes, and makes use of coherent low-energy in-line electron holography to obtain low-dose, high-contrast images of light element materials. We report here an overview of the instrumentation and first experimental results on gold nano-particles and carbon nano-fibers for system performance tests. Reconstructed images obtained from the holographic imaging mode of the new microscope show substantial image contrast and resolution compared to those acquired by SEM and BF-STEM modes, demonstrating the feasibility of high-contrast imaging via low-energy in-line electron holography. The prospect of utilizing the new microscope to image purified biological specimens at the individual particle level is discussed and electron optical issues and challenges to further improve resolution and contrast are considered.

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.

Low-energy electron holographic imaging of individual tobacco mosaic virions

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 deBroglie wavelength of approximately 1 Å do not impose radiation damage to biomolecules, the method has the potential for Angstrom resolution imaging of single biomolecules.

Structural Biology at The Single Particle Level: Imaging Tobacco Mosaic Virus by Low-Energy Electron Holography

Structural Biology at The Single Particle Level: Imaging Tobacco Mosaic Virus by Low-Energy Electron Holography

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.

Pulsed electron holography

A technique of pulsed low-energy electron holography is introduced, which allows for recording highly resolved holograms within reduced exposure times. Therefore, stacks of holograms are accumulated in a pulsed mode with individual acquisition times as short as 50 μs. Subsequently, these holograms are aligned and finally superimposed. The resulting holographic record reveals previously latent high-order interference fringes and thereby pushing interference resolution into the sub-nanometer regime. In view of the non-damaging character of low-energy electrons, the method is of particular interest for structural analysis of fragile biomolecules.

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.

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 object's shapes and distribution. Furthermore, a Moiré effect has been observed with free-standing graphene multi-layers.

Non-destructive imaging of an individual protein

The mode of action of proteins is to a large extent given by their ability to adopt different conformations. This is why imaging single biomolecules at atomic resolution is one of the ultimate goals of biophysics and structural biology. The existing protein database has emerged from X-ray crystallography, NMR or cryo-TEM investigations. However, these tools all require averaging over a large number of proteins and thus over different conformations. This of course results in the loss of structural information. Likewise it has been shown that even the emergent X-FEL technique will not get away without averaging over a large quantity of molecules. Here we report the first recordings of a protein at sub-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 ferritin exhibits an iron core, the holographic reconstructions could also be cross-validated against TEM images of the very same molecule by imaging the iron cluster inside the molecule while the protein shell is decomposed.

Low Energy Electron Holography of Charged Tip

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

Conducting, semiconducting and insulating objects observed by low-energy electron holography

To demonstrate the potential of low energy in line projection holography, we study the reconstruction of the experimental holograms of three electrically different objects: a conducting, a semiconducting and an insulating object. The reconstructions of these holograms provide meaningful results for a large range of magnification of the object. The comparison between the reconstructed images and the scanning electron microscopy (SEM) micrographs of the same objects shows that the shapes and the dimensions of the reconstructed objects are identical to those obtained by conventional SEM. So, the simple assumptions needed to the reconstruction are justified. The reconstructions show a 2 nm resolution and appear superior than the best obtained SEM micrographs, when available. We also show some limitations of the reconstruction process. We point out that both numerical artifacts and experimental conditions are responsible for these limitations.