Atomic Force Microscopy Force Distance Research Articles

Quantitative study of early-stage transient bacterial adhesion to bioactive glass and glass ceramics: atomic force microscopic observations

Antimicrobial potential of bioactive glass (BAG) makes it promising for implant applications, specifically overcoming the toxicity concerns associated with traditional antibacterial nanoparticles. The 58S composition of BAG (with high Ca and absence of Na) has been known to exhibit excellent bioactivity and antibacterial behaviour, but the mechanisms behind have not been investigated in detail. In this pioneering study, we are using Atomic Force Microscopy (AFM) to gain insights into 58S BAG’s adhesive interactions with planktonic cells of both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria; along with the impact of crystallinity on antibacterial properties. We have recorded greater bacterial inhibition by amorphous BAG compared to semi-crystalline glass–ceramics and stronger effect against gram-negative bacteria via conventional long-term antibacterial tests. AFM force distance curves has illustrated substantial bonding between bacteria and BAG within the initial one second (observed at a gap of 250 ms) of contact, with multiple binding events. Further, stronger adhesion of BAG with E.coli (~ 6 nN) compared to S. aureus (~ 3 nN) has been found which can be attributed to more adhesive nano-domains (size effect) distributed uniformly on E.coli surface. This study has revealed direct evidence of impact of contact time and 58S BAG’s crystalline phase on bacterial adhesion and antimicrobial behaviour. Current study has successfully demonstrated the mode and mechanisms of initial bacterial adhesion with 58S BAG. The outcome can pave the way towards improving the designing of implant surfaces for a range of biomedical applications.

Evaluation of extracellular polymeric substances matrix volume, surface roughness and bacterial adhesion property of oral biofilm

Background/purposeOral biofilms are highly structured bacterial colonies embedded in a highly hydrated extracellular polymeric substances (EPS) matrix. This study aimed to investigate the characteristics of oral biofilm at different stages of maturation. Materials and methodsOral multispecies biofilms were grown anaerobically from plaque bacteria on collagen coated hydroxyapatite discs in brain heart infusion broth for one and three weeks. The volume of live bacteria and EPS matrix of the biofilms were determined by using corresponding fluorescent probes and confocal laser scanning microscopy. Atomic force microscopy (AFM) was used to quantitatively probe and correlate cell surface adhesion force of biofilms. The surface roughness was quantified in terms of the root mean square average of the height deviations. Adhesion was measured from force–distance data for the retraction of the cell from the surface. ResultsThe volume of live bacteria and EPS of 3-week-old biofilms was higher than 1-week-old biofilms. The surface roughness value in 1-week-old biofilms was significantly higher than that in 3-week-old biofilms. AFM force–distance curve results showed that the adhesion force at the cell–cell interface was significantly more at-tractive than those at bacterial cells surface of both stages biofilms. Adhesion forces between the AFM tip and the surface of bacterial cell were fairly constant, whereas the cell–cell interface experienced greater adhesion forces in the biofilm's development. ConclusionAs oral biofilms become mature, EPS volume and cell–cell adhesion forces increase while the surface roughness decreases.

Sensing red blood cell nano-mechanics: Toward a novel blood biomarker for Alzheimer's disease.

Red blood cells (RBCs) are characterized by a remarkable elasticity, which allows them to undergo very large deformation when passing through small vessels and capillaries. This extreme deformability is altered in various clinical conditions, suggesting that the analysis of red blood cell (RBC) mechanics has potential applications in the search for non-invasive and cost-effective blood biomarkers. Here, we provide a comparative study of the mechanical response of RBCs in patients with Alzheimer’s disease (AD) and healthy subjects. For this purpose, RBC viscoelastic response was investigated using atomic force microscopy (AFM) in the force spectroscopy mode. Two types of analyses were performed: (i) a conventional analysis of AFM force–distance (FD) curves, which allowed us to retrieve the apparent Young’s modulus, E; and (ii) a more in-depth analysis of time-dependent relaxation curves in the framework of the standard linear solid (SLS) model, which allowed us to estimate cell viscosity and elasticity, independently. Our data demonstrate that, while conventional analysis of AFM FD curves fails in distinguishing the two groups, the mechanical parameters obtained with the SLS model show a very good classification ability. The diagnostic performance of mechanical parameters was assessed using receiving operator characteristic (ROC) curves, showing very large areas under the curves (AUC) for selected biomarkers (AUC > 0.9). Taken all together, the data presented here demonstrate that RBC mechanics are significantly altered in AD, also highlighting the key role played by viscous forces. These RBC abnormalities in AD, which include both a modified elasticity and viscosity, could be considered a potential source of plasmatic biomarkers in the field of liquid biopsy to be used in combination with more established indicators of the pathology.

Effects of substrate stiffness on the viscoelasticity and migration of prostate cancer cells examined by atomic force microscopy.

The stiffness of the extracellular matrix of tumour cells plays a key role in tumour cell metastasis. However, it is unclear how mechanical properties regulate the cellular response to the environmental matrix. In this study, atomic force microscopy (AFM) and laser confocal imaging were used to qualitatively evaluate the relationship between substrate stiffness and migration of prostate cancer (PCa) cells. Cells cultured on stiff substrates (35 kPa) undergone several interesting phenomena compared to those on soft substrates (3 kPa). Here, the stimulation generated by the stiff substrates triggered the F-actin skeleton to bundle its filaments, increasing the polarity index of the external contour of PCa cells. Analysis of AFM force–distance curves indicated that the elasticity of the cells cultured on 35 kPa substrates increased while the viscosity decreased. Wound-healing experiments showed that PCa cells cultured on 35 kPa substrates have higher migration potential. These phenomena suggested that the mechanical properties may be correlated with the migration of PCa cells. After actin depolymerisation, the elasticity of the PCa cells decreased while the viscosity increased, and the migration ability was correspondingly decreased. In conclusion, this study clearly demonstrated the relationship between substrate stiffness and the mechanical properties of cells in prostate tumour metastasis, providing a basis for understanding the changes in the biomechanical properties at a single-cell level.

Direct measurement of storage and loss behavior in AFM force–distance experiments using the modified Fourier transformation

Force–distance curve experiments are commonly performed in atomic force microscopy (AFM) to obtain the viscoelastic characteristics of materials, such as the storage and loss moduli or compliances. The classic methods used to obtain these characteristics consist of fitting a viscoelastic material model to the experimentally obtained AFM data. Here, we demonstrate a new method that utilizes the modified discrete Fourier transform to approximate the storage and loss behavior of a material directly from the data, without the need for a fit. Additionally, one may still fit a model to the resulting storage and loss behavior if a parameterized description of the material is desired. In contrast to fitting the data to a model chosen a priori, departing from a model-free description of the material's frequency behavior guides the selection of the model, such that the user may choose the one that is most appropriate for the particular material under study. To this end, we also include modified Fourier domain descriptions of commonly used viscoelastic models.

Multiple-fibre interferometry setup for probe sample interaction measurements in atomic force microscopy

Atomic force microscopy (AFM) often relies on the assumption that cantilever bending can be described by simple beam theory and that the displacement of the tip can be evaluated from the cantilever angle. Some more advanced metrological instruments use free-space or fibre interferometers for measuring the position of the cantilever apex directly, thereby simplifying the metrology traceability chain. The next logical development, covering measurements of both the cantilever apex position and its deformation due to lateral forces acting during different AFM measurement regimes, is presented in this paper. It is based on using a set of closely packed fibre interferometers that can be used to determine localised bending of the cantilever at different positions along the cantilever. This can be used for detection of cantilever deformation beyond classical beam theory, and can yield both better understanding of sources of uncertainty in individual AFM force–distance measurements and more accurate scanning in constant height mode in high-speed AFM applications.

Surfaces and Interfaces of Liquid Metal Core–Shell Nanoparticles under the Microscope

Eutectic gallium indium (EGaIn), a Ga-based liquid metal alloy holds great promise for designing next generation core-shell nanoparticles (CSNs). A shearing assisted ligand-stabilization method has shown promise as a synthetic method for these CSNs; however, determining the role of the ligand on stabilization demands an understanding of the surface chemistry of the ligand-nanoparticle interface. EGaIn CSNs have been created functionalized with aliphatic carboxylates of different chain length allowing a fundamental investigation on ligand stabilization of EGaIn CSNs. Raman and diffuse reflectance Fourier transform spectroscopies (DRIFTS) confirm reaction of the ligand with the oxide shell of the EGaIn nanoparticles. Changing the length of the alkyl chain in the aliphatic carboxylates (C2-C18) may influence the size and structural stability of EGaIn CSNs, which is easily monitored using atomic force microscopy (AFM). No matter how large the carboxylate ligand, there is no obvious effect on the size of the EGaIn CSNs, except the particle size got more uniform when coated with longer chain carboxylates. The AFM force distance (F-D) measurements are used to measure the stiffness of the carboxylate coated EGaIn CSN. In corroboration with DRIFTS analysis, the stiffness studies show that the alkyl chains undergo conformational changes upon compression.

Strong Attraction and Adhesion Forces of Dust Particles by System Voltages of Photovoltaic Modules

We report for the first time on direct measurements using atomic force microscopy (AFM) of electric field induced attraction and adhesion forces associated with soiling on photovoltaic (PV) modules. Real dust particles and silica spheres as surrogate to simulating dust particles were glued to AFM probe cantilevers. The electric field induced force (Fes) was measured via AFM force–distance ( f – z ) curves, where the electric field was generated by applying a voltage (Vs) to a simulated PV module. Fes and van der Waals (Fvw) force contributions could be separated with the f – z curves. The results show that Fes ∼2.5 μ N on dust particles is ∼5 times larger than Fvw ∼0.5 μ N at even Vs = −100 V (i.e., similar to large-module operating voltages). Fes increases by an order of magnitude as the applied potential increases from Vs = −100 V to Vs = −500 V. These adhesion forces are by far the strongest that we have measured using the AFM technique for “initial” contact of particles. Furthermore, unlike the more typical short-range forces of Fvw and liquid bridge, Fes extends sub-millimeters to a millimeter beyond the PV module surface, creating large attraction forces to even uncharged dust particles (i.e., via induced dipoles) in the air. These results indicate that the high voltages typically used with PV arrays today will attract more dust particles from the air, hold the dust particles to the surface very strongly, and potentially induce other PV module surface effects, all of which could increase the power production losses because of soiling.

Efficient Spatial Sampling for AFM-Based Cancer Diagnostics: A Comparison between Neural Networks and Conventional Data Analysis

Atomic force microscopy (AFM) in spectroscopy mode receives a lot of attention because of its potential in distinguishing between healthy and cancer tissues. However, the AFM translational process in clinical practice is hindered by the fact that it is a time-consuming technique in terms of measurement and analysis time. In this paper, we attempt to address both issues. We propose the use of neural networks for pattern recognition to automatically classify AFM force–distance (FD) curves, with the aim of avoiding curve-fitting with the Sneddon model or more complicated ones. We investigated the applicability of this method to the classification of brain cancer tissues. The performance of the classifier was evaluated with receiving operating characteristic (ROC) curves for the approach and retract curves separately and in combination with each other. Although more complex and comprehensive models are required to demonstrate the general applicability of the proposed approach, preliminary evidence is given for the accuracy of the results, and arguments are presented to support the possible applicability of neural networks to the classification of brain cancer tissues. Moreover, we propose a possible strategy to shorten measurement times based on the estimation of the minimum number of FD curves needed to classify a tissue with a confidence level of 0.005. Taken together, these results have the potential to stimulate the design of more effective protocols to reduce AFM measurement times and to get rid of curve-fitting, which is a complex and time-consuming issue that requires experienced staff with a strong data-analysis background.

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.

Crystallization of Probucol in Nanoparticles Revealed by AFM Analysis in Aqueous Solution.

The crystallization behavior of a pharmaceutical drug in nanoparticles was directly evaluated by atomic force microscopy (AFM) force curve measurements in aqueous solution. A ternary spray-dried sample (SPD) was prepared by spray drying the organic solvent containing probucol (PBC), hypromellose (HPMC), and sodium dodecyl sulfate (SDS). The amorphization of PBC in the ternary SPD was confirmed by powder X-ray diffraction (PXRD) and solid-state 13C NMR measurements. A nanosuspension containing quite small particles of 25 nm in size was successfully prepared immediately after dispersion of the ternary SPD into water. Furthermore, solution-state 1H NMR measurements revealed that a portion of HPMC coexisted with PBC as a mixed state in the freshly prepared nanosuspension particles. After storing the nanosuspension at 25 °C, a gradual increase in the size of the nanoparticles was observed, and the particle size changed to 93.9 nm after 7 days. AFM enabled the direct observation of the morphology and agglomeration behavior of the nanoparticles in water. Moreover, AFM force-distance curves were changed from (I) to (IV), depending on the storage period, as follows: (I) complete indentation within an applied force of 1 nN, (II) complete indentation with an applied force of 1-5 nN, (III) partial indentation with an applied force of 5 nN, and (IV) nearly no indentation with an applied force of 5 nN. This stiffness increase of the nanoparticles was attributed to gradual changes in the molecular state of PBC from the amorphous to the crystal state. Solid-state 13C NMR measurements of the freeze-dried samples demonstrated the presence of metastable PBC Form II crystals in the stored nanosuspension, strongly supporting the AFM results.

Extracting local surface charges and charge regulation behavior from atomic force microscopy measurements at heterogeneous solid-electrolyte interfaces.

We present a method to determine the local surface charge of solid-liquid interfaces from Atomic Force Microscopy (AFM) measurements that takes into account shifts of the adsorption/desorption equilibria of protons and ions as the cantilever tip approaches the sample. We recorded AFM force distance curves in dynamic mode with sharp tips on heterogeneous silica surfaces partially covered by gibbsite nano-particles immersed in an aqueous electrolyte with variable concentrations of dissolved NaCl and KCl at pH 5.8. Forces are analyzed in the framework of Derjaguin-Landau-Verwey-Overbeek (DLVO) theory in combination with a charge regulation boundary that describes adsorption and desorption reactions of protons and ions. A systematic method to extract the equilibrium constants of these reactions by simultaneous least-squared fitting to experimental data for various salt concentrations is developed and is shown to yield highly consistent results for silica-electrolyte interfaces. For gibbsite-electrolyte interfaces, the surface charge can be determined, yet, an unambiguous identification of the relevant surface speciation reactions is not possible, presumably due to a combination of intrinsic chemical complexity and heterogeneity of the nano-particle surfaces.

Atomic force microscopy and X-ray photoelectron spectroscopy evaluation of adhesion and nanostructure of thin Cr films

Chromium (Cr) thin films were deposited on float glass using electron beam (e-beam) physical vapor deposition and radio frequency (RF) magnetron sputtering techniques. Surface morphology of these Cr films was studied using atomic force microscopy (AFM). The e-beam deposited Cr films consisted of isolated surface mounds while in RF sputtered samples, these mounds combined to form larger islands. Lower surface adhesive properties were observed for e-beam deposited films, as determined from AFM force–distance curves, presumably due to the nanostructural differences. Similar amounts of adsorbed atmospheric carbonaceous contaminants and water vapor were detected on samples deposited using both methods with e-beam deposited samples having additional carbide species, as determined by X-ray photoelectron spectroscopy data. The dominant crystallographic plane in both e-beam deposited and RF sputtered Cr thin films was (110) of body-centered cubic Cr metal structure as determined from X-ray diffraction data. Weak (211) reflection was also observed in RF sputtered samples and was attributed to a different thin Cr film condensation and growth mechanism which resulted in nanostructural differences between films deposited using two different methods.

Structural evaluation of probucol nanoparticles in water by atomic force microscopy

Structural evaluation of probucol nanoparticles coground with polyvinylpyrrolidone K17 and sodium dodecyl sulfate for 90min was performed by solid-state nuclear magnetic resonance (NMR) spectroscopy and atomic force microscopy (AFM) with force–distance curve analysis. The results of solid-state NMR indicated that the cogrinding changed crystalline probucol to amorphous form. The number-averaged mean heights of probucol particles in the ground mixture (GM) suspension were determined by AFM to be 6 and 15nm for freshly prepared and 24h-stored samples, respectively. Nucleation and the subsequent crystal growth might have occurred after the GM was dispersed in water. The presence of probucol nanocrystals and agglomeration of the primary probucol nanoparticles were recognized by AFM force–distance curve analysis. AFM could be a promising tool to evaluate the structure of nanoparticles as well as their agglomeration behavior in aqueous media.

The Role of Growth Temperature in the Adhesion and Mechanics of Pathogenic L. monocytogenes: An AFM Study

The adhesion strengths of pathogenic L. monocytogenes EGDe to a model surface of silicon nitride were quantified using atomic force microscopy (AFM) in water for cells grown under five different temperatures (10, 20, 30, 37, and 40 °C). The temperature range investigated was chosen to bracket the thermal conditions in which L. monocytogenes survive in the environment. Our results indicated that adhesion force and energy quantified were at their maximum when the bacteria were grown at 30 °C. The higher adhesion observed at 30 °C compared to the adhesion quantified for bacterial cells grown at 37, 40, 20, and 10 °C was associated with longer and denser bacterial surface biopolymer brushes as predicted from fitting a model of steric repulsion to the approach distance-force data as well from the results of protein colorimetric assays. Theoretically predicted adhesion energies based on soft-particle DLVO theory agreed well with the adhesion energies computed from AFM force-distance retraction data (r(2) = 0.94); showing a minimum energy barrier to adhesion at 30 °C.

Thermal annealing effect on the nanomechanical properties and structure of P3HT:PCBM thin films

Nanostructured polymer–fullerene thin films are among the most prominent materials for application in high efficient polymer solar cells. Specifically, poly(3-hexylthiophene) (P3HT) and fullerene derivatives (PCBM) blends are used as the donor/acceptor materials forming a bulk heterojunction. Although P3HT:PCBM properties have been extensively studied, less light has been set on its nanomechanical properties, which affect the device service life. In this work Atomic Force Acoustic Microscopy (AFAM), Atomic Force Spectroscopy and Nanoindentation were used to study the effect of the fullerene presence and the annealing on the P3HT:PCBM nanomechanical behavior. The P3HT:PCBM thin films were prepared by spin coating on glass substrates and then annealed at 100 °C and 145 °C for 30 min. Large phase separation was identified by optical and Atomic Force Microscopy (AFM) for the annealed samples. Needle-like PCBM crystals were formed and an increase of the polymer crystallinity degree with the increase of the annealing temperature was confirmed by X-ray diffraction. AFAM characterization revealed the presence of aggregates close to stiff PCBM crystals, possibly consisting of amorphous P3HT material. AFM force–distance curves showed a continuous change in stiffness in the vicinity of the PCBM crystals, due to the PCBM depletion near its crystals, and the AFM indentation provided qualitative results about the changes in P3HT nanomechanical response after annealing.

Characterization of mitochondria isolated from normal and ischemic hearts in rats utilizing atomic force microscopy

Mitochondria play critical roles in both the life and the death of cardiac myocytes. Various factors, such as the loss of ATP synthesis and increase of ATP hydrolysis, impairment in ionic homeostasis, formation of reactive oxygen species (ROS), and release of proapoptotic proteins are related to the generation of irreversible damage. It has been proposed that the release of cytochrome c is caused by a swelling of the mitochondrial matrix triggered by the apoptotic stimuli. However, there is a controversy about whether or not the mitochondria, indeed, swell during apoptosis. The major advantages of atomic force microscopy (AFM) over conventional optical and electron microscopes for bio-imaging include the fact that no special coating and vacuum are required and imaging can be done in all environments—air, vacuum or aqueous conditions. In addition, AFM force–distance curve measurements have become a fundamental tool in the fields of surface chemistry, biochemistry, and material science. In this study, we used AFM to observe the morphological and property changes in heart mitochondria that were isolated from a rat myocardial infarction model. From the shape parameters of the mitochondria in the AFM topographic image, it seemed that myocardial infarction caused the mitochondrial swelling. Also, the results of force–distance measurements showed that the adhesion force of heart mitochondria was significantly decreased by myocardial in infarction. Therefore, we suggested that myocardial infarction might be the cause of mitochondrial swelling and the changes in outer membrane of heart mitochondria.

Investigation of the effect of CrO 42− and MoO 42− on carbon steel corrosion using AFM and electrochemical techniques

The effect of chromate () and molybdate () ions on the corrosion of carbon steel in 0.5 M NaCl solution has been studied using electrochemical measurements and atomic force microscopy (AFM) technique. Potentiodynamic polarization data suggest that both and have inhibition effect on carbon steel corrosion, and the inhibition efficiency increases with increase in concentrations of and ; at the same concentration, the inhibition efficiency of is higher than that of . The increase in concentrations of and anions causes a shift of the breakdown potential (Eb) in the positive direction, indicating the inhibitive effect of the added anions on the pitting attack. At the same concentrations, the breakdown potential of is higher than that of . Electrochemical impedance spectroscopy (EIS) tests reveal that the charge transfer resistance and passive film resistance increase with increase in concentrations of or ; at the same concentrations, as for the charge transfer resistance and passive film resistance were bigger than those of . AFM imaging technique shows that local corrosion was inhibited obviously after the addtion of or , and passive film of was much more compact than that of . AFM force–distance curves indicate that the passive film of is much stiffer than that of .

PH-induced changes in adsorbed β-lactoglobulin molecules measured using atomic force microscopy

We have used atomic force microscopy (AFM) imaging and force spectroscopy to study β-lactoglobulin (β-LG) food protein molecules adsorbed onto a mica surface. In particular, we have studied the effect of in situ changes in pH on several different properties: the topographical morphology of the adsorbed β-LG molecules, adhesion of the β-LG molecules to the underlying mica substrate, and the mechanical unfolding of the β-LG molecules. In AFM images, the structure of the adsorbed protein layer was observed to change dramatically with changes in pH. This result was consistent with the mechanical unfolding of single protein molecules within the adsorbed protein layer at different pH values performed using AFM. The short rupture length (∼50 nm) measured for the fully unfolded protein at an acidic pH value of 2.5 is in good agreement with the dominant single molecule population measured previously for this pH value. Unfolding β-LG molecules from the same protein layer at a neutral value of pH = 6.8 resulted primarily in longer rupture lengths, corresponding to dimers of β-LG. AFM force–distance curves collected at pH = 9 were dominated by a large repulsion between the AFM tip and the adsorbed protein layer, which is likely due to the extended nature of the molecules because of irreversible denaturation for pH values greater than 9. This work provides a novel insight into the mechanisms of protein adsorption onto surfaces and shows that AFM force spectroscopy is a promising tool for probing in situ conformational changes in single molecules under various conditions.

Measurement of polyamide and polystyrene adhesion with coated-tip atomic force microscopy

This work presents atomic force microscopy (AFM) measurements of adhesion forces between polyamides, polystyrene and AFM tips coated with the same materials. The polymers employed were polyamide 6 (PA6), PA66, PA12 and polystyrene (PS). All adhesion forces between the various unmodified or modified AFM tips and the polymer surfaces were in the range −1.5 to − 8 nN . The weakest force was observed for an unmodified AFM tip with a PS surface and the strongest was between a PS-coated tip and PS surface. The results point to both the benefits and drawbacks of coated-tip AFM force–distance measurements. Adhesion forces between the two most dissimilar (PA6–PS and PA66–PS) materials were significantly asymmetric, e.g., the forces were different depending on the relative placement of each polymer on the AFM tip or substrate. Materials with similar chemistry and intermolecular interactions yielded forces in close agreement regardless of placement on tip or substrate. Using experimental forces, we calculated the contact radii via four models: Derjaguin, Muller, and Toporov; Johnson, Kendall, and Roberts; parametric tip–force–distance relation; and a square pyramid–flat surface (SPFS) model developed herein. The SPFS model gave the most reasonable contact tip radius estimate. Hamaker constants calculated from the SPFS model using this radius agreed in both magnitude and trends with experiment and Lifshitz theory.