The Effect of the Endothelial Cell Cortex on Atomic Force Microscopy Measurements (original) (raw)
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Characterization of cell elasticity correlated with cell morphology by atomic force microscope
Journal of Biomechanics, 2012
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.
Microscopy Research and Technique, 2006
Cytoskeleton fibers form an intricate three-dimensional network to provide structure and function to microvessel endothelial cells. During accommodation to blood flowing, stress fiber bundles become more prominent and align with the direction of blood flow. This network either mechanically resists the applied shear stress (lateral force) or, if deformed, is dynamically remodeled back to a preferred architecture. However, the detailed response of these stress fiber bundles to applied lateral force at submicrometer scales are as yet poorly understood. In our in vitro study, the tip, topography probe in lateral force microscopy of atomic force microscopy, acted as a tool for exerting quantitative vertical and lateral force on the filaments of the cytoskeleton. Moreover, the authors developed a formula to calculate the value of lateral force exerted on every point of the filaments. The results show that cytoskeleton fibers of healthy tight junctions in rat cerebral microvessel endothelial cells formed a cross-type network, and were reinforced and elongated in the direction of scanning under lateral force of 15–42 nN. Under peroxidation (H2O2 of 300 μmol/L), the cytoskeleton remodeled at intercellular junctions, and changed over the meshwork structures into a dense bundle, that redistributed the stress. Once mechanical forces were exerted on an area, the cells shrank and lost morphologic tight junctions. It would be useful in our understanding of certain pathological processes, such as cerebral ischemia/reperfusion injury, which maybe caused by biomechanical forces and which are overlooked in current disease models. Microsc. Res. Tech., 2006. © 2006 Wiley-Liss, Inc.
Biophysical Journal, 2000
This paper describes the combined use of atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM) to examine the transmission of force from the apical cell membrane to the basal cell membrane. A Bioscope AFM was mounted on an inverted microscope, the stage of which was configured for TIRFM imaging of fluorescently labeled human umbilical vein endothelial cells (HUVECs). Variable-angle TIRFM experiments were conducted to calibrate the coupling angle with the depth of penetration of the evanescent wave. A measure of cellular mechanical properties was obtained by collecting a set of force curves over the entire apical cell surface. A linear regression fit of the force-indentation curves to an elastic model yields an elastic modulus of 7.22 Ϯ 0.46 kPa over the nucleus, 2.97 Ϯ 0.79 kPa over the cell body in proximity to the nucleus, and 1.27 Ϯ 0.36 kPa on the cell body near the edge. Stress transmission was investigated by imaging the response of the basal surface to localized force application over the apical surface. The focal contacts changed in position and contact area when forces of 0.3-0.5 nN were applied. There was a significant increase in focal contact area when the force was removed (p Ͻ 0.01) from the nucleus as compared to the contact area before force application. There was no significant change in focal contact coverage area before and after force application over the edge. The results suggest that cells transfer localized stress from the apical to the basal surface globally, resulting in rearrangement of contacts on the basal surface.
Journal of Microscopy, 1996
Rat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparation techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference.
Contribution of the nucleus to the mechanical properties of endothelial cells
Journal of Biomechanics, 2002
The cell nucleus plays a central role in the response of the endothelium to mechanical forces, possibly by deforming during cellular adaptation. The goal of this work was to precisely quantify the mechanical properties of the nucleus. Individual endothelial cells were subjected to compression between glass microplates. This technique allows measurement of the uniaxial force applied to the cell and the resulting deformation. Measurements were made on round and spread cells to rule out the influence of cell morphology on the nucleus mechanical properties. Tests were also carried out with nuclei isolated from cell cultures by a chemical treatment. The non-linear force-deformation curves indicate that round cells deform at lower forces than spread cells and nuclei. Finite-element models were also built with geometries adapted to actual morphometric measurements of round cells, spread cells and isolated nuclei. The nucleus and the cytoplasm were modeled as separate homogeneous hyperelastic materials. The models simulate the compression and yield the force-deformation curve for a given set of elastic moduli. These parameters are varied to obtain a best fit between the theoretical and experimental data. The elastic modulus of the cytoplasm is found to be on the order of 500 N/m 2 for spread and round cells. The elastic modulus of the endothelial nucleus is on the order of 5000 N/m 2 for nuclei in the cell and on the order of 8000 N/m 2 for isolated nuclei. These results represent an unambiguous measurement of the nucleus mechanical properties and will be important in understanding how cells perceive mechanical forces and respond to them. r
Microscopy Research and Technique
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.
Relative Microelastic Mapping of Living Cells by Atomic Force Microscopy
Biophysical Journal, 1998
The spatial and temporal changes of the mechanical properties of living cells reflect complex underlying physiological processes. Following these changes should provide valuable insight into the biological importance of cellular mechanics and their regulation. The tip of an atomic force microscope (AFM) can be used to indent soft samples, and the force versus indentation measurement provides information about the local viscoelasticity. By collecting force-distance curves on a time scale where viscous contributions are small, the forces measured are dominated by the elastic properties of the sample. We have developed an experimental approach, using atomic force microscopy, called force integration to equal limits (FIEL) mapping, to produce robust, internally quantitative maps of relative elasticity. FIEL mapping has the advantage of essentially being independent of the tip-sample contact point and the cantilever spring constant. FIEL maps of living Madine-Darby canine kidney (MDCK) cells show that elasticity is uncoupled from topography and reveal a number of unexpected features. These results present a mode of high-resolution visualization in which the contrast is based on the mechanical properties of the sample.
Atomic force microscopy probing of cell elasticity
Micron, 2007
Atomic force microscopy (AFM) has recently provided the great progress in the study of micro-and nanostructures including living cells and cell organelles. Modern AFM techniques allow solving a number of problems of cell biomechanics due to simultaneous evaluation of the local mechanical properties and the topography of the living cells at a high spatial resolution and force sensitivity. Particularly, force spectroscopy is used for mapping mechanical properties of a single cell that provides information on cellular structures including cytoskeleton structure. This entry is aimed to review the recent AFM applications for the study of dynamics and mechanical properties of intact cells associated with different cell events such as locomotion, differentiation and aging, physiological activation and electromotility, as well as cell pathology. Local mechanical characteristics of different cell types including muscle cells, endothelial and epithelial cells, neurons and glial cells, fibroblasts and osteoblasts, blood cells and sensory cells are analyzed in this paper.