Three-dimensional Atomic Force Microscopy Research Articles

Atomic-scale structure of interfacial water on gel and liquid phase lipid membranes.

Hydration of biological membranes is essential to a wide range of biological processes. In particular, it is intrinsically linked to lipid thermodynamic properties, which in turn influence key cell functions such as ion permeation and protein mobility. Experimental and theoretical studies of the surface of biomembranes have revealed the presence of an interfacial repulsive force, which has been linked to hydration or steric effects. Here, we directly characterise the atomic-scale structure of water near supported lipid membranes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine in their gel and liquid phase through three-dimensional atomic force microscopy (3D AFM). First, we demonstrate the ability to probe the morphology of interfacial water of lipid bilayers in both phases with sub-molecular resolution by using ultrasharp tips. We then visualise the molecular arrangement of water at the lipid surface at different temperatures. Our experiments reveal that water is organised in multiple hydration layers on both the solid-ordered and liquid-disordered lipid phases. Furthermore, we observe a monotonic repulsive force, which becomes relevant only in the liquid phase. These results offer new insights into the water structuring near soft biological surfaces, and demonstrate the importance of investigating it with vertical and lateral sub-molecular resolution.

Atomic structure and water arrangement on K-feldspar microcline (001).

The properties of clouds, such as their reflectivity or their likelihood to precipitate, depend on whether the cloud droplets are liquid or frozen. Thus, understanding the ice nucleation mechanisms is essential for the development of reliable climate models. Most ice nucleation in the atmosphere is heterogeneous, i.e., caused by ice nucleating particles such as mineral dusts or organic aerosols. In this regard, K-feldspar minerals have attracted great interest recently as they have been identified as one of the most important ice nucleating particles under mixed-phase cloud conditions. The mechanism by which feldspar minerals facilitate ice nucleation remains, however, elusive. Here, we present atomic force microscopy (AFM) experiments on microcline (001) performed in an ultrahigh vacuum and at the solid-water interface together with density functional theory (DFT) and molecular dynamics (MD) calculations. Our ultrahigh vacuum data reveal features consistent with a hydroxyl-terminated surface. This finding suggests that water in the residual gas readily reacts with the surface. Indeed, the corresponding DFT calculations confirm a dissociative water adsorption. Three-dimensional AFM measurements performed at the mineral-water interface unravel a layered hydration structure with two features per surface unit cell. A comparison with MD calculations suggests that the structure observed in AFM corresponds to the second hydration layer rather than the first water layer. In agreement with previous computation results, no ice-like structure is seen, questioning an explanation of the ice nucleation ability by lattice match. Our results provide an atomic-scale benchmark for the clean and water-covered microcline (001) plane, which is mandatory for understanding the ice nucleation mechanism on feldspar minerals.

Investigation of the interface state characteristics of the Al/Al2O3/Ge/p-Si heterostructure over a wide frequency range by capacitance and conductance measurements

In this work, Al/Al2O3/Ge/p-Si heterostructures were fabricated by e-beam thermal evaporation. The EDX (energy dispersive X-ray spectroscopy) map visually obtained the basic distribution information in a two-dimensional graph. Three-dimensional atomic Force Microscope (AFM) analysis of the roughness of the Al/Al2O3/Ge/p-Si heterostructure shows that the Al2O3 particle has approximately 4.35 nm. Quantitative analysis via histogram plots for Al2O3 interlayer grown on p-Si shows a Gaussian-like distribution, and it has a sharp profile between 0 and 2 nm on silicon, located around 1.2 nm. The current-voltage (I–V) and capacitance-conductance-voltage (C-G/ω-V) measurements were used to investigate the electrical properties of the structure at room temperature. The I–V measurements revealed two distinct linear regions, attributed to barrier inhomogeneity and distribution of interfacial state/trap densities (Dit). For this reason, we obtained Dit distributions by considering the voltage dependence of the ideality factor n(V) in two regions. Diode parameters acquired through different methods are comparatively presented with a brief literature review. Then, the C-G/ω-V measurements were conducted across a broad frequency range (from 0.3 kHz to 3 MHz), and these measurements revealed notable alterations in the electrical parameters with regard to frequency. In order to probe interface states/traps, we employed the conductance/admittance method, which provided satisfactory information about trap lifetime, following the low-high frequency (LF-HF) and Hill-Coleman methods. The effects of these states/traps on the measurements according to voltage, frequency, and energy level (Eit-Ev) are discussed in detail.

Halobacillus trueperi CT7: A spore-forming, gelatinase producing, salt-tolerant bacteria isolated from Çankırı Salt Mine

Halophilic microorganisms are an extreme group of organisms that can spread at high salt concentrations, and a significant part of them consists of halophilic bacteria. Salt mines are important sources where halophilic bacteria are detected. In this study, Halobacillus trueperi CT7, a halophilic bacterium, was isolated from Çankırı Salt Mine. It was determined that this strain obtained showed 98.16 % similarity to Halobacillus trueperi by DNA isolation and sequence analysis as well as biochemical analysis. In addition, two-dimensional (scanning electron microscopy) and three-dimensional (atomic force microscopy) images of Halobacillus trueperi were performed. In order to determine the industrial use potential of the microorganism, the minimum and maximum salt concentrations, temperature and pH ranges, as well as the enzyme activities, where the species can grow, were determined qualitatively. The interest in halophilic organisms for their use in extreme industrial processes is increasing day by day. It is thought that this study will contribute to future studies on halophilic bacteria.

Unraveling Spatial Charge Density Distributions at Electrode-Electrolyte Interfaces

Charge distributions at electrode-electrolyte interfaces play a crucial role in many electrochemical processes, some of which are electrocatalysis, supercapacitors, batteries etc., However, most experimental techniques, either microscopy or spectroscopy tools that are used to probe these systems, cannot provide any information about the interfacial spatial charge distribution. Techniques based on kelvin-probe force microscopy (KPFM) can provide some information, however they have a very limited depth of resolution and can only work with extremely dilute electrolytes. Recently, we developed a technique known as charge profiling three-dimensional (3D) atomic force microscopy (CP-3D-AFM) which could map out the charge densities at angstrom-depth resolution. The method is based on measuring 3D-AFM maps at different electrode potentials and further using electrostatic calculations to obtain the charge density depth profiles at the respective potentials. We used this method to measure charge distributions in highly ionic electrolyte systems and can explain their differential capacitance profiles. This CP-3D-AFM technique could enable to obtain molecular structural insights for a wide range of electrolyte systems and serve in designing principles for engineering effective electrode-electrolyte interfaces.

Solution Structure and Hydration Forces between Mica and Hydrophilic Versus Hydrophobic Surfaces

Solid–liquid interfaces are central to a range of interesting phenomena including colloidal aggregation, crystallization by particle attachment, catalysis, heterogeneous nucleation, water desalination, and biomolecular assembly. While three-dimensional atomic force microscopy (3D AFM) has emerged as a technique for resolving interfacial solution structure at the molecular scale, key challenges for data interpretation persist, most notably regarding the influence of the probe on the measured structure. Using the mica–water system as a case study, we investigate the effect of hydrophilic and hydrophobic probes on interfacial solution structure measured by 3D AFM. Data from hydrophilic silicon-based probes are in good agreement with molecular dynamics simulations, wherein the innermost water molecules adsorb preferentially at the surface ditrigonal cavity sites, followed by two additional ordered hydration layers. In contrast, the hydrophobic carbon-based probes detect vertical oscillatory features but do not show lateral patterning that matches the underlying mica lattice. At high ionic strength, up to six of these oscillatory features are observed extending 2 nm into the solution phase with an average spacing of 0.29 ± (0.04) nm. We also determine that the repulsive hydration force between mica and the hydrophilic probe depends on the nature and concentration of ions in solution. Specifically, solutions with stronger ion–water and ion–ion interactions produce a stronger repulsive hydration force as the probe approaches the surface. Based on these observations, we present a scheme for controlling the outcomes of particle aggregation and attachment by varying the solution conditions to tune the hydration force.

Three-dimensional ordering of water molecules reflecting hydroxyl groups on sapphire (001) and α-quartz (100) surfaces.

Water molecules on oxide surfaces influence the chemical reactivity and molecular adsorption behavior of oxides. Herein, three-dimensional atomic force microscopy (3D-AFM) and molecular dynamics simulations are used to visualize the surface hydroxyl (OH) groups and their hydration structures on sapphire (001) and α-quartz (100) surfaces at the atomic-scale. The obtained results revealed that the spatial density distributions and hydrogen-bonding strengths of surface OH groups affect their local hydration structures. In particular, the force curves obtained by 3D-AFM suggest that the hydration forces of water molecules intensify at sites where water molecules strongly interact with the surface OH groups. The insights obtained in this study deepen our understanding of the affinities of Al2O3 and SiO2 for water molecules and contribute to the use of 3D-AFM in the investigation of atomic-scale hydration structures on various surfaces, thereby benefiting a wide range of research fields dealing with solid-liquid interfaces.

Real-Space Charge Density Profiling of Electrode-Electrolyte Interfaces with Angstrom Depth Resolution.

The accumulation and depletion of charges at electrode-electrolyte interfaces is crucial for all types of electrochemical processes. However, the spatial profile of such interfacial charges remains largely elusive. Here we develop charge profiling three-dimensional (3D) atomic force microscopy (CP-3D-AFM) to experimentally quantify the real-space charge distribution of the electrode surface and electric double layers (EDLs) with angstrom depth resolution. We first measure the 3D force maps at different electrode potentials using our recently developed electrochemical 3D-AFM. Through statistical analysis, peak deconvolution, and electrostatic calculations, we derive the depth profile of the local charge density. We perform such charge profiling for two types of emergent electrolytes, ionic liquids, and highly concentrated aqueous solutions, observe pronounced sub-nanometer charge variations, and find the integrated charge densities to agree with those derived from macroscopic electrochemical measurements.

Hydration Structure at the Calcite-Water (10.4) Interface in the Presence of Rubidium Chloride.

Solid-liquid interfaces are of significant importance in a multitude of geochemical and technological fields. More specifically, the solvation structure plays a decisive role in the properties of the interfaces. Atomic force microscopy (AFM) has been used to resolve the interfacial hydration structure in the presence and absence of ions. Despite many studies investigating the calcite-water interface, the impact of ions on the hydration structure at this interface has rarely been studied. Here, we investigate the calcite-water interface at various concentrations (ranging from 0 to 5 M) of rubidium chloride (RbCl) using three-dimensional atomic force microscopy (3D AFM). We present molecularly resolved images of the hydration structure at the interface. Interestingly, the characteristic pattern of the hydration structure appears similar regardless of the RbCl concentration. The presence of the ions is detected in an indirect manner by more frequent contrast changes and slice displacements.

Computed Three-Dimensional Atomic Force Microscopy Images of Biopolymers Using the Jarzynski Equality.

Three-dimensional atomic force microscopy (3D-AFM) has resolved three-dimensional distributions of solvent molecules at solid–liquid interfaces at the subnanometer scale. This method is now being extended to the imaging of biopolymer assemblies such as chromosomes or proteins in cells, with the expectation of being able to resolve their three-dimensional structures. Here, we have developed a computational method to simulate 3D-AFM images of biopolymers by using the Jarzynski equality. It is found that some parts of the fiber structure of biopolymers are indeed resolved in the 3D-AFM image. The dependency of 3D-AFM images on the vertical scanning velocity is investigated, and optimum scanning velocities are found. It is also clarified that forces in nonequilibrium processes are measured in 3D-AFM measurements when the dynamics of polymers are slower than the scanning of the probe.

Automated measurement and analysis of sidewall roughness using three-dimensional atomic force microscopy

As semiconductor device architecture develops, from planar field-effect transistors (FET) to FinFET and gate-all-around (GAA), there is an increased need to measure 3D structure sidewalls precisely. Here, we present a 3-Dimensional Atomic Force Microscope (3D-AFM), a powerful 3D metrology tool to measure the sidewall roughness (SWR) of vertical and undercut structures. First, we measured three different dies repeatedly to calculate reproducibility in die level. Reproducible results were derived with a relative standard deviation under 2%. Second, we measured 13 different dies, including the center and edge of the wafer, to analyze SWR distribution in wafer level and reliable results were measured. All analysis was performed using a novel algorithm, including auto flattening, sidewall detection, and SWR calculation. In addition, SWR automatic analysis software was implemented to reduce analysis time and to provide standard analysis. The results suggest that our 3D-AFM, based on the tilted Z scanner, will enable an advanced methodology for automated 3D measurement and analysis.

Probing the hydration friction of ionic interfaces at the atomic scale.

Despite the extensive studies conducted in exploring friction in the aqueous environment, the mechanism of hydration friction remains not well understood. Herein, we directly probed hydration friction on mica-electrolyte interfaces with different hydrated alkali cations through a combination of three-dimensional atomic force microscopy and friction force microscopy. The atomic scale imaging of the hydration layers at the mica surface in different electrolyte solutions clearly revealed a correlation between the alkali cations and the structure of the hydration layers. Our detailed analysis showed that the hydration force was much higher at high ionic concentrations than that at low concentrations. The hydration friction coefficient was found to follow the trend K+< Na+< Li+< Cs+, which contrasts with the Hofmeister series, indicating that the hydration friction depends not only on the hydration strength of the alkali cations but also on the arrangement of the alkali cations at the interface. The results of this study provide deep insights into the origins of hydration friction, with potential implications for the development of new boundary lubrication in aqueous media.

Experimental investigation into nano-finishing of β-TNTZ alloy using magnetorheological fluid magnetic abrasive finishing process for orthopedic applications

This study describes the effect of magnetorheological fluid assisted magnetic abrasive finishing (MRAF) process on the surface topography of fine finished high strength biomedical grade β-phase Ti–Nb–Ta–Zr (β-TNTZ) alloy for orthopedic applications. β-type Ti–35Nb–7Ta–5Zr alloy exhibit high strength, better corrosion resistance and excellent bioactivity in comparison with Ti–6Al–4V alloy. The topographic features of finished surfaces (including surface roughness, skewness, and kurtosis), percentage change in surface roughness, and material removal have been studied to understand the influence of MRAF processing parameters, such as carbonyl iron particles proportion, diamond abrasive particles proportion, rotational speed of the abrasive tool, and work gap between workpiece and abrasive tool. Furthermore, MRAF finishing has been conducted using raster and spiral strategies. The topographic characteristics of the finished surfaces have been measured using a noncontact three-dimensional Surface Profilometer and atomic force microscopy. The results of the study showed that all the input process parameters have strongly influenced the surface characteristics in both quantitative (material removal) and qualitative measures (Surface roughness). The β-TNTZ have possed excellent bio-mechanical properties such as high compressive strength (1195 MPa), micro-hardness (515 HV), corrosion resistance, and excellent bioactivity. The best optimal condition to obtain lowest SR (9 nm) and highest MR (65 mg) was obtained in the case of rater path finishing strategy at CIP – 40%vol., Dv – 3.5%vol., Nt – 900 rpm, and Gp – 1 mm. The maximum percentage change in surface roughness (%ΔRa) was measured around 97.68% and 93.27% in raster and spiral path strategy, respectively. The minimum surface finish ranging about 9 nm has been achieved through the MRAF process. Further, the raster path strategy has been found more effective in producing negative skewness (Ssk), kurtosis (Sku) value less than 3, and minimum number of peaks density (Spd). The overall results of the study suggested that MRAF of β-TNTZ alloy is a good solution for obtaining fine-finished orthopedic devices while sustaining their tribological and wear properties.

Structural and mechanical characteristics of exosomes from osteosarcoma cells explored by 3D-atomic force microscopy.

Exosomes have recently gained interest as mediators of cell-to-cell communication and as potential biomarkers for cancer and other diseases. They also have potential as nanocarriers for drug delivery systems. Therefore, detailed structural, molecular, and biomechanical characterization of exosomes is of great importance for developing methods to detect and identify the changes associated with the presence of cancer and other diseases. Here, we employed three-dimensional atomic force microscopy (3D-AFM) to reveal the structural and nanomechanical properties of exosomes at high spatial resolution in physiologically relevant conditions. The substructural details of exosomes released from three different cell types were determined based on 3D-AFM force mapping. The resulting analysis revealed the presence of distinct local domains bulging out from the exosome surfaces, which were associated with the exosomal membrane proteins present on the outer surface. The nanomechanical properties of individual exosomes were determined from the 3D-force maps. We found a considerably high elastic modulus, ranging from 50 to 350 MPa, as compared to that obtained for synthetic liposomes. Moreover, malignancy-dependent changes in the exosome mechanical properties were revealed by comparing metastatic and nonmetastatic tumor cell-derived exosomes. We found a clear difference in their Young's modulus values, suggesting differences in their protein profiles and other exosomal contents. Exosomes derived from a highly aggressive and metastatic k-ras-activated human osteosarcoma (OS) cell line (143B) showed a higher Young's modulus than that derived from a nonaggressive and nonmetastatic k-ras-wildtype human OS cell line (HOS). The increased elastic modulus of the 143B cell-derived exosomes was ascribed to the presence of abundant specific proteins responsible for elastic fiber formation as determined by mass spectroscopy and confirmed by western blotting and ELISA. Therefore, we conclude that exosomes derived from metastatic tumor cells carry an exclusive protein content that differs from their nonmetastatic counterparts, and thus they exhibit different mechanical characteristics. Discrimination between metastatic and nonmetastatic malignant cell-derived exosomes would be of great importance for studying exosome biological functions and using them as diagnostic biomarkers for various tumor types. Our findings further suggest that metastatic tumor cells release exosomes that express increased levels of elastic fiber-associated proteins to preserve their softness.

Three-Dimensional Molecular Mapping of Ionic Liquids at Electrified Interfaces.

Electric double layers (EDLs), occurring ubiquitously at solid-liquid interfaces, are critical for electrochemical energy conversion and storage processes such as capacitive charging and redox reactions. However, to date the molecular-scale structure of EDLs remains elusive. Here we report an advanced technique, electrochemical three-dimensional atomic force microscopy (EC-3D-AFM), and use it to directly image the molecular-scale EDL structure of an ionic liquid under different electrode potentials. We observe not only multiple discrete ionic layers in the EDL on a graphite electrode but also a quasi-periodic molecular density distribution within each layer. Furthermore, we find pronounced 3D reconfiguration of the EDL at different voltages, especially in the first layer. Combining the experimental results with molecular dynamics simulations, we find potential-dependent molecular redistribution and reorientation in the innermost EDL layer, both of which are critical to EDL capacitive charging. We expect this mechanistic understanding to have profound impacts on the rational design of electrode-electrolyte interfaces for energy conversion and storage.

Subnanometer Interfacial Forces in Three-Dimensional Atomic Force Microscopy: Water and Octane near a Mica Surface

This work was funded by the Ministerio de Ciencia e Innovacion (Spain) under grants PID2019-106801GB-I00, MAT2016-76507-R, FIS2017-86007-C3, and FPU2015/0248 (P.T.) and through the “Maria de Maeztu” Programme for Units of Excellence in R&D (CEX2018-000805-M) and the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement no.721874 (SPM2.0).

Improvements in fundamental performance of in-liquid frequency modulation atomic force microscopy.

In-liquid frequency modulation atomic force microscopy (FM-AFM) has been used for visualizing subnanometer-scale surface structures of minerals, organic thin films and biological systems. In addition, three-dimensional atomic force microscopy (3D-AFM) has been developed by combining it with a three-dimensional (3D) tip scanning method. This method enabled the visualization of 3D distributions of water (i.e. hydration structures) and flexible molecular chains at subnanometer-scale resolution. While these applications highlighted the unique capabilities of FM-AFM, its force resolution, speed and stability are not necessarily at a satisfactory level for practical applications. Recently, there have been significant advancements in these fundamental performances. The force resolution was dramatically improved by using a small cantilever, which enabled the imaging of a 3D hydration structure even in pure water and made it possible to directly compare experimental results with simulated ones. In addition, the improved force resolution allowed the enhancement of imaging speed without compromising spatial resolution. To achieve this goal, efforts have been made for improving bandwidth, resonance frequency and/or latency of various components, including a high-speed phase-locked loop (PLL) circuit. With these improvements, now atomic-resolution in-liquid FM-AFM imaging can be performed at ∼1 s/frame. Furthermore, a Si-coating method was found to improve stability and reproducibility of atomic-resolution imaging owing to formation of a stable hydration structure on a tip apex. These improvements have opened up new possibilities of atomic-scale studies on solid-liquid interfacial phenomena by in-liquid FM-AFM.

Subnanometer-scale imaging of nanobio-interfaces by frequency modulation atomic force microscopy.

Recently, there have been significant advancements in dynamic-mode atomic force microscopy (AFM) for biological applications. With frequency modulation AFM (FM-AFM), subnanometer-scale surface structures of biomolecules such as secondary structures of proteins, phosphate groups of DNAs, and lipid-ion complexes have been directly visualized. In addition, three-dimensional AFM (3D-AFM) has been developed by combining a high-resolution AFM technique with a 3D tip scanning method. This method enabled visualization of 3D distributions of water (i.e. hydration structures) with subnanometer-scale resolution on various biological molecules such as lipids, proteins, and DNAs. Furthermore, 3D-AFM also allows visualization of subnanometer-scale 3D distributions of flexible surface structures such as thermally fluctuating lipid headgroups. Such a direct local information at nano-bio interfaces can play a critical role in determining the atomic- or molecular-scale model to explain interfacial structures and functions. Here, we present an overview of these recent advancements in the dynamic-mode AFM techniques and their biological applications.

Atomic-Scale Three-Dimensional Local Solvation Structures of Ionic Liquids.

Room-temperature ionic liquids are promising media for next-generation energy devices because of their various superior characteristics. Because device performance is often dictated by the solvation structures at the solid-liquid interfaces, particularly at the local reactive sites, their atomistic pictures are in great demand. However, there has been no experimental technique for their three-dimensional solvation structures. Here, we first demonstrate the measurement of the atomic-scale ionic liquids using a recently established ultralow-noise three-dimensional frequency-modulation atomic force microscopy technique supported by molecular dynamics simulations. We conducted the experiments in protic and aprotic aqueous solutions and reveal that the aprotic solvation structure exhibits the higher site specificity, which resolves atomic-scale surface charge distribution on mica because of the absence of the H-bonding network. Our methodology is also applicable to pure liquids and would be a breakthrough for expanding their future applications.

Hydration layers at the graphite-water interface: Attraction or confinement

Water molecules at solid surfaces typically arrange in layers. The physical origin of the hydration layers is usually explained by two different reasons: (1) the attraction between the surface and water and (2) the water confinement due to the surface. While the attraction is specific to the particular solid, the confinement is a general property of surfaces; a differentiation between the two effects is, therefore, critical for research on interactions at aqueous interfaces. Here, we investigate the graphite-water interface, which is a widely used model system where the solid-water attraction is often considered to be negligible. Similar to previous studies, we observe hydration layers using three-dimensional atomic force microscopy at the graphite-water interface. We explain why the confinement could cause the formation of hydration layers even in the absence of attraction between surface and water by employing Monte Carlo simulations. Using additional molecular dynamics simulations, we continue to show that at ambient conditions, however, the confinement alone does not cause the formation of layers at the graphite-water interface. We thereby demonstrate that there is a significant graphite-water attraction.