Atomic Force Microscopy Approach Research Articles

Light-induced modulation of viscoelastic properties in azobenzene polymers.

Photo-induced isomerization of azobenzene molecules drives mass migrations in azopolymer samples. The resulting macroscopic directional photo-deformation of the material morphology has found many applications in literature, although the fundamental mechanisms behind this mass transfer are still under debate. Hence, it is of paramount importance to find quantitative observables that could drive the community toward a better understanding of this phenomenon. In this regard, azopolymer mechanical properties have been intensively studied, but the lack of a nanoscale technique capable of quantitative viscoelastic measurements has delayed the progress in the field. Here, we use bimodal atomic force microscopy (AFM) as a powerful technique for nanomechanical characterizations of azopolymers. With this multifrequency AFM approach, we map the azopolymer local elasticity and viscosity, with high resolution, after irradiation. We find that, while in the (previously) illuminated region, a general photo-softening is measured; locally, the Young modulus and the viscosity depend upon the inner structuring of the illuminating light spot. We then propose a possible interpretation based on a light-induced expansion plus a local alignment of the polymer chains (directional hole-burning effect), which explains the experimental observations. The possibility to access, in a reliable and quantitative way, both Young modulus and viscosity could trigger new theoretical-numerical investigations on the azopolymer mass migration dynamics since, as we show, both parameters can be considered measurable. Furthermore, our results provide a route for engineering the nanomechanical properties of azopolymers, which could find interesting applications in cell mechanobiology research.

Atomic Force Microscopy of Viruses: Stability, Disassembly, and Genome Release.

In atomic force microscopy (AFM), the probe is a nanometric tip located at the end of a microcantilever which palpates the specimen under study as a blind person manages a walking stick. In this way, AFM allows obtaining nanometric resolution images of individual protein shells, such as viruses, in liquid milieu. Beyond imaging, AFM also enables not only the manipulation of single protein cages but also the evaluation of each physicochemical property which is able of inducing any measurable mechanical perturbation to the microcantilever that holds the tip. In this chapter, we start revising some recipes for adsorbing protein shells on surfaces and how the geometrical dilation of tips can affect to the AFM topographies. This work also deals with the abilities of AFM to monitor TGEV coronavirus under changing conditions of the liquid environment. Subsequently, we describe several AFM approaches to study cargo release, aging, and multilayered viruses with single indentation and fatigue assays. Finally, we comment on a combined AFM/fluorescence application to study the influence of crowding on GFP packed within individual P22 bacteriophage capsids.

Quantification of a Neurological Protein in a Single Cell Without Amplification.

Proteins are key biomolecules that not only play various roles in the living body but also are used as biomarkers. If these proteins can be quantified at the level of a single cell, understanding the role of proteins will be deepened and diagnosing diseases and abnormality will be further upgraded. In this study, we quantified a neurological protein in a single cell using atomic force microscopy (AFM). After capturing specifically disrupted-in-schizophrenia 1 (DISC1) in a single cell onto a microspot immobilizing the corresponding antibody on the surface, force mapping with AFM was followed to visualize individual DISC1. Although a large variation of the number of DISC1 in a cell was observed, the average number is 4.38 × 103, and the number agrees with the ensemble-averaged value. The current AFM approach for the quantitative analysis of proteins in a single cell should be useful to study molecular behavior of proteins in depth and to follow physiological change of individual cells in response to external stimuli.

An AFM Approach Applied in a Study of α-Crystallin Membrane Association: New Insights into Lens Hardening and Presbyopia Development.

The lens of the eye loses elasticity with age, while α-crystallin association with the lens membrane increases with age. It is unclear whether there is any correlation between α-crystallin association with the lens membrane and loss in lens elasticity. This research investigated α-crystallin membrane association using atomic force microscopy (AFM) for the first time to study topographical images and mechanical properties (breakthrough force and membrane area compressibility modulus (KA), as measures of elasticity) of the membrane. α-Crystallin extracted from the bovine lens cortex was incubated with a supported lipid membrane (SLM) prepared on a flat mica surface. The AFM images showed the time-dependent interaction of α-crystallin with the SLM. Force spectroscopy revealed the presence of breakthrough events in the force curves obtained in the membrane regions where no α-crystallin was associated, which suggests that the membrane’s elasticity was maintained. The force curves in the α-crystallin submerged region and the close vicinity of the α-crystallin associated region in the membrane showed no breakthrough event within the defined peak force threshold, indicating loss of membrane elasticity. Our results showed that the association of α-crystallin with the membrane deteriorates membrane elasticity, providing new insights into understanding the molecular basis of lens hardening and presbyopia.

Effect of pH on the Surface Layer of Molecular Crystals at the Solid-Liquid Interface.

Dissolution of solid matter into aqueous solution is one of the most challenging physicochemical aspects related to drug development. While influenced by several parameters, the effect of pH remains the most important one to be fully understood. The dissolution process is essentially controlled by activity at the surface of the molecular crystals, which is difficult to characterize experimentally. To address this, a combination of in situ atomic force microscopy (AFM) with molecular dynamics (MD) simulation is reported. AFM allows for direct visualization of the crystal surface of basic and acidic model compounds (carvedilol and ibuprofen) in contact with an aqueous medium with varying pH. A dramatic increase in surface mobility in the solid-liquid interface could be observed experimentally as a function of pH. The in situ AFM approach opens up for a more detailed understanding of the behavior of particulate matter in solution with importance at different levels, ranging from engineering aspects related to crystallization, and biological considerations related to bioavailability of the final drug product.

Simultaneous Deconvolution of In‐Plane and Out‐of‐Plane Forces of HOPG at the Atomic Scale under Ambient Conditions by Multifrequency Atomic Force Microscopy

AbstractMultifrequency atomic force microscopy (AFM) is shown to be an excellent tool for imaging crystal structures at atomic resolution in different spatial directions. However, determining the forces between single atoms remains challenging, particularly in air under ambient conditions. Developed here is a trimodal AFM approach that simultaneously acquires torsional and flexural frequency‐shift images and spectroscopic data to transfer these observables into in‐plane and out‐of‐plane forces between single bonds of highly oriented pyrolytic graphite (HOPG) at atomic resolution in air under ambient conditions based on the Fourier method. It is found that the cantilever mean deflection is an excellent indicator to understand that strong attractive interactions between the tip and the surface of HOPG in dynamic AFM imply a local lift of the topmost carbon layer when using higher eigenmodes for the topographical feedback. Cross‐talk between torsional and flexural‐oscillation modes is shown to be negligible. Interestingly, significant differences are observed in the in‐plane forces depending on the orientation of the carbon bonds relative to the direction of torsional oscillation.

A fast first-principles approach to model atomic force microscopy on soft, adhesive, and viscoelastic surfaces

Quantitative atomic force microscopy (AFM) on soft polymers remains challenging due to the lack of easy-to-use computational models that accurately capture the physics of the interaction between the tip and sticky, viscoelastic samples. In this work, we enhance Attard’s continuum mechanics-based model, arguably the most rigorous contact model for adhesive viscoelastic samples, via three key enabling strategies. First, the original model’s formalism is rearranged to enable a fast and explicit solution of the model’s ordinary differential equations (ODEs). Second, the deformed surface is reconstructed using a complete set of optimized orthogonal basis functions as opposed to Attard’s original, computationally expensive radial discretization. Third, the model’s governing ODEs are solved using a multi-step numerical method to further stabilize the solution when using for soft and sticky samples. Implementing these enhancements, enhanced Attard’s model (EAM) is more stable, 3+ orders of magnitude faster, and equally accurate when compared to the original model. These facilitate EAM’s inclusion into simulations of various AFM operating modes. We demonstrate EAM based simulations of quasi-static force spectroscopy and amplitude modulation AFM approach curves on soft sticky polymer surfaces. On a typical desktop computer, simulation of an amplitude modulation approach curve with EAM takes less than a minute as compared to ≈15 h by the original Attard’s model. We expect EAM to be of interest to the AFM community because it facilitates the inclusion of rigorous models of tip-sample contact in simulations on polymer samples. EAM is available as part of the VEDA set of simulation tools deployed on nanoHUB.org cyber-infrastructure.

Structural and Mechanical Characterization of Viruses with AFM.

Microscopes are used to characterize small objects with the help of probes that interact with the specimen, such as photons and electrons in optical and electron microscopies, respectively. In atomic force microscopy (AFM) the probe is a nanometric tip located at the end of a micro cantilever which palpates the specimen under study as a blind person manages a walking stick. In this way AFM allows obtaining nanometric resolution images of individual protein shells, such as viruses, in liquid milieu. Beyond imaging, AFM also enables not only the manipulation of single protein cages, but also the characterization of every physicochemical property able of inducing any measurable mechanical perturbation to the microcantilever that holds the tip. In this chapter we start revising some recipes for adsorbing protein shells on surfaces. Then we describe several AFM approaches to study individual protein cages, ranging from imaging to spectroscopic methodologies devoted for extracting physical information, such as mechanical and electrostatic properties. We also explain how a convenient combination of AFM and fluorescence methodologies entails monitoring genome release from individual viral shells during mechanical unpacking.

Nanoscale characterization of squaraine-fullerene-based photovoltaic active layers by atomic force microscopy mechanical and electrical property mapping

The efficiency of organic solar cells can be increased by careful control of the nanoscale morphology of a dispersed bulk heterojunction device. Atomic force microscopy (AFM) has often been used to characterize morphology but debate persists over the value of traditional AFM measurements since the technique only addresses the active layer topography, and provides insufficient contrast to differentiate between components in a well-mixed composite. Using newer Kelvin Probe Force Microscopy (KPFM) and Quantitative Nanomechanical Mapping (QNM) modes, we demonstrate contrast due to differing elastic modulus and surface potentials between donor and acceptor materials and highlight the value of these techniques to understand critical materials properties as part of a comprehensive nanomorphology study. We test the value of our approach using blends of each of two anilinic squaraines with phenyl-C61-butyric acid methylester. These two squaraine materials differ in chemical compatibility with the standard fullerene acceptor. We vary annealing conditions for our blended films and use the described AFM approaches to demonstrate changing domain sizes, which are affected by chemical compatibility with the fullerene. We demonstrate how KPFM measurements go beyond QNM to provide contrast between materials with reproducibility at a higher image resolution. With the ability to measure contrast between donor and acceptor material, we make a strong case for non-destructive microscopy data to measure effects of variations in annealing temperature on squaraine film morphology, which we confirm influences device performance and efficiency. These conclusions are important for informing material selection for long-term use of associated commercial devices in the field.

Force Spectroscopy on a Cell Drum: AFM Measurements on the Basolateral Side of Cells via Inverted Cell Cultures.

The elasticity of a cell is one of the most critical measures of the difference between cancerous cells and healthy cells: cancer cells tend to be softer than healthy cells, and highly invasive cells tend to be more elastic than less aggressive cells. In this work, we present the novel "bottom-up" cell force spectroscopy method for the biophysical characterization of cancer cells, in which an atomic force microscopy (AFM) tip approach from the backside of a net-shaped culture substrate exposing the basolateral cell membrane drum, and compare it with the conventional "top-down" AFM measurements. We used two different human pancreatic carcinoma cell lines, PaTu8988S and PaTu8988T. Our bottom-up AFM tip approach provided a more statistically synchronized distribution of the measured elastic moduli of the cells, demonstrating its superior applicability for the clinical use of force spectroscopy, which is not attainable with the conventional top-down AFM approach.

Improvement of the Correlative AFM and ToF-SIMS Approach Using an Empirical Sputter Model for 3D Chemical Characterization.

Technological progress has spurred the development of increasingly sophisticated analytical devices. The full characterization of structures in terms of sample volume and composition is now highly complex. Here, a highly improved solution for 3D characterization of samples, based on an advanced method for 3D data correction, is proposed. Traditionally, secondary ion mass spectrometry (SIMS) provides the chemical distribution of sample surfaces. Combining successive sputtering with 2D surface projections enables a 3D volume rendering to be generated. However, surface topography can distort the volume rendering by necessitating the projection of a nonflat surface onto a planar image. Moreover, the sputtering is highly dependent on the probed material. Local variation of composition affects the sputter yield and the beam-induced roughness, which in turn alters the 3D render. To circumvent these drawbacks, the correlation of atomic force microscopy (AFM) with SIMS has been proposed in previous studies as a solution for the 3D chemical characterization. To extend the applicability of this approach, we have developed a methodology using AFM-time-of-flight (ToF)-SIMS combined with an empirical sputter model, "dynamic-model-based volume correction", to universally correct 3D structures. First, the simulation of 3D structures highlighted the great advantages of this new approach compared with classical methods. Then, we explored the applicability of this new correction to two types of samples, a patterned metallic multilayer and a diblock copolymer film presenting surface asperities. In both cases, the dynamic-model-based volume correction produced an accurate 3D reconstruction of the sample volume and composition. The combination of AFM-SIMS with the dynamic-model-based volume correction improves the understanding of the surface characteristics. Beyond the useful 3D chemical information provided by dynamic-model-based volume correction, the approach permits us to enhance the correlation of chemical information from spectroscopic techniques with the physical properties obtained by AFM.

Reconstruction of atomic force microscopy image using compressed sensing

Atomic Force Microscopy (AFM) is one of the most popular and advanced tools for ultra high-resolution imaging and nanomanipulation of nano-scale matter. But AFM imaging typically takes a long time. High-speed and high-precision AFM measurement has attracted wide attention in recent several years. In traditional AFM, simple reduction in the number of measurement points may lose essential sample topography information. To resolve such problems, an AFM image reconstruction method based on Compressed Sensing (CS) theory is applied to reduce image acquisition time without cutting down the image quality. The benefit of using CS approach in AFM is shortening the imaging time, minimizing the interaction with the sample, and finally avoiding sample damage in AFM. Three kinds of testing samples with high and low frequency components were examined by a scanning electron microscope (SEM) and by AFM. An orthogonal Matching Pursuit (OMP) algorithm is employed to reconstruct an AFM image with different sampling rates. Subsequently the reconstruction results of sample topography images are analyzed and evaluated. Using the CS approach in AFM can greatly improve the AFM imaging process. Experimental results show that the obtained reconstructed images have different resolution and quality, depending on the surface morphology of the sample and sampling rates.

Combining confocal and atomic force microscopy to quantify single-virus binding to mammalian cell surfaces.

Over the past five years, atomic force microscopy (AFM)-based approaches have evolved into a powerful multiparametric tool set capable of imaging the surfaces of biological samples ranging from single receptors to membranes and tissues. One of these approaches, force-distance curve-based AFM (FD-based AFM), uses a probing tip functionalized with a ligand to image living cells at high-resolution and simultaneously localize and characterize specific ligand-receptor binding events. Analyzing data from FD-based AFM experiments using appropriate probabilistic models allows quantification of the kinetic and thermodynamic parameters that describe the free-energy landscape of the ligand-receptor bond. We have recently developed an FD-based AFM approach to quantify the binding events of single enveloped viruses to surface receptors of living animal cells while simultaneously observing them by fluorescence microscopy. This approach has provided insights into the early stages of the interaction between a virus and a cell. Applied to a model virus, we probed the specific interaction with cells expressing viral cognate receptors and measured the affinity of the interaction. Furthermore, we observed that the virus rapidly established specific multivalent interactions and found that each bond formed in sequence strengthened the attachment of the virus to the cell. Here we describe detailed procedures for probing the specific interactions of viruses with living cells; these procedures cover tip preparation, cell sample preparation, step-by-step FD-based AFM imaging and data analysis. Experienced microscopists should be able to master the entire set of protocols in 1 month.

Atomic Force Microscopy of Protein Shells: Virus Capsids and Beyond.

In Atomic Force Microscopy (AFM) the probe is a nanometric tip located at the end of a microcantilever which palpates the specimen under study as a blind person uses a white cane. In this way AFM allows obtaining nanometric resolution images of individual protein shells, such as viruses, in liquid milieu. Beyond imaging, AFM also enables the manipulation of single protein cages, and the characterization a variety physicochemical properties able of inducing any measurable mechanical perturbation to the microcantilever that holds the tip. In this chapter we start revising some recipes for adsorbing protein shells on surfaces. Then we describe several AFM approaches to study individual protein cages, ranging from imaging to spectroscopic methodologies devoted to extracting physical information, such as mechanical and electrostatic properties.

Atomic force microscopy of virus shells.

Microscopes are used to characterize small objects with the help of probes that interact with the specimen, such as photons and electrons in optical and electron microscopies, respectively. In atomic force microscopy (AFM), the probe is a nanometric tip located at the end of a microcantilever which palpates the specimen under study just as a blind person manages a walking stick. In this way, AFM allows obtaining nanometric resolution images of individual protein shells, such as viruses, in a liquid milieu. Beyond imaging, AFM also enables not only the manipulation of single protein cages, but also the characterization of every physicochemical property capable of inducing any measurable mechanical perturbation to the microcantilever that holds the tip. In the present revision, we start revising some recipes for adsorbing protein shells on surfaces. Then, we describe several AFM approaches to study individual protein cages, ranging from imaging to spectroscopic methodologies devoted to extracting physical information, such as mechanical and electrostatic properties. We also explain how a convenient combination of AFM and fluorescence methodologies entails monitoring genome release from individual viral shells during mechanical unpacking.

Generation, manipulation and characterization of molecules by atomic force microscopy

Using atomic manipulation, one can dissociate, form and rearrange bonds, as well as alter the conformation or charge state of molecules. The molecular structures of reactants, intermediates and products are revealed at unprecedented resolution by using atomic force microscopy (AFM) and a suitably functionalized tip. Our present capabilities of manipulation and imaging of molecules by AFM approach the level of control predicted by Richard P. Feynman in his famous lecture ‘There's plenty of room at the bottom’, in which he described how molecules and materials might be formed by attaching and detaching individual atoms at will. In this Review, we discuss recent progress and the future prospects of molecule generation by atom manipulation and molecular characterization by AFM. Atomic force microscopy (AFM) enables the imaging and manipulation of individual molecules at atomic resolution. This Review addresses experimental considerations, including operating modes and choices for tips and substrates. Examples are presented in which AFM is used to image molecules and induce bond formation or dissociation.

Elastic and viscoelastic characterization of inhomogeneous polymers by bimodal atomic force microscopy

The elastic and viscoelastic responses of inhomogeneous polymers upon interacting with an atomic force microscopy (AFM) probe are simultaneously characterized by a bimodal AFM approach namely the amplitude- and frequency-modulation (AM–FM) method. In this approach, the AFM probe is operated in the AM mode at the first flexural frequency and in the FM mode at a higher flexural frequency. The AM mode provides information about the viscoelasticity of polymers in terms of the mechanical loss tangent, whereas the modulus of polymers is obtained as a function of the frequency shift of flexural frequencies in both modes. For a glassy polymer blend, the AM–FM method provides a consistent result in both the elastic modulus and loss tangent in comparison with those obtained by other methods. Moreover, a significant improvement of the contrast and lateral resolution in the AM–FM modulus image can be observed. However, the current approach shows a substantial increase in the modulus of rubbery polymers.

Adhesion of Pseudomonas fluorescens biofilms to glass, stainless steel and cellulose.

The adhesion of colloidal probes of stainless steel, glass and cellulose to Pseudomonas fluorescens biofilms was examined using atomic force microscopy (AFM) to allow comparisons between surfaces to which biofilms might adhere. Biofilm was grown on a stainless steel substrate and covered most of the surface after 96h. AFM approach and retraction curves were obtained when the biofilm was immersed in a tryptone/soy medium. On approach, all the colloidal probes experienced a long non-contact phase more than 100nm in length, possibly due to the steric repulsion by extracellular polymers from the biofilm and hydrophobic effects. Retraction data showed that the adhesion varied from position to position on the biofilm. The mean value of adhesion of glass to the biofilm (48±7nN) was the greatest, followed by stainless steel (30±7nN) and cellulose (7.8±0.4nN). The method allows understanding of adhesion between the three materials and biofilm, and development of a better strategy to remove the biofilm from these surfaces relevant to different industrial applications.

Nanomechanics of Cells and Biomaterials Studied by Atomic Force Microscopy.

The behavior and mechanical properties of cells are strongly dependent on the biochemical and biomechanical properties of their microenvironment. Thus, understanding the mechanical properties of cells, extracellular matrices, and biomaterials is key to understanding cell function and to develop new materials with tailored mechanical properties for tissue engineering and regenerative medicine applications. Atomic force microscopy (AFM) has emerged as an indispensable technique for measuring the mechanical properties of biomaterials and cells with high spatial resolution and force sensitivity within physiologically relevant environments and timescales in the kPa to GPa elastic modulus range. The growing interest in this field of bionanomechanics has been accompanied by an expanding array of models to describe the complexity of indentation of hierarchical biological samples. Furthermore, the integration of AFM with optical microscopy techniques has further opened the door to a wide range of mechanotransduction studies. In recent years, new multidimensional and multiharmonic AFM approaches for mapping mechanical properties have been developed, which allow the rapid determination of, for example, cell elasticity. This Progress Report provides an introduction and practical guide to making AFM-based nanomechanical measurements of cells and surfaces for tissue engineering applications.

Specific binding at the cellulose binding module-cellulose interface observed by force spectroscopy.

The need for effective enzymatic depolymerization of cellulose has stimulated an interest in interactions between protein and cellulose. Techniques utilized for quantitative measurements of protein-cellulose noncovalent association include microgravimetry, calorimetry, and atomic force microscopy (AFM), none of which differentiate between specific protein-cellulose binding and nonspecific adhesion. Here, we describe an AFM approach that differentiates nonspecific from specific interactions between cellulose-binding modules (CBMs) and cellulose. We demonstrate that the "mismatched" interaction between murine galectin-3, a lectin with no known affinity for cellulose, and cellulose shows molecular recognition force microscopy profiles similar to those observed during the interaction of a "matched" clostridial CBM3a with the same substrate. We also examine differences in binding probabilities and rupture profiles during CBM-cellulose binding experiments in the presence and absence of a blocking agent-a substrate specific for CBM that presumably blocks binding sites. By comparison of the behavior of the two proteins, we separate specific (i.e., blockable) and nonspecific adhesion events and show that both classes of interaction exhibit nearly identical rupture forces (45 pN at ∼0.4 nN/s). Our work provides an important caveat for the interpretation of protein-carbohydrate binding by force spectroscopy; delineation of the importance of such interactions to other classes of binding warrants further study.