Using Knock-Out Mutants to Investigate the Adhesion of Staphylococcus aureus to Abiotic Surfaces

The adhesion of Staphylococcus aureus to abiotic surfaces is crucial for establishing device-related infections. With a high number of single-cell force spectroscopy measurements with genetically modified S. aureus cells, this study provides insights into the adhesion process of the pathogen to abiotic surfaces of different wettability. Our results show that S. aureus utilizes different cell wall molecules and interaction mechanisms when binding to hydrophobic and hydrophilic surfaces. We found that covalently bound cell wall proteins strongly interact with hydrophobic substrates, while their contribution to the overall adhesion force is smaller on hydrophilic substrates. Teichoic acids promote adhesion to hydrophobic surfaces as well as to hydrophilic surfaces. This, however, is to a lesser extent. An interplay of electrostatic effects of charges and protein composition on bacterial surfaces is predominant on hydrophilic surfaces, while it is overshadowed on hydrophobic surfaces by the influence of the high number of binding proteins. Our results can help to design new models of bacterial adhesion and may be used to interpret the adhesion of other microorganisms with similar surface properties.     

原子力显微镜(AFM)在石英薄片表面形貌分析中的应用

介绍了研究石英中杂质赋存状态的重要性以及原子力显微镜在石英表面分析中的应用,结果通过原子力显微镜我们能够看到石英表面颗粒分布均匀,结构致密,颗粒没有大尺度的起伏等表面结构,并且利用它高的分辨率得到了石英表面纳米级别的微观形貌,为后续的确定杂质赋存状态做了准备。     

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.     

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.     

The Effect of Ethanol on Disassembly of Amyloid-β1-42 Pentamer Revealed by Atomic Force Microscopy and Gel Electrophoresis

The most common type of dementia, Alzheimer’s disease, is associated with senile plaques formed by the filamentous aggregation of hydrophobic amyloid-β (Aβ) in the brains of patients. Small oligomeric assemblies also occur and drugs and chemical compounds that can interact with such assemblies have attracted much attention. However, these compounds need to be solubilized in appropriate solvents, such as ethanol, which may also destabilize their protein structures. As the impact of ethanol on oligomeric Aβ assembly is unknown, we investigated the effect of various concentrations of ethanol (0 to 7.2 M) on Aβ pentameric assemblies (Aβp) by combining blue native-PAGE (BN-PAGE) and ambient air atomic force microscopy (AFM). This approach was proven to be very convenient and reliable for the quantitative analysis of Aβ assembly. The Gaussian analysis of the height histogram obtained from the AFM images was correlated with band intensity on BN-PAGE for the quantitative estimation of Aβp. Our observations indicated up to 1.4 M (8.3%) of added ethanol can be used as a solvent/vehicle without quantitatively affecting Aβ pentamer stability. Higher concentration induced significant destabilization of Aβp and eventually resulted in the complete disassembly of Aβp.     

Mechanical Investigations on Agar Gels Using Atomic Force Microscopy: Effect of Deuteration

The isotopic effect of exchanging deuterium with hydrogen on the mechanical and surface properties of agar gel is examined. The elastic modulus of the D₂O gels obtained by AFM nanoindentation is significantly higher (factor of ≈1.5–2) than the modulus found in H₂O agar gels. Furthermore, the modulus is independent of loading rate. Surface imaging reveals that the surface roughness gets progressively smaller with increasing agar concentration. All these data suggest that the isotopic replacement of deuterium enhances the mechanical properties of the agar gel, with significant advantages in its use as a biphasic scaffold.

     

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.     

用原子力显微镜观察菁染料聚集体

对应用原子力显微镜观察菁染料聚集体的文献进行了评述 ,在此基础上采用原子力显微镜观察了 2个感绿增感染料的聚集体     

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.     

原子力显微镜研究星形SEBS聚集体的表面形态变化

采用原子力显微镜观察不同条件下星型苯乙烯-乙烯-丁烯-苯乙烯嵌段共聚物（简称SEBS）的甲苯溶液旋涂所得膜的表面形态,发现SEBS溶液质量浓度对膜表面形态起支配作用,随着SEBS质量浓度的增加,聚集体结构由单个大分子线团变为小球、二维网络以及连续膜;不同溶度参数的溶剂可以改变表面聚集体的尺寸,随着溶度参数的降低,旋涂膜表面聚集体的高度不断下降,溶剂更易渗透至聚合物大分子链间;随着热处理温度的提高,聚集体向完美小球颗粒转化;超声波处理后立即旋涂所得的膜表面形态上会出现大量气泡和气孔。对比相同条件下,线型SEBS与星型SEBS所得的AFM图可以发现,星型SEBS由于空间位阻的影响,分子链运动能力差,所以较线型SEBS聚集态的尺寸小。     

Mass Spectrometric Identification of Proteins Enhanced by the Atomic Force Microscopy Immobilization Surface

An approach to highly-sensitive mass spectrometry detection of proteins after surface-enhanced concentrating has been elaborated. The approach is based on a combination of mass spectrometry and atomic force microscopy to detect target proteins. (1) Background: For this purpose, a technique for preliminary preparation of molecular relief surfaces formed as a result of a chemical or biospecific concentration of proteins from solution was developed and tested on several types of chip surfaces. (2) Methods: mass spectrometric identification of proteins using trailing detectors: ion trap, time of flight, orbital trap, and triple quadrupole. We used the electrospray type of ionization and matrix-assisted laser desorption/ionization. (3) Results: It is shown that when using locally functionalized atomically smooth surfaces, the sensitivity of the mass spectrometric method increases by two orders of magnitude as compared with measurements in solution. Conclusions: It has been demonstrated that the effective concentration of target proteins on specially prepared surfaces increases the concentration sensitivity of mass spectrometric detectors—time-of-flight, ion trap, triple quadrupole, and orbital ion trap in the concentration range from up to 10⁻¹⁵ M.     

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.     

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.     

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.     

Determining Surface Potential of the Bitumen‐Water Interface at Nanoscale Resolution using Atomic Force Microscopy

Atomic force microscopy (AFM) was used to measure the surface forces between a silicon nitride AFM tip and a deposited layer of Athabasca bitumen; the measurements were carried out in pure water (pH 6.0–6.5) and 1 mM KCl solution (pH 9). An AFM pyramidal‐shaped tip was moved stepwise using an operator‐controlled offset (10 nm per step) and the tip‐bitumen colloidal forces were measured at each location. Surface charge densities at the bitumen‐water interface were calculated from the measured colloidal forces using a theoretical model that combined both electrostatic and van der Waals forces for a conical tip‐flat substrate system. Fitted values of the bitumen surface charge density ranged from –0.002 to –0.004 C/m² in water (pH 6.0–6.5), and –0.005 to –0.022 C/m² in KCl solution (pH 9); the variation of local charge density along the bitumen surface appeared random. Bitumen surface potentials were also calculated from the surface charge densities using the Graham equation; the values ranged from –90 to –130 mV in water, and –45 to –110 mV in KCl solution. This study suggests the presence of bitumen surface domains of different surface charge densities/surface potentials. The domains are estimated to have characteristic sizes of 20 to 40 nm or less.     

Molecular Recognition of Surface Trans-Sialidases in Extracellular Vesicles of the Parasite Trypanosoma cruzi Using Atomic Force Microscopy (AFM)

Trans-sialidases (TS) are important constitutive macromolecules of the secretome present on the surface of Trypanosoma cruzi (T. cruzi) that play a central role as a virulence factor in Chagas disease. These enzymes have been related to infectivity, escape from immune surveillance and pathogenesis exhibited by this protozoan parasite. In this work, atomic force microscopy (AFM)-based single molecule-force spectroscopy is implemented as a suitable technique for the detection and location of functional TS on the surface of extracellular vesicles (EVs) released by tissue-culture cell-derived trypomastigotes (Ex-TcT). For that purpose, AFM cantilevers with functionalized tips bearing the anti-TS monoclonal antibody mAb 39 as a sense biomolecule are engineered using a covalent chemical ligation based on vinyl sulfonate click chemistry; a reliable, simple and efficient methodology for the molecular recognition of TS using the antibody-antigen interaction. Measurements of the breakdown forces between anti-TS mAb 39 antibodies and EVs performed to elucidate adhesion and forces involved in the recognition events demonstrate that EVs isolated from tissue-culture cell-derived trypomastigotes of T. cruzi are enriched in TS. Additionally, a mapping of the TS binding sites with submicrometer-scale resolution is provided. This work represents the first AFM-based molecular recognition study of Ex-TcT using an antibody-tethered AFM probe.     

Lipid Self-Assemblies under the Atomic Force Microscope

Lipid model membranes are important tools in the study of biophysical processes such as lipid self-assembly and lipid–lipid interactions in cell membranes. The use of model systems to adequate and modulate complexity helps in the understanding of many events that occur in cellular membranes, that exhibit a wide variety of components, including lipids of different subfamilies (e.g., phospholipids, sphingolipids, sterols…), in addition to proteins and sugars. The capacity of lipids to segregate by themselves into different phases at the nanoscale (nanodomains) is an intriguing feature that is yet to be fully characterized in vivo due to the proposed transient nature of these domains in living systems. Model lipid membranes, instead, have the advantage of (usually) greater phase stability, together with the possibility of fully controlling the system lipid composition. Atomic force microscopy (AFM) is a powerful tool to detect the presence of meso- and nanodomains in a lipid membrane. It also allows the direct quantification of nanomechanical resistance in each phase present. In this review, we explore the main kinds of lipid assemblies used as model membranes and describe AFM experiments on model membranes. In addition, we discuss how these assemblies have extended our knowledge of membrane biophysics over the last two decades, particularly in issues related to the variability of different model membranes and the impact of supports/cytoskeleton on lipid behavior, such as segregated domain size or bilayer leaflet uncoupling.     

Antibacterial Peptide NP-6 Affects Staphylococcus aureus by Multiple Modes of Action

Our previous study extracted and identified an antibacterial peptide that was named NP-6. Herein, we investigated the physicochemical properties of NP-6, and elucidated the mechanisms underlying its antimicrobial activity against Staphylococcus aureus. The results showed that the hemolysis activity of NP-6 was 2.39 ± 0.13%, lower than Nisin A (3.91 ± 0.43%) at the same concentration (512 µg/mL). Negligible cytotoxicity towards RAW264.7 cells was found when the concentration of NP-6 was lower than 512 µg/mL. In addition, it could keep most of its activity in fetal bovine serum. Moreover, transmission electron microscopy, confocal laser scanning microscopy, and flow cytometry results showed that NP-6 can destroy the integrity of the bacterial cell membrane and increase the membrane permeability. Meanwhile, NP-6 had binding activity with bacterial DNA and RNA in vitro and strongly inhibited the intracellular β-galactosidase activity of S. aureus. Our findings suggest that NP-6 could be a promising candidate against S. aureus.     

Reconsideration of Dynamic Force Spectroscopy Analysis of Streptavidin-Biotin Interactions

To understand and design molecular functions on the basis of molecular recognition processes, the microscopic probing of the energy landscapes of individual interactions in a molecular complex and their dependence on the surrounding conditions is of great importance. Dynamic force spectroscopy (DFS) is a technique that enables us to study the interaction between molecules at the single-molecule level. However, the obtained results differ among previous studies, which is considered to be caused by the differences in the measurement conditions. We have developed an atomic force microscopy technique that enables the precise analysis of molecular interactions on the basis of DFS. After verifying the performance of this technique, we carried out measurements to determine the landscapes of streptavidin-biotin interactions. The obtained results showed good agreement with theoretical predictions. Lifetimes were also well analyzed. Using a combination of cross-linkers and the atomic force microscope that we developed, site-selective measurement was carried out, and the steps involved in bonding due to microscopic interactions are discussed using the results obtained by site-selective analysis.