Reliable preparation and regeneration of well-defined single-atom tips through laser annealing.

Single-atom tips (SATs) have crucial scientific and technological applications, such as in scanning probe microscopy and charged particle beam technology. We reported a reliable method of preparing and regenerating noble metal-covered W(111) SATs through laser annealing at approximately 1000 K under ultrahigh vacuum. The field emission patterns obtained during laser heating revealed the self-assembly process of a pyramidal tip. The SATs can be regenerated through laser annealing tens of times with little change in sharpness, indicating a long lifetime. Various pyramidal SATs can be generated and regenerated using visible-light, near-infrared, mode-locked, and continuous-wave lasers at different polarizations relative to the tip axis. The generation of well-defined pyramidal SATs through laser annealing can facilitate various applications of SATs.

Low-cost, open-source XYZ nanopositioner for high-precision analytical applications.

Nanoscale positioning has numerous applications in both academia and industry. A growing number of applications require devices with long working distances and nanoscale resolutions. Friction-inertia piezoelectric positioners, which are based on the stick-slip mechanism, achieve both nanometer resolution and centimeter-scale travel. However, the requirements of complex preload mechanism, precision machining, and precise assembly increase the cost of conventional friction-inertia nanopositioners. Herein we present the design of an open-source XYZ-axis nanopositioning system. Utilizing a magnet-based stick-slip driving mechanism, the proposed XYZ nanopositioner provides several advantages, including sub-nanometer resolution, a payload capacity of up to 12 kg (horizontal), compact size, low cost, and easy assembly; furthermore, the system is adjustment-free. The performance tests validate the precision of the system in both scanning and stepping operation modes. Moreover, the resonant spectra affirm the rigidity and dynamic response of the mechanism. In addition, we demonstrate the practical applications of this nanopositioner in various measurement techniques, including scanning electron microscopy, vibrometry, and atomic force microscopy. Furthermore, we present 11 variations of the nanopositioner designs that are either compatible with ultra-high-vacuum systems and other existing systems, 3D printable, or hacking commercial linear slides.

Investigating states of gas in water encapsulated between graphene layers.

Conventionally, only two states are assumed to exist in water: well-dispersed gas monomers and gas bubbles. Rarely is this paradigm explored experimentally. To close this gap, here we used transmission electron microscopy (TEM) to study degassed water, deionized water, and gas-supersaturated water encapsulated in graphene liquid cells. While neither degassed water nor deionized water yielded specific features, two major microscopic structures were evident in gas-supersaturated water: (1) polycrystalline nanoparticles formed of gas molecules and (2) a high density of tiny cells. Dark-field TEM imaging revealed that water molecules surrounding each cell form crystalline structures-a surprising discovery of a clathrate state in gas-supersaturated water that may help resolve several long-standing puzzles. Overall, this study suggests that water may form a matrix that actively interacts with gas molecules in complex and subtle ways.

Effects of Dissolved Gases on the Amyloid Fibril Morphology.

The onset or progression of numerous neurodegenerative diseases occurs due to aggregation of proteins that ultimately form fibrils. The assembly and morphology of fibrils are susceptible to environmental factors. In this work, we used atomic force microscopy (AFM) to investigate the effects of dissolved nitrogen and oxygen molecules on the morphology of fibrils formed by a hydrophobic amyloid peptide implicated in amyotrophic lateral sclerosis, 15 repeats of glycine-alanine, on a highly oriented pyrolytic graphite substrate. We started with preformed fibril solutions that were then diluted with buffers of different gas conditions, resulting in the aggregation of the fibrils into different morphologies that were revealed by AFM after adsorption on the substrate. Straight fibrils were observed in both degassed and ambient buffers, but a stronger lateral association was seen in degassed buffers. Smaller and softer fibrils were observed in O2-supersaturated buffers, and plaque-like fibril aggregates of considerably large size were evident in N2-supersaturated buffers. In overnight incubation experiments, we observed changes in both the morphology and height of the fibril aggregates, and their evolution varied with different gas conditions. These findings indicate that the gas type and concentration affect the aggregation of amyloid fibrils and may facilitate the development of biomaterial applications and treatments for amyloid-related diseases.

A horizontal-type scanning near-field optical microscope with torsional mode operation toward high-resolution and non-destructive imaging of soft materials.

We design and build a horizontal-type aperture based scanning near-field optical microscope (a-SNOM) with superior mechanical stability toward high-resolution and non-destructive topographic and optical imaging. We adopt the torsional mode in AFM (atomic force microscopy) operation to achieve a better force sensitivity and a higher topographic resolution when using pyramidal a-SNOM tips. The performance and stability of the AFM are evaluated through single-walled carbon nanotube and poly(3-hexyl-thiophene) nanowire samples. An optical resolution of 93 nm is deduced from the a-SNOM imaging of a metallic grating. Finally, a-SNOM fluorescence imaging of soft lipid domains is successfully achieved without sample damage by our horizontal-type a-SNOM instrument with torsional mode AFM operation.

Emerging Roles of Air Gases in Lipid Bilayers.

Recent studies indicate that changing the physical properties of lipid bilayers may profoundly change the function of membrane proteins. Here, the effects of dissolved nitrogen and oxygen molecules on the mechanical properties and stability of lipid bilayers are investigated using differential confocal microscopy, atomic force microscopy, and molecular dynamics simulations. All experiments evidence the presence of dissolved air gas in lipid bilayers prepared without gas control. The lipid bilayers in degassed solutions are softer and less stable than those in ambient solutions. High concentrations of nitrogen increase the bending moduli and stability of the lipid bilayers and impede phase separation in ternary lipid bilayers. The effect of oxygen is less prominent. Molecular dynamics simulations indicate that higher nitrogen affinity accounts for increased rigidity. These findings have fundamental and wide implications for phenomena related to lipid bilayers and cell membranes, including the origin of life.

Direct comparison between subnanometer hydration structures on hydrophilic and hydrophobic surfaces via three-dimensional scanning force microscopy.

Investigating interfacial water ordering on solid surfaces with different hydrophobicities is fundamentally important. Here, we prepared hydrophilic mica substrates with some areas covered by mildly hydrophobic graphene layers and studied the resulting hydration layers using three-dimensional (3D) force measurements based on frequency-modulation atomic force microscopy. Hydration layers of 0.3-0.6 nm were detected on bare graphene regions; these layers were considerably larger than the spacing measured on mica (0.2-0.3 nm). On the graphene-covered regions, we also observed the formation of special ordered structures of adsorbates over time, on which, surprisingly, no prominent hydration layers were detected. Based on these findings, we present one possible scenario to describe the formation process of the ordered interfacial structures and the enhanced oscillation period in the force profiles. This work also demonstrates the capability and significance of 3D force measurements in probing hydration behaviors on a heterogeneous substrate with a lateral resolution smaller than several nanometers.

Galectin-1 Restricts Vascular Smooth Muscle Cell Motility Via Modulating Adhesion Force and Focal Adhesion Dynamics.

Vascular smooth muscle cell (VSMC) migration play a key role in the development of intimal hyperplasia and atherosclerosis. Galectin-1 (Gal-1) is a redox-sensitive ß-galactoside-binding lectin expressed in VSMCs with intracellular and extracellular localizations. Here we show that VSMCs deficient in Gal-1 (Gal-1-KO) exhibited greater motility than wild type (WT) cells. Likewise, Gal-1-KO-VSMC migration was inhibited by a redox-insensitive but activity-preserved Gal-1 (CSGal-1) in a glycan-dependent manner. Gal-1-KO-VSMCs adhered slower than WT cells on fibronectin. Cell spreading and focal adhesion (FA) formation examined by phalloidin and vinculin staining were less in Gal-1-KO-VSMCs. Concomitantly, FA kinase (FAK) phosphorylation was induced to a lower extent in Gal-1-KO cells. Analysis of FA dynamics by nocodazole washout assay demonstrated that FA disassembly, correlated with FAK de-phosphorylation, was faster in Gal-1-KO-VSMCs. Surface plasmon resonance assay demonstrated that CSGal-1 interacted with α5ß1integrin and fibronectin in a glycan-dependent manner. Chemical crosslinking experiment and atomic force microscopy further revealed the involvement of extracellular Gal-1 in strengthening VSMC-fibronectin interaction. In vivo experiment showed that carotid ligation-induced neointimal hyperplasia was more severe in Gal-1-KO mice than WT counterparts. Collectively, these data disclose that Gal-1 restricts VSMC migration by modulating cell-matrix interaction and focal adhesion turnover, which limits neointimal formation post vascular injury.

Lateral Force Microscopy of Interfacial Nanobubbles: Friction Reduction and Novel Frictional Behavior.

Atomic force microscopy is used to conduct single-asperity friction measurements at a water-graphite interface. Local mapping of the frictional force, which is based on the degree of the cantilever twisting, shows nearly friction-free when a tip scans over a nanobubble. Surprisingly, apart from being gapless, the associated friction loop exhibits a tilt in the cantilever twisting versus the tip's lateral displacement with the slope depending on the loading force. The sign of the slope reverses at around zero loading force. In addition, the measured normal and lateral tip-sample interactions exhibit unison versus tip-sample separation. Theoretical analysis, based on the balance of forces on the tip originated from the capillary force of the nanobubble and the torsion of the cantilever, offers quantitative explanations for both the tilted friction loop and the unison of force curves. The analysis may well apply in a wider context to the lateral force characterization on cap-shaped fluid structures such as liquid droplets on a solid substrate. This study further points to a new direction for friction reduction between solids in a liquid medium.

Xenon gas field ion source from a single-atom tip.

Focused ion beam (FIB) systems have become powerful diagnostic and modification tools for nanoscience and nanotechnology. Gas field ion sources (GFISs) built from atomic-size emitters offer the highest brightness among all ion sources and thus can improve the spatial resolution of FIB systems. Here we show that the Ir/W(111) single-atom tip (SAT) can emit high-brightness Xe+ ion beams with a high current stability. The ion emission current versus extraction voltage was analyzed from 150 K up to 309 K. The optimal emitter temperature for maximum Xe+ ion emission was ∼150 K and the reduced brightness at the Xe gas pressure of 1 × 10-4 torr is two to three orders of magnitude higher than that of a Ga liquid metal ion source, and four to five orders of magnitude higher than that of a Xe inductively coupled plasma ion source. Most surprisingly, the SAT emitter remained stable even when operated at 309 K. Even though the ion current decreased with increasing temperature, the current at room temperature (RT) could still reach over 1 pA when the gas pressure was higher than 1 × 10-3 torr, indicating the feasibility of RT-Xe-GFIS for application to FIB systems. The operation temperature of Xe-SAT-GFIS is considerably higher than the cryogenic temperature required for the helium ion microscope (HIM), which offers great technical advantages because only simple or no cooling schemes can be adopted. Thus, Xe-GFIS-FIB would be easy to implement and may become a powerful tool for nanoscale milling and secondary ion mass spectroscopy.

Three-dimensional surface topography of graphene by divergent beam electron diffraction.

There are only a handful of scanning techniques that can provide surface topography at nanometre resolution. At the same time, there are no methods that are capable of non-invasive imaging of the three-dimensional surface topography of a thin free-standing crystalline material. Here we propose a new technique-the divergent beam electron diffraction (DBED) and show that it can directly image the inhomogeneity in the atomic positions in a crystal. Such inhomogeneities are directly transformed into the intensity contrast in the first-order diffraction spots of DBED patterns and the intensity contrast linearly depends on the wavelength of the employed probing electrons. Three-dimensional displacement of atoms as small as 1 angstrom can be detected when imaged with low-energy electrons (50-250 eV). The main advantage of DBED is that it allows visualization of the three-dimensional surface topography and strain distribution at the nanometre scale in non-scanning mode, from a single shot diffraction experiment.

High-sensitivity imaging with lateral resonance mode atomic force microscopy.

In the operation of a dynamic mode atomic force microscope, a micro-fabricated rectangular cantilever is typically oscillated at or near its mechanical resonance frequency. Lateral bending resonances of cantilevers are rarely used because the resonances are not expected to be detected by the beam-deflection method. In this work, we found that micro-cantilevers with a large tip produced an out-of-plane displacement in lateral resonance (LR), which could be detected with the beam-deflection method. Finite-element analysis indicated that the presence of a large tip is the major source of the out-of-plane coupling for the LR. We also imaged a heterogeneous sample by operating a cantilever in LR, torsional resonance, and tapping modes. LR mode yielded a small deformation and noise level in the height maps as well as a high contrast and small noise level in the phase maps. LR mode also had a resonance frequency that was orders of magnitude higher than that of tapping mode. Operation with LR mode may have the benefits of high-speed scanning, high-sensitivity imaging, and mapping of in-plane mechanical properties of the sample surface. In general, LR mode may become a powerful new atomic force microscopy technique for characterizing sample materials.

High-Resolution Characterization of Preferential Gas Adsorption at the Graphene-Water Interface.

The contact of water with graphene is of fundamental importance and of great interest for numerous promising applications, but how graphene interacts with water remains unclear. Here we used atomic force microscopy (AFM) to investigate hydrophilic mica substrates with some regions covered by mechanically exfoliated graphene layers in water. In water containing air gas close to the saturation concentration (within ∼40%), cap-shaped nanostructures (or interfacial nanobubbles) and ordered-stripe domains were observed on graphene-covered regions but not on pure mica regions. These structures did not appear on graphene when samples were immersed in highly degassed water, indicating that their formation was caused by the adsorption of gas dissolved in water. Thus, atomically thin graphene, even at a narrow width of 20 nm, changes the local surface chemistry of a highly hydrophilic substrate. Furthermore, surface hydrophobicity significantly affects gas adsorption, which has broad implications for diverse phenomena in water.

Optical imaging module for astigmatic detection system.

In this paper, an optical imaging module design for an astigmatic detection system (ADS) is presented. The module is based on a commercial optical pickup unit (OPU) and it contains a coaxial illuminant for illuminating a specimen. Furthermore, the imaging module facilitates viewing the specimen and the detection laser spot of the ADS with a lateral resolution of approximately 1 µm without requiring the removal of an element of the OPU. Two polarizers and one infrared filter are used to eliminate stray laser light in the OPU and stray light produced by the illuminant. Imaging modules designed for digital versatile disks (DVDs) and Blu-ray DVDs were demonstrated. Furthermore, the module can be used for imaging a small cantilever with approximate dimensions of 2 µm (width) × 5 µm (length), and therefore, it has the potential to be used in high-speed atomic force microscopy.

Nucleation processes of nanobubbles at a solid/water interface.

Experimental investigations of hydrophobic/water interfaces often return controversial results, possibly due to the unknown role of gas accumulation at the interfaces. Here, during advanced atomic force microscopy of the initial evolution of gas-containing structures at a highly ordered pyrolytic graphite/water interface, a fluid phase first appeared as a circular wetting layer ~0.3 nm in thickness and was later transformed into a cap-shaped nanostructure (an interfacial nanobubble). Two-dimensional ordered domains were nucleated and grew over time outside or at the perimeter of the fluid regions, eventually confining growth of the fluid regions to the vertical direction. We determined that interfacial nanobubbles and fluid layers have very similar mechanical properties, suggesting low interfacial tension with water and a liquid-like nature, explaining their high stability and their roles in boundary slip and bubble nucleation. These ordered domains may be the interfacial hydrophilic gas hydrates and/or the long-sought chemical surface heterogeneities responsible for contact line pinning and contact angle hysteresis. The gradual nucleation and growth of hydrophilic ordered domains renders the original homogeneous hydrophobic/water interface more heterogeneous over time, which would have great consequence for interfacial properties that affect diverse phenomena, including interactions in water, chemical reactions, and the self-assembly and function of biological molecules.

The Glycine-Alanine Dipeptide Repeat from C9orf72 Hexanucleotide Expansions Forms Toxic Amyloids Possessing Cell-to-Cell Transmission Properties.

Hexanucleotide expansions, GGGGCC, in the non-coding regions of the C9orf72 gene were found in major frontotemporal lobar dementia and amyotrophic lateral sclerosis patients (C9FTD/ALS). In addition to possible RNA toxicity, several dipeptide repeats (DPRs) are translated through repeat-associated non-ATG-initiated translation. The DPRs, including poly(GA), poly(GR), poly(GP), poly(PR), and poly(PA), were found in the brains and spinal cords of C9FTD/ALS patients. Among the DPRs, poly(GA) is highly susceptible to form cytoplasmic inclusions, which is a characteristic of C9FTD/ALS. To elucidate DPR aggregation, we used synthetic (GA)15 DPR as a model system to examine the aggregation and structural properties in vitro. We found that (GA)15 with 15 repeats fibrillates rapidly and ultimately forms flat, ribbon-type fibrils evidenced by transmission electron microscopy and atomic force microscopy. The fibrils are capable of amyloid dye binding and contain a characteristic cross-ß sheet structure, as revealed by x-ray scattering. Furthermore, using neuroblastoma cells, we demonstrated the neurotoxicity and cell-to-cell transmission property of (GA)15 DPR. Overall, our results show the structural and toxicity properties of GA DPR to facilitate future DPR-related therapeutic development.

Atomic Force Microscopy Characterization of Protein Fibrils Formed by the Amyloidogenic Region of the Bacterial Protein MinE on Mica and a Supported Lipid Bilayer.

Amyloid fibrils play a crucial role in many human diseases and are found to function in a range of physiological processes from bacteria to human. They have also been gaining importance in nanotechnology applications. Understanding the mechanisms behind amyloid formation can help develop strategies towards the prevention of fibrillation processes or create new technological applications. It is thus essential to observe the structures of amyloids and their self-assembly processes at the nanometer-scale resolution under physiological conditions. In this work, we used highly force-sensitive frequency-modulation atomic force microscopy (FM-AFM) to characterize the fibril structures formed by the N-terminal domain of a bacterial division protein MinE in solution. The approach enables us to investigate the fibril morphology and protofibril organization over time progression and in response to changes in ionic strength, molecular crowding, and upon association with different substrate surfaces. In addition to comparison of the fibril structure and behavior of MinE1-31 under varying conditions, the study also broadens our understanding of the versatile behavior of amyloid-substrate surface interactions.

Interface-induced ordering of gas molecules confined in a small space.

The thermodynamic properties of gases have been understood primarily through phase diagrams of bulk gases. However, observations of gases confined in a nanometer space have posed a challenge to the principles of classical thermodynamics. Here, we investigated interfacial structures comprising either O2 or N2 between water and a hydrophobic solid surface by using advanced atomic force microscopy techniques. Ordered epitaxial layers and cap-shaped nanostructures were observed. In addition, pancake-shaped disordered layers that had grown on top of the epitaxial base layers were observed in oxygen-supersaturated water. We propose that hydrophobic solid surfaces provide low-chemical-potential sites at which gas molecules dissolved in water can be adsorbed. The structures are further stabilized by interfacial water. Here we show that gas molecules can agglomerate into a condensed form when confined in a sufficiently small space under ambient conditions. The crystalline solid surface may even induce a solid-gas state when the gas-substrate interaction is significantly stronger than the gas-gas interaction. The ordering and thermodynamic properties of the confined gases are determined primarily according to interfacial interactions.

Self-assembly of MinE on the membrane underlies formation of the MinE ring to sustain function of the Escherichia coli Min system.

The pole-to-pole oscillation of the Min proteins in Escherichia coli results in the inhibition of aberrant polar division, thus facilitating placement of the division septum at the midcell. MinE of the Min system forms a ring-like structure that plays a critical role in triggering the oscillation cycle. However, the mechanism underlying the formation of the MinE ring remains unclear. This study demonstrates that MinE self-assembles into fibrillar structures on the supported lipid bilayer. The MinD-interacting domain of MinE shows amyloidogenic properties, providing a possible mechanism for self-assembly of MinE. Supporting the idea, mutations in residues Ile-24 and Ile-25 of the MinD-interacting domain affect fibril formation, membrane binding ability of MinE and MinD, and subcellular localization of three Min proteins. Additional mutations in residues Ile-72 and Ile-74 suggest a role of the C-terminal domain of MinE in regulating the folding propensity of the MinD-interacting domain for different molecular interactions. The study suggests a self-assembly mechanism that may underlie the ring-like structure formed by MinE-GFP observed in vivo.