Force-distance Curve Measurements Research Articles

Correlation between plant cell wall stiffening and root extension arrest phenotype in the combined abiotic stress of Fe and Al.

The plasticity and growth of plant cell walls (CWs) remain poorly understood at the molecular level. In this work, we used atomic force microscopy (AFM) to observe elastic responses of the root transition zone of 4-day-old Arabidopsis thaliana wild-type and almt1-mutant seedlings grown under Fe or Al stresses. Elastic parameters were deduced from force-distance curve measurements using the trimechanic-3PCS framework. The presence of single metal species Fe2+ or Al3+ at 10 µM exerts no noticeable effect on the root growth compared with the control conditions. On the contrary, a mix of both the metal ions produced a strong root-extension arrest concomitant with significant increase of CW stiffness. Raising the concentration of either Fe2+ or Al3+ to 20 µM, no root-extension arrest was observed; nevertheless, an increase in root stiffness occurred. In the presence of both the metal ions at 10 µM, root-extension arrest was not observed in the almt1 mutant, which substantially abolishes the ability to exude malate. Our results indicate that the combination of Fe2+ and Al3+ with exuded malate is crucial for both CW stiffening and root-extension arrest. However, stiffness increase induced by single Fe2+ or Al3+ is not sufficient for arresting root growth in our experimental conditions.

Non-raft submicron domain formation in cholesterol-containing lipid bilayers induced by polyunsaturated phosphatidylethanolamine

Domain formation in “HLC” ternary lipid bilayers, comprising a high transition temperature (High-Tm) lipid, a Low-Tm lipid, and cholesterol (Chol), has been extensively studied as raft-resembling systems. Recently, we reported the formation of submicron domains in an “LLC” lipid bilayer, encompassing Low-Tm phosphatidylethanolamine (PE), Low-Tm phosphatidylcholine (PC), and Chol. We hypothesized that the formation of this unique domain is driven by polyunsaturated PE. In this study, we explored the effects of the degree of PE unsaturation and the double bond distribution at the sn-position on the mechanism of formation and the composition of submicron domains. Supported lipid bilayers (SLBs), comprising PE with various degrees of unsaturation, monounsaturated PC (POPC), and Chol, were investigated using fluorescence microscopy, atomic force microscopy, and the force–distance curve measurement. The area fraction of submicron domains in PE+POPC+Chol-SLB increased with the PE concentration and degree of unsaturation of the PE acyl chain. The results indicated that the submicron domains were enriched with polyunsaturated PE and were in the liquid-disordered-like state, whereas their surrounding regions were in the liquid-ordered-like state. Segregation of polyunsaturated PE from the Chol-containing region generated submicron domains in the LLC lipid bilayer. We propose a mechanism for the formation of these submicron domains based on molecular interactions involving the hydrophobic and hydrophilic parts of the bilayer membrane.

Surface Roughness of Cu-Bearing Stainless Steel Affects Its Contact-Killing Efficiency by Mediating the Interfacial Interaction with Bacteria.

Numerous studies have found that the surface topography affects the material antibacterial properties by reducing the attachment of bacteria on the surfaces without influencing the viability of the adhered cells. For Cu-bearing alloys with excellent contact-killing properties, bacterial adhesion on the surface is also accompanied by short-range interactions which regulate the toxic effects of the material surface against bacterial cells. Thus, the surface topography of Cu-bearing alloys, as an important factor dominating the exposure level of bacteria on the surfaces, should affect the subsequent contact-killing efficiency. In this work, our major focus was on the regulation mechanism of the surface features on the material-bacterial interactions. We correlated the surface properties including different surface roughnesses of Cu-bearing stainless steel (SS) with the bacterial damage pattern and attempted to clarify the role of surface roughness in mediating the contact-killing behavior of Cu-bearing SS. The results of both atomic force microscopy and scanning electron microscopy investigations showed that E. coli cells experienced the most rapid physical and mechanical damages after incubating with the diamond-polished Cu-bearing SS surface. The bacterial cells noticeably stiffened and the adhesion force significantly increased, as evidenced by force-distance curve measurements. Because of the enhanced hydrophobicity and higher surface potential of the diamond-polished surface, which strengthened the Lewis acid-base attractive forces and weakened the electrostatic barrier between the bacteria and the surface, a higher exposure surface for bacteria was generated. Furthermore, the contact-induced charge transfer, manifested by Cu ion burst release, and reactive oxygen species overexpression contribute to an efficient contact-killing process.

Calibration of T-shaped atomic force microscope cantilevers using the thermal noise method.

The tip-sample interaction force measurements in atomic force microscopy (AFM) provide information about materials' properties with nanoscale resolution. The T-shaped cantilevers used in Torsional-Harmonic AFM allow measuring the rapidly changing tip-sample interaction forces using the torsional (twisting) deflections of the cantilever due to the off-axis placement of the sharp tip. However, it has been difficult to calibrate these cantilevers using the commonly used thermal noise-based calibration method as the mechanical coupling between flexural and torsional deflections makes it challenging to determine the deflection sensitivities from force-distance curves. Here, we present thermal noise-based calibration of these T-shaped AFM cantilevers by simultaneously analyzing flexural and torsional thermal noise spectra, along with deflection signals during a force-distance curve measurement. The calibration steps remain identical to the conventional thermal noise method, but a computer performs additional calculations to account for mode coupling. We demonstrate the robustness of the calibration method by determining the sensitivity of calibration results to the laser spot position on the cantilever, to the orientation of the cantilever in the cantilever holder, and by repeated measurements. We validated the quantitative force measurements against the known unfolding force of a protein, the I91 domain of titin, which resulted in consistent unfolding force values among six independently calibrated cantilevers.

Mapping the surface potential, charge density and adhesion of cellulose nanocrystals using advanced scanning probe microscopy

Cellulose nanocrystals (CNC) are the focus of significant attention in the broad area of sustainable technologies for possessing many desirable properties such as a large surface area, high strength and stiffness, outstanding colloidal stability, excellent biocompatibility and biodegradability, low weight and abundance in nature. Yet, a fundamental understanding of the micro- and nanoscale electrical charge distribution on nanocellulose still remains elusive. Here we present direct quantification and mapping of surface charges on CNCs at ambient condition using advanced surface probe microscopy techniques such as Kelvin probe force microscopy (KPFM), electrostatic force microscopy (EFM) and force-distance (F–D) curve measurements. We show by EFM measurements that the surface charge in the solid-state (as contrasted with liquid dispersions) present at ambient condition on CNCs provided by Innotech Alberta is intrinsically negative and the charge density is estimated to be 13 μC/cm2. These charges also result in CNCs having two times the adhesive force exhibited by SiO2 substrates in adhesion mapping studies. The origin of negative surface charge is likely due to the formation of CNCs through sulfuric acid hydrolysis where sulfate half esters groups remained on the surface (Johnston et al., 2018).

Effect of the adhesion force on the equation of adhesive wear and the generation process of wear elements in adhesive wear of metals

The effects of the adhesion force on the equation of adhesive wear were investigated by examining the relationship between the adhesion force of actual sliding surfaces and the number of wear elements generated at a junction. The number of wear elements and their generation mechanism in the elementary wear processes were determined by nanoscale observations and by analysis of the sliding surfaces by scanning electron microscopy and atomic-force microscopy. In addition, the adhesion force between actual sliding metal surfaces was measured by a method combining friction-force microscopy and force–distance curve measurement. The adhesion force between metal-probe cantilevers of nine different metals and the surfaces of blocks of the same metal were measured. We propose a modified equation for adhesive wear that takes into account the effect of the adhesion force, and we discuss the precise process for the generation of wear elements.

Study of ultrasound-assisted nanomachining on monocrystalline silicon

This study constructed an ultrasound-assisted AFM nanomachining platform by integrating an atomic force microscope (AFM) with a quartz crystal microbalance, which was subsequently used in experiments for determining the microscopic phenomena of ultrasound-assisted nanomachining. Force-distance curve measurement was used to establish a normal force measurement model, obtaining the downforce strengths during ultrasound-assisted nanomachining. Subsequently, machining experiments were conducted on monocrystalline silicon to explore the effects of various experimental parameters (e.g., machining speed and normal force) on machining depth. The experiments showed that the ultrasound-assisted AFM nanomachining platform constructed in this study is effective. After ultrasound was incorporated, monocrystalline silicon specimens made of brittle material could be more effectively processed under various experimental parameters. Moreover, the performance of ultrasound-assisted nanomachining was superior to that of general nanomachining.

Novel microscopy-based approaches to study the film formation mechanism and associated mechanical response in polymer lattices.

In this paper, we present the results from novel microscopy-based approaches aimed at providing further insight into the mechanism of film formation and associated mechanical response in polymer lattices. Firstly, a 'simple' methodology, combining the use of variable pressure scanning electron microscopy and a recently introduced enhanced coolstage (-50 to +50°C), was successfully developed and not only used to study dynamic processes, e.g. different stages of latex film formation, but also for high-resolution imaging of 'freeze-dried' structures. By using the enhanced freeze-drying capability of the system, it was also possible to preserve the structure and features of the studied system with minimum shrinkage and distortion and in the case of polymer lattices at a desired stage of film formation. Moreover, specimens can then be readily imaged, without the need of conductive coatings and at much lower chamber gas pressures, thus minimizing the beam skirting effects and allowing higher resolutions to be achieved. The second and final part of our study consider the mechanical response of the studied latex dried under different conditions, with the particular emphasis on the effects of drying rate [% relative humidity (RH)]. Atomic force microscopy force distance curve measurements revealed that while the %RH did not have an effect on the structures formed, it did have an effect on the adhesive properties of the studied system. It is strongly believed that the methodologies developed and used here can be applied to other material systems, including biologicals and pharmaceuticals.

Intermolecular Interaction of Polymer Brushes Containing Phosphorylcholine and Inverse-Phosphorylcholine.

Choline phosphate (CP) is a phosphobetaine-type zwitterionic functional group, referred to as inverse phosphorylcholine (PC) due to the reverse orientation of a positively charged quaternary amine and anionic phosphate in contrast to PC lipids in nature. The A unique dipole paring between CP and PC groups has attracted much attention in the biointerface research field. Herein, to evaluate the molecular interaction between the CP and PC groups in water, force-distance curve measurements using scanning probe microscopy (SPM) with a PC-group-functionalized cantilever was carried out on the surface of polymer brushes bearing the CP groups. Three types of methacrylate monomers bearing CP with ethyl (Et), methoxyethyl (MOE), and isopropyl (iPr) phosphates were synthesized in 42-71% yields, and polymerized by surface-initiated atom transfer radical polymerization to form polymer brushes on silicon wafers. The surface free energy of CP-polymer brushes with Et, MOE, and iPr was estimated to be 64.0, 61.4, and 57.4 mN m-1, respectively, based on contact angle measurements. Force-distance curve measurements of polymer brushes having a CP group was conducted in water at 25 °C by SPM using a spherical probe produced by attaching a silica particle (SiP; d = 25 μm) covered with PC or CP groups to a tipless cantilever. Adhesion force larger than 14 nN was observed between the CP-polymer brushes and PC-SiP, whereas PC-polymer brushes revealed extremely low adhesion force of less than 0.6 nN with PC-SiP and propylsilane-modified SiP. The specific attractive molecular interaction between CP and PC groups was quantitatively evaluated.

Jumping-mode atomic force microscopy in studying the electromechanical properties of soft samples

A new approach is proposed for the nondestructive piezoresponse measuring of soft, loose, and fragile nanostructures with simultaneous characterization of their mechanical properties. The new mode is based on HybriD mode atomic force microscopy (fast force–distance curve measurements with real-time processing of the obtained data). The piezoelectric and mechanical properties of diphenylalanine peptide tubes with diameters of less than 100 nm are measured for the first time.

Active damping by Q-control for fast force-distance curve measurements in atomic force microscopy.

This paper investigates the benefit of active damping by an analog Q-control circuit for measuring fast force-distance curves in atomic force microscopy. By active damping of the cantilever oscillation after snap-off, the down-ring time-constant is reduced significantly from 385 μs to 23 μs. Experimental results demonstrate that the number of force-distance curves per second can be increased by a factor of more than 30.

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.

Radial stiffness characteristics of the overlap regions of sarcomeres in isolated skeletal myofibrils in pre-force generating state

We have studied the stiffness of myofilament lattice in sarcomeres in the pre-force generating state, which was realized by a relaxing reagent, BDM (butane dione monoxime). First, the radial stiffness for the overlap regions of sarcomeres of isolated single myofibrils was estimated from the resulting decreases in diameter by osmotic pressure applied with the addition of Dextran. Then, the radial stiffness was also estimated from force-distance curve measurements with AFM technology. The radial stiffness for the overlap regions thus obtained was composed of a soft and a rigid component. The soft component visco-elastically changed in a characteristic fashion depending on the physiological conditions of myofibrils, suggesting that it comes from cross-bridge structures. BDM treatments significantly affected the soft radial component of contracting myofibrils depending on the approach velocity of cantilever: It was nearly equal to that in the contracting state at high approach velocity, whereas as low as that in the relaxing state at low approach velocity. However, comparable BDM treatments greatly suppressed the force production and the axial stiffness in contracting glycerinated muscle fibers and also the sliding velocity of actin filaments in the in vitro motility assay. Considering that BDM shifts the cross-bridge population from force generating to pre-force generating states in contracting muscle, the obtained results strongly suggest that cross-bridges in the pre-force generating state are visco-elastically attached to the thin filaments in such a binding manner that the axial stiffness is low but the radial stiffness significantly high similar to that in force generating state.

AFM force distance curve measurement for surface investigation of polymers compound blend with metal nanoparticles

The atomic force microscopy (AFM) has been used to perform surface force measurements in contact mode to investigate surface properties of model systems in the nanoscale. Two different polymers compound are observed include Polydimethylsiloxane (PDMS) and Polyvinylidene fluoride (PVDF). Both polymers compound are blended with metal nanoparticles consist of gold and silver by compare with the pristine polymer. The results from adhesion force and surface roughness tests are inversed. The adhesion force is increased with the NPs concentration whereas the surface roughness is decreased. Hydrophobic and hydrophilic properties of the polymer have been studied by employing contact angle. However, variation of contact angle of polymer nanocomposites is confirmed the hydrophobicity of PDMS while PVDF is certainly different around 70-77° of Ag NPs and 70-81° of Au NPs.

Design methodology for nano-engineered surfaces to control adhesion: Application to the anti-adhesion of particles

With increasing demand for means of controlling surface adhesion in various applications, including the semiconductor industry, optics, micro/nanoelectromechanical systems, and the medical industry, nano-engineered surfaces have attracted much attention. This study suggests a design methodology for nanostructures using the Derjaguin approximation in conjunction with finite element analysis for the control of adhesion forces. The suggested design methodology was applied for designing a nano-engineered surface with low-adhesion properties. To verify this, rectangular and sinusoidal nanostructures were fabricated and analyzed using force-distance curve measurements using atomic force microscopy and centrifugal detachment testing. For force-distance curve measurements, modified cantilevers with tips formed with atypical particles were used. Subsequently, centrifugal detachment tests were also conducted. The surface wettability of rectangular and sinusoidal nanostructures was measured and compared with the measured adhesion force and the number of particles remaining after centrifugal detachment tests.

Polyurethane Coatings Based on Chemically Unmodified Fractionated Lignin

New lignin-based thermoset polyurethane (PU) coatings with high lignin content are presented in this work. These materials were obtained by extracting a sol fraction from a raw kraft lignin sample with the bioderived solvent 2-methyltetrahydrofuran (MeTHF), and subsequently directly cross-linking the MeTHF-soluble lignin fraction with a toluene diisocyanate (TDI)-based polyisocyanate at different weight ratios. A thorough characterization of the PU materials highlighted their improved thermal stability, better film forming ability and higher hydrophobic character compared with the un-cross-linked lignin precursor. Additionally, force–distance curve measurements by atomic force microscopy were employed to determine the elastic modulus of the PU materials. Finally, the lignin-based PU materials were found to exhibit high adhesion on different substrates, including glass, wood and metals. The results of this study demonstrate that the direct reaction of chemically unmodified fractionated lignin with polyisoc...

Label-free and quantitative evaluation of cytotoxicity based on surface nanostructure and biophysical property of cells utilizing AFM

In this study, the four commonly used cytotoxicity assays and the mechanical properties as evaluated by atomic force microscopy (AFM) were compared in a cellular system. A cytotoxicity assay is the first and most essential test to evaluate biocompatibility of various toxic substances. Many of the cytotoxicity methods require complicated and labor-intensive process, as well as introduce experimental error. In addition, these methods cannot provide instantaneous and quantitative cell viability information. AFM has become an exciting analytical tool in medical, biological, and biophysical research due to its unique abilities. AFM-based force–distance curve measurements precisely measure the changes in the biophysical properties of the cell. Therefore, we observed the morphological changes and mechanical property changes in L929 cells following sodium lauryl sulfate (SLS) treatment utilizing AFM. AFM imaging showed that the toxic effects of SLS changed not only the spindle-like shape of L929 cells into a round shape, but also made a rough cell surface. As the concentration of SLS was increased, the surface roughness of L929 cell was increased, and stiffness decreased. We confirmed that inhibition of proliferation clearly increased with increases in SLS concentration based on results from MTT, WST, neutral red uptake, and LIVE/DEAD viability/cytotoxicity assays. The estimated IC50 value by AFM analysis was similar to those of other conventional assays and was included within the 95% confidence interval range. We suggest that an AFM quantitative analysis of the morphological and biophysical changes in cells can be utilized as a new method for evaluating cytotoxicity.

Evaluation of Interaction Between Histidine Binding Cu2+ Ion and Histidine by Atomic Force Microscopy

This paper presents a direct interaction force measurement between histidine molecules using AFM force-distance curve measurement. AFM force-distance curves between the histidine-modified cantilever and substrate in the different conditions with or without intercalating Cu2+ ion were measured and interpreted via Gaussian curve fitting analyses. The adhesion force between histidine molecules was shown to be 110 pN under the presence of Cu2+. The result was compareable to the measured adhesion force about 0 pN, which was measured by the removal of Cu2+ ion with the addition of EDTA. The result indicated the direct histidine-histidie interaction was difficult without the role of the bridigible ionic component. From the results, the possibility of direct measurement on chemical affinities between biomolecules was suggested by using AFM force-distance curve analyses. Especially, the current approach showed the possible affinity measurement techniques that elucidate the role of bridge ions.

Quantitative and qualitative analysis of heart mitochondria for evaluating the degree of myocardial injury utilizing atomic force microscopy

Mitochondrial dysfunction plays a central role in mediating both the necrotic and apoptotic components of reperfusion injury. Because mitochondrial swelling is one of the most important indicators of the beginning of mitochondrial permeability transition, quantification of morphological changes in mitochondria would be useful in evaluating the degree of IR injury, as well as the protective effects of various therapies. In this study, we characterized the morphological changes in heart mitochondria caused by the duration and severity of ischemia utilizing particle shape analysis on atomic force microscopy (AFM) topographic images. We also simultaneously investigated the nano-mechanical changes in rat heart mitochondria by injury using force–distance curve measurements. Rats were randomly divided into 3 groups: control group (n=3), myocardial ischemia without reperfusion (PI group, n=3), and myocardial ischemia with reperfusion (IR group, n=4). Normal mitochondria appeared ellipsoidal with a mean area of 3551±1559nm2 and mean perimeter of 217.54±52.09nm (n=60). The mean area and perimeter of mitochondria in the IR groups increased to 28,181±21,248nm2 and 595.74±234.29nm (n=40, p<0.0001 vs. control group, respectively), maintaining oval in shape. But, in the PI group, all parameters showed significant differences compared to parameters of the control group (n=35, p<0.0001). In particular, the mean axial ratio and roundness were significantly different from those in the IR group. Mitochondria in the PI group looked more spherical than those of control and IR groups. Adhesion force is the force before the last event on the retraction half of force–distance curve measurements, corresponding to the point where the tip and the surface loose contact. The adhesion forces of heart mitochondria in the IR and PI groups significantly decreased to 19.56±1.08nN (n=30, p<0.0001) and 18.65±3.18nN (n=30, p<0.0001), compared to normal mitochondria which had an adhesion force of 27.64±0.88nN (n=30). Adhesion force is governed by the attractive portion of the interacting forces between the surface atoms of the contacts. From the morphological and nano-mechanical changes in heart mitochondria, we suggested that the outer membranes of mitochondria were broken by myocardial ischemic injury before they became swollen, and the swelling might be correlated with the ischemic injury. We inferred that the breakage of membranes leads to uptake of water and matrix swelling. As a result, shape measurement parameters for the quantitative analysis of mitochondrial swelling could be very effective for evaluating the myocardial injury.

Real-Time Monitoring of the Effects of Telmisartan on Angiotensin II-Induced Mechanical Changes in Live Mesangial Cells Using Atomic Force Microscopy

Background/Aims: Recent studies have shown that angiotensin II (Ang II) type 1 receptor blockers (ARB) may provide renal protection independent of their blood pressure-lowering effect. However, evidence for this comes from indirect methods, such as genetic or protein expression studies. In this study, we hypothesized that telmisartan, a specific ARB, applied to Ang II-stimulated mesangial cell (MC) would exert a renoprotective effect via modulation of MCs’ mechanical properties. Methods: We investigated the effect of telmisartan on Ang II-induced changes in MCs utilizing real-time atomic force microscopy (AFM) imaging and force-distance curve measurements. Results: Real-time AFM images of live MCs demonstrated that cells contracted towards the center after Ang II exposure, and telmisartan treatment abolished this change. Cellular spring constants showed that telmisartan prevented Ang II-induced MC stiffening (Ang II: 0.109 ± 0.019 N/m, Ang II + telmisartan: 0.051 ± 0.016 N/m, p < 0.005). Telmisartan-treated MCs had a significantly lower adhesion force than those of the control group (control: 0.49 ± 0.22 nN, telmisartan: 0.22 ± 0.06 nN, Ang II: 0.40 ± 0.25nN, Ang II + telmisartan: 0.27 ± 0.14 nN, p < 0.005). These results demonstrate that the dynamic contraction and mechanical properties of Ang II-stimulated MCs are restored by telmisartan. Conclusions: We report for the first time the use of AFM force-distance curves on live MCs to directly monitor changes in surface adhesion and stiffness of cells after treatment with telmisartan in real time.