Cell Mechanics and Adhesion- Atomic Force Microscopy-Total Internal Reflection Fluorescence Microscopy(AFM-TIRFM) (original) (raw)
Related papers
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.
Force and compliance measurements on living cells using atomic force microscopy (AFM
Biological Procedures Online, 2004
We describe the use of atomic force microscopy (AFM) in studies of cell adhesion and cell compliance. Our studies use the interaction between leukocyte function associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) as a model system. The forces required to unbind a single LFA-1/ICAM-1 bond were measured at different loading rates. This data was used to determine the dynamic strength of the LFA-1/ICAM-1 complex and characterize the activation potential that this complex overcomes during its breakage. Force measurements acquired at the multiple- bond level provided insight about the mechanism of cell adhesion. In addition, the AFM was used as a microindenter to determine the mechanical properties of cells. The applications of these methods are described using data from a previous study.
Journal of Microbiological Methods, 2011
Atomic force microscopy (AFM) is a technique that has long been employed in materials science, but is now increasingly being used in the biological sciences. AFM provides excellent topographical information on prokaryotic and eukaryotic cell surfaces, and the extracellular material produced by the cells. It helps to generate important data on the mechanical properties of cells, such as hardness and elasticity. AFM can also be used to measure the strength of adhesion, attraction, and repulsion forces between cells and surfaces or even between individual molecules. Additionally, by combining AFM with other complementary techniques such as fluorescence microscopy or Raman spectroscopy, the chemistry of given surface structures can be identified. This review aims to provide an update on the AFM techniques currently used in cell biology studies, along with a description of the range of recently developed research methodologies in which AFM plays a key role.
Nanomechanical Investigation of Soft Biological Cell Adhesion using Atomic Force Microscopy
Cellular and Molecular Bioengineering, 2014
Mechanical coupling between living cells is a complex process that is important for a variety of biological processes. In this study the effects of specific biochemical treatment on cell-to-cell adhesion and single cell mechanics were systematically investigated using atomic force microscopy (AFM) single cell force spectroscopy. Functionalised AFM tipless cantilevers were used for attaching single suspended cells that were brought in contact with substrate cells. Cell-to-cell adhesion parameters, such as maximum unbinding force (F max) and work or energy of detachment (W D), were extracted from the retraction force-displacement (F-d) curves. AFM indentation experiments were performed by indenting single cells with a spherical microbead attached to the cantilever. Hertzian contact model was applied to determine the elastic modulus (E) of single cells. Following treatment of the cells with neutralising antibody for epithelial (E)-cadherin, F max was increased by 25%, whereas W D decreased by 11% in response to a 43% increase in E. The results suggest that although the adhesion force between cells was increased after treatment, the energy of adhesion was decreased due to the reduced displacement separation as manifested by the loss of elastic deformation. Conclusively, changes in single cell mechanics are important underlying factors contributing to cell-to-cell adhesion and hence cytomechanical characterization is critical for cell adhesion measurements.
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.
Influencing Factors in Atomic Force Microscopy Based Mechanical Characterization of Biological Cells
Experimental Techniques, 2017
AFM-based single cell force spectroscopy is increasingly being used to understand many biological health problems like malaria, cancer etc. A reliable diagnostics needs, accurate measurement and a clear understanding of the influencing factors, which may otherwise sour the measured biomechanics. Results from any successful experimentation should be repeatable and error free. For this, a deeper understanding of the sources of uncertainties, which may affect the results is necessary. In order to assure the accuracy of evaluated properties and to avoid misinterpretation of the experimental data, we have categorized the common causes of uncertainties in an AFM force spectroscopy, based on their sources of origin and discussed possible remedies to them. The present work discusses the assumptions involved in AFM-based biomechanical studies (assumptions in contact model, data analysis, instrument calibration etc.) and their implications in overall estimations of mechanical biomarkers like stiffness, and adhesive strength. Advantages and disadvantages of simultaneous measurement of stiffness and adhesiveness from single force-indentation data have also been discussed.
The Effect of the Endothelial Cell Cortex on Atomic Force Microscopy Measurements
Biophysical Journal, 2013
We examined whether the presence of the cell cortex might explain, in part, why previous studies using atomic force microscopy (AFM) to measure cell modulus (E) gave higher values with sharp tips than for larger spherical tips. We confirmed these AFM findings in human umbilical vein endothelial cells (HUVEC) and Schlemm's canal (SC) endothelial cells with AFM indentation % 400 nm, two cell types with prominent cortices (312 5 65 nm in HUVEC and 371 5 91 nm in SC cells). With spherical tips, E (kPa) was 0.71 5 0.16 in HUVEC and 0.94 5 0.06 in SC cells. Much higher values of E were measured using sharp tips: 3.23 5 0.54 in HUVEC and 6.67 5 1.07 in SC cells. Previous explanations for this difference such as strain hardening or a substrate effect were shown to be inconsistent with our measurements. Finite element modeling studies showed that a stiff cell cortex could explain the results. In both cell types, Latrunculin-A greatly reduced E for sharp and rounded tips, and also reduced the ratio of the values measured with a sharp tip as compared to a rounded tip. Our results suggest that the cell cortex increases the apparent endothelial cell modulus considerably when measured using a sharp AFM tip.
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.
Combined atomic force microscopy and side-view optical imaging for mechanical studies of cells
Nature Methods, 2009
The mechanical rigidity of cells and adhesion forces between cells play an important role in a variety of biological processes including cell differentiation, proliferation, and tissue organization. Atomic force microscopy (AFM) has emerged as a powerful tool to quantify these mechanical properties and adhesion forces at the cellular level. Here we demonstrate an instrument that combines AFM with a side-view fluorescent imaging path that enables direct imaging of cellular deformation and cytoskeletal rearrangements along the axis of loading. With this instrument, we were able to directly observe cell shape under load, correlate changes in shape with force-induced ruptures, and image formation of membrane tethers during cell-cell adhesion measurements. Additionally, we observed cytoskeletal reorganization and stress fiber formation while measuring the contractile force of an individual cell. This instrument provides a useful tool for understanding the role of mechanics in biological processes.
Probing elasticity and adhesion of live cells by atomic force microscopy indentation
European Biophysics Journal, 2008
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 forcedisplacement curves measured in the AFM indentations is not a trivial task. The present work shows that these forcedisplacement 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 indentercell work of adhesion.