Atomic Force Microscopy Force-distance Curves Research Articles

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.

Few-shot deep learning for AFM force curve characterization of single-molecule interactions

Deep learning (DL)-based analytics has the scope to transform the field of atomic force microscopy (AFM) with regard to fast and bias-free measurement characterization. For example, AFM force-distance curves can help estimate important parameters of binding kinetics, such as the most probable rupture force, binding probability, association, and dissociation constants, as well as receptor density on live cells. Other than the ideal single-rupture event in the force-distance curves, there can be no-rupture, double-rupture, or multiple-rupture events. The current practice is to go through such datasets manually, which can be extremely tedious work for the experimentalists. We address this issue by adopting a few-shot learning approach to build sample-efficient DL models that demonstrate better performance than shallow ML models while matching the performance of moderately trained humans. We also release our AFM force curve dataset and annotations publicly as a benchmark for the research community.

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.

New insights into external layers of cyanobacteria and microalgae based on multiscale analysis of AFM force-distance curves

External layers, the outermost structures around cells, perform essential eco-physiological functions to support cyanobacteria and microalgae in aquatic environments. These layers have been recognized as adaptations to turbulence, a ubiquitous and inherent physical process occurring in the environments of most cyanobacteria and microalgae. However, the underlying biophysical mechanism of these layers is still poorly understood. Force measurements were performed directly on the external layers of eight living cyanobacterial and green algal strains in situ using atomic force microscopy (AFM). We developed a wavelet analysis method based on a multiscale decomposition of derivative force-distance curves to quantify the elastic responses of various external layers upon mechanical deformation. Such analysis has the advantages of detecting singularities and distinguishing the biomechanical contributions of each external layer. The elastic modulus of the same type of external layer follows the same statistical distribution. However, the elastic response among different types of external layers is challenged by our method, indicating the heterogeneity of the mechanical properties of inner and outer layers in multilayer strains. This discrepancy was due to the thickness and texture of each external layer, especially the chemical presence of ribose, hydroxyproline and glutamic acid. This study highlights a new way to elucidate more precise information about external layers and provides a biophysical mechanistic explanation for the functioning of the various external layers to protect cyanobacterial and microalgal cells in a turbulent environment.

Graphene–enhanced differentiation of neuroblastoma mouse cells mediated by poly-D-lysine

We compared the proliferation and differentiation of mouse neuroblastoma Neuro 2A cell line on single layer graphene and glass substrates. Quantitative and qualitative analysis of the cell proliferation and differentiation were performed, considering also the effect of a common adhesion factor, namely polylysine. We observed that on graphene substrates the cells proliferate faster with respect to glass; additionally, the presence of the adhesion factor enhances the difference and, remarkably, boosts the cell differentiation on the graphene-based interface. To understand the mechanism underlying a different cell behavior on the same adhesion coating, we carried out a physicochemical investigation of the studied interfaces (glass and graphene, bare and polylysine coated) by several techniques. In particular, we employed infrared spectroscopy to gain information on polylysine conformation, and atomic force microscopy force-distance curves to study adhesion properties at the surface. The results indicate that polylysine has an enhanced binding affinity for graphene, as well as a different molecular arrangement on graphene with respect to glass. These properties act as surface cues to trigger the cell response.

Cryo-TEM and AFM Observation of the Time-Dependent Evolution of Amorphous Probucol Nanoparticles Formed by the Aqueous Dispersion of Ternary Solid Dispersions.

In this study, the time-dependent evolution of amorphous probucol nanoparticles was characterized by cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy (AFM). The nanoparticles were formed by dispersing ternarysoliddispersions of probucol in water. Spray drying and cogrinding were used to prepare a spray-dried sample (SPD) and two ground-mixture samples (GM(I) and GM(II)) of probucol (PBC) form I and form II/hypromellose/sodium dodecyl sulfate ternary solid dispersions. The amorphization of PBC in the SPDs and GMs was confirmed using powder X-ray diffraction (PXRD) and solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. Additionally, differential scanning calorimetry showed that relatively small amounts of PBC nuclei or PBC-rich domains remained in both GMs. Then, the physical stability of drug nanoparticles formed after aqueous dispersion in the SPD and GM suspensions during storage at 40 °C was characterized. Cryogenic transmission electron microscopy was used to monitor the evolution of the amorphous PBC nanoparticles in the SPD and GM suspensions during storage. Spherical nanoparticles smaller than 30 nm were observed in all of the suspensions just after dispersion. The size of the particles in the SPD suspension gradually increased but remained on the order of nanometers and retained their spherical shape during storage. In contrast, both GM suspensions evolved through three morphologies, spherical nanoparticles that gradually increased in size, needle-like nanocrystals, and micrometer-sized crystals with various shapes. The evolution of the nanoparticles suggested that their stability in the GM suspension was lower than in the SPD suspension. PXRD analysis of the freeze-dried suspensions of the particles showed that the PBC in the nanoparticles of the SPD suspension was in the amorphous state just after dispersion, while a small fraction of the PBC in the nanoparticles of the GM suspension exhibited a crystal phase and selectively crystallized to its initial crystal form during storage. AFM force-distance curves also demonstrated the existence of crystal phase PBC in the spherical nanoparticles in the GM suspension just after dispersion. The molecular state of PBC in the ternary solid dispersion was dependent on the preparation method (either completely amorphized or incompletely amorphized with residual nuclei or drug-rich domains) and determined the potential mechanisms of PBC nanoparticle evolution after aqueous dispersion. These findings confirm the importance of the molecular state on the particle evolution and the physical stability of the drug nanoparticles in the suspension. Cryo-TEM and AFM measurements provide more direct insight for designing solid dispersion formulations to produce stable amorphous drug nanosuspensions that efficiently improve the solubility and bioavailability of poorly water-soluble drugs.

Physicochemical Tools: Toward a Detailed Understanding of the Architecture of Targeted Radiotherapy Nanoparticles.

Targeted radiotherapy is proving to be an effective alternative to external beam radiotherapy for cancer treatment. Gold nanoparticles are biocompatible, commercially available, and readily functionalized, which makes them perfect candidates for the delivery of cytotoxic radionuclides labeled with antibodies to proteins abnormally expressed on cancer tissue. However, there is a lack of information regarding the efficacy of the successive modification steps involved in the functionalization process, as well as of the actual final state of the nanoparticles prior to preclinical tests, which results in a very inefficient screening and that will further impact on biological barriers, such as half-life interactions with serum proteins. Here, gold nanoparticles (15 nm diameter) were functionalized with linkers for antibody and radionuclide conjugation, following a well-stablished method. Successful coating of the gold nanoparticles was demonstrated using state-of-the-art physicochemical techniques, which include AF4-UV-ICPMS-MALS, Raman spectroscopy, and force-distance spectroscopy, which have led to an accurate description of the hydrodynamic diameter of the functionalized NPs and also about the adhesion energy and elastic properties of the modified NPs. Successive steps involved in the coating led to an organic shell of 12 nm diameter and no nanoparticle aggregation was observed. This may be a consequence of a decrease (or even the total absence) in water adsorption on the metal surface and/or of the organic labeling, that decreases the surface tension of the particles as estimated from the atomic force microscopy force-distance curves. Radiolabeling of gold nanoparticles prescreened using these physicochemical tools with 177Lu resulted in >75% efficiency.

A fully-automated neural network analysis of AFM force-distance curves for cancer tissue diagnosis

Atomic Force Microscopy (AFM) has the unique capability of probing the nanoscale mechanical properties of biological systems that affect and are affected by the occurrence of many pathologies, including cancer. This capability has triggered growing interest in the translational process of AFM from physics laboratories to clinical practice. A factor still hindering the current use of AFM in diagnostics is related to the complexity of AFM data analysis, which is time-consuming and needs highly specialized personnel with a strong physical and mathematical background. In this work, we demonstrate an operator-independent neural-network approach for the analysis of surgically removed brain cancer tissues. This approach allowed us to distinguish—in a fully automated fashion—cancer from healthy tissues with high accuracy, also highlighting the presence and the location of infiltrating tumor cells.

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.

Polyvinylpyrrolidone surface modification with SiOx containing amorphous hydrogenated carbon (a-C:H/SiOx) and nitrogen-doped a-C:H/SiOx films using Hall-type closed drift ion beam source

In this study SiOx containing amorphous hydrogenated carbon (a-C:H/SiOx) and nitrogen-doped a-C:H/SiOx (a-C:H:N/SiOx) films were deposited on polyvinylpyrrolidone (PVP) templates of variable thickness using a Hall-type closed drift ion beam source with constant irradiation parameters. A detailed surface characterization was followed using atomic force microscopy (AFM) topography images, surface morphology parameters, height distribution histograms and bearing ratio curves with hybrid parameters. The AFM analysis directly showed that the a-C:H/SiOx/PVP and a-C:H:N/SiOx/PVP composite films represent different morphologies with characteristic surface textures. Surface adhesive properties were evaluated by measuring the force required to separate the AFM tip from the surface by means of AFM force-distance curves. The variance in adhesion force detected was lower for a-C:H/SiOx/PVP composite films due to lower structural homogeneity of the surfaces. Fourier transform infrared spectroscopy analysis was performed to study the blend behavior of PVP upon a-C:H/SiOx and a-C:H:N/SiOx direct ion beam deposition. It was determined that interfacial interactions of PVP with the direct ion beam induced changes in the carbonyl group of the PVP and are dependent on the carrier gas used for the synthesis of the amorphous hydrogenated carbon films.

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.

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.

Measuring a Stabilization Constant Between Two Bio-Molecules using Atomic Force Microscopy

Stabilization force constants are known to play an important role in biomolecular functions that carry out delicate structural conformation changes along a reaction coordinate during bio-molecular activation. Atomic force microscopy (AFM) has been used as a tool for probing protein-ligand interactions at the single molecular level. We developed a method that converts AFM force-distance curves into intermolecular force-distance curves. This method was applied to a model enzyme-inhibitor system of 5’ methylthioadenosine/S-adenosylhomocysteine nucleo-sidase (MTN) and its transition state analogue homocysteinyl Immucillin A (HIA).

The effect of humidity on nano-scaled domain switching in LiNbO3 single crystal

This paper deals with the influence of relative humidity on nano-scaled domain switching caused by the electric field of atomic force microscopy (AFM) in a LiNbO3 single crystal. Experimental results show that the switched domain size under AFM tip does not increase monotonously with relative humidity. The domain radius increases slightly in lower relative humidity conditions (&amp;lt; 40%), and decreases dramatically in higher relative humidity conditions (40–99%). The domain is not created under a relative humidity of 99%. Experiments on the AFM force-distance curve prove that there is a water bridge between the tip and the sample surface, whose size increases with the rise of humidity. The influence of water bridges on the effective electric field acting on the domain switching in the sample is discussed.

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.

The Degradation and Adsorption Behaviors of Enzyme on Poly(butylene succinate) Single Crystals

The enzymatic degradation behavior of poly(butylene succinate) (PBS) single crystals with a lipase from Pseudomonas cepacia (lipase PS) is monitored using atomic force microscopy (AFM) in phosphate buffer at pH 6.8 and 40 degrees C. In-situ AFM results show that enzymatic degradation of the single crystal starts from the crystal edges rather than the chain-folded surfaces and the lamellar thickness remains constant during the whole degradation process. Total internal reflection fluorescence microscopy (TIRFM) is used for the first time to study the adsorption behavior of lipase onto the PBS crystal surface. The results clearly show that the enzyme molecules preferentially adsorb on the lateral surfaces of the single crystal but not on the chain-folded surfaces. AFM force-distance curve measurements and force-volume imaging obtained using a lipase-immobilized AFM tip show that small and large adhesive forces exist in the flat-on and edge-on areas of a PBS banded spherulite, respectively, which correspond to the chain-folded surface and lateral edges of a single crystal.