Scanning Probe Microscopy Characterization of Biomolecules enabled by Mass-Selective, Soft-landing Electrospray Ion Beam Deposition.

Scanning probe microscopy (SPM), in particular at low temperature (LT) under ultra-high vacuum (UHV) conditions, offers the possibility of real-space imaging with resolution reaching the atomic level. However, its potential for the analysis of complex biological molecules has been hampered by requirements imposed by sample preparation. Transferring molecules onto surfaces in UHV is typically accomplished by thermal sublimation in vacuum. This approach however is limited by the thermal stability of the molecules, i.e. not possible for biological molecules with low vapour pressure. Bypassing this limitation, electrospray ionisation offers an alternative method to transfer molecules from solution to the gas-phase as intact molecular ions. In soft-landing electrospray ion beam deposition (ESIBD), these molecular ions are subsequently mass-selected and gently landed on surfaces which permits large and thermally fragile molecules to be analyzed by LT-UHV SPM. In this concept, we discuss how ESIBD+SPM prepares samples of complex biological molecules at a surface, offering controls of the molecular structural integrity, three-dimensional shape, and purity. These achievements unlock the analytical potential of SPM which is showcased by imaging proteins, peptides, DNA, glycans, and conjugates of these molecules, revealing details of their connectivity, conformation, and interaction that could not be accessed by any other technique.

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.

Identifying the origin of local flexibility in a carbohydrate polymer.

Correlating the structures and properties of a polymer to its monomer sequence is key to understanding how its higher hierarchy structures are formed and how its macroscopic material properties emerge. Carbohydrate polymers, such as cellulose and chitin, are the most abundant materials found in nature whose structures and properties have been characterized only at the submicrometer level. Here, by imaging single-cellulose chains at the nanoscale, we determine the structure and local flexibility of cellulose as a function of its sequence (primary structure) and conformation (secondary structure). Changing the primary structure by chemical substitutions and geometrical variations in the secondary structure allow the chain flexibility to be engineered at the single-linkage level. Tuning local flexibility opens opportunities for the bottom-up design of carbohydrate materials.

Visualizing Chiral Interactions in Carbohydrates Adsorbed on Au(111) by High-Resolution STM Imaging.

Carbohydrates are the most abundant organic material on Earth and the structural "material of choice" in many living systems. Nevertheless, design and engineering of synthetic carbohydrate materials presently lag behind that for protein and nucleic acids. Bottom-up engineering of carbohydrate materials demands an atomic-level understanding of their molecular structures and interactions in condensed phases. Here, high-resolution scanning tunneling microscopy (STM) is used to visualize at submolecular resolution the three-dimensional structure of cellulose oligomers assembled on Au(1111) and the interactions that drive their assembly. The STM imaging, supported by ab initio calculations, reveals the orientation of all glycosidic bonds and pyranose rings in the oligomers, as well as details of intermolecular interactions between the oligomers. By comparing the assembly of D- and L-oligomers, these interactions are shown to be enantioselective, capable of driving spontaneous enantioseparation of cellulose chains from its unnatural enantiomer and promoting the formation of engineered carbohydrate assemblies in the condensed phases.

Cryo-EM samples of gas-phase purified protein assemblies using native electrospray ion-beam deposition.

An increasing number of studies on biomolecular function indirectly combine mass spectrometry (MS) with imaging techniques such as cryo electron microscopy (cryo-EM). This approach allows information on the homogeneity, stoichiometry, shape, and interactions of native protein complexes to be obtained, complementary to high-resolution protein structures. We have recently demonstrated TEM sample preparation via native electrospray ion-beam deposition (ES-IBD) as a direct link between native MS and cryo-EM. This workflow forms a potential new route to the reliable preparation of homogeneous cryo-EM samples and a better understanding of the relation between native solution-phase and native-like gas-phase structures. However, many aspects of the workflow need to be understood and optimized to obtain performance comparable to that of state-of-the-art cryo-EM. Here, we expand on the previous discussion of key factors by probing the effects of substrate type and deposition energy. We present and discuss micrographs from native ES-IBD samples with amorphous carbon, graphene, and graphene oxide, as well as landing energies in the range between 2 and 150 eV per charge.

Catalyzing Bond-Dissociation in Graphene via Alkali-Iodide Molecules.

Atomic design of a 2D-material such as graphene can be substantially influenced by etching, deliberately induced in a transmission electron microscope. It is achieved primarily by overcoming the threshold energy for defect formation by controlling the kinetic energy and current density of the fast electrons. Recent studies have demonstrated that the presence of certain species of atoms can catalyze atomic bond dissociation processes under the electron beam by reducing their threshold energy. Most of the reported catalytic atom species are single atoms, which have strong interaction with single-layer graphene (SLG). Yet, no such behavior has been reported for molecular species. This work shows by experimentally comparing the interaction of alkali and halide species separately and conjointly with SLG, that in the presence of electron irradiation, etching of SLG is drastically enhanced by the simultaneous presence of alkali and iodine atoms. Density functional theory and first principles molecular dynamics calculations reveal that due to charge-transfer phenomena the CC bonds weaken close to the alkali-iodide species, which increases the carbon displacement cross-section. This study ascribes pronounced etching activity observed in SLG to the catalytic behavior of the alkali-iodide species in the presence of electron irradiation.

Fast Molecular Compression by a Hyperthermal Collision Gives Bond-Selective Mechanochemistry.

Using electrospray ion beam deposition, we collide the complex molecule Reichardt's dye (C_{41}H_{30}NO^{+}) at low, hyperthermal translational energy (2-50 eV) with a Cu(100) surface and image the outcome at single-molecule level by scanning tunneling microscopy. We observe bond-selective reaction induced by the translational kinetic energy. The collision impulse compresses the molecule and bends specific bonds, prompting them to react selectively. This dynamics drives the system to seek thermally inaccessible reactive pathways, since the compression timescale (subpicosecond) is much shorter than the thermalization timescale (nanosecond), thereby yielding reaction products that are unobtainable thermally.

Exploring the Molecular Conformation Space by Soft Molecule-Surface Collision.

Biomolecules function by adopting multiple conformations. Such dynamics are governed by the conformation landscape whose study requires characterization of the ground and excited conformation states. Here, the conformational landscape of a molecule is sampled by exciting an initial gas-phase molecular conformer into diverse conformation states, using soft molecule-surface collision (0.5-5.0 eV). The resulting ground and excited molecular conformations, adsorbed on the surface, are imaged at the single-molecule level. This technique permits the exploration of oligosaccharide conformations, until now, limited by the high flexibility of oligosaccharides and ensemble-averaged analytical methods. As a model for cellulose, cellohexaose chains are observed in two conformational extremes, the typical "extended" chain and the atypical "coiled" chain-the latter identified as the gas-phase conformer preserved on the surface. Observing conformations between these two extremes reveals the physical properties of cellohexaose, behaving as a rigid ribbon that becomes flexible when twisted. The conformation space of any molecule that can be electrosprayed can now be explored.

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.

Carbohydrate Self-Assembly at Surfaces: STM Imaging of Sucrose Conformation and Ordering on Cu(100).

Saccharides are ubiquitous biomolecules, but little is known about their interaction with, and assembly at, surfaces. By combining preparative mass spectrometry with scanning tunneling microscopy, we have been able to address the conformation and self-assembly of the disaccharide sucrose on a Cu(100) surface with subunit-level imaging. By employing a multistage modeling approach in combination with the experimental data, we can rationalize the conformation on the surface as well as the interactions between the sucrose molecules, thereby yielding models of the observed self-assembled patterns on the surface.

Chemical Analysis of Complex Surface-Adsorbed Molecules and Their Reactions by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry.

Desorption/ionization induced by neutral SO2 clusters (DINeC) is used for mass spectrometry (MS) of surface-adsorbed molecules. The method is shown to be a surface-sensitive analysis tool capable of detecting molecular adsorbates in a wide range of molecular weights as well as their reactions on surfaces, which are otherwise difficult to access. Two different surface/adsorbate systems prepared by means of electrospray ion beam deposition (ES-IBD) were investigated: For the peptide angiotensin II on gold, intact molecules were desorbed from the surface when deposited by soft landing ES-IBD. By comparison to the well-controlled amount of substance deposited by ES-IBD, the sensitivity of DINeC-MS was shown to be on the order of 0.1% of a monolayer coverage, corresponding to femtomoles of analyte. Depending on deposition and sample conditions, the original state of charge of the molecules could be retrieved. Reaction of the adsorbed molecules both with surface atoms as well as with coadsorbed D2O was monitored. Rhodamine 6G was also desorbed as an intact molecule when deposited with kinetic energies below 50 eV. For higher deposition energy, fragmentation of the dye molecules was observed by means of DINeC-MS.

The classical and quantum dynamics of molecular spins on graphene.

Controlling the dynamics of spins on surfaces is pivotal to the design of spintronic and quantum computing devices. Proposed schemes involve the interaction of spins with graphene to enable surface-state spintronics and electrical spin manipulation. However, the influence of the graphene environment on the spin systems has yet to be unravelled. Here we explore the spin-graphene interaction by studying the classical and quantum dynamics of molecular magnets on graphene. Whereas the static spin response remains unaltered, the quantum spin dynamics and associated selection rules are profoundly modulated. The couplings to graphene phonons, to other spins, and to Dirac fermions are quantified using a newly developed model. Coupling to Dirac electrons introduces a dominant quantum relaxation channel that, by driving the spins over Villain's threshold, gives rise to fully coherent, resonant spin tunnelling. Our findings provide fundamental insight into the interaction between spins and graphene, establishing the basis for electrical spin manipulation in graphene nanodevices.

Active conformation control of unfolded proteins by hyperthermal collision with a metal surface.

The physical and chemical properties of macromolecules like proteins are strongly dependent on their conformation. The degrees of freedom of their chemical bonds generate a huge conformational space, of which, however, only a small fraction is accessible in thermal equilibrium. Here we show that soft-landing electrospray ion beam deposition (ES-IBD) of unfolded proteins allows to control their conformation. The dynamics and result of the deposition process can be actively steered by selecting the molecular ion beam's charge state or tuning the incident energy. Using these parameters, protein conformations ranging from fully extended to completely compact can be prepared selectively on a surface, as evidenced on the subnanometer/amino acid resolution level by scanning tunneling microscopy (STM). Supported by molecular dynamics (MD) simulations, our results demonstrate that the final conformation on the surface is reached through a mechanical deformation during the hyperthermal ion surface collision. Our experimental results independently confirm the findings of ion mobility spectrometry (IMS) studies of protein gas phase conformations. Moreover, we establish a new route for the processing of macromolecular materials, with the potential to reach conformations that would be inaccessible otherwise.

Atomic-scale observation of multiconformational binding and energy level alignment of ruthenium-based photosensitizers on TiO2 anatase.

Dye-sensitized solar cells constitute a promising approach to sustainable and low-cost solar energy conversion. Their overall efficiency crucially depends on the effective coupling of the photosensitizers to the photoelectrode and the details of the dye's energy levels at the interface. Despite great efforts, the specific binding of prototypical ruthenium-based dyes to TiO2, their potential supramolecular interaction, and the interrelation between adsorption geometry and electron injection efficiency lack experimental evidence. Here we demonstrate multiconformational adsorption and energy level alignment of single N3 dyes on TiO2 anatase (101) revealed by scanning tunnelling microscopy and spectroscopy. The distinctly bound molecules show significant variations of their excited state levels associated with different driving forces for photoelectron injection. These findings emphasize the critical role of the interfacial coupling and suggest that further designs of dye-sensitized solar cells should target a higher selectivity in the dye-substrate binding conformations in order to ensure efficient electron injection from all photosensitizers.

Chemical modification of graphene via hyperthermal molecular reaction.

Chemical functionalization of graphene is achieved by hyperthermal reaction with azopyridine molecular ions. The one-step, room temperature process takes place in high vacuum (10(-7) mbar) using an electrospray ion beam deposition (ES-IBD) setup. For ion surface collisions exceeding a threshold kinetic energy of 165 eV, molecular cation beams of 4,4'-azobis(pyridine) covalently attach to chemical vapor deposited (CVD) graphene. A covalent functionalization degree of 3% of the carbon atoms of graphene is reached after 3-5 h of ion exposure of 2 × 10(14) azopyridinium/cm(2) of which 50% bind covalently. This facile approach for the controlled modification of graphene extends the scope of candidate species that would not otherwise react via existing conventional methods.

Cryo-EM of soft-landed ß-galactosidase: Gas-phase and native structures are remarkably similar.

Native mass spectrometry (MS) has become widely accepted in structural biology, providing information on stoichiometry, interactions, homogeneity, and shape of protein complexes. Yet, the fundamental assumption that proteins inside the mass spectrometer retain a structure faithful to native proteins in solution remains a matter of intense debate. Here, we reveal the gas-phase structure of ß-galactosidase using single-particle cryo-electron microscopy (cryo-EM) down to 2.6-Å resolution, enabled by soft landing of mass-selected protein complexes onto cold transmission electron microscopy (TEM) grids followed by in situ ice coating. We find that large parts of the secondary and tertiary structure are retained from the solution. Dehydration-driven subunit reorientation leads to consistent compaction in the gas phase. By providing a direct link between high-resolution imaging and the capability to handle and select protein complexes that behave problematically in conventional sample preparation, the approach has the potential to expand the scope of both native mass spectrometry and cryo-EM.

A close look at proteins: submolecular resolution of two- and three-dimensionally folded cytochrome c at surfaces.

Imaging of individual protein molecules at the single amino acid level has so far not been possible due to the incompatibility of proteins with the vacuum environment necessary for high-resolution scanning probe microscopy. Here we demonstrate electrospray ion beam deposition of selectively folded and unfolded cytochrome c protein ions on atomically defined solid surfaces in ultrahigh vacuum (10(-10) mbar) and achieve unprecedented resolution with scanning tunneling microscopy. On the surface folded proteins are found to retain their three-dimensional structure. Unfolded proteins are observed as extended polymer strands displaying submolecular features with resolution at the amino acid level. On weakly interacting surfaces, unfolded proteins refold into flat, irregular patches composed of individual molecules. This suggests the possibility of two-dimensionally confined folding of peptides of an appropriate sequence into regular two-dimensional structures as a new approach toward functional molecular surface coatings.

The quantum magnetism of individual manganese-12-acetate molecular magnets anchored at surfaces.

The high intrinsic spin and long spin relaxation time of manganese-12-acetate (Mn(12)) makes it an archetypical single molecular magnet. While these characteristics have been measured on bulk samples, questions remain whether the magnetic properties replicate themselves in surface supported isolated molecules, a prerequisite for any application. Here we demonstrate that electrospray ion beam deposition facilitates grafting of intact Mn(12) molecules on metal as well as ultrathin insulating surfaces enabling submolecular resolution imaging by scanning tunneling microscopy. Using scanning tunneling spectroscopy we detect spin excitations from the magnetic ground state of the molecule at an ultrathin boron nitride decoupling layer. Our results are supported by density functional theory based calculations and establish that individual Mn(12) molecules retain their intrinsic spin on a well chosen solid support.

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.