Real-time Atomic Force Microscopy Research Articles

A real-time AFM study on crystal nucleation and growth in nanodroplets of isotactic polypropylene

Isotactic polypropylene (iPP) nanodroplets were prepared by using the classical droplet method in this study. The formation of nanodroplets allowed the controlled observation of polymer nucleation as well as access to crystal growth at exceptionally high supercooling in iPP. Three cases including the heterogeneous nucleation and fast crystallization in iPP droplets, the formation of multiple independent homogeneous nuclei within a single droplet and a single nucleus within a single droplet were detected by using atomic force microscopy (AFM) during gradually cooling after remelting the nanodroplets. Moreover, it is found that when the volume of droplet is larger than the value of ca. 130000 nm3, the first case was observed. Otherwise, the latter two cases appeared. The temperature at which the onset of nucleation was observed in individual droplets was found to be mainly dependent on height of the droplets when the size scale of the droplet is comparable to the size of the critical nucleus in at least one dimension, which indicates the nucleation behavior under confinement.

Epithelial–Mesenchymal Transition Enhances Nanoscale Actin Filament Dynamics of Ovarian Cancer Cells

Epithelial ovarian cancer cells enhance their ability to migrate and invade through the epithelial-mesenchymal transition (EMT), resulting in cell seeding and metastasis in the peritoneal cavity and onto adjacent organ surfaces. It has been speculated that cytoskeletal dynamics, such as those of the actin filament, play a role in enhanced cell motility; however, direct evidence has not been provided. Herein, we have directly measured pico- to nanonewton-scale mechanical forces generated by actin dynamics of ovarian cancer SKOV-3 cells upon binding of integrin α5β1 to fibronectin (FN), i.e., formation of a focal adhesion, using real-time atomic force microscopy (AFM) in a force spectroscopy mode. The dendrimer surface chemistry through which FN was immobilized on the AFM probe surfaces further enhanced the sensitivity of the force measurement by 1.5-fold. Post-EMT SKOV-3 cells, induced by transforming growth factor-β, generated larger focal adhesion mechanical forces (17 and 41 nN before and after EMT, respectively) with migration faster than that of pre-EMT cells. Importantly, 22% of the forces transmitted through a single FN-integrin α5β1 pair from post-EMT cells were shown to be sufficient to rupture the binding between FN and integrin α5β1 on the cells, a result which is not observed on pre-EMT cells. This implies that post-EMT cells, by generating forces strong enough to break the FN-integrin binding, migrate and metastasize beyond the ovary, whereas pre-EMT cancer cells are confined in the ovary without such force generation. These results demonstrate quantitative and direct evidence for the role of actin dynamics in the enhanced motility of post-EMT ovarian cancer cells, providing a fundamental insight into the mechanism of ovarian cancer metastasis.

Real time atomic force microscopy imaging during nanogap formation by electromigration

We present real time atomic force microscopy imaging during nanogap fabrication by feedback controlled electromigration of a gold nanowire. The correlated measurements of electrical resistance and atomic force microscopy reveal that the major structural changes appear at the early stage of the process. Moreover, despite important morphological changes, the resistance of the nanowire shows a weak increase of just a few ohms. The detailed analysis of the atomic force microscopy images clearly shows that the electromigration process is strongly influenced by the initial microstructure of the nanowire.

Controlling a Single DNA Molecule in an Electric Field by Means of In Situ Atomic Force Microscopy

DNA is the bio-polymer containing the genetic information needed for the development and functioning for all living organisms. It has a polymeric structure consisting of units called nucleotides, each consisting of a non-polar, hydrophobic interior (the base pairs) and polar, hydrophilic exterior which is negatively charged due to the phosphate groups along the backbone of the DNA. Its heterogeneous properties permit DNA molecule to interact with other molecules and different types of substrates at the same time. It is important to understand the DNA morphology on flat surface that serves as a template for DNA based sensors and microarray applications, particularly under an electric field. Taken together, the ability to deposit a single DNA molecule on the electrode is essential to increase its specificity. In our study, we optimize the conditions in order to control a single DNA molecule adsorbing and desorbing to/from the electrode. Simultaneously dynamic imaging enables us to analyze its morphological behavior by real-time Electrochemical Atomic Force Microscopy (EC-AFM) in aqueous conditions.

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.

Host responses of a marine bacterium, Roseobacter denitrificans OCh114, to phage infection

RDJLΦ1 is a marine siphophage infecting Roseobacter denitrificans OCh114. In this study, host responses of R. denitrificans OCh114 to phage infection were investigated through in situ real-time atomic force microscopy (AFM) and proteomics approaches. As seen from the AFM observations, during phage infection processes, depression areas appeared on the host cell surface in a few minutes after infection and expanded in both diameter and depth over time and finally led to the collapse of host cells within 30min. The two-dimensional polyacrylamide gel electrophoresis revealed significant changes in the proteomic composition of the host cells during infection. The expression of 91 proteins, including some involved in DNA transcription regulation and substrate transportation, was changed with at least twofold up- or downregulation as compared to the control without phage infection. This observed rapid lysis of host cells and the great changes in protein expression caused by phage infection added more perspectives to the documented important roles of viruses in mediating carbon cycling in the ocean.

Photoinduced mass-transport based holographic recording of surface relief gratings in amorphous selenium films

Surface relief gratings formation in amorphous selenium thin films in two recording configurations with light intensity modulation were studied in situ by real-time atomic force microscopy and diffraction efficiency measurements. We report observation of mass transport effect in films induced by band-gap irradiation when the light polarization of the recording beams has a component along the light intensity gradient (“p-p” scheme of recording) that allows obtaining giant stable gratings in this versatile chalcogenide material. On the contrary, only a pure scalar weak grating caused by photoinduced volume shrinkage is obtained in the “s-s” recording configuration, even for long-term irradiation.

Study of polymeric additive effect on calcium oxalate dihydrate crystal growth using real-time atomic force microscopy

Microscopic events associated with crystal growth and characterization of the growth hillocks on the (1 0 0) and (1 0 1) faces of COD were examined by atomic force microscopy. The (1 0 0) and (1 0 1) faces of COD developed elliptical and triangular hillocks and pits, respectively. Each face exhibited hillocks with step sites that can be assigned to specific crystal planes, enabling direct determination of the growth rates along specific crystallographic directions. The addition of macromolecules with anionic side chains, poly- l-aspartate, poly- l-glutamate, and polyacrylate resulted in inhibition of growth on the hillock step planes. The magnitude of their effect depended on the macromolecule structures and identity of the step site. The isotropic shape of the COD hillocks mimicked the shape of the resulting macroscopic COD crystals based on step-specific binding of the macromolecules to the COD crystal, with stronger step pinning along the [0 1 0] direction than in the [0 0 1] direction. Electrostatic matching between the crystal faces and additives according to the ionic array of calcium oxalate in the COD structure was found to be responsible for the preferential binding of the macromolecules to terraces.

Antimicrobial Protegrin-1 Forms Amyloid-Like Fibrils with Rapid Kinetics Suggesting a Functional Link

Protegrin-1 (PG-1) is an 18 residues long, cysteine-rich β-sheet antimicrobial peptide (AMP). PG-1 induces strong cytotoxic activities on cell membrane and acts as a potent antibiotic agent. Earlier we reported that its cytotoxicity is mediated by its channel-forming ability. In this study, we have examined the amyloidogenic fibril formation properties of PG-1 in comparison with a well-defined amyloid, the amyloid-β (Aβ1–42) peptide. We have used atomic force microscopy (AFM) and thioflavin-T staining to investigate the kinetics of PG-1 fibrils growth and molecular dynamics simulations to elucidate the underlying mechanism. AFM images of PG-1 on a highly hydrophilic surface (mica) show fibrils with morphological similarities to Aβ1–42 fibrils. Real-time AFM imaging of fibril growth suggests that PG-1 fibril growth follows a relatively fast kinetics compared to the Aβ1–42 fibrils. The AFM results are in close agreement with results from thioflavin-T staining data. Furthermore, the results indicate that PG-1 forms fibrils in solution. Significantly, in contrast, we do not detect fibrillar structures of PG-1 on an anionic lipid bilayer 2-dioleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; only small PG-1 oligomers can be observed. Molecular dynamics simulations are able to identify the presence of these small oligomers on the membrane bilayer. Thus, our current results show that cytotoxic AMP PG-1 is amyloidogenic and capable of forming fibrils. Overall, comparing β-rich AMPs and amyloids such as Aβ, in addition to cytotoxicity and amyloidogenicity, they share a common structural motif, and are channel forming. These combined properties support a functional relationship between amyloidogenic peptides and β-sheet-rich cytolytic AMPs, suggesting that amyloids channels may have an antimicrobial function.

Direct observation of stepwise movement of a synthetic molecular transporter

Controlled motion at the nanoscale can be achieved by using Watson-Crick base-pairing to direct the assembly and operation of a molecular transport system consisting of a track, a motor and fuel, all made from DNA. Here, we assemble a 100-nm-long DNA track on a two-dimensional scaffold, and show that a DNA motor loaded at one end of the track moves autonomously and at a constant average speed along the full length of the track, a journey comprising 16 consecutive steps for the motor. Real-time atomic force microscopy allows direct observation of individual steps of a single motor, revealing mechanistic details of its operation. This precisely controlled, long-range transport could lead to the development of systems that could be programmed and routed by instructions encoded in the nucleotide sequences of the track and motor. Such systems might be used to create molecular assembly lines modelled on the ribosome.

High magnetic gradient environment causes alterations of cytoskeleton and cytoskeleton-associated genes in human osteoblasts cultured in vitro

The effects of a high magnetic gradient environment (HMGE) on the cytoskeletal architecture and genes associated with the cytoskeleton in osteoblasts (MC3T3-E1 and MG-63 cells) were investigated using confocal microscopy, real-time polymerase chain reaction (PCR) and atomic force microscopy (AFM). The findings showed that, under diamagnetic levitation conditions, the architecture and average height of the cytoskeleton and surface roughness in osteoblasts were dramatically altered. HMGE affects cytoskeleton arrangement and cytoskeleton-associated gene expression.

Real-time atomic force microscopy imaging of photoinduced surface deformation in AsxSe100−x chalcogenide films

We present direct measurements of the kinetics of surface relief gratings (SRGs) formation in amorphous AsxSe100−x (20≤xAs≤50) thin films. SRGs are induced in different holographic schemes of recording using near-band-gap light and their growth is further facilitated by illumination with an interference pattern and observed in real time by in situ atomic force microscopy. It is found that the kinetics of SRG formation depends upon film composition and incident light polarization. The light-stimulated vectorial surface deformations are maximized for Se-rich glasses and increase even further by additional illumination during recording.

Kinetics of interaction of HIV fusion protein (gp41) with lipid membranes studied by real-time AFM imaging

One of the most important steps in the process of viral infection is a fusion between cell membrane and virus, which is mediated by the viral envelope glycoprotein. The study of activity of the glycoprotein in the post-fusion state is important for understanding the progression of infection. Here we present a first real-time kinetic study of the activity of gp41 (the viral envelope glycoprotein of human immunodeficiency virus—HIV) and its two mutants in the post-fusion state with nanometer resolution by atomic force microscopy (AFM). Tracking the changes in the phosphatidylcholine (PC) and phosphatidylcholine–phosphatidylserine (PC:PS) membrane integrity over one hour by a set of AFM images revealed differences in the interaction of the three types of protein with zwitterionic and negatively charged membranes. A quantitative analysis of the slow kinetics of hole formation in the negatively charged lipid bilayer is presented. Specifically, analysis of the rate of roughness change for the three types of proteins suggests that they exhibit different types of kinetic behavior.

Real-time atomic force microscopy in lubrication condition

We have studied frictional force and wear problem in real-time atomic force microscopy in contact-mode using a resonator type mechanical scanner allegedly reported. The fast scanning may cause wear in the sample surface or the tip, and may deteriorate the image quality. Mineral oil was used to make a lubricious surface on a polycarbonate sample, and it was found that the interfacial frictional force was decreased. A Si tip which was coated with a hydrophobic film by means of chemical modification was confirmed to diminish the frictional force in the fast scanning process. The resultant image quality was improved due to reduced friction and wear.

Interaction of reducible polypeptide gene delivery vectors with supported lipid bilayers: pore formation and structure–function relationships

Real-time atomic force microscopy (AFM) in aqueous buffer has been used to probe interactions of synthetic disulfide-linked polypeptide gene delivery vectors with supported phospholipid bilayers. Disruption of the membranes was apparent in AFM, and the extent of surface heterogeneity and hole (pore) formation was evaluated by depth and area-profiling image analysis. The overall extent of membrane disruption varied with the reducible polycation peptide sequence and block structure and was found to be correlated with the overall degree of transgene expression in two representative cell lines.

Lability Landscape and Protease Resistance of Human Insulin Amyloid: A New Insight into Its Molecular Properties

Amyloid formation is a universal behavior of proteins central to many important human pathologies and industrial processes. The extreme stability of amyloids towards chemical and proteolytic degradation is an acquired property compared to the precursor proteins and is a major prerequisite for their accumulation. Here, we report a study on the lability of human insulin amyloid as a function of pH and amyloid ageing. Using a range of methods such as atomic force microscopy, thioflavin T fluorescence, circular dichroism, and gas-phase electrophoretic mobility macromolecule analysis, we probed the propensity of human insulin amyloid to propagate or dissociate in a wide span of pH values and ageing in a low concentration regime. We generated a three-dimensional amyloid lability landscape in coordinates of pH and amyloid ageing, which displays three distinctive features: (i) a maximum propensity to grow near pH 3.8 and an age corresponding to the inflection point of the growth phase, (ii) an abrupt cutoff between growth and disaggregation at pH 8–10, and (iii) isoclines shifted towards older age during the amyloid growth phase at pH 4–9, reflecting the greater stability of aged amyloid. Thus, lability of amyloid strongly depends on the ionization state of insulin and on the structure and maturity of amyloid fibrils. The stability of insulin amyloid towards protease K was assessed by using real-time atomic force microscopy and thioflavin T fluorescence. We estimated that amyloid fibrils can be digested both from the free ends and within the length of the fibril with a rate of ca 4 nm/min. Our results highlight that amyloid structures, depending on solution conditions, can be less stable than commonly perceived. These results have wide implications for understanding the propagation of amyloids via a seeding mechanism as well as for understanding their natural clearance and dissociation under solution conditions unfavorable for amyloid formation in biological systems and industrial applications.

Real-Time In Situ Atomic Force Microscopy Imaging of Bismuth Crystal Growth

We have performed real-time atomic force microscopy (AFM) imaging of bismuth crystals that were grown under electrochemical control at low overpotentials to ensure a slow growth rate and allow in situ observation of the growth. A two step chronoamperometric potential was applied to a boron-doped diamond (BDD) working electrode with a short high overpotential, -0.4 V (2 s), to nucleate the bismuth, and then a long low overpotential for slow growth, -0.32 V (4.4 h). Growth rates of individual crystals and detailed growth mechanisms could be followed in real time because of the slow crystal growth. The close proximity of the AFM tip and tip holder to the working electrode appears to hinder the diffusion of bismuth to the BDD surface, as evidenced by the significantly lower density of crystals under the cantilever as compared to the rest of the electrode, therefore slowing down the growth process.

In situ x-ray studies of native and Mo-seeded surface nanostructuring during ionbombardment of Si(100)

Native and Mo-seeded nanostructuring of the Si(100) surface duringAr+ ion bombardment is investigated by means of real-time grazing-incidence small-angle x-rayscattering and atomic force microscopy. During off-axis bombardment at roomtemperature, the native early-stage growth kinetics of nanoripples on the surface is foundto be in reasonable overall agreement with theoretical predictions, particularlywhen an ion impact induced lateral mass redistribution term is included. Fornormal-incidence bombardment at room temperature, a native short wavelengthsmoothing of the amorphized Si surface is observed, suggesting that ion impactinduced lateral mass redistribution dominates the Bradley–Harper instability.During 5% Mo-seeded normal-incidence bombardment at temperatures up to450 °C, nanodots form with heights decreasing as the substrate temperature increases.This trend is counter to that typically observed for the growth of largecone structures on metals and suggests that the primary effect of thermalenergy here is in promoting surface smoothing, rather than increasing diffusionof seed atoms to form protective clusters. During seeded bombardment at650 °C the surface remains crystalline and surface corrugations exhibit dynamic scalingcharacteristic of surface diffusion-driven instabilities. This is the same behavior as is foundin the absence of seeding and its presence suggests that at this concentration seeding doesnot play a large role during normal-incidence bombardment of the Si surface at hightemperatures.

Real time monitoring of the in situ growth of silver nanoparticles in a polymer film under ambient conditions

Direct monitoring of the formation and growth of nanoparticles by microscopy in real time is of fundamental interest in understanding the chemical and self assembly processes involved. Such studies are difficult to implement in solution, but have been carried out on solid substrates, mostly under specialized conditions, including ultra high vacuum. The present study illustrates the facility with which in situ growth of metal nanoparticles in thin polymer films under ambient conditions can be monitored by real time atomic force microscopy. Formation of silver nanoparticles inside spin-coated thin films of poly(vinylpyrrolidone) containing silver nitrate, under ambient conditions, is revealed by the emergence and growth of surface plasmon resonance absorption extending over several hours. Atomic force microscopy allows 'direct observation' of structures growing near the surface; individual nanostructures can be monitored in the case of very thin films. A plausible mechanism is proposed for the chemical reactions occurring inside the film with the polymer itself acting as the reducing agent leading to the formation and growth of the nanoparticles. The present study opens up new avenues to carry out investigations into the mechanisms and kinetics of nanoparticle growth.

Enzymatic Degradation of Monolayer for Poly(lactide) Revealed by Real-Time Atomic Force Microscopy: Effects of Stereochemical Structure, Molecular Weight, and Molecular Branches on Hydrolysis Rates

The influences of the stereochemical structure, the molecular weight, and the number of molecular branches for poly(lactide) (PLA) on enzymatic hydrolysis rates of PLA monolayers were studied by atomic force microscopy (AFM) and the Langmuir-Blodgett (LB) technique. Monolayers of six kinds of PLA with different molecular weights, stereochemical structure, and numbers of molecular branches were prepared by LB techniques and then characterized by AFM in air. The PLA molecules covered homogeneously with a silicon substrate and did not form lamellar crystals in the monolayer. We determined the initial hydrolysis rate of PLA monolayers in presence of proteinase K by volumetric analysis from the continuous AFM height images. The presence of D-lactyl unit reduced the hydrolysis rate of the monolayer. The hydrolysis rate for the linear PLLA samples increased with a decrease in the molecular weight. In contrast, the rates of erosion for branched PLLA monolayers were independent of the molecular weight of samples. The erosion rate of branched PLLA monolayers was found to be dependent on the average molecular weight of PLLA segment in branched molecules, not on the overall molecular weight of samples. From these results, furthermore, the hydrolysis mode of PLAs by proteinase K is discussed.