Imaging proteins at the single molecule level, by Jean-Nicolas Longchamp, Stephan Rauschenbach, Sabine Abb, Conrad Escher, Tatiana Latychevskaia, Klaus Kern, Hans-Werner Fink - PNAS - read more

PNAS, February 14, 2017, vol. 114 no. 7

All established methods for obtaining structural information about proteins require averaging over thousands or even millions of molecules, be it due to radiation damage or a too weak signal from just one individual protein.  Since proteins are flexible objects that can assume distinct conformations often associated with different functions, averaging over a large ensemble of proteins leaves these associated structural details undiscovered.
However, very recently it has become possible for the first time in the history of structural biology to image just one individual protein by low-energy electron holography.

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CHARGE TRANSPORT MEASUREMENTS ON GRAPHENE
CLEANED BY PALLADIUM-METAL CATALYSIS by Davide Pietro Rocco

This thesis investigates the electronic properties of graphene rendered ultraclean by a palladium-metal catalysis. Additionally Raman spectroscopy is carried out to further investigate the properties of the ultraclean graphene. The research objectives can be summarized in the following way:
• Implementing a 4-point set-up that makes use of a palladium-metal catalytic process to obtain clean graphene
• Observation of the catalytic annealing of the PMMA in the change of the sheet resistance of graphene
• Investigating the properties of the ultraclean graphene by Raman spectroscopy - read more

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Low-energy electron holographic imaging of individual tobacco mosaic virions by Jean-Nicolas Longchamp, Tatiana Latychevskaia, Conrad Escher and Hans-Werner Fink - read more

Appl. Phys. Lett. 107, 133101 (2015)

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.

In relation to the above article: Milestone single-biomolecule imaging technique may advance drug design by Zhengzheng Zhang, the American Institute of Physics, an AIP publication

Atomically resolved structural determination of graphene and its point defects by employing extrapolation assisted phase retrieval by Tatiana Latychevskaia and Hans-Werner Fink, Appl. Phys. Lett. 106, 021908 (2015) - read more

We demonstrate that the structure of a nanocrystal can unambiguously be recovered from its diffraction pattern alone by applying a direct phase retrieval procedure not relying on prior information of the object shape. Individual point defects in the atomic lattice are clearly apparent. We summarize the conditions for this phase retrieval method and show that the diffraction pattern can be extrapolated beyond the original record to even reveal formerly not visible Bragg peaks. Such extrapolated wave field pattern leads to enhanced spatial resolution in the reconstruction.

Coherent microscopy at resolution beyond diffraction limit using post-experimental data extrapolation, by Tatiana Latychevskaia and Hans-Werner Fink, Appl. Phys. Lett. 103, 204105 (2013) read more

Conventional microscopic records represent intensity distributions whereby local sample information is mapped onto local information at the detector. In coherent microscopy, the superposition principle of waves holds; field amplitudes are added, not intensities. This non-local representation is spread out in space and interference information combined with wave continuity allows extrapolation beyond the actual detected data. Established resolution criteria are thus circumvented and hidden object details can retrospectively be recovered from just a fraction of an interference pattern.

Graphene Unit Cell Imaging by Holographic Coherent Diffraction, by Jean-Nicolas Longchamp, Tatiana Latychevskaia, Conrad Escher, Hans-Werner Phys. Rev. Lett. 110, 255501 (2013) read more

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.

Low-energy electron transmission imaging of clusters on free-standing graphene by Jean-Nicolas Longchamp, Tatiana Latychevskaia, Conrad Escher, Hans-Werner Fink, read more

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 by J.-N. Longchamp, T. Latychevskaia, C. Escher, and H.-W. Fink, read more

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.

When holography meets coherent diffraction imaging. We discovered a mathematical relationship between a hologram and a diffraction pattern allowing us to merge the two prominent phase retrieval methods into one leading to fast and unambiguous object reconstructions while not requiring the oversampling condition anymore. read more

Coherent low-energy electron diffraction of individual nanometer sized objects. The first coherent diffraction pattern of an individual free-standing carbon nanotube using low-energy electrons has been obtained. read more

Fabrication and characterization of low aberration micrometre-sized electron lenses. We have developed methods for the routine fabrication of micron sized electron lenses and evaluated their performance in a dedicated UHV vacuum system. A detailed evaluation of the spherical aberrations shows that these lenses have a quality that can otherwise only be reached by elaborate magnetic objective lenses in modern electron microscopes.  read more

Pulsed electron holography has been invented to circumvent the disturbing effects of residual vibrations, drift and ac-magnetic fields limiting the coherence of the low energy electron wave. It has been possible to acquire a hologram of a single DNA within 10 micro-seconds. A set of several 100 such holograms of one and the same molecule is used to build up a record with a high signal to noise level. For this, the maximum of the cross correlation between subsequently acquired holograms is computed prior to shifting and superimposing the whole set of pulsed holograms.

Ref.: Master thesis of Matthias Germann

Quantitative Radiation Damage studies in Low Energy Electron Point Source Microscopy. While it seemed qualitatively apparent for quite some time that electron waves, associated with kinetic energies below 300eV, introduce little respectively non-detectable radiation damage on such fragile biological objects like an individual DNA molecule, a quantitative study of radiation damage has only recently been attempted.  

A set of holograms of a freshly prepared DNA sample has been acquired and the cross correlation between subsequent taken records been computed. Thereafter, the DNA molecule has been subject to continues radiation for one hour corresponding to a total effective dose of several orders of magnitude larger than what is considered tolerable in high energy TEM studies of biological samples.
Following this, another set of pulsed holograms has been taken. A cross correlation coefficient close to unity between the set of holograms taken at the beginning of the experiment and that following one hour of continues radiation, indicates that a free-standing DNA molecule has not been damaged in such a way that it causes structural changes on a scale associated with the present interference resolution of pulsed holography which is in the sub-nanometer regime.

It should be noted that similar experiments have been carried out at slightly higher energies, namely above 300 eV. Under these conditions, a single DNA decomposes within a few 10 seconds of observation time. Future experiments should reveal if there is a well defined threshold for damage or if there exist discrete windows between a few 10 and a few 100 eV.

Ref.: Master thesis of Matthias Germann Phys. Rev. Lett. 104, 095501 (2010)

Solution of the Twin Image Problem in Holography. The reconstruction of a hologram is the final step that leads to the three dimensional shape of the scattered object wave that eventually reveals the structural detail of the object. While the quality of the reconstructed image depends on the quality of detection of amplitude and phase of the object wave in the holographic record, one intrinsic problem remains. It is the so-called “twin image problem” recognized since the invention of holography by Dennis Gabor. It is a consequence of the foundation of coherent optics that the out of focus conjugated twin is entangled with the wanted image and obscures its recognition.

This long standing problem of coherent optics has recently been solved and provides object reconstructions no longer obscured by the twin image.

Ref.: Phys. Rev. Lett. 98, 233901 (2007)

A more popular written account of the above described work can be found here:

June 8, 2007: Molecular holograms are coming into focus: New Scientist comments on the twin image removal.

June 19, 2007: Getting rid of the twin image that plagues holography: PhysOrg comments on the twin image removal.

Energetics of a single DNA Molecule: Exploring the dynamics of individual DNA molecules has been subject of various theoretical studies and models over many decades, but only during the last 10 years the ability to actually carry out experiments on individual molecules has become routine. However, direct insight into the energetics on a single molecule level has not been obtained so far. We have carried out  temperature dependent experiments with individual lambda-DNA molecules and provide the first quantitative information about the internal energetics of a single molecule in solution. From the mean square displacement of the center of mass of a DNA random coil we derive the diffusion coefficient from Einstein equation and from its temperature dependence the activation energy for diffusion to be roughly 1/6 eV. Following these control experiment we determine the activation energy associated with the transition from the straightened to the lowest free energy random coil configuration. The statistics of a total of 600 measurements on individual lambda-DNA molecules at 12 different temperatures reveals an activation free energy for the transition between these two prominent configurations of 1/4 eV. This indicates that the DNA random coil equilibrium configuration is not entirely given by entropy in contrast to current models.