Atomic Force Microscopy Reveals Complexity Underlying General Secretory System Activity

The translocation of specific polypeptide chains across membranes is an essential activity for all life forms. The main components of the general secretory (Sec) system of E. coli include integral membrane translocon SecYEG, peripheral ATPase SecA, and SecDF, an ancillary complex that enhances polypeptide secretion by coupling translocation to proton motive force. Atomic force microscopy (AFM), a single-molecule imaging technique, is well suited to unmask complex, asynchronous molecular activities of membrane-associated proteins including those comprising the Sec apparatus. Using AFM, the dynamic structure of membrane-external protein topography of Sec system components can be directly visualized with high spatial-temporal precision. This mini-review is focused on AFM imaging of the Sec system in near-native fluid conditions where activity can be maintained and biochemically verified. Angstrom-scale conformational changes of SecYEG are reported on 100 ms timescales in fluid lipid bilayers. The association of SecA with SecYEG, forming membrane-bound SecYEG/SecA translocases, is directly visualized. Recent work showing topographical aspects of the translocation process that vary with precursor species is also discussed. The data suggests that the Sec system does not employ a single translocation mechanism. We posit that differences in the spatial frequency distribution of hydrophobic content within precursor sequences may be a determining factor in mechanism selection. Precise AFM investigations of active translocases are poised to advance our currently vague understanding of the complicated macromolecular movements underlying protein export across membranes.     

Energetic Material/Polymer Interaction Studied by Atomic Force Microscopy

The interactions of energetic materials and polymers have important implications in safety, long‐term storage, and performance of explosives and explosive mixtures. Atomic force microscopy was used to investigate adhesion forces at the molecular scale of nine energetic materials, organic explosives and energetic salts, on eleven common polymers (polyethylene, polyvinylalcohol, poly(vinyl chloride), polycarbonate, polystyrene, poly(methyl methacrylate), styrene‐butadiene rubber, poly(4‐vinyl phenol), poly(2,6‐dimethylphenylene oxide), poly(2,6‐diphenyl‐p‐phenylene oxide) (Tenax®), and polytetrafluoroethylene (Teflon®)). Teflon was the least adhesive polymer to all energetic materials, while no distinct trend could be elucidated among the other polymers or energetic materials.     

Preparation of DOPC and DPPC Supported Planar Lipid Bilayers for Atomic Force Microscopy and Atomic Force Spectroscopy

Cell membranes are typically very complex, consisting of a multitude of different lipids and proteins. Supported lipid bilayers are widely used as model systems to study biological membranes. Atomic force microscopy and force spectroscopy techniques are nanoscale methods that are successfully used to study supported lipid bilayers. These methods, especially force spectroscopy, require the reliable preparation of supported lipid bilayers with extended coverage. The unreliability and a lack of a complete understanding of the vesicle fusion process though have held back progress in this promising field. We document here robust protocols for the formation of fluid phase DOPC and gel phase DPPC bilayers on mica. Insights into the most crucial experimental parameters and a comparison between DOPC and DPPC preparation are presented. Finally, we demonstrate force spectroscopy measurements on DOPC surfaces and measure rupture forces and bilayer depths that agree well with X-ray diffraction data. We also believe our approach to decomposing the force-distance curves into depth sub-components provides a more reliable method for characterising the depth of fluid phase lipid bilayers, particularly in comparison with typical image analysis approaches.     

Nanomechanical Atomic Force Microscopy to Probe Cellular Microplastics Uptake and Distribution

The concerns regarding microplastics and nanoplastics pollution stimulate studies on the uptake and biodistribution of these emerging pollutants in vitro. Atomic force microscopy in nanomechanical PeakForce Tapping mode was used here to visualise the uptake and distribution of polystyrene spherical microplastics in human skin fibroblast. Particles down to 500 nm were imaged in whole fixed cells, the nanomechanical characterization allowed for differentiation between internalized and surface attached plastics. This study opens new avenues in microplastics toxicity research.     

Atomic Force Microscopy as a Characterization Tool for Contact Lenses: Indentation Tests and Grain Analysis

Nondestructive methods for testing mechanical properties of soft contact lenses allow quality control and testing to be performed on same sample by considering various parameters. A novel alternative to conventional mechanical test technique for contact lenses is presented implementing atomic force microscopy (AFM) and force spectroscopy (FS) technique as pico-indentation and mechanical characterization tool. This technique gives nN force resolution with producing nm range elastic deformation. FS and AFM topography results are evaluated simultaneously to compare mechanical and topographical properties of contact lenses such as Young's modulus, grain size evaluation, surface roughness, and adhesion force. Mechanical properties of soft contact lenses were reported by considering equilibrium water content of contact lens.     

Interfacial Interpretation of Autohesion of Ethylene/1-Octene Copolymers by Atomic Force Microscopy

The T-peel fractured surfaces of bonded films of ethylene/1-octene copolymers with different 1-octene contents were characterized using atomic force microscopy (AFM) and analyzed by fractal analysis. The AFM images showed strong dependence on the bonding temperature, peel rate, and the 1-octene content visually. This dependence has been demonstrated quantitatively by the fractal analyses which quantified an irregular surface by fractal dimensions and characteristic sizes. Two regimes showing fractal features were identified for each surface. In Regime I (higher magnifications) the welding and the following T-peel fracture procedures did little to change the fractal dimensions compared with the original surfaces before welding. But there were significant changes in Regime II (lower magnification) before welding and after T-peel fracture tests. The length scale that separated these two regimes is of the same order as that of polyethylene lamellar crystal structures. This suggests that the amorphous chains interdiffused across the interface while unmelted interfacial crystal structures remain essentially unaltered during the autohesion process. A “stitch-welding” autohesion mechanism was proposed to describe the bonding process in which only chains in the amorphous portions could interdiffuse. During the T-peel fracture tests, a crystal structure on the interface is either pulled over to the other side of the interface due to the interdiffused chains, remains unchanged, or is used as an anchor to pull a crystal structure from the other side of the interface. The characteristic sizes at which the fractal characteristics emerge were shown to be larger for the surfaces fractured at higher peel rates, which corresponds to higher fracture energy. This suggests that the appearance of fractal behavior at larger scales requires higher fracture energies. The characteristic sizes and fractal dimensions were also shown to depend on the molecular structure.     

Neuron Biomechanics Probed by Atomic Force Microscopy

Mechanical interactions play a key role in many processes associated with neuronal growth and development. Over the last few years there has been significant progress in our understanding of the role played by the substrate stiffness in neuronal growth, of the cell-substrate adhesion forces, of the generation of traction forces during axonal elongation, and of the relationships between the neuron soma elastic properties and its health. The particular capabilities of the Atomic Force Microscope (AFM), such as high spatial resolution, high degree of control over the magnitude and orientation of the applied forces, minimal sample damage, and the ability to image and interact with cells in physiologically relevant conditions make this technique particularly suitable for measuring mechanical properties of living neuronal cells. This article reviews recent advances on using the AFM for studying neuronal biomechanics, provides an overview about the state-of-the-art measurements, and suggests directions for future applications.     

Interfacial Interpretation of Autohesion of Ethylene/1-Octene Copolymers by Atomic Force Microscopy

The T-peel fractured surfaces of bonded films of ethylene/1-octene copolymers with different 1-octene contents were characterized using atomic force microscopy (AFM) and analyzed by fractal analysis. The AFM images showed strong dependence on the bonding temperature, peel rate, and the 1-octene content visually. This dependence has been demonstrated quantitatively by the fractal analyses which quantified an irregular surface by fractal dimensions and characteristic sizes. Two regimes showing fractal features were identified for each surface. In Regime I (higher magnifications) the welding and the following T-peel fracture procedures did little to change the fractal dimensions compared with the original surfaces before welding. But there were significant changes in Regime II (lower magnification) before welding and after T-peel fracture tests. The length scale that separated these two regimes is of the same order as that of polyethylene lamellar crystal structures. This suggests that the amorphous chains interdiffused across the interface while unmelted interfacial crystal structures remain essentially unaltered during the autohesion process. A “stitch-welding” autohesion mechanism was proposed to describe the bonding process in which only chains in the amorphous portions could interdiffuse. During the T-peel fracture tests, a crystal structure on the interface is either pulled over to the other side of the interface due to the interdiffused chains, remains unchanged, or is used as an anchor to pull a crystal structure from the other side of the interface. The characteristic sizes at which the fractal characteristics emerge were shown to be larger for the surfaces fractured at higher peel rates, which corresponds to higher fracture energy. This suggests that the appearance of fractal behavior at larger scales requires higher fracture energies. The characteristic sizes and fractal dimensions were also shown to depend on the molecular structure.     

The Use of Atomic Force Microscopy in Investigating Particle Caking Mechanisms

Spray‐dried materials are being used increasingly in industries such as food, detergent and pharmaceutical manufacture. Spray‐dried sodium carbonate is an important product that has a great propensity to cake; its moisture‐sorption properties are very different to the crystalline and amorphous species, with a great affinity for atmospheric moisture. This work demonstrates how the noncontact surface analysis of individual particles using atomic force microscopy can highlight the possible mechanisms of unwanted agglomeration. The nondestructive nature of this method allows cycling of localised humidity in situ and repeated scanning of the same particle area. The resulting topography and phase scans showed that humidity cycling caused changes in the distribution of material phases that were not solely dependent on topographical changes.     

Maximum Force Technique for the Measurement of the Surface Tension of a Small Droplet by AFM

An atomic force microscope (AFM) is used to measure the meniscus force on a vertical quartz rod as the rod is pulled through an air/liquid interface. A fluid bridge forms between the liquid and the base of the rod as the rod is withdrawn from the liquid. The force reaches a maximum as the bridge necks down and finally detaches from the rod. The maximum force on the rod is independent of the material of the rod and can be used to calculate the surface tension of the liquid. Alternately, if the surface tension of the liquid is known, the maximum force of the meniscus can be used to calibrate the spring constant of the AFM cantilever. The contact angle of the liquid on the rod was calculated as the rod was inserted into the liquid droplet. Contact angle hysteresis was observed. Results are presented of the measurement of the meniscus force of water, 10^?3 M cetyl trimethyl ammonium bromide (CTAB) and tetradecane as the rod is withdrawn from the liquid.     

Maximum Force Technique for the Measurement of the Surface Tension of a Small Droplet by AFM

An atomic force microscope (AFM) is used to measure the meniscus force on a vertical quartz rod as the rod is pulled through an air/liquid interface. A fluid bridge forms between the liquid and the base of the rod as the rod is withdrawn from the liquid. The force reaches a maximum as the bridge necks down and finally detaches from the rod. The maximum force on the rod is independent of the material of the rod and can be used to calculate the surface tension of the liquid. Alternately, if the surface tension of the liquid is known, the maximum force of the meniscus can be used to calibrate the spring constant of the AFM cantilever. The contact angle of the liquid on the rod was calculated as the rod was inserted into the liquid droplet. Contact angle hysteresis was observed. Results are presented of the measurement of the meniscus force of water, 10^-3 M cetyl trimethyl ammonium bromide (CTAB) and tetradecane as the rod is withdrawn from the liquid.     

Atomic Force Microscopy (AFM) Applications in Arrhythmogenic Cardiomyopathy

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disorder characterized by progressive replacement of cardiomyocytes by fibrofatty tissue, ventricular dilatation, cardiac dysfunction, arrhythmias, and sudden cardiac death. Interest in molecular biomechanics for these disorders is constantly growing. Atomic force microscopy (AFM) is a well-established technic to study the mechanobiology of biological samples under physiological and pathological conditions at the cellular scale. However, a review which described all the different data that can be obtained using the AFM (cell elasticity, adhesion behavior, viscoelasticity, beating force, and frequency) is still missing. In this review, we will discuss several techniques that highlight the potential of AFM to be used as a tool for assessing the biomechanics involved in ACM. Indeed, analysis of genetically mutated cells with AFM reveal abnormalities of the cytoskeleton, cell membrane structures, and defects of contractility. The higher the Young’s modulus, the stiffer the cell, and it is well known that abnormal tissue stiffness is symptomatic of a range of diseases. The cell beating force and frequency provide information during the depolarization and repolarization phases, complementary to cell electrophysiology (calcium imaging, MEA, patch clamp). In addition, original data is also presented to emphasize the unique potential of AFM as a tool to assess fibrosis in cardiac tissue.     

Martensitic Relief Observation by Atomic Force Microscopy in Yttria-Stabilized Zirconia

The tetragonal to monoclinic (t–m) phase transformation of zirconia has been the object of extensive investigations of the past 20 years and is now recognized as being of martensitic nature. However, martensitic transformation has only been observed by transmission electron microscopy or indirect methods. Though the benefit on the fracture toughness and crack resistance was the main interest, the transformation is now considered for its consequences on the degradation of the material. The use of atomic force microscopy reported here allowed the observation of the first stages of martensite relief growth and of new martensitic features.     

Eukaryotic CRFK Cells Motion Characterized with Atomic Force Microscopy

We performed a time-lapse imaging with atomic force microscopy (AFM) of the motion of eukaryotic CRFK (Crandell-Rees Feline Kidney) cells adhered onto a glass surface and anchored to other cells in culture medium at 37 °C. The main finding is a gradient in the spring constant of the actomyosin cortex along the cells axis. The rigidity increases at the rear of the cells during motion. This observation as well as a dramatic decrease of the volume suggests that cells may organize a dissymmetry in the skeleton network to expulse water and drive actively the rear edge.     

Probing Specific Interaction Forces Between Human IgG and Rat Anti-Human IgG by Self-Assembled Monolayer and Atomic Force Microscopy

Interaction forces between biological molecules such as antigen and antibody play important roles in many biological processes, but probing these forces remains technically challenging. Here, we investigated the specific interaction and unbinding forces between human IgG and rat anti-human IgG using self assembled monolayer (SAM) method for sample preparation and atomic force microscopy (AFM) for interaction force measurement. The specific interaction force between human IgG and rat anti-human IgG was found to be 0.6–1.0 nN, and the force required for unbinding a single pair of human IgG and rat anti-human IgG was calculated to be 144 ± 11 pN. The results are consistent with those reported in the literatures. Therefore, SAM for sample preparation combined with AFM for interaction measurement is a relatively simple, sensitive and reliable technique to probe specific interactions between biological molecules such as antigen and antibody.     

Adhesion Maps Using Scanning Force Microscopy Techniques

A technique has been developed that allows a real-time measurement of the lift-off force required to remove a scanning force microscope tip from a substrate. Both topography and adhesion maps are obtained simultaneously, allowing the correlation between topography and adhesion properties to be studied. Quantitative values of important adhesion parameters can be extracted from these data. A number of examples are given which illustrate the utility of this technique.     

Extended defects in natural diamonds: An Atomic Force Microscopy investigation

Surfaces of natural diamonds etched in high-pressure experiments in H₂O, CO₂ and H₂O–NaCl fluids were investigated using Atomic Force Microscopy. Partial dissolution of the crystals produced several types of surface features including the well-known trigons and hillocks and revealed several new types of defects. The most remarkable ones are assigned to twins of several types. The observation of abundant microtwins, ordering of hillocks, and presence of defects presumably related to knots of branched dislocations suggests importance of post-growth deformation events on formation of diamond microstructure. This work confirms previous reports of ordering of extended defects in some deformed diamonds. In addition, the current study shows that natural diamonds deform not only by slip, but also by mechanical twinning. The dominant mechanism should depend on pressure–temperature–stress conditions during diamond transport from the formation domain to the Earth surface.     

Resolving Intra- and Inter-Molecular Structure with Non-Contact Atomic Force Microscopy

A major challenge in molecular investigations at surfaces has been to image individual molecules, and the assemblies they form, with single-bond resolution. Scanning probe microscopy, with its exceptionally high resolution, is ideally suited to this goal. With the introduction of methods exploiting molecularly-terminated tips, where the apex of the probe is, for example, terminated with a single CO, Xe or H₂ molecule, scanning probe methods can now achieve higher resolution than ever before. In this review, some of the landmark results related to attaining intramolecular resolution with non-contact atomic force microscopy (NC-AFM) are summarised before focussing on recent reports probing molecular assemblies where apparent intermolecular features have been observed. Several groups have now highlighted the critical role that flexure in the tip-sample junction plays in producing the exceptionally sharp images of both intra- and apparent inter-molecular structure. In the latter case, the features have been identified as imaging artefacts, rather than real intermolecular bonds. This review discusses the potential for NC-AFM to provide exceptional resolution of supramolecular assemblies stabilised via a variety of intermolecular forces and highlights the potential challenges and pitfalls involved in interpreting bonding interactions.     

原子力显微镜在纳米材料表面形貌及粒度的研究

在探测纳米尺寸上、分子水平上的表面形貌,原子力显微镜是最先进的测试工具之一。文章主要是在室温大气条件下,用原子力显微镜分别对液相化学沉淀法制备的羟基磷灰石（HAP）粉体,压片后在马福炉中用不同的温度和时间烧结,得到不同烧结状态的样品表面进行扫描成像,并用原子力显微镜分析软件分析发现羟基磷灰石样品颗粒的平均尺寸随温度升高而增大的变化趋势。并用RISE-2008型激光粒度分析仪测样品颗粒粒度作对比测试印证原子力显微镜测试结果的可靠性。