Simultaneous Atomic Force Microscopy Research Articles

Force Feedback Controls Motor Activity and Mechanical Properties of Self-Assembling Branched Actin Networks

Branched actin networks--created by the Arp2/3 complex, capping protein, and a nucleation promoting factor--generate and transmit forces required for many cellular processes, but their response to force is poorly understood. To address this, we assembled branched actin networks in vitro from purified components and used simultaneous fluorescence and atomic force microscopy to quantify their molecular composition and material properties under various forces. Remarkably, mechanical loading of these self-assembling materials increases their density, power, and efficiency. Microscopically, increased density reflects increased filament number and altered geometry but no change in average length. Macroscopically, increased density enhances network stiffness and resistance to mechanical failure beyond those of isotropic actin networks. These effects endow branched actin networks with memory of their mechanical history that shapes their material properties and motor activity. This work reveals intrinsic force feedback mechanisms by which mechanical resistance makes self-assembling actin networks stiffer, stronger, and more powerful.

Simultaneous Scanning Ion Conductance Microscopy and Atomic Force Microscopy with Microchanneled Cantilevers.

We combined scanning ion conductance microscopy (SICM) and atomic force microscopy (AFM) into a single tool using AFM cantilevers with an embedded microchannel flowing into the nanosized aperture at the apex of the hollow pyramid. An electrode was positioned in the AFM fluidic circuit connected to a second electrode in the bath. We could thus simultaneously measure the ionic current and the cantilever bending (in optical beam deflection mode). First, we quantitatively compared the SICM and AFM contact points on the approach curves. Second, we estimated where the probe in SICM mode touches the sample during scanning on a calibration grid and applied the finding to image a network of neurites on a Petri dish. Finally, we assessed the feasibility of a double controller using both the ionic current and the deflection as input signals of the piezofeedback. The experimental data were rationalized in the framework of finite elements simulations.

Vibrational characteristics of triethylamine–Mn(II) complex self-assembled with the formation of hydrophobic core in comparison with those of diethylamine–Mn(II) complex

Properties of diethylamine–MnCl2 and triethylamine–MnCl2 complexes formed in aqueous solutions have been investigated by electronic absorption and infrared spectroscopy, and by simultaneous atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). Formation of compact hydrophobic core is found by AFM/SNOM for the TEA–MnCl2 complex in contrast with DEA–MnCl2 one. In the assemblies of both complexes Mn(II) oxidation to Mn(III) was monitored by the appearance of bands of MnOH stretching and bending vibrations in the IR spectra near 665 and 750cm−1, respectively. In assemblies of DEA–MnCl2 complex Mn(II) has been oxidized by O2. While in the presence of O2 Mn(II) was retained in the non-oxidized state in the TEA–MnCl2 complex self-organized with the formation of the hydrophobic core. Vibrational characteristics of these complexes are considered depending on different states of Mn and interaction with dioxygen. A broadened signal with the width of 80mT found by ESR spectroscopy supports the assembled state of Mn(II) in the complex with TEA in the compact hydrophobic core.

Relationship between cell stiffness and stress fiber amount, assessed by simultaneous atomic force microscopy and live-cell fluorescence imaging.

Actomyosin stress fibers, one of the main components of the cell’s cytoskeleton, provide mechanical stability to adherent cells by applying and transmitting tensile forces onto the extracellular matrix (ECM) at the sites of cell–ECM adhesion. While it is widely accepted that changes in spatial and temporal distribution of stress fibers affect the cell’s mechanical properties, there is no quantitative knowledge on how stress fiber amount and organization directly modulate cell stiffness. We address this key open question by combining atomic force microscopy with simultaneous fluorescence imaging of living cells, and combine for the first time reliable quantitative parameters obtained from both techniques. We show that the amount of myosin and (to a lesser extent) actin assembled in stress fibers directly modulates cell stiffness in adherent mouse fibroblasts (NIH3T3). In addition, the spatial distribution of stress fibers has a second-order modulatory effect. In particular, the presence of either fibers located in the cell periphery, aligned fibers or thicker fibers gives rise to reinforced cell stiffness. Our results provide basic and significant information that will help design optimal protocols to regulate the mechanical properties of adherent cells via pharmacological interventions that alter stress fiber assembly or via micropatterning techniques that restrict stress fiber spatial organization.Electronic supplementary materialThe online version of this article (doi:10.1007/s10237-015-0706-9) contains supplementary material, which is available to authorized users.

Atomic species identification at the (101) anatase surface by simultaneous scanning tunnelling and atomic force microscopy

Anatase is a pivotal material in devices for energy-harvesting applications and catalysis. Methods for the accurate characterization of this reducible oxide at the atomic scale are critical in the exploration of outstanding properties for technological developments. Here we combine atomic force microscopy (AFM) and scanning tunnelling microscopy (STM), supported by first-principles calculations, for the simultaneous imaging and unambiguous identification of atomic species at the (101) anatase surface. We demonstrate that dynamic AFM-STM operation allows atomic resolution imaging within the material's band gap. Based on key distinguishing features extracted from calculations and experiments, we identify candidates for the most common surface defects. Our results pave the way for the understanding of surface processes, like adsorption of metal dopants and photoactive molecules, that are fundamental for the catalytic and photovoltaic applications of anatase, and demonstrate the potential of dynamic AFM-STM for the characterization of wide band gap materials.

Opto-mechanical probe for combining atomic force microscopy and optical near-field surface analysis.

We have developed a new easy-to-use probe that can be used to combine atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). We show that, using this device, the evanescent field, obtained by total internal reflection conditions in a prism, can be visualized by approaching the surface with the scanning tip. Furthermore, we were able to obtain simultaneous AFM and SNOM images of a standard test grating in air and in liquid. The lateral resolution in AFM and SNOM mode was estimated to be 45 and 160 nm, respectively. This new probe overcomes a number of limitations that commercial probes have, while yielding the same resolution.

Graphene Nanopore Support System for Simultaneous High-Resolution AFM Imaging and Conductance Measurements

Accurately defining the nanoporous structure and sensing the ionic flow across nanoscale pores in thin films and membranes has a wide range of applications, including characterization of biological ion channels and receptors, DNA sequencing, molecule separation by nanoparticle films, sensing by block co-polymers films, and catalysis through metal–organic frameworks. Ionic conductance through nanopores is often regulated by their 3D structures, a relationship that can be accurately determined only by their simultaneous measurements. However, defining their structure–function relationships directly by any existing techniques is still not possible. Atomic force microscopy (AFM) can image the structures of these pores at high resolution in an aqueous environment, and electrophysiological techniques can measure ion flow through individual nanoscale pores. Combining these techniques is limited by the lack of nanoscale interfaces. We have designed a graphene-based single-nanopore support (∼5 nm thick with ∼20 nm pore diameter) and have integrated AFM imaging and ionic conductance recording using our newly designed double-chamber recording system to study an overlaid thin film. The functionality of this integrated system is demonstrated by electrical recording (<10 pS conductance) of suspended lipid bilayers spanning a nanopore and simultaneous AFM imaging of the bilayer.

Single molecule binding dynamics measured with atomic force microscopy

We present a new method to analyse simultaneous Topography and RECognition Atomic Force Microscopy data such that it becomes possible to measure single molecule binding rates of surface bound proteins. We have validated this method on a model system comprising a S-layer surface modified with Strep-tagII for binding sites and strep-tactin bound to an Atomic Force Microscope tip through a flexible Poly-Ethylene-Glycol linker. At larger distances, the binding rate is limited by the linker, which limits the diffusion of the strep-tactin molecule, but at lateral distances below 3 nm, the binding rate is solely determined by the intrinsic molecular characteristics and the surface geometry and chemistry of the system. In this regime, Kon as determined from single molecule TREC data is in agreement with Kon determined using traditional biochemical methods.

Cross talk between matrix elasticity and mechanical force regulates myoblast traction dynamics

Growing evidence suggests that critical cellular processes are profoundly influenced by the cross talk between extracellular nanomechanical forces and the material properties of the cellular microenvironment. Although many studies have examined either the effect of nanomechanical forces or the material properties of the microenvironment on biological processes, few have investigated the influence of both. Here, we performed simultaneous atomic force microscopy and traction force microscopy to demonstrate that muscle precursor cells (myoblasts) rapidly generate a significant increase in traction when stimulated with a local 10 nN force. Cells were cultured and nanomechanically stimulated on hydrogel substrates with controllable local elastic moduli varying from ∼16–89 kPa, as confirmed with atomic force microscopy. Importantly, cellular traction dynamics in response to nanomechanical stimulation only occurred on substrates that were similar to the elasticity of working muscle tissue (∼64–89 kPa) as opposed to substrates mimicking resting tissue (∼16–51 kPa). The traction response was also transient, occurring within 30 s, and dissipating by 60 s, during constant nanomechanical stimulation. The observed biophysical dynamics are very much dependent on rho-kinase and myosin-II activity and likely contribute to the physiology of these cells. Our results demonstrate the fundamental ability of cells to integrate nanoscale information in the cellular microenvironment, such as nanomechanical forces and substrate mechanics, during the process of mechanotransduction.

Resiliency of the plasma membrane and actin cortex to large‐scale deformation

The tight coupling between the plasma membrane and actin cortex allows cells to rapidly change shape in response to mechanical cues and during physiological processes. Mechanical properties of the membrane are critical for organizing the actin cortex, which ultimately governs the conversion of mechanical information into signaling. The cortex has been shown to rapidly remodel on timescales of seconds to minutes, facilitating localized deformations and bundling dynamics that arise during the exertion of mechanical forces and cellular deformations. Here, we directly visualized and quantified the time-dependent deformation and recovery of the membrane and actin cortex of HeLa cells in response to externally applied loads both on- and off-nucleus using simultaneous confocal and atomic force microscopy. The local creep-like deformation of the membrane and actin cortex depends on both load magnitude and duration and does not appear to depend on cell confluency. The membrane and actin cortex rapidly recover their initial shape after prolonged loading (up to 10 min) with large forces (up to 20 nN) and high aspect ratio deformations. Cytoplasmic regions surrounding the nucleus are shown to be more resistant to long-term creep than nuclear regions. These dynamics are highly regulated by actomyosin contractility and an intact actin cytoskeleton. Results suggest that in response to local deformations, the nucleus does not appear to provide significant resistance or play a major role in cell shape recovery. The membrane and actin cortex clearly possess remarkable mechanical stability, critical for the transduction of mechanical deformation into long term biochemical signals and cellular remodeling.

Mechanical Manipulation of Photophysical Properties of Single Conjugated Polymer Nanoparticles

Results on simultaneous atomic force microscopy (AFM) and fluorescence microspectroscopy on single nanoparticles of the conjugated polymer poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) are presented. Single particle fluorescence spectra and defocused images confirm that the particles are composed of large numbers of randomly oriented conjugated segments (chromophores) with a wide distribution of the segment lengths. Mechanical contact of the particle surface with a sharp tip of the AFM probe causes reversible modification of the particle fluorescence intensity and spectra. The majority of the particles show an increase of fluorescence intensity and blue spectral shift upon contact with the tip. The fluorescence increase is correlated with the particle size and is most pronounced for the smallest particles. The phenomena are interpreted in terms of local disturbance of the MEH-PPV chain conformation by the probe tip.

Computational study of tip apex symmetry characterization in high-resolution atomic force microscopy

Simultaneous atomic force microscopy (AFM)/scanning tunnelling microscopy (STM) is a powerful combination of two complementary techniques that enables high-resolution measurement of surface physical and chemical properties. Although blind characterization of the chemical structure of a surface or probe tip is extremely challenging, recent experimental reports suggest that measurement of the tip bonding symmetry is possible when it interacts with a well-defined sample, such as an adsorbed molecule. In order to gain understanding into the relationship between the symmetry observed in the experiments and the actual tip structure, we explore computationally the mechanism through which the tip apex electronic density and angular stiffness is revealed in AFM images. Our study is based on idealized 1-atom models of the surface, which allow us to isolate the effects of probe size and reactivity. Our results show that image symmetry is directly related to tip symmetry and that the contrast diminishes with larger or more reactive sample atoms. We also find that that the mechanics of the probe–sample interaction are not trivial even for the most idealized models, and that the electronic density distribution around the apex atom is also more complex than what can be inferred from the experimental images.

Room Temperature Discrimination of Adsorbed Molecules and Attachment Sites on the Si(111)–7 × 7 Surface Using a qPlus Sensor

In this paper, we show that simultaneous noncontact atomic force microscopy (nc-AFM) and scanning tunneling microscopy (STM) is a powerful tool for molecular discrimination on the Si(111)-7 × 7 surface, even at room temperature. Using density functional theory modeling, we justify this approach and show that the force response allows us to distinguish straightforwardly between molecular adsorbates and common defects, such as vacancies. Finally, we prove that STM/nc-AFM method is able to determine attachment sites of molecules deposited on semiconductor surface at room temperature.

Phospholipase-Catalyzed Hydrolysis in an Artificial Cell Membrane in the Presence of Melittin

Biomimicry involves the use of the structure and function of biological systems as models for the design and engineering of materials and machines. An artificial cell membrane was developed using biomembrane components, and the membrane, formed by a lipid bilayer, was analyzed using surface plasmon resonance (SPR) to monitor hydrolysis by phospholipase (PL). The simultaneous atomic force microscope (AFM) images show that PL catalyzed the nanometer-scale hydrolysis of the artificial lipid biomembranes through enzymatic hydrolysis. In addition, it was confirmed that the combination of PL and melittin allowed the control of enzyme hydrolysis for the degradation of the lipid bilayer. Regarding the expected activating effect of melittin on hydrolysis, no difference with respect to the non-treated lipid membrane was observed in the AFM images. It is assumed that the partitioning of melittin into the membrane might prevent the binding or hydrolysis of Phospholipase A2 (PLA2). This study provides basic knowledge on a new approach for patterning biomimicking lipid membranes on a nano-scale.

Fluidfm for Force Controlled Electrophysiology

The Fluid Force Microscope (FluidFM) is a recently developed technology based on atomic force microscopy (AFM). The uniqueness of this technology consists in hollow cantilevers connected to an external liquid reservoir. Different cantilever designs have been fabricated, with different tip shapes and apertures. The FluidFM allows for precise control on both the force exerted on a sample and on the hydrodynamic pressure at cantilever's aperture. Here we present the FluidFM adapted for electrophysiology experiments, in particular for whole-cell patch-clamp and for scanning ion conductance microscopy (SICM).Patch-clamp is considered the gold standard for electrophysiology measurements. We investigated on the formation of a force-controlled high electric resistance seal between FluidFM aperture and cell membrane (gigaseal). Exploiting the force control capabilities of the FluidFM, the probe is positioned on the cell membrane in a controlled fashion simplifying the most critical step of traditional patch-clamp. Whole-cell configuration is now being explored.The same setup is suitable for SICM operated in far contact using the ion conductivity between substrate and tip as feedback signal. SICM adapts very well on soft tissues where AFM fails to preserve the integrity of the imaged sample. Our system enables simultaneous AFM and SICM.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Simultaneous Atomic Force and Confocal Fluorescence Microscopy for Correlated Nanometer Topographical and Optical Resolution

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Modulation of Step Heights of Thin Pb Films by the Quantum Size Effect Observed by Non-Contact Atomic Force Microscopy

Using a home-made Q-plus sensor, simultaneous scanning tunneling microscopy (STM) and atomic force microscopy (AFM) measurements were performed on the wedge-shaped Pb islands grown on Si(111)-7 × 7. Atomic resolved AFM images were observed. The contrast of AFM topography shows no dependence on the sample bias (tip is grounded), while the simultaneously obtained tunneling current image exhibits strong bias dependence due to quantum well states (QWS). Furthermore, In the AFM mode, neighboring Pb films with one monolayer (ML) thickness difference within the same Pb island show the same apparent height, which means that the apparent step heights of Pb films oscillate with a bilayer periodicity, being consistent with previous observations by helium atom scattering, x-ray diffraction, and STM. The possible reasons underlying the oscillation of apparent step heights in AFM topography are discussed.

Combined QCMD and AFM studies of lysozyme and poly-l-lysine–poly-galacturonic acid multilayers

A quartz crystal microbalance with dissipation monitoring (QCMD) has been used to monitor the adsorption and structure of lysozyme monolayers and multilayers, and poly-l-lysine (PLL)–polygalacturonic acid (PGalA) multilayers at a solid–liquid interface using freshly-cleaved mica as a substrate. QCMD measurements were complemented with atomic force microscopy (AFM). AFM images revealed that lysozyme formed incomplete monolayers and provided a basis for calculation of the thickness of the protein film. Comparative studies of adsorption onto standard and mica-coated quartz crystals showed higher areal mass adsorption and a longer-time adsorption process for mica-coated quartz crystals. Simultaneous AFM images and QCMD data were obtained for lysozyme, linear PLL–PGalA and 7nm PLL dendrimer-PGalA multilayers. The layer-by-layer deposited multilayer films exhibited viscoelastic properties and their growth followed a non-linear regime, associated with the PLL diffusion in and out of the film formation for linear PLL–PGalA films. For the PLL 7nm dendrimer-PGalA films the AFM images revealed marked changes in surface roughness during layer by layer deposition: these changes influence the interpretation of the QCMD data and provide additional information on the growth and structure of the multilayers.

An Approach to Visualize the Deformation of the Intermediate Filament Cytoskeleton in Response to Locally Applied Forces

The intermediate filament (IF) cytoskeleton plays an important role in integrating biomechanical pathways associated with the actin and microtubule cytoskeleton. Vimentin is a type III IF protein commonly found in fibroblast cells and plays a role in transmitting forces through the cytoskeleton. Employing simultaneous laser scanning confocal and atomic force microscopy (AFM), we developed a methodology to quantify the deformation of the GFP-vimentin-labeled IF cytoskeleton as a function of time in response to force application by the AFM. Over short times (seconds), IFs deformed rapidly and transmitted force throughout the entire cell in a highly complex and anisotropic fashion. After several minutes, mechanically induced displacements of IFs resemble basal movements. In well-adhered cells the deformation of IFs is highly anisotropic as they tend to deform away from the longitudinal axis of the cell. This study demonstrates that simultaneous AFM and LSCM can be employed to track the deformation and dissipation of force through the IF cytoskeleton.

Simultaneous Surface-Enhanced Raman Spectroscopy (SERS) and Atomic Force Microscopy (AFM) for Label-Free Physicochemical Analysis of Lipid Bilayers

Large-scale two-dimensional (2D) arrays of metallic nanostructures formed by thin-film evaporation over hexagonally close-packed polystyrene spheres are established substrates for surface-enhanced Raman spectroscopy (SERS). By using these substrates with an integrated atomic force microscopy (AFM) and inverted Raman spectroscopy system, simultaneous topographical imaging and high-sensitivity chemical mapping can be performed. In this paper, we have used this technique to investigate supported bilayers of long-chain fatty acids and phospholipids deposited by the Langmuir–Blodgett (LB) and spin-coating techniques. Nanosphere lithography (NSL) substrates created from 384 and 1002 nm polystyrene spheres and silver (Ag) deposition on glass and sapphire substrates were characterized for SERS in terms of their structure, distribution, and level of enhancement. SERS mappings of rhodamine 6G (R6G) and p-aminothiophenol (p-ATP) monolayers on the 384 nm substrates demonstrate high and uniform enhancement at a micrometer scale. The enhancement was sufficiently high to enable measurement of SERS spectra for arachidic acid (AA) and dipalmitoylphosphatidylcholine (DPPC) layers on sapphire/Ag substrates. The roughness of these substrates (&lt;2 nm) was lower than for glass/Ag (∼5 nm); therefore, simultaneous to SERS it was possible to measure the topography of the samples by AFM and determine the number of layers of AA and DPPC. This study shows the potential of the combined AFM/SERS technique for spectral and topographical characterization of phospholipid bilayers. This may prove to be an interesting approach for further studies with more complex heterogeneous lipid mixtures aiming to measure spatially resolved features such as microdomains.