用原子力显微镜研究阿莫西林对沙门氏菌和单核增生性李斯特菌的抗菌活性

目的:探测阿莫西林作用于沙门氏菌(G)和单核增生性李斯特菌(G~+)后2种菌体的形貌和生物力学特性的变化,探讨阿莫西林的抗菌活性和抗菌机理。方法:通过平板菌落计数法测细菌的失活率,利用原子力显微镜(AFM)对药物作用后细菌的表面形貌及细胞的硬度、粘附力做定性和定量分析。结果:平板菌落计数得,25μg/mL的阿莫西林作用1h后,沙门氏菌的失活率较李斯特菌的失活率大。AFM测量显示,与低浓度阿莫西林作用后,沙门细菌表面出现孔洞,而李斯特菌表面出现裂缝,力曲线测量显示,药物作用后针尖和细胞壁之间的粘附力明显增加,而杨氏模量(E)显著降低。结论:结合AFM图像可知形貌与生物力学特性的变化反映细胞壁的变化,细胞壁的成分由均一性变为异质性从而导致细菌的粘附力增加即F_(native)E_(amoxicillin)通过以上分析进一步探讨阿莫西林的杀菌机理和不同的细菌对阿莫西林的敏感程度。这些AFM数据为阿莫西林的临床应用提供可视化的数据支持。     

猪笼草蜡质滑移区表面反粘附特性的研究

利用环境扫描电子显微镜(ESEM)和原子力显微镜(AFM)表征红瓶猪笼草蜡质滑移区表面微观形貌,并提取粗糙度的相关参数。利用AFM分别在低载荷和高载荷下对蜡质区表面同一区域进行扫描,在不同条件下的扫描形貌一致,且扫描后的探针针尖上未发现附着污染物。利用胶体探针技术在无针尖的探针悬臂上粘附15 μm SiO2小球,模拟单根刚毛与猪笼草蜡质区表面的接触,并测试蜡质区表面的粘附力和摩擦力,并与不同粗糙度的抛光纸表面做对照。考虑到表面物理化学性质对其粘附特性的重要影响,利用接触角测量仪测量蜡质区表面和同粗糙度范围抛光纸表面对水和二碘甲烷的表观接触角并利用二滴法计算其表面能。研究结果表明：蜡质滑移区表面单个蜡质晶体具有力学稳定性,不会因脱落而污染昆虫的粘附器官,污染学说不成立;表面微粗糙度能有效地减小界面间的接触面积,降低了蜡质滑移区表面的粘附力和摩擦力;蜡质滑移区超疏水特性和低表面能是降低表面粘附力和摩擦力的另一个重要因素,两者共同作用形成了猪笼草蜡质滑移区的反粘附特性。     

非共振轻敲模式原子力显微镜的研究

原子力显微镜(AFM)是在微纳尺度观测以及操纵样品的重要工具。与传统的接触模式和轻敲模式AFM相比,非共振轻敲模式AFM因为其控制力精度高并可同步获取多种机械特性等优势得到了广泛的应用。采用基于背景相减和同步算法相结合的方法搭建了一套自制的非共振轻敲模式AFM,并对位置检测电路建立了通用噪声仿真模型,优化了位置检测电路噪声,从而优化了最小可控力的精度,使其最小可控力小于50 pN。接着通过对标准硅材料栅格进行形貌表征验证了系统的成像性能,以及对复合材料的形貌、粘附力、形变等多种力学性质表征,验证了系统及成像方法的有效性。     

考虑摩擦力及试样表面倾角的原子力显微镜扫描模式

原子力显微镜(Atomic force microscopes,AFM)接触模式下的测量结果因受样本表面倾角和针尖一样本表面间摩擦力的影响而存在较大的测量误差.为避免针尖-表面间的摩擦力对AFM测量试样表面形貌的影响,并能够准确测量表面倾角,提出了一种新的AFM工作模式--消除倾角和摩擦力影响模式.在这种工作模式中,扫描方向垂直悬臂的长轴方向,通过测量悬臂的竖向和横向偏转而得到针尖所受的竖向和横向力,并计算得到针尖-试样表面间的van der Waals力及试样表面局部倾角,然后结合针尖项点和扫描器的位置及针尖-试样表面间距可以得到试样表面形貌的测量结果.在上述工作模式下,针尖-试样表面间的摩擦力是可控的,能够避免针尖或试样的损伤.仿真结果证明了这种方法的可行性.     

硫醇自组装膜在金表面吸附行为的AFM力曲线研究

本文用原子力显微镜（AFM）对硫醇自组装金电极表面随机取点测得力曲线，并对力曲线粘附力数据进行统计分析，结果表明随自组装液中硫醇浓度改变，粘附力特征呈现出关联性的变化。力曲线所揭示的自组装膜吸附行为的变化规律和传统电化学方法交流阻抗（EIS）测试所得结论一致，表明AFM力曲线技术可应用于自组膜吸附行为的研究。     

AFM针尖"突跳"研究

为了研究原子力显微镜（AFM）“突跳”现象的产生机理，基于经典弹性理论和Lennard-Jones势能定律建立了AFM针尖与样品纳米接触的弹性模型。给出了在AFM针尖逐渐趋近样品表面的过程中，AFM针尖与样品间的粘着力、样品表面的轮廓曲线和样品表面的变形量随AFM针尖与样品表面间距的变化规律。分析了AFM“突跳”现象的产生机理和影响因素。研究表明，AFM“突跳”现象主要是由样品表面在粘着引力的作用下产生拉伸变形并与AFM针尖“突跳”接触引起的。     

原子力显微镜在生物分子力学性质方面的研究

原子力显微镜在生物纳米研究领域有广泛应用,包括对生物样品的形貌成像、超微结构、机械性能和相互作用等方面的研究。利用其非修饰和修饰探针进行样品扫描,可以得到样品表面形貌和样品表面某一特定点的力与距离的关系曲线,从而得到相关生物分子的力学性质。目前国际上应用原子力显微镜对生物分子力学特性方面的研究已经成为最热门的研究课题之一,在生物医学和临床医学方面有重要研究意义。本文简述了原子力显微镜的力曲线原理,并对近年来应用原子力显微镜在探测生物分子力学性质方面的研究进展进行了综述。     

单细胞结构与功能的原子力显微镜研究

首先强调了研究单细胞的生物学意义;介绍了原子力显微镜作为一种显微探测和操纵工具的主要特点及其在生命科学特别是在单细胞研究中的巨大优势.回顾了国内外有关AFM在单细胞表面形态与结构、活细胞的力学响应和生理过程的监控、细胞间粘附力的测量和肿瘤的科学诊断等研究中的应用情况;并结合自己在肿瘤细胞形态研究中的经验和体会对AFM在细胞研究中的深入研究进行了探讨.     

钛合金表面红细胞吸附特性AFM研究

采用原子力显微镜(AFM)观察吸附在钛合金基底上的红细胞表面形貌,研究红细胞、钛合金与探针之间的微摩擦力,对比分析红细胞表面和钛合金表面同探针之间的微摩擦力-距离关系曲线,及红细胞破损前后的探针与红细胞的微摩擦力-距离关系曲线。结果表明:在外力作用下,红细胞不仅细胞表面形貌受影响,而且其黏性增大,摩擦力增大,影响润滑效果;因红细胞表面柔润粗糙,且探针在活细胞表面所测得的微摩擦力为多种微观力的合力,从而使红细胞表面的摩擦力较大。     

类金刚石薄膜纳米摩擦性能的试验研究

利用摩擦力显微镜(FFM),对由等离子体增强化学气相法沉积的类金刚石(DLC)薄膜的纳米摩擦性能进行了试验研究。用原子力显微镜(AFM)观察了DLC薄膜样品的表面形貌,同时测定了其粘附力值。从外加载荷、扫描速度和湿度的角度分析了薄膜的摩擦特性。     

Probing molecular interactions and mechanical properties of microbial cell surfaces by atomic force microscopy

Knowledge of the surface properties of microbial cells is a key to gain a detailed understanding of their functions in the natural environment and to efficiently exploit them in biotechnological processes. In this paper, we present force-distance curves recorded, by atomic force microscopy (AFM) in aqueous solutions, on various microbial samples: reconstituted S-layers, whole fungal spores and several bacterial strains. The approach and retraction curves exhibited important differences--depending on the type of microorganism, on the physiological state (dormancy versus germination) and on the environmental conditions (ionic strength)--which were shown to reflect differences in long-range surface forces, adhesion forces and mechanical properties. These data illustrate the great potential of AFM force measurements to elucidate the physical properties of microbial cells and to understand, at the molecular level, biointerfacial phenomena such as cell adhesion and cell aggregation.     

Artesunate induced morphological and mechanical changes of Jurkat cell studied by AFM

In this study, we have used atomic force microscopy (AFM) to study the morphology and mechanical property changes of Jurkat cells exposed to different concentrations of Artesunate (ART) for 24 h at single cellular level. Cell viability and proliferation assays were performed by using the Cell Counting Kit‐8. The concentration of ART, which resulted in the inhibition rate >50% was selected. The AFM images revealed that the cell membrane changed and the ultrastructure also became complex. Mechanical properties of individual cell were tracked with AFM‐based force spectroscopy. The force curves revealed that when a cell was exposed to the ART, the mechanical properties changed obviously. Treated cells had a lower adhesion force of 416.8±37.9 pN, whereas control group had a higher adhesion force of 1064.2±97.0 pN. The Young's modulus decreased to nearly one‐third, from control group of 0.648±0.037 kPa to treated group of 0.254±0.035 kPa and the stiffness increased to nearly 1.5 times, from control group of 1.231±0.084 mN/m to treated group of 1.917±0.137 mN/m. These results suggest that ART can inhibit the proliferation of Jurkat and induce changes in the morphological structure and mechanical properties of Jurkat cells. The high resolution and high sensitivity of AFM can be used to detect morphological and mechanical properties of cells exposed to ART. The AFM may be developed to be a useful tool for detecting the cell death and evaluating the anti‐carcinogen efficacy against tumor cell. SCANNING 31: 83–89, 2009. © 2009 Wiley Periodicals, Inc.     

Morphology, mechanical properties and viability of encapsulated cells

The morphological and mechanical properties of encapsulated yeast cells (Saccharomyces cerevisiae) have been investigated by atomic force microscopy (AFM). Single living cells have been coated through the alternate deposition of oppositely charged polyelectrolyte (PE) layers. The properties of cells coated by different numbers of PE layers and from PE solutions of different ionic strength have been investigated. AFM imaging indicates an increase in PE coating stability when decreasing the solution ionic strength. The Young's moduli of the different examined systems have been evaluated through a quantitative analysis of force-distance curves by using the Hertz-Sneddon model. The analysis indicates an increase in hybrid system stiffness when lowering the ionic strength of the PE solution. An evaluation of the viability of encapsulated cells was obtained by confocal laser scanning microscopy (CLSM) measurements. CLSM analysis indicates that cells preserve their subcellular structure and duplication capability after encapsulation. By coupling AFM and CLSM data, a correlation between local stiffness and duplication rate was obtained.     

Drift-free atomic force microscopy measurements of cell height and mechanical properties

Atomic force microscopy (AFM) is used to study the morphological and mechanical properties of living cells. However, experiments performed over minutes to hours are subject to significant instrumental drift. The main sources of drift are the cantilever's geometrical asymmetry and bimorphic construction. We developed a simple software Stick-and-Move (SaM) routine for AFM that eliminates drift by continuously referencing the sample position to the substrate while acquiring force-distance curves. Control experiments show no drift over 15 min at an acquisition rate of 0.1 Hz. As a proof of concept, we applied the SaM to study the response of rat astrocytes to osmotic stress, observing dimensional and constitutive changes during volume regulation.     

Ultrastructural investigation of intact orbital implant surfaces using atomic force microscopy

This study examined the surface nanostructures of three orbital implants: nonporous poly(methyl methacrylate) (PMMA), porous aluminum oxide and porous polyethylene. The morphological characteristics of the orbital implants surfaces were observed by atomic force microscopy (AFM). The AFM topography, phase shift and deflection images of the intact implant samples were obtained. The surface of the nonporous PMMA implant showed severe scratches and debris. The surface of the aluminum oxide implant showed a porous structure with varying densities and sizes. The PMMA implant showed nodule nanostructures, 215.56 ± 52.34 nm in size, and the aluminum oxide implant showed crystal structures, 730.22 ± 341.02 nm in size. The nonporous PMMA implant showed the lowest roughness compared with other implant biomaterials, followed by the porous aluminum oxide implant. The porous polyethylene implant showed the highest roughness and severe surface irregularities. Overall, the surface roughness of orbital implants might be associated with the rate of complications and cell adhesion. SCANNING 33: 211–221, 2011. © 2011 Wiley Periodicals, Inc.     

Investigation of integrin expression on the surface of osteoblast-like cells by atomic force microscopy

The transforming growth factor β1 (TGF-β1) is a human cytokine which has been demonstrated to modulate cell surface integrin repertoire. In this work integrin expression in response to TGF-β1 stimulation has been investigated on the surface of human osteoblast-like cells. We used atomic force microscopy (AFM) and confocal laser scanning microscopy to assess integrin expression and to evaluate their distribution over the dorsal side of the plasma membrane. AFM probes have been covalently functionalised with monoclonal antibodies specific to the β1 integrin subunit. Force curves have been collected in order to obtain maps of the interaction between the immobilized antibody and the respective cell membrane receptors. Adhesion peaks have been automatically detected by means of an ad hoc developed data analysis software. The specificity of the detected interactions has been assessed by adding free antibody in the solution and monitoring the dramatic decrease in the recorded interactions. In addition, the effect of TGF-β1 treatment on both the fluorescence signal and the adhesion events has been tested. The level of expression of the β1 integrin subunit was enhanced by TGF-β1. As a further analysis, the adhesion force of the single living cells to the substrate was measured by laterally pushing the cell with the AFM tip and measuring the force necessary to displace it. The treatment with TGF-β1 resulted in a decrease of the cell/substrate adhesion force. Results obtained by AFM have been validated by confocal laser scanning microscopy thus demonstrating the high potential of the AFM technique for the investigation of cell surface receptors distribution and trafficking at the nanoscale.     

Atomic force microscope imaging and force measurements at electrified and actively corroding interfaces: challenges and novel cell design

We report the design of an improved electrochemical cell for atomic force microscope measurements in corrosive electrochemical environments. Our design improvements are guided by experimental requirements for studying corrosive reactions such as selective dissolution, dealloying, pitting corrosion, and∕or surface and interface forces at electrified interfaces. Our aim is to examine some of the limitations of typical electrochemical scanning probe microscopy (SPM) experiments and in particular to outline precautions and cell-design elements, which must necessarily be taken into account in order to obtain reliable experimental results. In particular, we discuss electrochemical requirements for typical electrochemical SPM experiments and introduce novel design features to avoid common issues such as crevice formations; we discuss the choice of electrodes and contaminations from ions of reference electrodes. We optimize the cell geometry and introduce standard samples for electrochemical AFM experiments. We have tested the novel design by performing force-distance spectroscopy as a function of the applied electrochemical potential between a bare gold electrode surface and a SAM-coated AFM tip. Topography imaging was tested by studying the well-known dealloying process of a Cu(3)Au(111) surface up to the critical potential. Our design improvements should be equally applicable to in situ electrochemical scanning tunneling microscope cells.     

Nanostructural analysis by atomic force microscopy followed by light microscopy on the same archival slide

Integrated information on ultrastructural surface texture and chemistry increasingly plays a role in the biomedical sciences. Light microscopy provides access to biochemical data by the application of dyes. Ultrastructural representation of the surface structure of tissues, cells, or macromolecules can be obtained by scanning electron microscopy (SEM). However, SEM often requires gold or coal coating of biological samples, which makes a combined examination by light microscopy and SEM difficult. Conventional histochemical staining methods are not easily applicable to biological material subsequent to such treatment. Atomic force microscopy (AFM) gives access to surface textures down to ultrastructural dimensions without previous coating of the sample. A combination of AFM with conventional histochemical staining protocols for light microscopy on a single slide is therefore presented. Unstained cores were examined using AFM (tapping mode) and subsequently stained histochemically. The images obtained by AFM were compared with the results of histochemistry. AFM technology did not interfere with any of the histochemical staining protocols. Ultrastructurally analyzed regions could be identified in light microscopy and histochemical properties of ultrastructurally determined regions could be seen. AFM-generated ultrastructural information with subsequent staining gives way to novel findings in the biomedical sciences. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Membrane deformation of living glial cells using atomic force microscopy

Using atomic force microscopy (AFM) it has been possible to detect actin filaments that are beneath the cell membrane of living cells despite the fact that the AFM tip is applied to the surface of the cell. To determine whether the AFM tip actually penetrates or deforms the cell membrane we determined whether an intracellularly trapped fluorescent indicator was lost from cells during AFM. Using epi-fluorescence illumination to monitor the presence of fluo-3 in the cell, we found that AFM did not cause dye leakage from the cell. Further, force–distance curves indicated that standard tips did not penetrate the membrane while sharper Supertips^TM did. In addition, the physiology of cells was found to be unaffected by AFM with standard tips since volume regulatory signal transduction mechanisms were intact in such studies. Thus, traditional AFM tips deform the cell membrane in order to reveal the presence of subcellular structures.     

Substrate influence on cell shape and cell mechanics: HepG2 cells spread on positively charged surfaces

A human hepatoma cell line (HepG2) was cultured on positively and negatively charged polyelectrolytes. Cell/surface adhesion and cell shape evolution were followed with quartz microbalance with dissipation (QCM‐D) and optical microscopy as a function of time, respectively. In particular, substrates coated with poly(ethyleneimine) (PEI) led to fast cell attachment and further spreading, with average maximum frequency Δf = 79 Hz and dissipation ΔD = 40 × 10^?6. On the contrary, no cell spreading was observed on poly(sodium‐4‐styrenesulfonate) (PSS), with Δf = 33 Hz and ΔD = 4.5 × 10^?6. Atomic force microscopy (AFM) was used to investigate the influence of cell shape on its mechanical properties. Considering the cells as an homogenous solid material, the corresponding elastic modulus was estimated using the Hertz model. The elastic modulus was calculated at the central part of the cell, and the average values obtained were 191 ± 14 Pa and 941 ± 58 Pa for cells adsorbed on PSS and PEI, respectively. Thus, different cell–substrate interaction implied different cell mechanical properties reflected in a higher elastic modulus for stronger cell/substrate interaction. The combination of QCM‐D, AFM, and optical microscopy allowed the online study of the cell adhesion process, and the mechanical properties of the adhered cells. Microsc. Res. Tech. 2009. © 2009 Wiley‐Liss, Inc.