AFM Indentation Research Articles

Anisotropy vs isotropy in living cell indentation with AFM

The measurement of local mechanical properties of living cells by nano/micro indentation relies on the foundational assumption of locally isotropic cellular deformation. As a consequence of assumed isotropy, the cell membrane and underlying cytoskeleton are expected to locally deform axisymmetrically when indented by a spherical tip. Here, we directly observe the local geometry of deformation of membrane and cytoskeleton of different living adherent cells during nanoindentation with the integrated Atomic Force (AFM) and spinning disk confocal (SDC) microscope. We show that the presence of the perinuclear actin cap (apical stress fibers), such as those encountered in cells subject to physiological forces, causes a strongly non-axisymmetric membrane deformation during indentation reflecting local mechanical anisotropy. In contrast, axisymmetric membrane deformation reflecting mechanical isotropy was found in cells without actin cap: cancerous cells MDA-MB-231, which naturally lack the actin cap, and NIH 3T3 cells in which the actin cap is disrupted by latrunculin A. Careful studies were undertaken to quantify the effect of the live cell fluorescent stains on the measured mechanical properties. Using finite element computations and the numerical analysis, we explored the capability of one of the simplest anisotropic models – transverse isotropy model with three local mechanical parameters (longitudinal and transverse modulus and planar shear modulus) – to capture the observed non-axisymmetric deformation. These results help identifying which cell types are likely to exhibit non-isotropic properties, how to measure and quantify cellular deformation during AFM indentation using live cell stains and SDC, and suggest modelling guidelines to recover quantitative estimates of the mechanical properties of living cells.

Mechanical characterization of polyethylene-based thermoplastic composite materials for radiation shielding

Abstract Polyethylene is a hydrogen-rich polymer and has been used in making composite materials for radiation shielding. It has been shown that the radiation shielding properties of polyethylene are enhanced through the incorporation of multifunctional nanostructured materials into the matrix of the polymer. In this study, composites of boron nitride added to injection-molding grade high-density polyethylene (HDPE) were prepared and physically tested for their radiation shielding capabilities. Also, the mechanical properties of these composites were also investigated using the novel technique of AFM indentation. The goal is to create a composite material which exhibits advantageous properties in areas besides radiation shielding, i.e., structural or mechanical properties, which does not inhibit the shielding properties to provide multifunctionality which pure polyethylene materials lack.

Effect of APTMS modification on multiwall carbon nanotube reinforced epoxy nanocomposites

In this present investigation, we have studied the effect of chemical functionalization, and mechanical dispersion of carbon nanotube (CNT) reinforced epoxy resin. The chemical function of the CNT has been done by silane treatment. Silane modification has been done first by acid treatment on ball-milled CNT followed by chemical treatment of amine-based silane. FTIR spectra exhibit that silane chemically interacts with the CNT and epoxy resin. Mechanical dispersion of CNT in the epoxy was performed in the planetary ball mill. Ball milled CNT shows some degree of exfoliation. Mechanical properties of ball-milled and silane modified CNT reinforced epoxy is characterized in terms of fracture toughness and tensile modulus. Silane-modified CNT/epoxy shows an increase of 38% and 42% compared to the neat resin for fracture toughness and tensile modulus values respectively. Increase in the mechanical properties for silane modified CNT/epoxy is due to the excellent adhesion between CNT-matrix as well as significant dispersion of CNT in the epoxy resin provided by silane molecule as observed from TEM images. AFM indentation on the nanocomposites exhibits the presence of CNT agglomerates through adhesion and corresponding modulus map at the nanoscale level. SEM images indicate an increase in surface roughness with CNT inclusion which relates to the existence of an extrinsic mechanism. Additionally, better adhesion between CNT and matrix due to silane modification indicate a higher Tg value of silane modified CNT/epoxy than ball milled CNT/epoxy.

An alternative approach for the Young’s modulus determination of biological samples regarding AFM indentation experiments

AFM indentation is a powerful method for the nanomechanical characterization of biological samples and biomaterials. The nanoindentation technique is based on the experimentally obtained load—indentation data which are processed using models of applied mechanics. In this paper, an alternative procedure for the Young’s modulus calculation regarding indentation experiments is presented. In the proposed approach a simple equation which relates the work done by the indenter on the sample of interest and on a hypothetical reference sample is derived and used for the Young’s modulus determination. The new method is applied for the construction of Young’s modulus maps for a human cell line and the results are compared to the results obtained by the classic approach.

Culture of Mesenchymal Stem Cells in a Hydrogel Model of Vocal Fold Lamina Propria.

Stem cell injection has been proposed as an alternative approach for the restoration of vocal fold (VF) function in patients with VF scarring. To assess the therapeutic efficacy of this treatment strategy, we evaluated the behaviors of human mesenchymal stem cells (hMSCs) in hydrogels derived from thiolated hyaluronic acid (HA-SH) and poly(ethylene glycol) diacrylate (PEG-DA) entrapping assembled collagen fibrils (abbreviated as HPC gels). Three hydrogel formulations with varying amounts of collagen (0, 1 and 2 mg/mL) but a fixed HA-SH (5 mg/mL) and PEG-DA (2 mg/mL) concentration, designated as HPC0, HPC1 and HPC2, were investigated. The HPC gels exhibit similar pore sizes (35-50 nm) and AFM indentation moduli (~175 Pa), although the elastic shear modulus for HPC1 (~32 Pa) is lower than HPC0 and HPC2 (~55 Pa). Although HPC1 and HPC2 gels both promoted the development of an elongated cell morphology, greater cell spreading was observed in HPC2 than in HPC1 by day 7. At the transcript level, cells cultured in HPC1 and HPC2 gels had an increased expression of fibronectin and integrin β1, but a decreased expression of tissue inhibitor of metalloproteinase-1, collagen types I/III and HA synthase-1 when compared to cells cultured in HPC0 gels. Cellular expression of connective tissue growth factor was also elevated in HPC1 and HPC2 cultures. Importantly, the HPC2 hydrogels promoted a signficant up-regulation of matrix metalloproteinase 1, transforming growth factor β1, and epithelial growth factor receptor, indicating an increased tissue turnover. Overall, hMSCs cultured in HPC2 gels adopt a phenotype reminiscent of cells involved in the wound healing process, providing a platform to study the effectiveness of therapeutic stem cell treatments for VF scarring.

Confined Synthesis of Two-Dimensional Covalent Organic Framework Thin Films within Superspreading Water Layer.

The confined synthesis of two-dimensional covalent organic framework (2D COF) thin films was developed by using thin superspreading water on the hydrogel immersed under oil as reactor. Through loading two monomers into oil and hydrogel, respectively, COF thin films are synthesized at the oil/water/hydrogel interface. This strategy provides a new way for synthesis of freestanding 2D COF thin films. Detailed characterizations of the COF thin films reveal homogeneous topography, large area, controllable thickness from 4 to 150 nm, and crystallinity with certain orientation. Young's modulus of COF film is measured by AFM indentation as 25.9 ± 0.6 GPa, showing good mechanical properties. On the basis of the freestanding COF films, a nanofilter membrane and photoelectrochemical sensors for Ru3+ were successfully developed. Moreover, the strategy was extended to the synthesis of crystalline zeolitic imidazolate framework-8 thin film, which exhibited high application potential.

Atomic force microscopy methodology and AFMech Suite software for nanomechanics on heterogeneous soft materials

Atomic force microscopy has proven to be a valuable technique to characterize the mechanical and morphological properties of heterogeneous soft materials such as biological specimens in liquid environment. Here we propose a 3-step method in order to investigate biological specimens where heterogeneity hinder a quantitative characterization: (1) precise AFM calibration, (2) nano-indentation in force volume mode, (3) array of finite element simulations built from AFM indentation events. We combine simulations to determine internal geometries, multi-layer material properties, and interfacial friction. In order to easily perform this analysis from raw AFM data to simulation comparison, we propose a standalone software, AFMech Suite comprising five interacting interfaces for simultaneous calibration, morphology, adhesion, mechanical, and simulation analysis. We test the methodology on soft hydrogels with hard spherical inclusions, as a soft-matter model system. Finally, we apply the method on E. coli bacteria supported on soft/hard hydrogels to prove usefulness in biological field.

Grand Canonical Investigation of the Quasi Liquid Layer of Ice: Is It Liquid?

In this study, the solid-vapor equilibrium and the quasi liquid layer (QLL) of ice Ih exposing the basal and primary prismatic faces were explored by means of grand canonical molecular dynamics simulations with the monatomic mW potential. For this model, the solid-vapor equilibrium was found to follow the Clausius-Clapeyron relation in the range examined, from 250 to 270 K, with a Δ Hsub of 50 kJ/mol in excellent agreement with the experimental value. The phase diagram of the mW model was constructed for the low pressure region around the triple point. The analysis of the crystallization dynamics during condensation and evaporation revealed that, for the basal face, both processes are highly activated, and in particular cubic ice is formed during condensation, producing stacking-disordered ice. The basal and primary prismatic surfaces of ice Ih were investigated at different temperatures and at their corresponding equilibrium vapor pressures. Our results show that the region known as QLL can be interpreted as the outermost layers of the solid where a partial melting takes place. Solid islands in the nanometer length scale are surrounded by interconnected liquid areas, generating a bidimensional nanophase segregation that spans throughout the entire width of the outermost layer even at 250 K. Two approaches were adopted to quantify the QLL and discussed in light of their ability to reflect this nanophase segregation phenomena. Our results in the μVT ensemble were compared with NPT and NVT simulations for two system sizes. No significant differences were found between the results as a consequence of model system size or of the working ensemble. Nevertheless, certain advantages of performing μVT simulations in order to reproduce the experimental situation are highlighted. On the one hand, the QLL thickness measured out of equilibrium might be affected because of crystallization being slower than condensation. On the other, preliminary simulations of AFM indentation experiments show that the tip can induce capillary condensation over the ice surface, enlarging the apparent QLL.

ANALYSIS OF THE MATERIAL REMOVAL RATE IN MAGNETIC ABRASIVE FINISHING OF THIN FILM COATED PYREX GLASS

The material removal rate (MRR) during precision finishing/polishing is a key factor, which dictates the process performance. Moreover, the MRR or wear rate is closely related to the material/part reliability. For nanoscale patterning and/or planarization on nano-order thickness coatings, the prediction and in-process monitoring of the MRR is necessary, because the process is not characterizable due to size effects and material property/process condition variations as a result of the coating/substrate interactions. The purpose of this research was to develop a practical methodology for the prediction and in-process monitoring of MRR during nanoscale finishing of coated surfaces. Using a specially designed magnetic abrasive finishing (MAF) and acoustic emission (AE) monitoring setup, experiments were carried out on indium-zinc-oxide (IZO) coated Pyrex glasses. After a given polishing time interval, AFM indentation was conducted for each workpiece sample to measure the adhesion force variations of the coating layers (IZO), which are directly related to the MRR changes. The force variation and AE monitoring data were compared to the MRR calculated form the surface measurement (Nanoview) results. The experimental results demonstrate strong correlations between AFM indentation and MRR measurement data. In addition, the monitored AE signals show sensitivity of the material structure variations of the coating layer, as the polishing progresses.

Axisymmetric Contact Problem for a Flattened Cell: Contributions of Substrate Effect and Cell Thickness to the Determination of Viscoelastic Properties by Using AFM Indentation

Nanoindentation technology has proven to be an effective method to investigate the viscoelastic properties of biological cells. The experimental data obtained by nanoindentation are frequently interpreted by Hertz contact model. However, in order to validate Hertz contact model, some studies assume that cells have infinite thickness which does not necessarily represent the real situation. In this study, a rigorous contact model based upon linear elasticity is developed for the interpretation of indentation tests of flattened cells. The cell, normally bonded to the Petri dish, is initially treated as an elastic layer of finite thickness perfectly fixed to a rigid substrate. The theory of linear elasticity is utilized to solve this contact issue and then the solutions are extended to viscoelastic situation which is regarded as a good indicator for mechanical properties of biological cells. To test the present model, AFM-based creep test has been conducted on living human hepatocellular carcinoma cell (SMMC-7721 cell) and its fullerenol-treated counterpart. The results indicate that the present model could not only describe very well the creep behavior of SMMC-7721 cells, but also curb overestimation of the mechanical properties due to substrate effect.

Breaking a virus: Identifying molecular level failure modes of a viral capsid by multiscale modeling

We use coarse-grained (CG) simulations to study the deformation of empty Cowpea Chlorotic Mottle Virus (CCMV) capsids under uniaxial compression, from the initial elastic response up to capsid breakage. Our CG model is based on the MARTINI force field and has been amended by a stabilizing elastic network, acting only within individual proteins, that was tuned to capture the fluctuation spectrum of capsid protein dimers, obtained from all atom simulations. We have previously shown that this model predicts force-compression curves that match AFM indentation experiments on empty CCMV capsids. Here we investigate details of the actual breaking events when the CCMV capsid finally fails. We present a symmetry classification of all relevant protein contacts and show that they differ significantly in terms of stability. Specifically, we show that interfaces which break readily are precisely those which are believed to form last during assembly, even though some of them might share the same contacts as other non-breaking interfaces. In particular, the interfaces that form pentamers of dimers never break, while the virtually identical interfaces within hexamers of dimers readily do. Since these units differ in the large-scale geometry and, most noticeably, the cone-angle at the center of the 5- or 6-fold vertex, we propose that the hexameric unit fails because it is pre-stressed. This not only suggests that hexamers of dimers form less frequently during the early stages of assembly; it also offers a natural explanation for the well-known β-barrel motif at the hexameric center as a post-aggregation stabilization mechanism. Finally, we identify those amino acid contacts within all key protein interfaces that are most persistent during compressive deformation of the capsid, thereby providing potential targets for mutation studies aiming to elucidate the key contacts upon which overall stability rests.

Mechanical Properties of Architectured Gelatin-Based Hydrogels on Different Hierarchical Levels

ABSTRACTPreparation of three-dimensionally architectured porous biomaterials can be achieved in a one-step process by stabilizing gelatin with L-lysine diisocyanate ethyl ester (LDI) in water. The reaction of gelatin with LDI in presence of water leads to the formation of oligourea bridges between gelatin molecules and oligourea chains grafted on gelatin. The number and the length of the bridges, as well as of the grafted chains strongly depend on the concentration of the LDI used for the stabilization, and this has huge influence on the mechanical properties of the material on different hierarchical levels. Higher LDI concentrations yield materials with increased deformation resistance in tensile tests due to the higher number of covalent and physical netpoints in the material. However, mechanical properties determined on the micro-level by AFM indentation showed the opposite trend, i.e. a decrease of Young’s modulus with increasing LDI content. This was interpreted by a decreasing number of shorter oligourea bridges between gelatin chains with decreasing LDI content.

Gas Permeability of Hyperthin Polyelectrolyte Multilayers Having Matched and Mismatched Repeat Units.

A series of polyelectrolyte multilayers (PEMs) has been fabricated using polyanions and polycations that have repeat units (i) similar in structure and composition (matched), (ii) partially similar in structure and composition (semimatched), and (iii) very different in structure and composition (mismatched). The primary aim of this investigation was to determine whether the matching of the polyelectrolytes can significantly influence the permeability properties of hyperthin PEMs. While matching, per se, was not found to be a key factor in defining membrane permeability, large differences in permeability were observed (the permeances of N2 varied by a factor of 20), which were correlated with the concentration of pendant aryl groups present, i.e., the greater the concentration of these groups, the higher the permeability. Analysis by AFM indentation measurements further revealed that high-permeability PEMs tend to be more compliant than low-permeability PEMs. These findings underscore the need for considering a broad range of polyelectrolyte combinations when optimizing a particular functional property of PEMs.

Elastic constants of lamellar and interlamellar regions in α and mesomorphic isotactic polypropylene by AFM indentation

ABSTRACTA study of the nanoscale mechanical properties of isotactic mesomorphic and semi‐crystalline polypropylene (iPP) is presented. Two iPPs produced with metallocene and Ziegler‐Natta catalyst polymerization are used. The resulting fibers are characterized by wide angle X‐ray scattering, small‐angle X‐ray scattering (SAXS), and Raman spectroscopy. The spatial variability of the percentage crystallinity is evaluated based on the SAXS data. AFM indentation is performed to measure the elastic modulus of the fibers in the direction perpendicular and parallel to the fiber axis. Since the AFM probing is performed on a scale larger than the lamellar thickness, a statistical analysis of the AFM and SAXS data is necessary to infer the elastic moduli of the α crystals and of the inter‐lamellar regions. The elastic modulus of the crystalline lamellae in the direction perpendicular to the c‐axis of the α crystal probed in compression is estimated at approximately 3.3 GPa, while the effective modulus of the interlamellar regions ranges from 1.5 to 2.2 GPa. The method proposed can be applied to other material systems with similar layered structure to measure elastic moduli or hardness on length scales smaller than the resolution of the indentation test. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43649.

AFM force indentation analysis on leukemia cells

To complement the existing literature, an automated batch analysis protocol was developed to perform unbiased data processes on AFM indentation data measured over leukemia cancer cells.

Quantifying cellular mechanics and adhesion in renal tubular injury using single cell force spectroscopy

Tubulointerstitial fibrosis represents the major underlying pathology of diabetic nephropathy where loss of cell-to-cell adhesion is a critical step. To date, research has predominantly focussed on the loss of cell surface molecular binding events that include altered protein ligation. In the current study, atomic force microscopy single cell force spectroscopy (AFM-SCFS) was used to quantify changes in cellular stiffness and cell adhesion in TGF-β1 treated kidney cells of the human proximal tubule (HK2). AFM indentation of TGF-β1 treated HK2 cells showed a significant increase (42%) in the elastic modulus (stiffness) compared to control. Fluorescence microscopy confirmed that increased cell stiffness is accompanied by reorganization of the cytoskeleton. The corresponding changes in stiffness, due to F-actin rearrangement, affected the work of detachment by changing the separation distance between two adherent cells. Overall, our novel data quantitatively demonstrate a correlation between cellular elasticity, adhesion and early morphologic/phenotypic changes associated with tubular injury. From the Clinical EditorDiabetes affects many patients worldwide. One of the long term problems is diabetic nephropathy. Here, the authors utilized atomic force microscopy single cell force spectroscopy (AFM- SCFS) to study cellular stiffness and cell adhesion after TGF1 treatment in human proximal tubule kidney cells. The findings would help further understand the overall disease mechanism in diabetic patients.

Determination of work of adhesion of biological cell under AFM bead indentation

Hertz contact theory has been widely used for the determination of cell elasticity based on AFM indentation experiments. In light of the adhesive contact between AFM tip and cell, this study applied Johnson–Kendall–Roberts (JKR) model to fit the indentation force–displacement (F–D) curves reported previously. A MIN6 cell has been modeled as first a sphere and then a flattened cell with different thicknesses. The results have shown that both basic JKR model and “generalized” JKR model can best describe the unloading force–displacement behaviors of the indentation curves. The Young׳s modulus of the cell and the work of adhesion of the cell–indenter interface are obtained. In comparison to the Hertzian contact model, the JKR model provides obviously better fitting to the experimental results, indicating that the adhesion is significant in the cell interaction.

Influence of Nanoscale Surface Roughness on Colloidal Force Measurements.

Forces between colloidal particles determine the performances of many industrial processes and products. Colloidal force measurements conducted between a colloidal particle AFM probe and particles immobilized on a flat substrate are valuable in selecting appropriate surfactants for colloidal stabilization. One of the features of inorganic fillers and extenders is the prevalence of rough surfaces-even the polymer latex particles, often used as model colloidal systems including the current study, have rough surfaces albeit at a much smaller scale. Surface roughness is frequently cited as the reason for disparity between experimental observations and theoretical treatment but seldom verified by direct evidence. This work reports the effect of nanoscale surface roughness on colloidal force measurements carried out in the presence of surfactants. We applied a heating method to reduce the mean surface roughness of commercial latex particles from 30 to 1 nm. We conducted force measurements using the two types of particles at various salt and surfactant concentrations. The surfactants used were pentaethylene glycol monododecyl ether, Pluronic F108, and a styrene/acrylic copolymer, Joncryl 60. In the absence of the surfactant, nanometer surface roughness affects colloidal forces only in high salt conditions when the Debye length becomes smaller than the surface roughness. The adhesion is stronger between colloids with higher surface roughness and requires a higher surfactant concentration to be eliminated. The effect of surface roughness on colloidal forces was also investigated as a function of the adsorbed surfactant layer structure characterized by AFM indentation and dynamic light scattering. We found that when the layer thickness exceeds the surface roughness, the colloidal adhesion is less influenced by surfactant concentration variation. This study demonstrates that surface roughness at the nanoscale can influence colloidal forces significantly and should be taken into account in colloidal dispersion formulations.

Decreased deformability of lymphocytes in chronic lymphocytic leukemia.

This paper reports the first study of stiffness/deformability changes of lymphocytes in chronic lymphocytic leukemia (CLL) patients, demonstrating that at the single cell level, leukemic metastasis progresses are accompanied by biophysical property alterations. A microfluidic device was utilized to electrically measure cell volume and transit time of single lymphocytes from healthy and CLL patients. The results from testing thousands of cells reveal that lymphocytes from CLL patients have higher stiffness (i.e., lower deformability), as compared to lymphocytes in healthy samples, which was also confirmed by AFM indentation tests. This observation is in sharp contrast to the known knowledge on other types of metastatic cells (e.g., breast and lung cancer cells) whose stiffness becomes lower as metastasis progresses.

Exploring the Elastic Behavior of Core–shell Organic–Inorganic Spherical Particles by AFM Indentation Experiments

Monodispersed polystyrene (PS)-silica core–shell composite particles were synthesized via the hydrolysis and condensation of tetraethoxysilane (TEOS) on PS colloids at acidic medium. The thickness of silica coating was controlled by the amount of the addition of TEOS during the shell growth process. Transmission electron microscopy results confirmed that a continuous amorphous network of homogenous coating of silica was formed on the PS colloids. After coating by silica, the particle diameter increased from ca. 221 nm for uncoated PS cores to ca. 243–286 nm for PS-silica composite particles observed by scanning electron microscopy, indicating that the silica shell thickness was 11–32 nm. The elastic behavior of the obtained products was investigated by means of atomic force microscopy. The elastic moduli of samples were calculated by fitting the retract curves in force-separation plots based on the Hertzian contact model. The average moduli were 4–8 GPa for the PS-silica composite particles which were much lower than the that of the pure silica (72–75 GPa) and closed to that of the PS cores (2.1 ± 0.5 GPa). The elastic moduli of the PS-silica hybrids increased with increasing of silica shell thickness, suggesting that the elasticity of the PS-silica composite particles might be attributed to the PS cores and the silica shell was stiffening the polymer cores. These results provide a basis for exploring the mechanical properties of core–shell PS-silica hybrids in the application of novel abrasives for chemical mechanical polishing.