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. low-energy electron holography | single protein imaging | preparative mass spectrometry | microscopy | structural biology M ost of the currently available information on structures of macromolecules and proteins has been obtained from either X-ray crystallography experiments or cryo-electron microscopy investigations by means of averaging over many molecules assembled into a crystal or over a large ensemble selected from low signal-tonoise ratio electron micrographs, respectively (1). Despite the impressive coverage of the proteome by the available data, a strong desire for acquiring structural information from just one individual molecule is emerging. The biological relevance of a protein lies in its structural dynamics, which are accompanied by distinct conformations. For a protein to fulfill its vital functions in a living organism, it cannot exist in just one single and fixed structure, but needs to be able to assume different conformations to carry out specific functions. Conceptually, at least two different conformations, just like in a simple switch, are needed. In view of oxygen transport to cells for example, binding oxygen in one specific conformation and releasing it again in a different conformation are needed. To address the "physics of proteins" as described by Hans Frauenfelder in his pioneering review (2), one needs to realize that proteins are complex systems assuming different conformations and exhibiting a rich free-energy landscape. The associated structural details, however, remain undiscovered when averaging is involved. Moreover, a large subset of the entirety of proteins, in particular from the important category of membrane proteins, is extremely difficult, if not impossible, to obtain in a crystalline form. If just one individual protein or protein complex can be analyzed in sufficient detail, those objects will finally become accessible.For a meaningful contribution to structural biology, a tool for single-molecule imaging must allow for observing an individual protein long enough to acquire a sufficient amount of data to reveal its structure without altering it. The strong inelastic scattering cross-section of high-energy ...

The phase problem is inherent to crystallographic, astronomical and optical imaging where only the intensity of the scattered signal is detected and the phase information is lost and must somehow be recovered to reconstruct the object's structure. Modern imaging techniques at the molecular scale rely on utilizing novel coherent light sources like X-ray free electron lasers for the ultimate goal of visualizing such objects as individual biomolecules rather than crystals. Here, unlike in the case of crystals where structures can be solved by model building and phase refinement, the phase distribution of the wave scattered by an individual molecule must directly be recovered. There are two well-known solutions to the phase problem: holography and coherent diffraction imaging (CDI). Both techniques have their pros and cons. In holography, the reconstruction of the scattered complex-valued object wave is directly provided by a well-defined reference wave that must cover the entire detector area which often is an experimental challenge. CDI provides the highest possible, only wavelength limited, resolution, but the phase recovery is an iterative process which requires some pre-defined information about the object and whose outcome is not always uniquely-defined. Moreover, the diffraction patterns must be recorded under oversampling conditions, a pre-requisite to be able to solve the phase problem. Here, we report how holography and CDI can be merged into one superior technique: holographic coherent diffraction imaging (HCDI). An inline hologram can be recorded by employing a modified CDI experimental scheme. We demonstrate that the amplitude of the Fourier transform of an inline hologram is related to the complex-valued visibility, thus providing information on both, the amplitude and the phase of the scattered wave in the plane of the diffraction pattern. With the phase information available, the condition of oversampling the diffraction patterns can be relaxed, and the phase problem can be solved in a fast and unambiguous manner. We demonstrate the reconstruction of various diffraction patterns of objects recorded with visible light as well as with low-energy electrons. Although we have demonstrated our HCDI method using laser light and low-energy electrons, it can also be applied to any other coherent radiation such as X-rays or high-energy electrons. ABSTRACTThe phase problem is inherent to crystallographic, astronomical and optical imaging where only the intensity of the scattered signal is detected and the phase information is lost and must somehow be recovered to reconstruct the object's structure. Modern imaging techniques at the molecular scale rely on utilizing novel coherent light sources like X-ray free electron lasers for the ultimate goal of visualizing such objects as individual biomolecules rather than crystals. Here, unlike in the case of crystals where structures can be solved by model building and phase refinement, the phase distribution of the wave scattered by an individual molecule mus...

Articles you may be interested in N-doped graphene-supported Pt and Pt-Ru nanoparticles with high electrocatalytic activity for methanol oxidation J. Renewable Sustainable Energy 5, 021405 (2013) 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.

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.

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.

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.

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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. As an example, 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.