原子力显微镜在高分子研究中的应用

原子力显微镜(AFM)作为一种新型纳米显微技术,具有分辨率高、样本制作方便等优点,在高分子研究中获得广泛的应用,本文综述了其在高分子研究领域中的一些应用进展。     

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.     

Atomic Force Microscope Techniques for Adhesion Measurements

The Atomic Force Microscope (AFM) has become a powerful apparatus for performing real-time, quantitative force measurements between materials. Recently the AFM has been used to measure adhesive interactions between probes placed on the AFM cantilever and sample surfaces. This article reviews progress in this area of adhesion measurement, and describes a new technique (Jump Mode) for obtaining adhesion maps of surfaces. Jump mode has the advantage of producing fast, quantitative adhesion maps with minimal memory usage.     

Atomic Force Microscope Techniques for Adhesion Measurements

The Atomic Force Microscope (AFM) has become a powerful apparatus for performing real-time, quantitative force measurements between materials. Recently the AFM has been used to measure adhesive interactions between probes placed on the AFM cantilever and sample surfaces. This article reviews progress in this area of adhesion measurement, and describes a new technique (Jump Mode) for obtaining adhesion maps of surfaces. Jump mode has the advantage of producing fast, quantitative adhesion maps with minimal memory usage.     

原子力显微镜在晶体生长机理研究中的应用

长期以来,晶体生长机理的研究大多是间接的理论分析。原子力显微镜具有原子、分子级分辨率且能在溶液等环境下工作,为我们提供了一个直接观测研究晶体生长界面过程的全新有效的工具。观察和研究的结果表明：关于光滑界面生长动力学的BCF模型、关于杂质对台阶生长阻碍作用的C-V模型等已受到冲击与挑战;关于晶面结构各向异性对晶体生长影响的研究已有新的内容;生物大分子晶体生长机理的研究面临极好的发展机遇。借助于原子力显微镜的观察研究工作,晶体生长理论可望有新的突破。     

Interfacially polymerized nanofiltration membranes: Atomic force microscopy and salt rejection studies

Interfacial polymerization is one of the main techniques for producing composite nanofiltration (NF) membranes. In this study, five NF membranes were produced through interfacial polymerization under different conditions of reactions, namely varying reaction time, as well as monomer concentrations. The membranes were then imaged using atomic force microscope (AFM). AFM images provided information of the average pore size, pore size distribution, and surface roughness. For some of the membranes, discrete pore sizes were visible. Increasing the reaction time resulted in decreasing water permeabilities but based on AFM imaging the pore size was of similar value. Increasing the monomer concentration also resulted in decreasing water permeabilities. However, based on AFM imaging the pore size differs considerably. Additional permeation experiments were also carried out using NaCl and Na₂SO₄ solutions with membranes identified as NF. By fitting the rejection data using a model such as the Donnan‐steric‐pore model, the variation in effective charge density of the membranes was also determined. The ability to tailor composite NF membranes with the right properties will significantly improve membrane performance. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 605–612, 2005     

Atomic force microscopy of cellulose membranes prepared from the N‐methylmorpholine‐N‐oxide/water solvent system

Cellulose membranes were obtained by solutions of cellulose being cast into a mixture of N‐methylmorpholine‐N‐oxide (NMMO) and water under different processing conditions. Atomic force microscopy (AFM) was used to investigate the surface structures of the membranes. The AFM method provided information on both the size and shape of the pores on the surface, as well as the roughness of the skin, through a computerized analysis of AFM micrographs. The results obtained showed that the surface morphologies were intrinsically associated with the permeation properties. For the cellulose membranes, increasing the NMMO concentration and the temperature of the coagulation bath led to higher fluxes and lower bovine serum albumin rejection. These were always correlated with higher values of the roughness parameters and larger pore sizes of the membrane surfaces. When the cellulose concentration of the casting solution was 11 wt %, the membrane showed a nodular structure with interconnected cavity channels between the agglomerated nodules. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 3389–3395, 2002     

AFM and Multiple Transmission-Reflection Infrared Spectroscopy (MTR-IR) Studies on Formation of Air-Stable Supported Lipid Bilayers

Supported lipid bilayers (SLBs) were prepared by deposition of unilamellar vesicles on a silicon substrate. Atomic force microscopy (AFM) and a new Multiple Transmission-Reflection Infrared Spectroscopy (MTR-IR) developed by us were used to trace the dynamic formation of lipid bilayers on the silicon surfaces. The evolution from deformation of vesicles to formation of bilayers can be distinguished clearly by AFM imaging. MTR-IR provided high quality infrared spectra of ultrathin lipid bilayers with high sensitivity and high signal to noise ratio (SNR). The structural and orientational changes during vesicle’s fusion were monitored with MTR-IR. MTR-IR shows superiority over other infrared approaches for ultrathin films on standard silicon wafers in view of its economy and high sensitivity. Both MTR-IR and AFM results were consistent with each other and they provided more information for understanding the self-assembling procedure of SLBs.     

Dynamic Assembly/Disassembly of Staphylococcus aureus FtsZ Visualized by High-Speed Atomic Force Microscopy

FtsZ is a key protein in bacterial cell division and is assembled into filamentous architectures. FtsZ filaments are thought to regulate bacterial cell division and have been investigated using many types of imaging techniques such as atomic force microscopy (AFM), but the time scale of the method was too long to trace the filament formation process. Development of high-speed AFM enables us to achieve sub-second time resolution and visualize the formation and dissociation process of FtsZ filaments. The analysis of the growth and dissociation rates of the C-terminal truncated FtsZ (FtsZt) filaments indicate the net growth and dissociation of FtsZt filaments in the growth and dissociation conditions, respectively. We also analyzed the curvatures of the full-length FtsZ (FtsZf) and FtsZt filaments, and the comparative analysis indicated the straight-shape preference of the FtsZt filaments than those of FtsZf. These findings provide insights into the fundamental dynamic behavior of FtsZ protofilaments and bacterial cell division.     

A New Tool for Adhesion Science: The Atomic Force Microscope

The recently-developed Atomic Force Microscope (AFM) has been used to study the problem of adhesive failure in an elastomer/glass system. Polymeric residues have been obtained in various conditions by peeling a polyurethane film from glass plates. Their observation with nanometric resolution reveals structures which have not been detected by the usual techniques. We present typical images which emphasize the importance of the nature of the substrate and the ageing conditions. The results are discussed in relation to the nature of the interphase and the failure mechanism. Furthermore, a particular mode of use of AFM is described to illustrate the potential of this technique as a locally-resolved spectroscopic tool.     

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.     

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.

     

Nanoscale Indentation of Polymer Systems Using the Atomic Force Microscope

The use of the atomic force microscope (AFM) to measure surface forces has been developed to optimize its operation as a surface imaging tool. This capability can potentially be extended to evaluate nanoscale material response to indentation and would be ideal for the evaluation of multi-component polymer systems, such as adhesives and composites. In this paper, previous work related to the development of the AFM as a nanoindentation device is reviewed, and a technique is proposed which allows the AFM to be used to probe local stiffness changes in polymer systems. Cantilever probes with spring constants ranging from 0.4-150 N m were used to investigate a number of polymer systems, including an elastomer, several polyurethane systems, thermally cured epoxies, a thermoplastic polymer-thermosetting polymer adhesive system, and a thermoplastic matrix composite.     

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.     

Orientation of the OmpF Porin in Planar Lipid Bilayers

The outer‐membrane protein OmpF is an abundant trimeric general diffusion porin that plays a central role in the transport of antibiotics and colicins across the outer membrane of E. coli. Individual OmpF trimers in planar lipid bilayers (PLBs) show one of two current–voltage asymmetries, thus implying that insertion occurs with either the periplasmic or the extracellular end first. A method for establishing the orientation of OmpF in PLB was developed, based on targeted covalent modification with membrane‐impermeant reagents of peripheral cysteine residues introduced near the periplasmic or the extracellular entrance. By correlating the results of the modification experiments with measurements of current asymmetry or the sidedness of binding of the antibiotic enrofloxacin, OmpF orientation could be quickly determined in subsequent experiments under a variety of conditions. Our work will allow the precise interpretation of past and future studies of antibiotic permeation and protein translocation through OmpF and related porins.     

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.     

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.     

Tailoring the Surface Chemistry of PEDOT:PSS to Promote Supported Lipid Bilayer Formation

This communication reports on a versatile and substrate-agnostic method to tune the surface chemistry of conducting polymers with the aim of bridging the chemical mismatch between bioelectronic devices and biological systems. As a proof of concept, the surface of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) is grafted with a short-chain oligoethylene glycol monolayer to favor the formation of cell-derived supported lipid bilayers (SLBs). This method is tuned to optimize the affinity between the supported lipid bilayer and the conducting polymer, leading to significant improvements in bilayer quality and therefore electronic readouts. To validate the impact of surface functionalization on the system's ability to transduce biological phenomena into quantifiable electronic signals, the activity of a virus commonly used as a surrogate for SARS-CoV-2 (mouse hepatitis virus) is monitored with and without surface treatment. The functionalized devices exhibit significant improvements in electronic output, stemming from the improved SLB quality, therefore strengthening the case for the use of such an approach in membrane-on-a-chip systems.