Structured Water Molecules on Membrane Proteins Resolved by Atomic Force Microscopy.

Water structuring on the outer surface of protein molecules called the hydration shell is essential as well as the internal water structures for higher-order structuring of protein molecules and their biological activities in vivo. We now show the molecular-scale hydration structure measurements of native purple membrane patches composed of proton pump proteins by a noninvasive three-dimensional force mapping technique based on frequency modulation atomic force microscopy. We successfully resolved the ordered water molecules localized near the proton uptake channels on the cytoplasmic side of the individual bacteriorhodopsin proteins in the purple membrane. We demonstrate that the three-dimensional force mapping can be widely applicable for molecular-scale investigations of the solid-liquid interfaces of various soft nanomaterials.

Molecular-scale quantitative charge density measurement of biological molecule by frequency modulation atomic force microscopy in aqueous solutions.

Surface charge distributions on biological molecules in aqueous solutions are essential for the interactions between biomolecules, such as DNA condensation, antibody-antigen interactions, and enzyme reactions. There has been a significant demand for a molecular-scale charge density measurement technique for better understanding such interactions. In this paper, we present the local electric double layer (EDL) force measurements on DNA molecules in aqueous solutions using frequency modulation atomic force microscopy (FM-AFM) with a three-dimensional force mapping technique. The EDL forces measured in a 100 mM KCl solution well agreed with the theoretical EDL forces calculated using reasonable parameters, suggesting that FM-AFM can be used for molecular-scale quantitative charge density measurements on biological molecules especially in a highly concentrated electrolyte.

Atom-resolved analysis of an ionic KBr(001) crystal surface covered with a thin water layer by frequency modulation atomic force microscopy.

An ionic KBr(001) crystal surface covered with a thin water layer was observed with a frequency modulation atomic force microscope (FM-AFM) with atomic resolution. By immersing only the tip apex of the AFM cantilever in the thin water layer, the Q-factor of the cantilever in probing the solid-liquid interface can be maintained as high as that of FM-AFM operation in air, leading to improvement of the minimum detection of a differential force determined by the noise. Two types of images with atom-resolved contrast were observed, possibly owing to the different types of ions (K(+) or Br(-)) adsorbed on the tip apex that incorporated into the hydration layers on the tip and on the sample surface. The force-distance characteristics at the solid-water interface were analyzed by taking spatial variation maps of the resonant frequency shift of the AFM cantilever with the high Q-factor. The oscillatory frequency shift-distance curves exhibited atomic site dependence. The roles of hydration and the ions on the tip and on the sample surface in the measurements were discussed.

Molecular-scale investigations of structures and surface charge distribution of surfactant aggregates by three-dimensional force mapping.

Surface charges on nanoscale structures in liquids, such as biomolecules and nano-micelles, play an essentially important role in their structural stability as well as their chemical activities. These structures interact with each other through electric double layers (EDLs) formed by the counter ions in electrolyte solution. Although static-mode atomic force microscopy (AFM) including colloidal-probe AFM is a powerful technique for surface charge density measurements and EDL analysis on a submicron scale in liquids, precise surface charge density analysis with single-nanometer resolution has not been made because of its limitation of the resolution and the detection sensitivity. Here we demonstrate molecular-scale surface charge measurements of self-assembled micellar structures, molecular hemicylinders of sodium dodecyl sulfate (SDS), by three-dimensional (3D) force mapping based on frequency modulation AFM. The SDS hemicylindrical structures with a diameter of 4.8 nm on a graphite surface were clearly imaged. We have succeeded in visualizing 3D EDL forces on the SDS hemicylinder surfaces and obtaining the molecular-scale charge density for the first time. The results showed that the surface charge on the trench regions between the hemicylinders was much smaller than that on the hemicylinder tops. The method can be applied to a wide variety of local charge distribution studies, such as spatial charge variation on a single protein molecule.

Development of multi-environment dual-probe atomic force microscopy system using optical beam deflection sensors with vertically incident laser beams.

We developed a dual-probe atomic force microscopy (DP-AFM) system with two cantilever probes that can be operated in various environments such as in air, vacuum, and liquid. The system employs the optical beam deflection method for measuring the deflection of each cantilever mounted on a probe scanner. The cantilever probes mounted on the probe scanners are attached to inertia sliders, which allow independent control of the probe positions. We constructed three types of probe scanners (tube, shear-piezo, and tripod types) and characterized their performance. We demonstrated AFM imaging in ambient air, vacuum, and ultrapure water, and also performed electrical measurement and pick-up manipulation of a Au nanorod using the DP-AFM system.

Visualization of hydration layers on muscovite mica in aqueous solution by frequency-modulation atomic force microscopy.

A three-dimensional interaction force mapping experiment was carried out on a muscovite mica surface in an aqueous solution using a high-resolution and low-thermal drift frequency-modulation atomic force microscope. By collecting oscillatory frequency shift versus distance curves at the mica∕solution interface, complicated hydration structures on the mica surface were visualized. Reconstructed two-dimensional frequency shift maps showed dot-like or honeycomb-like patterns at different tip-sample distances with a separation of 0.2 nm with each other, which agree well to the water molecule density maps predicted by a statistical-mechanical theory. Moreover, site-specific force versus distance curves showed a good agreement with theoretically calculated site-specific force curves by a molecular dynamics simulation. It is found that the first and second hydration layers give honeycomb-like and dot-like patterns in the two-dimensional frequency shift images, respectively, corresponding to the lateral distribution function in each layer.

Beyond the helix pitch: direct visualization of native DNA in aqueous solution.

The DNA double helix was first elucidated by J.D. Watson and F.H.C. Crick over a half century ago. However, no one could actually "see" the well-known structure ever. Among all real-space observation methods, only atomic force microscopy (AFM) enables us to visualize the biologically active structure of natural DNA in water. However, conventional AFM measurements often caused the structural deformation of DNA because of the strong interaction forces acting on DNA. Moreover, large contact area between the AFM probe and DNA hindered us from imaging sub-molecular-scale features smaller than helical periodicity of DNA. Here, we show the direct observation of native plasmid DNA in water using an ultra-low-noise AFM with the highly sensitive force detection method (frequency modulation AFM: FM-AFM). Our micrographs of DNA vividly exhibited not only overall structure of the B-form double helix in water but also local structures which deviate from the crystallographic structures of DNA without any damage. Moreover, the interaction force area in the FM-AFM was small enough to clearly discern individual functional groups within DNA. The technique was also applied to explore the synthesized DNA nanostructures toward the current nanobiotechnology. This work will be essential for considering the structure-function relationship of biomolecular systems in vivo and for in situ analysis of DNA-based nanodevices.

A procedure to determine the optimum imaging parameters for atomic/molecular resolution frequency modulation atomic force microscopy.

We propose a general procedure to determine the optimum imaging parameters (spring constant and oscillation amplitude) to obtain the optimum resolution in frequency modulation atomic force microscopy. We calculated the effective signal-to-noise ratio for various spring constants and oscillation amplitudes, based on the measurement of frequency shift and energy dissipation versus tip-sample distance curves, to find the optimum. We applied this procedure for imaging a lead phthalocyanine (PbPc) thin film on a MoS(2)(0001) substrate, and found that the optimum parameters were about 5 N/m and 20 nm, respectively. An improved signal-to-noise ratio was attained in a preliminary experiment using parameters which were close to the calculated optimum.

Visualizing water molecule distribution by atomic force microscopy.

Hydration structures at biomolecular surfaces are essential for understanding the mechanisms of the various biofunctions and stability of biomolecules. Here, we demonstrate the measurement of local hydration structures using an atomic force microscopy system equipped with a low-noise deflection sensor. We applied this method to the analysis of the muscovite mica/water interface and succeeded in visualizing a hydration structure that is site-specific on a crystal. Furthermore, at the biomolecule/buffer solution interface, we found surface hydration layers that are more packed than those at the muscovite mica/water interface.

True atomic-resolution imaging of (1014) calcite in aqueous solution by frequency modulation atomic force microscopy.

Calcite (CaCO3) is one of the most abundant minerals on earth and plays an important role in a wide range of different fields including, for example, biomineralization and environmental geochemistry. Consequently, surface processes and reactions such as dissolution and growth as well as (macro)molecule adsorption are of greatest interest for both applied as well as fundamental research. An in-depth understanding of these processes requires knowledge about the detailed surface structure in its natural state which is quite often a liquid environment. We have studied the most stable cleavage plane of calcite under liquid conditions using frequency modulation atomic force microscopy. Using this technique, we achieved true atomic-resolution imaging, demonstrating the high-resolution capability of frequency modulation atomic force microscopy in liquids. We could reproduce contrast features reported before using contact mode atomic force microscopy, originating from the protruding oxygen atom of the carbonate groups. Besides this contrast, however, our results, indeed, indicate that we obtain more detailed structural information, revealing the calcium sublattice of the (1014) cleavage plane.

Single atomic contact adhesion and dissipation in dynamic force microscopy.

By combining dynamic force microscopy experiments and first-principles calculations, we have studied the adhesion associated with a single atomic contact between a nanoasperity--the tip apex--and a semiconductor surface--the Ge(111)-c(2 x 8). The nanoasperity's termination has been atomically characterized by extensive comparisons of the measured short-range force at specific sites with the chemical forces calculated using many atomic models that vary in structure, composition, and relative orientation with respect to the surface. This thorough characterization has allowed us to explain the dissipation signal observed in atomic-resolution images and force spectroscopic measurements, as well as to identify a dissipation channel and the associated atomic processes.

Atom inlays performed at room temperature using atomic force microscopy.

The ability to manipulate single atoms and molecules laterally for creating artificial structures on surfaces is driving us closer to the ultimate limit of two-dimensional nanoengineering. However, experiments involving this level of manipulation have been performed only at cryogenic temperatures. Scanning tunnelling microscopy has proved, so far, to be a unique tool with all the necessary capabilities for laterally pushing, pulling or sliding single atoms and molecules, and arranging them on a surface at will. Here we demonstrate, for the first time, that it is possible to perform well-controlled lateral manipulations of single atoms using near-contact atomic force microscopy even at room temperature. We report the creation of 'atom inlays', that is, artificial atomic patterns formed from a few embedded atoms in the plane of a surface. At room temperature, such atomic structures remain stable on the surface for relatively long periods of time.

Atom-selective imaging and mechanical atom manipulation using the non-contact atomic force microscope.

We succeeded in distinguishing between oxygen and silicon atoms on an oxygen-adsorbed Si(111)7 x 7 surface, and also distinguished between silicon and tin atoms on Si(111)7 x 7-Sn intermixed and Si(111) square root(3) x square root(3)-Sn mosaic-phase surfaces using non-contact atomic force microscopy (NC-AFM) at room temperature. Atom species of individual atoms are specified from the number of each atom in NC-AFM images, the tip-sample distance dependence of NC-AFM images and/or the surface distribution of each atom. Further, based on the NC-AFM method but using soft nanoindentation, we achieved two kinds of mechanical vertical manipulation of individual atoms: removal of a selected Si adatom and deposition of a Si atom into a selected Si adatom vacancy on the Si(111)7 x 7 surface at 78 K. Here, we carefully and slowly indented a Si atom on top of a clean Si tip apex onto a predetermined Si adatom to remove the targeted Si adatom and onto a predetermined Si adatom vacancy to deposit a Si atom, i.e. to repair the targeted Si adatom vacancy. By combining the atom-selective imaging method with two kinds of mechanical atom manipulation, i.e. by picking up a selected atom species and by depositing that atom one by one at the assigned site, we hope to construct nanomaterials and nanodevices made from more than two kinds of atom species in the near future.

Mechanical vertical manipulation of selected single atoms by soft nanoindentation using near contact atomic force microscopy.

A near contact atomic force microscope operated at low-temperature is used for vertical manipulation of selected single atoms from the Si(111)-(7 x 7) surface. The strong repulsive short-range chemical force interaction between the closest atoms of both tip apex and surface during a soft nanoindentation leads to the removal of a selected silicon atom from its equilibrium position at the surface without additional perturbation of the (7 x 7) unit cell. Deposition of a single atom on a created vacancy at the surface is achieved as well. These manipulation processes are purely mechanical, since neither bias voltage nor voltage pulse is applied between probe and sample. Differences in the mechanical response of the two nonequivalent adatoms of the Si(111)-(7 x 7) with the load applied is also detected.