Atomic force microscopy of model lipid membranes

Supported lipid bilayers (SLBs) are biomimetic model systems that are now widely used to address the biophysical and biochemical properties of biological membranes. Two main methods are usually employed to form SLBs: the transfer of two successive monolayers by Langmuir–Blodgett or Langmuir–Schaefer techniques, and the fusion of preformed lipid vesicles. The transfer of lipid films on flat solid substrates offers the possibility to apply a wide range of surface analytical techniques that are very sensitive. Among them, atomic force microscopy (AFM) has opened new opportunities for determining the nanoscale organization of SLBs under physiological conditions. In this review, we first focus on the different protocols generally employed to prepare SLBs. Then, we describe AFM studies on the nanoscale lateral organization and mechanical properties of SLBs. Lastly, we survey recent developments in the AFM monitoring of bilayer alteration, remodeling, or digestion, by incubation with exogenous agents such as drugs, proteins, peptides, and nanoparticles.

Investigation of the Effect of Thermal Annealing on Poly(3‐hexylthiophene) Nanofibers by Scanning Probe Microscopy: From Single‐Chain Conformation and Assembly Behavior to the Interfacial Interactions with Graphene Oxide

Poly(3‐hexylthiophene) (P3HT) has been widely used in devices owing to its excellent properties and structural features. However, devices based on pure P3HT have not exhibited high performance. Strategies, such as thermal annealing and surface doping, have been used to improve the electrical properties of P3HT. In this work, different from previous studies, the effect of thermal annealing on P3HT nanofibers are examined, ranging from the single polymer chain conformation to chain packing, and the interfacial interactions with graphene oxide (GO) at nanoscale dimensions, by using scanning tunneling microscopy (STM), atomic force microscopy (AFM) and Kelvin probe force microscopy (KPFM). High‐resolution STM images directly show the conformational changes of single polymer chains after thermal annealing. The morphology of P3HT nanofibers and the surface potential changes of the P3HT nanofibers and GO is further investigated by AFM and KPFM at the nanoscale, which demonstrate that the surface potentials of P3HT decrease, whereas that of GO increases after thermal annealing. All of the results demonstrate the stronger interfacial interactions between P3HT and GO occur after thermal treatments due to the changes in P3HT chain conformation and packing order.     

Electrostatic interactions in protein adsorption probed by comparing lysozyme and succinylated lysozyme

In this paper, we present a nanoscale study of the supramolecular structure of the dehydrogenate polymer (ZL-DHP) lignin model compound. The combination of near-field scanning optical microscopy (NSOM or SNOM) and atomic force microscopy (AFM) has been utilized to explore physicochemical properties of the lignin model compound on a scale ranging from individual macromolecules to globular supramolecular assemblies. By utilizing NSOM in transmission mode, the optical inhomogeneity in the lignin supramolecular structure has been observed for the first time. In particular, the transmission-mode NSOM images reveal a combination of hollow and layered supramolecular globular structure in the lignin model compound. Through the paired use of TappingMode and pulsed-mode AFM, we have also confirmed the existence of regions with different rheological properties on the single lignin model compound supramolecular assembly.     

Quantitative mapping of elastic moduli at the nanoscale in phase separated polyurethanes by AFM

The micro phase separated nanoscale morphology of phase separated polyurethanes (PUs) was visualized by atomic force microscopy (AFM) height and phase imaging of smooth surfaces obtained by ultramicrotonomy. PUs were obtained from 4,4′-methylenbis (phenyl isocyanate) (MDI), 1,4-butanediol (BD) and poly(tetrahydrofurane) polyether polyol (PTHF). The segmented polyether PUs with varying stoichiometric ratio of the isocyanate and hydroxyl groups were prepared to investigate the effect of molar mass, as well as the type and number of end-groups on their morphology and mechanical performance.The PU samples studied show characteristic “fingerprint” AFM phase images. Novel dynamic imaging modes of AFM, including HarmoniX material mapping and Peak Force Tapping were used to assess the mechanical performance of phase separated polyurethanes quantitatively as a function of their molecular structure. The values of surface elastic moduli were determined with nanoscale resolution and were in excellent agreement for both AFM modes. While tensile testing provides a bulk average value for the elastic modulus of the elastomers, the novel AFM based elastic moduli mappings introduced enable the study of surface stiffness with nanoscale resolution in a quantitative way.     

原子力显微镜研究高分子超薄膜结晶机理及功能化调控

近十年来,随着功能高分子单晶(含单层或寡层片晶)工程及应用研究的不断深入,除了纳米尺度结晶形貌的表征以外,多功能原子力显微镜还被用于研究分子结构、结晶条件和后处理条件对功能高分子晶体性能(电、热、光、磁等)的影响,进一步还可采用扫描探针加工技术(机械刻蚀、电致刻蚀和热致刻蚀等)对其性能进行调控以构筑功能化聚集态结构和微图案.另一方面,超薄膜中单层或寡层片晶可为研究高分子结晶提供合适的模型体系,与原子力显微镜相结合,不但可以原位、实空间、高分辨地研究高分子的成核与生长过程(生长形态演变和生长动力学),还可以用于研究亚稳态折叠链片晶厚度和形态随热处理温度与时间的演化,从而加深对片晶内有序差异、片晶增厚与熔融行为和自诱导成核的认识.     

AFM for nanoscale microbe analysis

The nanoscale surface analysis of microbial cells represents a significant challenge of current microbiology and is critical for developing new biotechnological and biomedical applications. Using atomic force microscopy (AFM) topographic imaging, researchers can visualize the ultrastructure of live cells under physiological conditions and their subtle modifications upon cell growth or treatment with drugs. Chemical force microscopy, in which AFM tips are modified with specific functional groups, allows investigators to measure molecular forces and chemical properties of cell surfaces on a scale of only 25 functional groups. Molecular recognition imaging using AFM offers a means to localize specific receptors on cells, such as cell adhesion proteins or antibiotic binding sites. With this Highlight on AFM, it is hoped that more and more microbiologists and biophysicists will take advantage of this powerful, multifunctional nanotechnique.     

Fabrication of polymeric replicas of cell surfaces with nanoscale resolution

We report an approach for fabricating biomimetic surface replicas of cells with nanoscale resolution. Fixed cells serve as a template for a two-stage replica molding process. Cast from the template, the impression replica contains a reproduction of cellular topographical features indented into its surface, and cast from the impression replica, the relief replica contains a copy of these features protruding from its surface. Various polymers and cells can be utilized, and scanning electron microscopy, atomic force microscopy, and white light interferometry analyses confirm the replication of nanoscale features. These replicas are useful for investigating cellular function and for biomimetic tissue engineering.     

Quantitative thermodynamic analysis of sublimation rates using an atomic force microscope

Atomic force microscopy (AFM) has been successfully used to study the activation energy for evaporation of pentaerythritol tetranitrate (PETN) nanoislands formed by spin coating. These islands are annealed isothermally in the temperature range of 30-70 degrees C for a given time and are scanned with AFM in contact mode at room temperature. The volume of these islands does not change significantly up to about 35-40 degrees C indicating that sublimation is not significant below 40 degrees C. Above 40 degrees C, the islands start shrinking, and the rate of weight loss is analyzed as a function of temperature. The activation energy of evaporation using AFM was found to be similar to that for bulk PETN crystals using thermogravimetric analysis (TGA) at higher temperatures (110-135 degrees C). These results demonstrate that AFM is a useful tool to measure thermodynamic properties with a nanoscale probe.     

Comparative study of nanoscale surface structures of calcite microcrystals using FE-SEM, AFM, and TEM.

The bulk morphology and surface features that developed upon precipitation on micrometer-size calcite powders and millimeter-size cleavage fragments were imaged by three different microscopic techniques: field-emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM) of Pt-C replicas, and atomic force microscopy (AFM). Each technique can resolve some nanoscale surface features, but they offer different ranges of magnification and dimensional resolutions. Because sample preparation and imaging is not constrained by crystal orientation, FE-SEM and TEM of Pt-C replicas are best suited to image the overall morphology of microcrystals. However, owing to the decoration effect of Pt-C on the crystal faces, TEM of Pt-C replicas is superior at resolving nanoscale surface structures, including the development of new faces and the different microtopography among nonequivalent faces in microcrystals, which cannot be revealed by FE-SEM. In conjunction with SEM, Pt-C replica provides the evidence that crystals grow in diverse and face-specific modes. The TEM imaging of Pt-C replicas has nanoscale resolution comparable to AFM. AFM yielded quantitative information (e.g., crystallographic orientation and height of steps) of microtopographic features. In contrast to Pt-C replicas and SEM providing three-dimensional images of the crystals, AFM can only image one individual cleavage or flat surface at a time.     

两性离子自组装仿生表面的制备、 表征及抗黏附性能

设计与合成了磺酸甜菜碱型的两性离子化合物: N,N-二甲基氨甲酸乙酯基丙基三乙氧基硅烷磺酸内盐(SiNNS), 利用红外光谱(FTIR)和氢核磁共振波谱(¹H NMR)对其分子组成与结构进行了表征. 通过自组装技术将SiNNS分子构筑在玻璃基材表面, 形成了模拟细胞外层膜的仿生表面. 利用原子力显微镜(AFM)、 X光电子能谱(XPS)和接触角测量仪对表面的形貌特征、 化学组成和润湿性进行了表征. 以空白玻璃为对照样品, 研究了这一表面的防雾性能和抗细菌黏附性能. 结果表明, 所制备的两性离子自组装仿生表面具有超亲水性和水下超疏油特性, 其水滴接触角为9.2°, 水下油滴接触角接近180°; 与对照样品相比, 两性离子自组装表面具有优异的防雾性与抗细菌黏附性.     

Patterning Porous Networks through Self-Assembly of Programmed Biomacromolecules

Two-dimensional (2D) porous networks are of great interest for the fabrication of complex organized functional materials for potential applications in nanotechnologies and nanoelectronics. This review aims at providing an overview of bottom-up approaches towards the engineering of 2D porous networks by using biomacromolecules, with a particular focus on nucleic acids and proteins. The first part illustrates how the advancements in DNA nanotechnology allowed for the attainment of complex ordered porous two-dimensional DNA nanostructures, thanks to a biomimetic approach based on DNA molecules self-assembly through specific hydrogen-bond base pairing. The second part focuses the attention on how polypeptides and proteins structural properties could be used to engineer organized networks templating the formation of multifunctional materials. The structural organization of all examples is discussed as revealed by scanning probe microscopy or transmission electron microscopy imaging techniques.     

Native surface structure of natural soil particles determined by combining atomic force microscopy and X-ray photoelectron spectroscopy

A procedure was developed for handling natural soil particles and probing their native surface structure by atomic force microscopy (AFM) under water. This procedure was used to investigate the nanometer scale organisation of organic matter at the surface of sand particles taken from three soil horizons. The latter were selected for the contrasted properties of their organic matter, namely Podzol E and Bh horizons and a Cambisol A–B horizon. The presence of an adsorbed layer was visualised at the surface of Podzol Bh and Cambisol particles in the form of aggregated structures that interacted with the AFM probe. Surface analysis by X-ray photoelectron spectrometry (XPS) confirmed the carbonaceous nature of this adsorbed layer. Displacement of organic matter by the scanning probe was directly evidenced for Podzol Bh sand particles. Such displacement was not observed for Cambisol particles. A dramatic effect of drying on the concentration, nanometer scale distribution and properties of the adsorbed organic matter was clearly demonstrated by combining AFM imaging and XPS analysis. The procedure developed here gives access to direct, nanoscale information of the surface structure of sand particles and offers promising prospects for the characterisation of other environmentally-relevant particles in native conditions.     

Nanoscale tripodal 1,3,5,7-tetrasubstituted adamantanes for AFM applications

The synthesis of four novel nanoscale 1,3,5,7-tetrasubstituted adamantanes 22 and 25-27 designed for atomic force microscopy (AFM) applications is described. Each tetrahedrally shaped molecule incorporates a broad tripodal base made up of three identical legs that terminate with a sulfur-containing moiety, which is either a 4-acetylsulfanylmethylphenyl unit or else a (1,2,5-dithiazepan-1-yl)phenyl unit. The sulfur atoms are intended for eventual binding of the molecule multivalently to the apex of a gold-coated commercial AFM tip through formation of multiple S-Au bonds. In each molecule, the fourth terminus is a para-substituted benzoic acid methyl ester that is designed to scan the sample. We demonstrate that 27 is sufficiently large and rigid to be imaged by a conventional AFM tip. Adamantanes 22 and 25-27 may also find application as chemically well-defined nanoscale objects for calibration of AFM tips.     

Correlating polymer structure,dynamics, and function with atomic force microscopy

Since its development, atomic force microscopy (AFM) has become an indispensable tool for investigating fundamental and technological applications of polymer materials. The versatility of AFM imaging modes and operating conditions allows for nanoscale characterization of a range of dynamic processes, such as crystallization, phase separation, self assembly, and electronic transport. Advances in AFM technology, particularly high-speed and high-resolution imaging, enable investigation of polymer structure, function, and dynamics in real world conditions and across a range of relevant spatial and temporal scales. In this perspective, we highlight a collection of recent polymer studies that utilize AFM to correlate the function and structure of polymer films, with focus on its multiparametric imaging capabilities. As the complexity of polymer materials and morphologies continues to increase, AFM is well poised to meet the accompanying demand for nanoscale imaging and characterization.     

AFM phase imaging of thin films of electronically conducting polymer polybithiophene prepared by electrochemical potentiodynamic deposition

Nanoscale properties of thin films of conducting polymer polybithiophene (PBT) deposited under potentiostatic and potentiodynamic conditions were compared using ex-situ atomic force microscopy (AFM) and its extension called phase imaging (PI-AFM). While the morphologies of the films prepared using the two techniques were quite similar, the phase contrast measurements revealed a profound difference in the mechanisms of potentiostatic and potentiodynamic electropolymerization, as well as in the nanoscale crystallinity and grain structure of the resulting polymer films. The overall crystallinity and degree of order were always higher for films deposited at constant potential. The differences were especially pronounced at the early deposition stages (film thicknesses of ca. 10 nm).     

Physical characteristics of Saccharomyces cerevisiae

We examined the physical properties of the surrounding yeast cell walls by using atomic force microscopy (AFM). The yeast cells were prepared on a cleaned glass substrate for confocal microscopy (CM) observation and were mechanically trapped into a porous membrane for AFM measurement. The confocal image of the yeast cells was measured in air, meanwhile the AFM topography images of the cells were measured in both deionized (DI) water (pH = 6.9) and phosphate‐buffered saline (PBS) solution (pH = 7.4). No significant differences between the AFM topography images of the yeast cells measured in DI water and in PBS solution could be inferred. In order to get the quantitative information on the sample elasticity, the force curves between an AFM tip and the yeast cell have been measured. These curves were measured in both DI water and in PBS solution on the same yeast cell using the same AFM cantilever to get the reliable result. The contact region of the force curve in approach mode was then converted into force versus indentation curve, which would be fitted with Hertz–Sneddon model for the calculation of the elasticity. Analysis of the curves indicates that there is a difference of the Young's modulus values of the yeast cell in various environments. These data show that the salt buffer solution increases the rigidity of the biological system. Copyright © 2008 John Wiley & Sons, Ltd.     

Effect of Compatibilizer and Nucleation Agent on the Properties of Poly(lactic acid)/Polycarbonate (PLA/PC) Blends

Poly(lactic acid) (PLA) and polycarbonate (PC) blends were prepared by melt processing with a twin-screw extruder. Ethylene-maleic anhydride-glycidyl methacrylate terpolymer (EMG) as compatibilizer and talc as nucleation agent were added in PLA/PC blends. The effect of EMG and talc on the mechanical properties including tensile, flexural, Izod notched impact properties and heat deflection temperature (HDT) of PLA/PC blends were investigated. The morphologies were observed by scanning electron microscopy (SEM). The crystalline behavior of PLA/PC blends was analyzed by differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The nanoscale mechanical properties of PLA/PC blends were investigated by atomic force microscope (AFM). The results showed that the addition of EMG and talc simultaneously with annealing treatment is the most effective process.     

Combined spectroscopic and topographic characterization of nanoscale domains and their distributions of a redox protein on bacterial cell surfaces

Redox protein nanoscale domains on the cell surface of a bacterium, Shewanella oneidensis MR1, grown in the absence and presence of electron acceptors, is topographically characterized using combined atomic force microscopy (AFM) and confocal surface enhanced Raman scattering (SERS) spectroscopy. The protruding nanoscale domains on the outer membrane of S. oneidensis were observed, as was their disappearance upon exposure to electron acceptors such as oxygen, nitrate, fumarate, and iron nitrilotriacetate (FeNTA). Using SERS spectroscopy, a redox heme protein was identified as a major component of the cell surface domains. This conclusion was further confirmed by the disappearance of Raman vibrational frequencies, characteristic of heme proteins, upon exposure of the cells to electron acceptors. Our experimental results from our AFM imaging and SERS spectroscopy, consistent with the literature, suggest the protruding nanoscale surface domains as heme-containing secretions. Our results on the distributions of redox proteins on microbial cell surfaces will be helpful for a mechanistic understanding of the behaviors of surface proteins and their interactions with redox environments.     

Extracellular matrix-like surfactant polymers containing arginine-glycine-aspartic acid (RGD) peptides

We report on a novel series of biomimetic polymers exhibiting interfacial properties similar to the extracellular matrix. A series of well-defined surfactant polymers were synthesized by simultaneously incorporating arginine-glycine-aspartic acid (RGD) peptide, dextran oligosaccharide, and hexyl ligands with controlled feed ratios onto a poly(vinyl amine) (PVAm) backbone. The peptide sequence was H-GSSSGRGDSPA-NH(2) (Pep) having a hydrophilic extender at the amino terminus and capped carboxy terminus. The peptide-to-dextran (Pep:Dex) ratios were varied to create surfactants having 0, 25, 50, 75, and 100 mol-% peptide relative to dextran. The surfactants were characterized by IR, NMR and atomic force microscopy (AFM) for composition and surface active properties. AFM confirmed full surface coverage of PVAm(Pep)(100%) on graphite, and supported the mechanism of interdigitation of hexyl ligands between surfactant molecules within a specified range of hexyl chain densities. the attachment and growth of human pulmonary artery endothelial cells on the PVAm(Pep)(100%) surface was identical to the fibronectin positive control. Cell adhesion decreased dramatically with decreasing peptide density on the surfactant polymers. Molecular model of a peptide surfactant polymer, consisting of poly(vinyl amine) backbone with peptide, dextran oligosaccharide and hexyl branches coupled to the polymer chain.