Probing elasticity and adhesion of live cells by atomic force microscopy indentation

Atomic force microscopy (AFM) indentation has become an important technique for quantifying the mechanical properties of live cells at nanoscale. However, determination of cell elasticity modulus from the force–displacement curves measured in the AFM indentations is not a trivial task. The present work shows that these force–displacement curves are affected by indenter-cell adhesion force, while the use of an appropriate indentation model may provide information on the cell elasticity and the work of adhesion of the cell membrane to the surface of the AFM probes. A recently proposed indentation model (Sirghi, Rossi in Appl Phys Lett 89:243118, 2006), which accounts for the effect of the adhesion force in nanoscale indentation, is applied to the AFM indentation experiments performed on live cells with pyramidal indenters. The model considers that the indentation force equilibrates the elastic force of the cell cytoskeleton and the adhesion force of the cell membrane. It is assumed that the indenter-cell contact area and the adhesion force decrease continuously during the unloading part of the indentation (peeling model). Force–displacement curves measured in indentation experiments performed with silicon nitride AFM probes with pyramidal tips on live cells (mouse fibroblast Balb/c3T3 clone A31-1-1) in physiological medium at 37°C agree well with the theoretical prediction and are used to determine the cell elasticity modulus and indenter-cell work of adhesion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cell dynamic adhesion and elastic properties probed with cylindrical atomic force microscopy cantilever tips

Cell adhesion is required for essential biological functions such as migration, tissue formation and wound healing, and it is mediated by individual molecules that bind specifically to ligands on other cells or on the extracellular matrix. Atomic force microscopy (AFM) has been successfully used to measure cell adhesion at both single molecule and whole cell levels. However, the measurement of inherent cell adhesion properties requires a constant cell-probe contact area during indentation, a requirement which is not fulfilled in common pyramidal or spherical AFM tips. We developed a procedure using focused ion beam (FIB) technology by which we modified silicon pyramidal AFM cantilever tips to obtain flat-ended cylindrical tips with a constant and known area of contact. The tips were validated on elastic gels and living cells. Cylindrical tips showed a fairly linear force-indentation behaviour on both gels and cells for indentations >200 nm. Cylindrical tips coated with ligands were used to quantify inherent dynamic cell adhesion and elastic properties. Force, work of adhesion and elasticity showed a marked dynamic response. In contrast, the deformation applied to the cells before rupture was fairly constant within the probed dynamic range. Taken together, these results suggest that the dynamic adhesion strength is counterbalanced by the dynamic elastic response to keep a constant cell deformation regardless of the applied pulling rate.     

Drug-induced changes of cytoskeletal structure and mechanics in fibroblasts: an atomic force microscopy study

The effect of various drugs affecting the integrity of different components of the cytoskeleton on the elasticity of two fibroblast cell lines was investigated by elasticity measurements with an atomic force microscope (AFM). Disaggregation of actin filaments always resulted in a distinct decrease in the cell's average elastic modulus indicating the crucial importance of the actin network for the mechanical stability of living cells. Disruption or chemical stabilization of microtubules did not affect cell elasticity. For the f-actin-disrupting drugs different mechanisms of drug action were observed. Cytochalasins B and D and Latrunculin A disassembled stress fibers. For Cytochalasin D this was accompanied by an aggregation of actin within the cytosol. Jasplakinolide disaggregated actin filaments but did not disassemble stress fibers. Fibrous structures found in AFM images and elasticity maps of fibroblasts could be identified as stress fibers by correlation of AFM data and fluorescence images.     

肌动蛋白的原子力显微镜研究

原子力显微镜 (AFM )是一种能够在生理条件下对生物大分子、活细胞表面以及细胞膜下结构进行在体或离体研究的强有力的新型工具 ,具有原子级的成像分辨率和纳牛顿级的力测定功能。目前原子力显微镜已被广泛地应用于生物大分子、超分子体系的结构解析、动力学过程观察 ,分子力学研究及细胞功能鉴定。原子力显微镜能够通过尖锐探针扫描待测样品表面 ,收集被测样品表面地貌坐标数据从而对单分子或细胞进行成像或操作 ,并能通过移动探针、记录探针与样品之间的作用力 ,对生物大分子 (蛋白质、核酸和多糖等 )的结构力学特性进行分析以获取分子构象、功能及其相互关系的有用信息。肌动蛋白是一种细胞内普遍存在 ,具有广泛、复杂生理功能的重要蛋白质 ,原子力显微镜的各项功能已广泛地用于肌动蛋白结构、功能及动力学研究。通过综述原子力显微镜在肌动蛋白研究中的应用 ,阐明了原子力显微镜在现代生命科学研究中的重要意义及巨大应用前景。     

Spatial distribution of filament elasticity determines the migratory behaviors of a cell

Any cellular response leading to morphological changes is highly tuned to balance the force generated from structural reorganization, provided by actin cytoskeleton. Actin filaments serve as the backbone of intracellular force, and transduce external mechanical signal via focal adhesion complex into the cell. During migration, cells not only undergo molecular changes but also rapid mechanical modulation. Here we focus on determining, the role of spatial distribution of mechanical changes of actin filaments in epithelial, mesenchymal, fibrotic and cancer cells with non-migration, directional migration, and non-directional migration behaviors using the atomic force microscopy. We found 1) non-migratory cells only generated one type of filament elasticity, 2) cells generating spatially distributed two types of filament elasticity showed directional migration, and 3) pathologic cells that autonomously generated two types of filament elasticity without spatial distribution were actively migrating non-directionally. The demonstration of spatial regulation of filament elasticity of different cell types at the nano-scale highlights the coupling of cytoskeletal function with physical characters at the sub-cellular level, and provides new research directions for migration related disease.     

On atomic force microscopy and the constitutive behavior of living cells

Atomic force microscopy (AFM) is one of many new technologies available to study the mechanical properties and mechanobiological responses of living cells. Despite the widespread usage of this technology, there has been little attempt to develop new theoretical frameworks to interpret the associated data. Rather, most analyses rely on the classical Hertz solution for the indentation of an elastic half-space within the context of linearized elasticity. In contrast, we propose a fully nonlinear, constrained mixture model for adherent cells that allows one to account separately for the contributions of the three primary structural constituents of the cytoskeleton. Moreover, we extend a prior solution for a small indentation superimposed on a finite equibiaxial extension by incorporating in this mixture model for the special case of an initially random distribution of constituents (actin, intermediate filaments, and microtubules). We submit that this theoretical framework will allow an improved interpretation of indentation force-depth data from a sub-class of atomic force microscopy tests and will serve as an important analytical check for future finite element models. The latter will be necessary to exploit further the capabilities of both atomic force microscopy and nonlinear mixture theories for cell behavior.     

Direct monitoring of drug‐induced mechanical response of individual cells by atomic force microscopy

Mechanical characteristics of individual cells play a vital role in many biological processes and are considered as indicators of the cells’ states. Disturbances including methyl‐β‐cyclodextrin (MβCD) and cytochalasin D (cytoD) are known to significantly affect the state of cells, but little is known about the real‐time response of single cells to these drugs in their physiological condition. Here, nanoindentation‐based atomic force microscopy (AFM) was used to measure the elasticity of human embryonic kidney cells in the presence and absence of these pharmaceuticals. The results showed that depletion of cholesterol in the plasma membrane with MβCD resulted in cell stiffening whereas depolymerization of the actin cytoskeleton by cytoD resulted in cell softening. Using AFM for real‐time measurements, we observed that cells mechanically responded right after these drugs were added. In more detail, the cell´s elasticity suddenly increased with increasing instability upon cholesterol extraction while it is rapidly decreased without changing cellular stability upon depolymerizing actin cytoskeleton. These results demonstrated that actin cytoskeleton and cholesterol contributed differently to the cell mechanical characteristics.     

Physiological role of gap-junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation

Hemichannels in the overlapping regions of apposing cells plasma membranes join to form gap junctions and provide an intercellular communication pathway. Hemichannels are also present in the nonjunctional regions of individual cells and their activity is gated by several agents, including calcium. However, their physiological roles are unknown. Using techniques of atomic force microscopy (AFM), fluorescent dye uptake assay, and laser confocal immunofluorescence imaging, we have examined the extracellular calcium-dependent modulation of cell volume. In response to a change in the extracellular physiological calcium concentration (1.8 to 相似文献   

Characterization of cell elasticity correlated with cell morphology by atomic force microscope

Biomechanical properties of cells have been identified as an important factor in a broad range of biological processes. Based on measurements of mechanical properties by atomic force microscopy (AFM) particularly cell elasticity has been linked with human diseases, such as cancer. AFM has been widely used as a nanomechanical tool to probe the elasticity of living cells, however, standard methods for characterizing cell elasticity are still lacking. The local elasticity of a cell is conventionally used to represent the mechanical property of the cell. However, since cells have highly heterogeneous regions, elasticity mapping over the entire cell, rather than at a few points of measurement, is required. Using human aortic endothelial cells (HAECs) as a model, we have developed in this study a new method to evaluate cell elasticity more quantitatively. Based on the height information of the cell, a new characterization method was proposed to evaluate the elasticity of a cell. Using this method, elasticities of cells on different substrates were compared. Results showed that the elasticity of HAECs on softer substrate also has higher value compared to those on harder substrate given a certain height where the statistical distribution analysis confirmed that higher actin filaments density was located. Thus, the elasticity of small portions of a cell could not represent the entire cell property and may lead to invalid characterization. In order to gain a more comprehensive and detailed understanding of biomechanical properties for future clinical use, elasticity and cell morphology should therefore be correlated with discussion.     

Application of atomic force microscopy in cancer research

Atomic force microscopy (AFM) allows for nanometer-scale investigation of cells and molecules. Recent advances have enabled its application in cancer research and diagnosis. The physicochemical properties of live cells undergo changes when their physiological conditions are altered. These physicochemical properties can therefore reflect complex physiological processes occurring in cells. When cells are in the process of carcinogenesis and stimulated by external stimuli, their morphology, elasticity, and adhesion properties may change. AFM can perform surface imaging and ultrastructural observation of live cells with atomic resolution under near-physiological conditions, collecting force spectroscopy information which allows for the study of the mechanical properties of cells. For this reason, AFM has potential to be used as a tool for high resolution research into the ultrastructure and mechanical properties of tumor cells. This review describes the working principle, working mode, and technical points of atomic force microscopy, and reviews the applications and prospects of atomic force microscopy in cancer research.     

Depth-sensing analysis of cytoskeleton organization based on AFM data

Atomic force microscopy is a common technique used to determine the elastic properties of living cells. It furnishes the relative Young’s modulus, which is typically determined for indentation depths within the range 300–500 nm. Here, we present the results of depth-sensing analysis of the mechanical properties of living fibroblasts measured under physiological conditions. Distributions of the Young’s moduli were obtained for all studied cells and for every cell. The results show that for small indentation depths, histograms of the relative values of the Young’s modulus described the regions rich in the network of actin filaments. For large indentation depths, the overall stiffness of a whole cell was obtained, which was accompanied by a decrease of the modulus value. In conclusion, the results enable us to describe the non-homogeneity of the cell cytoskeleton, particularly, its contribution linked to actin filaments located beneath the cell membrane. Preliminary results showing a potential application to improve the detection of cancerous cells, have been presented for melanoma cell lines.     

Macromolecular crowding directs extracellular matrix organization and mesenchymal stem cell behavior

Microenvironments of biological cells are dominated in vivo by macromolecular crowding and resultant excluded volume effects. This feature is absent in dilute in vitro cell culture. Here, we induced macromolecular crowding in vitro by using synthetic macromolecular globules of nm-scale radius at physiological levels of fractional volume occupancy. We quantified the impact of induced crowding on the extracellular and intracellular protein organization of human mesenchymal stem cells (MSCs) via immunocytochemistry, atomic force microscopy (AFM), and AFM-enabled nanoindentation. Macromolecular crowding in extracellular culture media directly induced supramolecular assembly and alignment of extracellular matrix proteins deposited by cells, which in turn increased alignment of the intracellular actin cytoskeleton. The resulting cell-matrix reciprocity further affected adhesion, proliferation, and migration behavior of MSCs. Macromolecular crowding can thus aid the design of more physiologically relevant in vitro studies and devices for MSCs and other cells, by increasing the fidelity between materials synthesized by cells in vivo and in vitro.     

原子力显微镜在细胞弹性研究中的应用

细胞表面的力学性质会随着细胞所处环境的不同而发生改变,它的变化间接反映出胞内复杂的生理过程。原子力显微镜（atomic force microscope,AFM）能以高的灵敏度和分辨率检测活体细胞,通过利用赫兹模型分析力曲线可以获得细胞的弹性信息。本文简介了原子力显微镜的工作原理与工作模式,着重介绍利用AFM力曲线检测细胞弹性的方法及其在细胞运动、细胞骨架、细胞黏附、细胞病理等方面的应用成果,表明AFM已经成为细胞弹性研究中十分重要的显微技术。     

Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

In the mammary gland, epithelial cells are embedded in a ‘soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for β-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of β-casein expression required both laminin signalling and a ‘soft' extracellular matrix, as is the case in normal tissues in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of β-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues' unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.     

Mechanical sensing of the penetration of various nanoneedles into a living cell using atomic force microscopy

Mechanical responses during insertion of a silicon nanoneedle into a living melanocyte were observed by using an atomic force microscope (AFM). In order to study the dependence of the mechanical response on the shape of the nanoneedle, we prepared various shapes of silicon AFM tips by focused-ion beam (FIB) etching. The force curves showed increases up to 0.65-1.9 nN after contact on the cell surface, and then the force dropped corresponding with the penetration of the needle through the cell membrane. The force required for penetration was significantly smaller than that using a normal pyramidal tip. The force curves with a cylindrical tip showed a shorter indenting distance before penetration than that with the cone-shaped tip. It is considered that the information about the geometry of penetrating material leads to the development of more suitable micro- and nano-materials to insert into a living cell for cell surgery.     

Probing the biophysical properties of tumor cells during mitosis by atomic force microscopy

Mitosis is an important physiological event accompanying with dramatic changes of cellar biophysical properties. Failure of mitosis results in cell death or chromosome aneuploidy. In this study, we used atomic force microscopy to probe and compare the biophysical properties of tumor cells at different stages during mitosis. The rounding forces of MCF-7 cells oscillated during mitosis. At anaphase, the average elasticity of cells was higher than that at other phases. Cholesterol depletion with M\(\upbeta \)CD led to an increase in the average elasticity, whereas the average roughness of membrane surface decreased at the absence of cholesterol. Our study indicated that the distribution of actin filaments could affect the biophysical properties of tumor cells and cellular morphology during mitosis. Furthermore, the biophysical properties of tumor cells were also regulated by membrane cholesterol during mitosis. This work provides a new detection approach for monitoring tumor cell development at single cell level.     

Temporal analysis of vascular smooth muscle cell elasticity and adhesion reveals oscillation waveforms that differ with aging

A spectral analysis approach was developed for detailed study of time‐resolved, dynamic changes in vascular smooth muscle cell (VSMC) elasticity and adhesion to identify differences in VSMC from young and aged monkeys. Atomic force microscopy (AFM) was used to measure Young’s modulus of elasticity and adhesion as assessed by fibronectin (FN) or anti‐beta 1 integrin interaction with the VSMC surface. Measurements demonstrated that VSMC cells from old vs. young monkeys had increased elasticity (21.6 kPa vs. 3.5 kPa or a 612% increase in elastic modulus) and adhesion (86 pN vs. 43 pN or a 200% increase in unbinding force). Spectral analysis identified three major frequency components in the temporal oscillation patterns for elasticity (ranging from 1.7 × 10^?3 to 1.9 × 10^?2 Hz in old and 8.4 × 10^?4 to 1.5 × 10^?2 Hz in young) and showed that the amplitude of oscillation was larger (P < 0.05) in old than in young at all frequencies. It was also observed that patterns of oscillation in the adhesion data were similar to the elasticity waveforms. Cell stiffness was reduced and the oscillations were inhibited by treatment with cytochalasin D, ML7 or blebbistatin indicating the involvement of actin–myosin‐driven processes. In conclusion, these data demonstrate the efficacy of time‐resolved analysis of AFM cell elasticity and adhesion measurements and that it provides a uniquely sensitive method to detect real‐time functional differences in biomechanical and adhesive properties of cells. The oscillatory behavior suggests that mechanisms governing elasticity and adhesion are coupled and affected differentially during aging, which may link these events to changes in vascular stiffness.