Mounting of Escherichia coli spheroplasts for AFM imaging

The cytoplasmic membrane of Escherichia coli (E. coli) is the location of numerous, chemically specific transporters and recognition elements. Investigation of this membrane in vivo by atomic force microscopy (AFM) requires removal of the cell wall and stable immobilization of the spheroplast. AFM images demonstrate that spheroplasts can be secured with warm gelatin applied to the mica substrate just before the addition of a spheroplast suspension. The resulting preparation can be repeatedly imaged by AFM over the course of several hours. Confocal fluorescence imaging confirms the association of the spheroplasts with the gelatin layer. Gelatin molecules are known to reorder into a network after heating. Entrapment within this gelatin network is believed to be responsible for the immobilization of spheroplasts on mica.     

Measuring cell surface elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM

Enteroaggregative Escherichia coli (EAEC) is pathogenic and produces severe diarrhea in humans. A mutant of EAEC that does not produce dispersin, a cell surface protein, is not pathogenic. It has been proposed that dispersin imparts a positive charge to the bacterial cell surface allowing the bacteria to colonize on the negatively charged intestinal mucosa. However, physical properties of the bacterial cell surface, such as rigidity, may be influenced by the presence of dispersin and may contribute to pathogenicity. Using the system developed in our laboratory for mounting and imaging bacterial cells by atomic force microscopy (AFM), in liquid, on gelatin coated mica surfaces, studies were initiated to measure cell surface elasticity. This was carried out in both wild type EAEC, that produces dispersin, and the mutant that does not produce dispersin. This was accomplished using AFM force-distance (FD) spectroscopy on the wild type and mutant grown in liquid or on solid medium. Images in liquid and in air of both the wild-type and mutant grown in liquid and on solid media are presented. This work represents an initial step in efforts to understand the pathogenic role of the dispersin protein in the wild-type bacteria.     

Vacuolar structures can be identified by AFM elasticity mapping

Fluid-filled organelles like vesicles, endosomes and pinosomes are inevitable parts of cellular signalling and transport. Endothelial cells, building a barrier between blood and tissue, can form vacuolar organelles. These structures are implicated in upregulated fluid transport across the endothelium under inflammatory conditions. Vacuolar organelles have been described by transmission electron microscopy so far. Here, we present a method that images and mechanically characterizes intracellular structures in whole cells by atomic force microscopy (AFM). After crosslinking the cellular proteins with the fixative glutaraldehyde, plasma membrane depressions become observable, which are scattered around the cell nucleus. Nanomechanical analysis identifies them as spots of reduced stiffness. Scanning electron microscopy confirms their pit-like appearance. In addition, fluorescence microscopy detects an analogous pattern of protein-poor spots, thereby confirming mechanical rigidity to arise from crosslinked proteins. This AFM application opens up a mechanical dimension for the investigation of intracellular organelles.     

Effect of crosslinking on the elasticity of polyelectrolyte multilayer films measured by colloidal probe AFM

A homemade colloidal probe atomic force microscope was used to perform nanoindentation with a spherical probe of 5 microm in diameter, at different approach velocities in order to extract the Young's modulus, E0, of poly(L-lysine)/hyaluronan (PLL/HA) films. This parameter is of prime importance to control cellular adhesion. The films were either kept in their native form or cross-linked with a mixture of 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) and N-hydrosulfosuccinimide (sulfo-NHS), where the EDC concentration was varied from 1 up to 100 mg mL(-1) (approximately from 5 to 500 mM). A model based on Hertz mechanics was used to account for the interactions between film and probe. It is shown that the Young's modulus varies with the approach velocity for the native (PLL/HA) films, whereas for cross-linked ones, E0 is independent from the velocity over the whole range investigated. It is found that for native films, E0 takes a value of 3 kPa at low approach velocities, a velocity domain that should be relevant in cellular adhesion processes. The Young's modulus increases with the EDC concentration used to cross-link the films and levels off at a value of about 400 kPa for EDC concentrations exceeding 40 mg mL(-1). Thus, it is possible by crosslinking PLL/HA films to control their elastic properties with the aim to alter their behavior as to the cellular adhesion.     

表面活性剂及复配在云母片上吸附的AFM研究

借助原子力显微镜对十二烷基硫酸钠(SDS)/十六烷基三甲基溴化铵(CTAB)及其复配溶液在云母表面的吸附以及吸附后不同的表面形貌和微摩擦特性进行了研究,探讨了摩擦力与表面形貌的关系。结果表明,SDS与CTAB表面活性剂分子都以纳米级颗粒吸附于云母表面;摩擦力受表面形貌的影响,但不成一一对应关系;在0.8和2倍临界胶束浓度(CMC)下,SDS/CTAB=1∶1复配溶液在云母表面吸附后的润滑效应较其它配比好。     

A new approach to the study of the E. coli nucleoid

E. coli were examined by the freeze-fracture thaw-fix technique, embedded in thin fibrin gels. After glutaraldehyde fixation the bacterial nucleoid was found spread out over the surrounding fibrin. Addition of calcium and uranyl acetate to the fixative preserved the nucleoid in compact form. The spread nucleoid was then examined against a smooth mica background after freeze-thaw and osmotic lysis. These spreads were critical-point dried, rotary shadowed with platinum–carbon and viewed as stereo-pair micrographs. Structures seen are tentatively interpreted as clusters of polyribosomes, extended DNA, and supercoiled DNA complexed with proteins or polyamines. After osmotic lysis, glutaraldehyde alone preserves the nucleoid in compact form. Only where strands are broken, in freeze-fracture or freeze-thaw lysis, must uranyl acetate be added to the fixative to preserve a compact structure.     

Nano-wear of the diamond AFM probing tip under scratching of silicon, studied by AFM

In order to improve such a widely used microtribological testing procedure as surface scratching by an AFM diamond tip, an experimental study has been carried out using single-crystalline silicon as the tested material. Wear of the AFM diamond tip under scratching was observed by a decrease in the scratch depth with increasing wear cycles and by the direct imaging of the diamond tip shape using a Si₃N₄ AFM tip. It was shown that the current widely used experimental method, which assumes the diamond tip to be non-wearable, introduces uncontrollable error into the obtained values for the tested material's wear rate. The harder the tested material, the larger may be the tip wear, and, therefore, the bigger may be its effect on the obtained wear rate values. The specific wear rates for the diamond tip and a silicon wafer were estimated to be 1.4 × 10^-9 and 4.5 × 10^-4 mm³/(N m), respectively.     

Lateral Displacement of an AFM Tip Observed by In-Situ TEM/AFM Combined Microscopy: The Effect of the Friction in AFM

When the lateral displacement of an AFM tip due to friction is comparable to or larger than the scan size, for example during atomic-scale friction measurement, the interpretation of the friction image is different from the situation where the scan size is much larger than the lateral displacement of the tip and the image is a simple direct mapping of the friction value. This is because, due to the lateral displacement of the tip, the tip is not at the position where the scan indicates, as can be clearly observed by an in-situ TEM/AFM combined microscopy and atomic-scale friction analysis. This lateral displacement of the tip at the nanometer scale affects the shape of the force-distance curve. We discuss the effect of the tip lateral displacement in AFM data and its normal load dependence.     

Shape Evaluation of HV-Indents by AFM

Vickers-type hardness patterns were investigated by atomic force microscopy. The resulting bitmap images were analyzed to evaluate the elastic relaxation of the impression. A procedure, which is suitable for the comparison of the shape of the residual hardness impression and that of the Vickers pyramid, is presented. With the help of difference images can be verified that the strongest relaxation takes place at the tip and at the horizontal edges of the Vickers pyramid.     

Calibration of tapping AFM cantilevers and uncertainty estimation: Comparison between different methods

We present a comparative study between three different methods for the spring constant calibration of silicon beam-shaped Atomic Force Microscope (AFM) cantilevers, used in tapping AFM mode in air. The geometries of these levers can be quite different from the standard rectangular cross section. We examine a method that combines the knowledge of cantilever dimensions and eigenfrequencies (Cleveland formula), the Sader method and we build cantilever models based on Finite Element Analysis (FEA). We demonstrate that with accurate measurement of dimensions, resonance frequency and quality factor, the Cleveland formula yields a combined cantilever stiffness uncertainty of approximately ±7% and the Sader method an uncertainty of ±5%. We also use FEA to show that when trying to approximate a realistic trapezoidal 3D tipped geometry, there exists a systematic overestimation in cantilever stiffness of ±2%, compared to when considering a simple rectangular cross section. Our constructed FE models are able to account for inhomogeneities in material properties as well as the influence of the added reflective coating in the cantilever stiffness estimation.     

Study of the sensitivity and resonant frequency of the torsional modes of an AFM cantilever with a sidewall probe based on a nonlocal elasticity theory

A relationship based on a nonlocal elasticity theory is developed to investigate the torsional sensitivity and resonant frequency of an atomic force microscope (AFM) with assembled cantilever probe (ACP). This ACP comprises a horizontal cantilever and a vertical extension, and a tip located at the free end of the extension, which makes the AFM capable of topography at sidewalls of microstructures. First, the governing differential equations of motion and boundary conditions for dynamic analysis are obtained by a combination of the basic equations of nonlocal elasticity theory and Hamilton's principle. Afterward, a closed‐form expression for the sensitivity of vibration modes has been obtained using the relationship between the resonant frequency and contact stiffness of cantilever and sample. These analysis accounts for a better representation of the torsional behavior of an AFM with sidewall probe where the small‐scale effect are significant. The results of the proposed model are compared with those of classical beam theory. The results show that the sensitivities and resonant frequencies of ACP predicted by the nonlocal elasticity theory are smaller than those obtained by the classical beam theory. Microsc. Res. Tech. 78:408–415, 2015. © 2015 Wiley Periodicals, Inc.     

Temperature-dependent imaging of living cells by AFM

Characterization of lateral organization of plasma membranes is a prerequisite to the understanding of membrane structure-function relationships in living cells. Lipid-lipid and lipid-protein interactions are responsible for the existence of various membrane microdomains involved in cell signalization and in numerous pathologies. Developing approaches for characterizing microdomains associate identification tools like recognition imaging with high-resolution topographical imaging. Membrane properties are markedly dependent on temperature. However, mesoscopic scale topographical information of cell surface in a temperature range covering most of cell biology experimentation is still lacking. In this work we have examined the possibility of imaging the temperature-dependent behavior of eukaryotic cells by atomic force microscopy (AFM). Our results establish that the surface of living CV1 kidney cells can be imaged by AFM, between 5 and 37 degrees C, both in contact and tapping modes. These first temperature-dependent data show that large cell structures appeared essentially stable at a microscopic scale. On the other hand, as shown by contact mode AFM, the surface was highly dynamic at a mesoscopic scale, with marked changes in apparent topography, friction, and deflection signals. When keeping the scanning conditions constant, a progressive loss in the image contrast was however observed, using tapping mode, on decreasing the temperature.     

检测纳米微粒粒径方法的研究

用光子相关法、原子力显微镜和扫描电镜三种测试方法测定了同一标准样品的粒径，比较了三种测试方法在纳米粒径检测方面的特点。光子相关法给出纳米微粒的平均粒径和多分散系数，而原子力显微镜和扫描电镜在测定粒径的同时直接观察到微粒的外貌。     

十二相AFM整流控制系统的组成及应用

介绍了全数字十二相AFM控制器的软、硬件组成情况，以及使用该控制器构成大功率直流电动机调速控制系统时的整体结构，由于控制器结构灵活，易于在原有系统上进行投资小、收效高的技术改造，目前已开始在工矿企业大量使用。     

用原子力显微镜观察水流处理后的DNA图案

原子力显微镜(Atomic force microscopy,AFM)由于自身的优势,在生物领域内应用越来越广泛。同时,DNA分子由于其稳定的物理化学性质而成为纳米领域的重要实验材料,近阶段把它作为模板应用在构建纳米线等方面的研究越来越多,而怎样建立有规则图样的DNA模板就成为一个关键问题。本文介绍用原子力显微镜观察液流操纵后的DNA规则图案。     

Comparison of different aminofunctionalization strategies for attachment of single antibodies to AFM cantilevers

Atomic force microscopy (AFM) has developed into a key technique for elucidation of biological systems on the single molecular level. In particular, molecular recognition force microscopy has proven to be a powerful tool for the investigation of biological interactions under near physiological conditions. For this purpose, ligands are tethered to AFM tips and the interaction forces with cognate receptors on the sample surface are measured with pico-Newton accuracy. In the first step of tip functionalization, amino groups are typically introduced on the initially inert AFM tip. Several methods have been developed to reproducibly adjust the desired low density of amino groups on the tip surface, i.e. esterification with ethanolamine, gas-phase silanization with aminopropyl-triethoxysilane (APTES), or treatment with aminophenyl-trimethoxysilane (APhS) in toluene solution. In the present study, the usefulness of these methods for attachments of antibodies to AFM tips was characterized by a standardized test system, in which biotinylated IgG was bound to the tip and a dense monolayer of avidin on mica served as test sample. All three methods of aminofunctionalization were found fully satisfactory for attachment of single antibodies to AFM tips, only in a parallel macroscopic assay on silicon nitride chips a minor difference was found in that APTES appeared to yield a slightly lower surface density of amino groups.     

Elasticity mapping of living fibroblasts by AFM and immunofluorescence observation of the cytoskeleton

Using the force mapping mode of atomic force microscopy (AFM), we measured spatial distribution of elastic moduli of living mouse fibroblasts (NIH3T3) in a physiological condition. The nuclear portion of the cellular surface is about 10 times softer than the surroundings. Stiffer fibers are confirmed in the elastic images. In order to investigate origin of the softer nuclear portion and the stiffer fibers, we fixed the identical cells imaged by the AFM, and carried out immunofluorescence observation for three types of cytoskeletal filaments--actin filaments, microtubules, and intermediate filaments, using confocal laser scanning microscopy (CLSM). A comparison between the AFM and the CLSM images revealed that the elasticity of the cells was concerned not only with the distribution of actin network, but also with intermediate filaments, whereas microtubules had no large effect on the measured elasticity.     

A constitutive model for transversely isotropic foams, and its application to the indentation of balsa wood

An elastic-plastic constitutive model for transversely isotropic compressible solids (foams) has been developed. A quadratic yield surface with four parameters and one hardening function is proposed. Associated plastic flow is assumed and the yield surface evolves in a self-similar manner calibrated by the uniaxial compressive (or tensile) response of the cellular solid in the axial direction. All material constants in the model (elastic and plastic) can be determined from a combination of a total of four uniaxial and shear tests. The model is used to predict the indentation response of balsa wood to a conical indenter. For the three cone angles considered in this study, very good agreement is found between the experimental measurements and the finite element (FE) predictions of the transversely isotropic cellular solid model. On the other hand, an isotropic foam model is shown to be inadequate to capture the indentation response.