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.     

AFM Investigation on Ox‐LDL‐Induced Changes in Cell Spreading and Cell‐Surface Adhesion Property of Endothelial Cells

The integrity and adhesion properties of endothelium play vital roles during atherosclerosis. It is well known that oxidized low‐density lipoprotein (Ox‐LDL) influences many physiological activities or mechanical properties of endothelial cells. However, the effects of Ox‐LDL on the integrity and nonspecific adhesion properties of endothelial cells are still unclear. In this study, using the topographical imaging and force measurement functions of atomic force microscopy (AFM), we found that Ox‐LDL can transiently weaken the integrity of endothelium by impairing cell spreading of endothelial cells and decrease the attachment of irrelevant blood cells to endothelium by impairing the nonspecific adhesion property of endothelial cells. The AFM‐based data provide important information for understanding the effects of Ox‐LDL on endothelial cells or during atherogenesis. SCANNING 35: 119‐126, 2013. © 2012 Wiley Periodicals, Inc.     

Changes in the biomechanical properties of a single cell induced by nonthermal atmospheric pressure micro‐dielectric barrier discharge plasma

Mechanical properties of a single cell are closely related to the fate and functions of the cell. Changes in mechanical properties may cause diseases or cell apoptosis. Selective cytotoxic effects of nonthermal atmospheric pressure micro‐dielectric barrier discharge (DBD) plasma have been demonstrated on cancer cells. In this work, changes in the mechanical properties of a single cell induced by nonthermal atmospheric pressure micro‐DBD plasma were investigated using atomic force microscopy (AFM). Two cervical cancer cell lines (HeLa and SiHa) and normal human fibroblast cells (HFBs) were exposed to micro‐DBD plasma for various exposure times. The elasticity of a single cell was determined by force–distance curve measurement using AFM. Young's modulus was decreased by plasma treatment for all cells. The Young's modulus of plasma‐treated HeLa cells was decreased by 75% compared to nontreated HeLa cells. In SiHa cells and HFBs, elasticity was decreased slightly. Chemical changes induced by the plasma treatment, which were observed by Raman spectroscopy, were also significant in HeLa cells compared to SiHa cells and HFBs. These results suggested that the molecular changes induced by micro‐DBD plasma were related to cell mechanical changes.     

Toward a better modulus at shallow indentations—Enhanced tip and sample characterization for quantitative atomic force microscopy

Approximations of the geometry of indenting probes, particularly when using shallow indentations on soft materials, can lead to the erroneous reporting of mechanical data in atomic force microscopy (AFM). Scanning electron microscopy (SEM) identified a marked change in geometry toward the tip apex where the conical probe assumes a near linear flat-punch geometry. Polydimethylsiloxane (PDMS) is a ubiquitous elastomer within the materials and biological sciences. Its elastic modulus is widely characterized but the data are dispersed and can display orders of magnitude disparity. Herein, we compare the moduli gathered from a range of analytical techniques and relate these to the molecular architecture identified with AFM. We present a simple method that considers sub-100 nm indentations of PDMS using the Hertz and Sneddon contact mechanics models, and how this could be used to improve the output of shallow indentations on similarly soft materials, such as polymers or cells.     

Surface chemistry-mechanical property relationship of low density polyethylene: an IR+visible sum frequency generation spectroscopy and atomic force microscopy study

Surface-specific IR+visible sum frequency generation (SFG) spectroscopy was used to obtain chemical composition of two polymer surfaces. The SFG surface vibrational spectrum of pure low density polyethylene and that of a commercial sample of the same kind of polymer, which contains additives, are markedly different. This correlates well with the very different surface mechanical properties, i.e., stiffness (indicative of the elastic modulus) and friction, which were measured by atomic force microscopy (AFM) on the same polymer surfaces. The surface of CLDPE is dominated by methoxy (−OCH₃) contained additives, segregated from the bulk, which explains a lower stiffness, adhesion and friction of the surface, as measured by AFM. This revised version was published online in August 2006 with corrections to the Cover Date.     

The morphological and biomechanical changes of keratocytes cultured on modified p (HEMA‐MMA) hydrogel studied by AFM

The poor integration with host cornea tissue and the low mechanical properties of pHEMA hydrogel for artificial cornea remains a difficult problem to solve. A modified pHEMA hydrogel, MMA copolymerized and type‐I collagen and bFGF immobilized, was previously prepared in an attempt to solve the problems. In this study, the cytotoxicity of Col/bFGF‐p (HEMA‐MMA) and p (HEMA‐MMA) was studied by cell adhesion assay and atomic force microscopy (AFM). The results of cell adhesion assay show that the attachment of keratocytes on the modified membrane is much higher than that of the unmodified membrane. This indicates that the material after modification have better cell–material interaction. The AFM images reveal that the morphology of keratocytes cultured on different substrate is obviously different. The cell cultured on modified membrane presented a completely elongated and spindle‐shape morphology. The force?distance indicates that the biomechanical of keratocytes changes significantly after culturing on different substrates. The adhesion force (2328±523 pN) and Young's modulus (0.51±0.125 kPa) of the cell cultured on modified membrane are much higher, and the stiffness (0.08±0.022 mN/m) is lower than those of the cell cultured on unmodified membrane. These results show that the cytotoxicity of Col/bFGF‐p (HEMA‐MMA) for keratocytes is much improved. SCANNING 31: 246–252, 2009. © 2010 Wiley Periodicals, Inc.     

人脐静脉内皮细胞形貌与表面粘附力的原子力显微镜表征

目的:探讨原子力显微镜(AFM)在研究人脐静脉内皮细胞(ECV304)表面形貌、超微结构及纳米机械性质等方面的应用,讨论ECV304超微结构和机械性质与其功能的关系。方法:利用AFM对ECV304细胞的表面形貌及生物机械性质进行表征与测量。结果:在AFM下观察到用普通光学显微镜难以观察到的ECV304细胞的独特的形态结构,如细胞骨架、伪足及细胞边缘微丝等。ECV304细胞呈现长梭形、多角形、圆形等多种形态,细胞表面平均粗糙度为320.52±75.98 nm,表面均匀分布微绒毛,细胞周围有铺展的圆盘状物质。力曲线定量分析得出针尖与细胞表面的非特异性粘附力为75±14 pN。结论:通过AFM成像和力曲线测量表明,ECV304细胞呈圆形,多角形,梭形等多种形态,针尖与细胞膜表面问的粘附力比较小,约75±14pN。     

Mechanical properties of L929 cells measured by atomic force microscopy: Effects of anticytoskeletal drugs and membrane crosslinking

To shed light on the architecture of the cytoskeleton, we used the atomic force microscope (AFM) to measure the elasticity, viscoelasticity, and plasticity of L929 cells. The initial elastic response (Young's modulus ～ 4,000 Pa) of the cells to an applied force was followed by a slow compression of the cytoskeleton (τ_1/2 ≈ 10 s). When force application was terminated, the cytoskeleton underwent a sudden partial decompression and a subsequent slow, incomplete recovery. The role of the cytoskeletal elements in cell mechanics was accessed in AFM measurements carried out on cells treated with cytochalasin D, nocodazole, or col-cemid. Cytochalasin D treatment reduced both elasticity (～45%) and cytoplasmic viscosity (～65%), whereas cells treated with nocodazole or colcemid exhibited a marked increase in elasticity (～100%) and a slight increase in viscosity (～15%). The AFM force measurements also provided evidence that the cell membrane and the cytoskeleton are mechanically coupled. Tightly adherent cells were stiffer than cells that were loosely attached. Moreover, cells crosslinked with either glutaraldehyde, 3,3 ‘-dithiobis’sul-fosuccinimidylpropionate] (DTSSP), or Concanavalin A were more rigid than untreated cells. It is of interest that cells crosslinked with Concanavalin A, but not DTSSP, displayed plastic behaviors that may reflect the induction of cytoskeletal reorganization by Concanavalin A.     

Analysis of simulated scanning of atomic-scale silicon surface by atomic force microscopy

This study constructs a contact-mode atomic force microscopy (AFM) simulation measurement model with constant force mode to simulate and analyze the outline scanning measurement by AFM. The simulation method is that when the probe passes the surface of sample, the action force of the atom of sample received by the atom of the probe can be calculated by using Morse potential. Through calculation, the equivalent force on the cantilever of probe can be acquired. By using the deflection angle equation for the cantilever of probe developed and inferred by this study, the deflection angle of receiving action force can be calculated. On the measurement point, as the deflection angle reaches a fixed deflection angle, the scan height of this simulation model can be acquired. By scanning in the right order, the scan curve of the simulation model can be obtained. By using this simulation measurement model, this study simulates and analyzes the scanning of atomic-scale surface outline. Meanwhile, focusing on the tip radii of different probes, the concept of sensitivity analysis is employed to investigate the effects of the tip radius of probe on the atomic-scale surface outline. As a result, it is found from the simulation on the atomic-scale surface that within the simulation scope of this study, when the tip radius of probe is greater than 12 nm, the effects of single atom on the scan curve of AFM can be better decreased or eliminated.     

Study of morphological and mechanical features of multinuclear and mononuclear SW480 cells by atomic force microscopy

This article studies the morphological and mechanical features of multinuclear and mononuclear SW480 colon cancer cells by atomic force microscopy to understand their drug‐resistance. The SW480 cells were incubated with the fullerenol concentrations of 1 mg/ml and 2 mg/ml. Morphological and mechanical features including the height, length, width, roughness, adhesion force and Young's modulus of three multinuclear cell groups and three mononuclear cell groups were imaged and analyzed. It was observed that the features of multinuclear cancer cells and mononuclear cancer cells were significantly different after the treatment with fullerenol. The experiment results indicated that the mononuclear SW480 cells were more sensitive to fullerenol than the multinuclear SW480 cells, and the multinuclear SW480 cells exhibited a stronger drug‐resistance than the mononuclear SW480 cells. This work provides a guideline for the treatments of multinuclear and mononuclear cancer cells with drugs.     

Polysaccharide properties probed with atomic force microscopy

In recent years, polysaccharides have been extensively studied using atomic force microscopy (AFM). Owing to its high lateral and vertical resolutions and ability to measure interaction forces in liquids at pico‐ or nano‐Newton level, the AFM is an excellent tool for characterizing biopolymers. The first imaging studies showed the morphology of polysaccharides, but gradually more quantitative image analysis techniques were developed as the AFM grew easier to use in aqueous liquids and in non‐contact modes. Recently, AFM has been used to stretch polysaccharides and characterize their physicochemical properties by application of appropriate polymer stretching models, using a technique called single‐molecule force spectroscopy. From application of such models as the wormlike chain, freely jointed chain, extensible‐freely jointed chain, etc., properties such as the contour length, persistence length and segment elasticity or spring constant can be calculated for polysaccharides. The adhesion between polysaccharides and surfaces has been quantified with AFM, and this application is particularly useful for studying polysaccharides on microbial and other types of cells, because their adhesion is controlled by biopolymer characteristics. This review presents a synthesis of the theory and techniques currently in use to probe the physicochemical properties of polysaccharides with AFM.     

Indentation modulus and hardness of viscoelastic thin films by atomic force microscopy: A case study

We propose a nanoindentation technique based on atomic force microscopy (AFM) that allows one to deduce both indentation modulus and hardness of viscoelastic materials from the force versus penetration depth dependence, obtained by recording the AFM cantilever deflection as a function of the sample vertical displacement when the tip is pressed against (loading phase) and then removed from (unloading phase) the surface of the sample. Reliable quantitative measurements of both indentation modulus and hardness of the investigated sample are obtained by calibrating the technique through a set of different polymeric samples, used as reference materials, whose mechanical properties have been previously determined by standard indentation tests. By analyzing the dependence of the cantilever deflection versus time, the proposed technique allows one to evaluate and correct the effect of viscoelastic properties of the investigated materials, by adapting a post-experiment data processing procedure well-established for standard depth sensing indentation tests. The technique is described in the case of the measurement of indentation modulus and hardness of a thin film of poly(3,4-ethylenedioxythiophene) doped with poly(4-styrenesulfonate), deposited by chronoamperometry on an indium tin oxide (ITO) substrate.     

Application of atomic force microscopy in bacterial research

The atomic force microscope (AFM) has evolved from an imaging device into a multifunctional and powerful toolkit for probing the nanostructures and surface components on the exterior of bacterial cells. Currently, the area of application spans a broad range of interesting fields from materials sciences, in which AFM has been used to deposit patterns of thiol‐functionalized molecules onto gold substrates, to biological sciences, in which AFM has been employed to study the undesirable bacterial adhesion to implants and catheters or the essential bacterial adhesion to contaminated soil or aquifers. The unique attribute of AFM is the ability to image bacterial surface features, to measure interaction forces of functionalized probes with these features, and to manipulate these features, for example, by measuring elongation forces under physiological conditions and at high lateral resolution (<1 Å). The first imaging studies showed the morphology of various biomolecules followed by rapid progress in visualizing whole bacterial cells. The AFM technique gradually developed into a lab‐on‐a‐tip allowing more quantitative analysis of bacterial samples in aqueous liquids and non‐contact modes. Recently, force spectroscopy modes, such as chemical force microscopy, single‐cell force spectroscopy, and single‐molecule force spectroscopy, have been used to map the spatial arrangement of chemical groups and electrical charges on bacterial surfaces, to measure cell–cell interactions, and to stretch biomolecules. In this review, we present the fascinating options offered by the rapid advances in AFM with emphasizes on bacterial research and provide a background for the exciting research articles to follow. SCANNING 32: 74–96, 2010. © 2010 Wiley Periodicals, Inc.     

Quantification of the lateral detachment force for bacterial cells using atomic force microscope and centrifugation

To determine the lateral detachment force for individual bacterial cells, a quantitative method using the contact mode of an atomic force microscope (AFM) was developed in this study. Three key factors for the proposed method, i.e. scan size, scan rate and cantilever choice, were evaluated and optimized. The scan size of 40×40 μm² was optimal for capturing sufficient number of adhered cells in a microscopic field and provide adequate information for cell identification and detachment force measurement. The scan rate affected the measurement results significantly, and was optimized at 40 μm/s considering both force measurement accuracy and experimental efficiency. The hardness of applied cantilevers also influenced force determination. The proposed protocol for cantilever selection is to use those with the lowest spring constant first and then step up to a harder cantilever until all cells are detached. The lateral detachment force of Escherichia coli cells on polished stainless steel and a glass-slide coated with poly-l-lysine were measured as 0.763±0.167 and 0.639±0.136 nN, respectively. The results showed that the established method had good repeatability and sensitivity to various bacteria/substrata combinations. The detachment force quantified by AFM (0.639±0.136 nN) was comparable to that measured by the centrifugation method (1.12 nN).     

Measuring biomaterials mechanics with atomic force microscopy. 1. Influence of the loading rate and applied force (pyramidal tips)

Atomic force microscopy (AFM) is today an established tool in imaging and determination of mechanical properties of biomaterials. Due to their complex organization, those materials show intricate properties such as viscoelasticity. Therefore, one has to consider that the loading rate at which the sample is probed will lead to different mechanical response (properties). In this work, we studied the dependence of the mechanical properties of endothelial cells on the loading rate using AFM in force spectroscopy mode. We employed a sharp, four‐sided pyramidal indenter and loading rates ranging from 0.5 to 20 μm/s. In addition, by variation of the load (applied forces from 100 to 10,000 pN), the dependence of the cell properties on indentation depth could be determined. We then showed that the mechanical response of endothelial cells depends nonlinearly on the loading rate and follows a weak power‐law. In addition, regions of different viscous response at varying indentation depth could be determined. Based on the results we obtained, a general route map for AFM users for design of cell mechanics experiments was described.     

Assembling and imaging of his-tag green fluorescent protein on mica surfaces studied by atomic force microscopy and fluorescence microscopy

The adsorption of his-tag green fluorescent protein (GFPH(6)) onto the mica surfaces has been studied by atomic force microscopy (AFM) and laser confocal fluorescence microscopy. By controlling the adsorption conditions, separated single GFPH(6) and GFPH(6) monolayer can be adsorbed and formed on mica surfaces. In present experiments, based on the AFM measurement, we found that the adsorbed GFPH(6) was bound on the mica surface with its beta-sheets. The formed GFPH(6) monolayer on mica surfaces was flat, uniform, and stable. Some applications of the formed monolayer have been demonstrated. The formed monolayer can be used as a substrate for DNA imaging and AFM mechanical lithography.     

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。     

Prototype cantilevers for quantitative lateral force microscopy

Prototype cantilevers are presented that enable quantitative surface force measurements using contact-mode atomic force microscopy (AFM). The "hammerhead" cantilevers facilitate precise optical lever system calibrations for cantilever flexure and torsion, enabling quantifiable adhesion measurements and friction measurements by lateral force microscopy (LFM). Critically, a single hammerhead cantilever of known flexural stiffness and probe length dimension can be used to perform both a system calibration as well as surface force measurements in situ, which greatly increases force measurement precision and accuracy. During LFM calibration mode, a hammerhead cantilever allows an optical lever "torque sensitivity" to be generated for the quantification of LFM friction forces. Precise calibrations were performed on two different AFM instruments, in which torque sensitivity values were specified with sub-percent relative uncertainty. To examine the potential for accurate lateral force measurements using the prototype cantilevers, finite element analysis predicted measurement errors of a few percent or less, which could be reduced via refinement of calibration methodology or cantilever design. The cantilevers are compatible with commercial AFM instrumentation and can be used for other AFM techniques such as contact imaging and dynamic mode measurements.     

Evaluation of a nonlinear Hertzian‐based model reveals prostate cancer cells respond differently to force than normal prostate cells

Understanding how the mechanical properties of cells alter with disease may help with the development of novel diagnostics and treatment regimes. The emergence of tools such as the atomic force microscope (AFM) has enabled us to physically measure the mechanical properties of cells. However, suitable models for the analysis of real experimental data are either absent, or fail to provide a simple analysis tool in which experimental data can be analyzed quickly and reliably. The Hertz model has been widely used to study AFM data on living cells, however it makes assumptions that are untrue for cells, namely that cells behave as linear elastic bodies. This article presents and evaluates an alternative nonlinear Hertz model, which allows the Young's modulus to vary according to a second order polynomial function of indentation depth. Evaluation of the model revealed that prostate cancer cells (PC3) responded more uniformly to force compared to the normal PNT2 cells. Also, more energy (J) was needed to deform the normal prostate cells compared to the prostate cancer cells. Finally, the model described here suggests that overall the normal prostate cells behave in a more linear fashion to applied force compared to the prostate cancer cells. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

A symmetrical stretching stage for electrical atomic force microscopy

We present a remotely-controlled device for sample stretching, designed for use with atomic force microscopy (AFM) and providing electrical connection to the sample. Such a device enables nanoscale investigation of electrical properties of thin gold films deposited on polydimethylsiloxane (PDMS) substrate as a function of the elongation of the structure. Stretching and releasing is remotely controlled with use of a dc actuator. Moreover, the sample is stretched symmetrically, which gives an opportunity to perform AFM scans in the same site without a time-consuming finding procedure. Electrical connections to the sample are also provided, enabling Kelvin probe force microscopy (KPFM) investigations. Additionally, we present results of AFM imaging using the stretching stage.