Observation of geometric structure of collagen molecules by atomic force microscopy

Atomic force microscopy was used to study the geometric structure of collagen fibrils and molecules of rat calcanean tendon tissues. The authors found that the diameter of the fibrils ranged from 124 to 170 nm, and their geometric form suggested a helical winding with spectral period from 59.4 to 61.7 nm, close to the band dimensions reported by electron microscopy. At high magnification, the surface of these bands revealed images that probably correspond to the almost crystalline array of collagen molecules, with the triple helix structure almost visible. The typical helix width is 1.43 nm, with main periods of 1.15 and 8.03 nm, very close to the dimensions reported by X-ray diffraction.     

Probing buried carbon nanotubes within polymer-nanotube composite matrices by atomic force microscopy

Multi-walled carbon nanotubes (MW-CNT) inside a polyamide-6 (PA6)-MW-CNT composite were visualized by atomic force microscopy (i) in a field-assisted intermittent contact and (ii) in the tunneling (TUNA) mode. Individual buried MW-CNTs were clearly discerned within the PA6 matrix. An average diameter of 33 ± 5 nm of the MW-CNTs was determined based on field-assisted intermittent contact mode AFM images, which is consistent with the expected size of PA6-coated MW-CNTs. Single well dispersed MW-CNTs that are located in the sub-surface region of the composite were also observed in the TUNA mode. These new AFM approaches circumvent the tedious sample preparation based on ultramicrotoming required for high resolution electron microscopy studies to obtain “in-depth” morphological information and hence are expected to facilitate the analysis of CNT-based and other nanocomposites in the future.     

Comparison of antibody--antigen interactions on collagen measured by conventional immunological techniques and atomic force microscopy

We have developed a means of using atomic force microscopy (AFM) to repeatedly localize a small area of interest (4 x 4 microm(2)) within a 0.5-cm(2) area on a heterogeneous sample, to obtain and localize high-resolution images and force measurements on nonideal samples (i.e., samples that better reflect actual biological systems, not prepared on atomically flat surfaces). We demonstrate the repeated localization and measurement of unbinding forces associated with antibody--antigen (ab--ag) interactions, by applying AFM in air and in liquid to visualize and measure polyclonal ab--ag interactions, using chicken collagen as a model system. We demonstrate that molecular interactions, in the form of ab--ag complexes, can be visualized by AFM when secondary antibodies are conjugated to 20-nm colloidal gold particles. We then compare those results with established immunological techniques, to demonstrate broader application of AFM technology to other systems. Data from AFM studies are compared with results obtained using immunological methods traditionally employed to investigate ab--ag interactions, including enzyme-linked immunosorbent assay, immunoblotting, and in situ immunofluorescence. Finally, using functionalized AFM tips with a flexible tether [poly(ethylene glycol) 800] to which a derivatized antibody was attached, we analyzed force curve data to measure the unbinding force of collagen antibody from its antigen, obtaining a value of approximately 90 +/- 40 pN with a MatLab code written to automate the analyses of force curves obtained in force--volume mode. The methodology we developed for embedded collagen sections can be readily applied to the investigation of other receptor--ligand interactions.     

Single molecule charging by atomic force microscopy

AFM/KPM charging and charge mapping of polyamine charge carriers in a PMMA matrix is reported. Selective charging of the designed charge carrier is demonstrated at concentrations down to a single molecule. This works constitutes electrochemical charging and detection of single redox-active organic molecules in low dielectric matrices by probe microscopy.     

Halo-like structures studied by atomic force microscopy

Nanometer-sized clusters of copper have been produced in a hollow cathode sputtering source and deposited on SiO_x. Halo-like structures consisting of micrometer sized protrusions in the silicon oxide surface surrounded by thin rings of smaller particles are observed. The area in between seems to be depleted of particles. We propose that the halo-like structures are a result of electrostatic forces acting between the incoming charged clusters and charged regions on the surface. A simple computer simulation supports this suggestion.     

Measuring molecular weight by atomic force microscopy

Absolute-molecular-weight distribution of cylindrical brush molecules were determined using a combination of the Langmuir Blodget (LB) technique and Atomic Force Microscopy (AFM). The LB technique gives mass density of a monolayer, i.e., mass per unit area, whereas visualization of individual molecules by AFM enables accurate measurements of the molecular density, i.e., number of molecules per unit area. From the ratio of the mass density to the molecular density, one can determine the absolute value for the number average molecular weight. Assuming that the structure of brush molecules is uniform along the backbone, the length distribution should be virtually identical to the molecular weight distribution. Although we used only brush molecules for demonstration purpose, this approach can be applied for a large variety of molecular and colloidal species that can be visualized by a microscopic technique.     

Measurement of the elastic modulus of single bacterial cellulose fibers using atomic force microscopy

The ability of the atomic force microscope to measure forces with subnanonewton sensitivity at nanometer-scale lateral resolutions has led to its use in the mechanical characterization of nanomaterials. Recent studies have shown that the atomic force microscope can be used to measure the elastic moduli of suspended fibers by performing a nanoscale three-point bending test, in which the center of the fiber is deflected by a known force. We extend this technique by modeling the deflection measured at several points along a suspended fiber, allowing us to obtain more accurate data, as well as to justify the mechanical model used. As a demonstration, we have measured a value of 78 +/- 17 GPa for Young's modulus of bacterial cellulose fibers with diameters ranging from 35 to 90 nm. This value is considerably higher than previous estimates, obtained by less direct means, of the mechanical strength of individual cellulose fibers.     

Preservation of bone collagen from the late Cretaceous period studied by immunological techniques and atomic force microscopy

Late Cretaceous avian bone tissues from Argentina demonstrate exceptional preservation. Skeletal elements are preserved in partial articulation and suspended in three dimensions in a medium-grained sandstone matrix, indicating unusual perimortem taphonomic conditions. Preservation extends to the microstructural and molecular levels. Bone tissues respond to collagenase digestion and histochemical stains. In situ immunohistochemistry localizes binding sites for avian collagen antibodies in fossil tissues. Immunohistochemical studies do not, however, guarantee the preservation of molecular integrity. A protein may retain sufficient antigenicity for antibody binding even though degradation may render it incapable of original function. Therefore, we have applied atomic force microscopy to address the integrity and functionality of retained organic structures. Collagen pull-off measurements not only support immunochemical evidence for collagen preservation for antibody recognition but also imply preservation of the whole molecular integrity. No appreciable differences in collagen pull-off properties were measured between fossil and extant bone samples under physiological conditions.     

Quantitative study of filled rubber microstructure by optical and atomic force microscopy

A comprehensive approach is proposed for studying the microstructure of filled rubbers by optical and atomic force microscopy (AFM). The optical results are found to be dependent on the illumination angle. Algorithms based on the mathematical morphology are developed for the processing of optical images (removing scratches, identifying agglomerates). AFM-images are treated by a segmentation method which separates a continuous surface into segments that match filler. Parameters of secondary filler structures and the size of an area of homogeneous filler dispersion are obtained from the analysis of the segmented images. Seven carbon black filled rubbers with different mixing times are investigated. The combination of AFM with optical imaging techniques makes it possible to perform a quantitative structural analysis at scales from tens of nanometers to tens of microns, and to establish the relationship between the mixing time and the filler microstructure over the whole range of filler peculiarities.     

Characterization of elastomeric blends by atomic force microscopy

The bulk mechanical properties of a blend of elastomers are found to depend on the micro and nano scale morphology of the phases of the materials in the blend. In this study, we examine the phase morphology of blends of incompatible elastomers using Atomic Force Microscopy (AFM). Specifically, nanoindentation and Tapping Mode AFM (TMAFM) imaging techniques are used as experimental tools for mapping the composition of unfilled elastomeric blends. Depending on the composition of the blend, either co‐continuous or discontinuous domain/matrix morphology is observed. To identify the different components in bromobutyl (BIIR)/natural rubber (NR) blends, nanoscale indentation measurements were made on the observed phase‐separated regions. Results from force mode AFM and mechanical measurements of bulk NR and BIIR are used to assist in the interpretation of the TMAFM results for the BIIR/NR blends. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 492–503, 2006     

Pull-off force measurements between rough surfaces by atomic force microscopy

Direct measurements of the pull-off (adhesion) forces between pharmaceutical particles (beclomethasone dipropionate, a peptide-type material, and lactose) with irregular geometry and rough polymeric surfaces (series of polypropylene coatings, polycarbonate, and acrylonitrile-butadiene-styrene) were carried out using the atomic force microscope. These measurements showed that roughness of the interacting surfaces is the significant factor affecting experimentally measured pull-off forces. A broad distribution of pull-off force values was noted in the measurements, caused by a varying adhesive contact area for a particle located on rough substrate. The possibility of multiple points of contact between irregularly shaped pharmaceutical particles and substrate surfaces is demonstrated with nanoindentations of the particle in a fluoro-polymer film. Force-distance curves showing the "sawtooth" pattern are additional evidence that particles make contact with substrates at more than one point. Reduced adhesion of 10- to 14-microm-diameter lactose and peptide material particles to the polypropylene coatings with a roughness of 194 nm was found in this study. Similar pull-off force versus roughness relationships are also reported for the model spherical particles, silanized glass particle with a size of 10 microm and polystyrene particle with a diameter of 9 microm, in contact with polypropylene coatings of varying roughness characteristics. It was found that the model recently proposed by Rabinovich et al. (J. Colloid Interface Sci. 232, 1-16 (2000)) closely predicts the pull-off forces for glass and lactose particles. On the other hand, the adhesion of the peptide material and polystyrene particle to polypropylene is underestimated by about an order of magnitude with the theoretical model, in which the interacting substrates are treated as rigid materials. The underestimate is attributed to the deformation of the peptide material and polystyrene particles.     

Investigation of aerosol particles by atomic force microscopy

AFM has been applied for studying morphology and size distribution of nanometer-sized particles adsorbed on flat surfaces. In order to optimize imaging of these ultrafine particles different substrates were evaluated with respect to their roughness and stability under the influence of the sensing tip. Moreover, a method for calculating particle volumes from the three-dimensional AFM data is described. This greatly enhances the information content of AFM images, because a large number of particles in the raw data can be evaluated automatically in order to derive information on size distribution or surface coverage. This evaluation method has also been applied successfully to quantitatively describe changes on particles induced by different humidity of the surrounding atmosphere. Received: 15 July 1996 / Revised: 18 December 1996 / Accepted: 3 January 1997     

High-resolution atomic force microscopy study of hexaglycylamide epitaxial structures on graphite

Two types of hexaglycylamide (HGA) epitaxial lamellar structures coexisting on the surface of highly oriented pyrolytic graphite (HOPG) exposed to water solutions were studied by high-resolution atomic force microscopy (AFM). Lamellae are distinguished by growth direction and by morphology. The lamellae of the first type (L1) produced by depositions from more dilute solutions are close-packed with a period of ～5.2 nm, twice the HGA molecular length, and form highly ordered domains morphologically similar to the lamellar domains of alkanes. The less-ordered lamellae of the second type (L2) appear at intermediate and large HGA concentrations and demonstrate variable lamellar width, morphological diversity, and a tendency to merge. The interlamellar separation in the domains of close-packed L2 lamellae varies with the discrete increment ～2.5 nm; the most frequently observed value is ～7.5-8.0 nm corresponding to the triple HGA molecular length. The growth directions of lamellae of each type have sixfold rotational symmetry indicating epitaxy with graphite; however, the rosettes of L1 and L2 lamellae orientations are misaligned by 30°. The molecular modeling of possible HGA epitaxial packing arrangements on graphite and their classification have been conducted, and the energetically preferable structures are selected. On this basis, the structural models of the L1 and L2 lamellae are proposed explaining the experimentally observed peculiarities as follows: (1) the L1 and L2 lamellae are respectively parallel and antiparallel β-sheets with two HGA molecules in the unit cell oriented normally to the lamellae boundaries, (2) HGA molecules in L1 and L2 lamellae have different orientations with respect to the graphite lattice, respectively along the directions <1120> and <1010>, (3) L1 lamella is the assembly of two hydrogen-bonded parallel β-sheets oriented head-to-head, (4) L2 lamellae are assemblies of several molecular rows (antiparallel β-sheets) cross-linked by hydrogen bonds. The AFM observations indicate that the covering of the hydrophobic graphite by the dense, closely packed, well-ordered monolayers of hydrophilic oligopeptide is possible.     

Elasticity of cardiac cells on the polymer substrates with different stiffness: an atomic force microscopy study

Influences of substrate stiffness on mechanical properties of cardiac myocytes and fibroblasts were investigated by cell elasticity measurement with atomic force microscopy. The cells were cultured on collagen-coated polyacrylamide substrates with gradient rigidity. While cardiac myocytes showed no evident change in cell elasticity on different substrates, cardiac fibroblasts displayed the non-monotonic dependence on substrate stiffness with a maximum elastic modulus. Moreover, the elasticity change of cardiac fibroblasts with substrates stiffness was found to be regulated by actin filaments. Study of the effect of substrate stiffness on cell elasticity for different cardiac cells provides new information for the better understanding of cardiac physiology and pathology.     

Young's moduli of surface-bound liposomes by atomic force microscopy force measurements

Mechanical properties of layers of intact liposomes attached by specific interactions on solid surfaces were studied by atomic force microscopy (AFM) force measurements. Force-distance measurements using colloidal probe tips were obtained over liposome layers and used to calculate Young's moduli by using the Hertz contact theory. A classical Hertz model and a modified Hertz one have been used to extract Young's moduli from AFM force curves. The modified model, proposed by Dimitriadis, is correcting for the finite sample thickness since Hertz's classical model is assuming that the sample is infinitely thick. Values for Young's moduli of 40 and 8 kPa have been obtained using the Hertz model for one and three layers of intact liposomes, respectively. Young's moduli of approximately 3 kPa have been obtained using the corrected Hertz model for both one and three layers of surface-bound liposomes. Compression work performed by the colloidal probe to compress these liposome layers has also been calculated.     

Thin liquid films studied by atomic force microscopy

Since its invention twenty years ago the atomic force microscope (AFM) has become one of the most important instruments in colloid and interface science. The ability of tracing force profiles between single particles or particles and flats in liquid environment makes it a tool-of-choice for investigating thin liquid films. In this paper we review experimental work on confined Newtonian and non-Newtonian liquids using the AFM.     

Confined polymer melts studied by atomic force microscopy

Using an atomic force microscope (AFM) the interaction between an AFM tip and different planar solid surfaces have been measured across a long-chain poly(dimethyl siloxane) (PDMS, M_W = 18,000 g/mol), a short-chain PDMS (M_W = 4200 g/mol), a poly(ethylmethyl siloxane) (PEMS, M_W = 16,800 g/mol), and a diblock copolymer consisting of one PDMS and one PEMS block (PDMS-b-PEMS, M_W = 15,100 g/mol). The interaction changed significantly during the first 10 h after immersing the solids in the polymer melt. This demonstrates that the time scale of structural changes at a solid surface is much slower than in the bulk. On mica and silicon oxide both polymers formed an immobilized “pinned” layer beyond which a monotonically decaying repulsive force was observed. Attractive forces were observed with short-chain PDMS on silicon oxide and PEMS on mica and silicon oxide. On the basal plane of graphite PEMS caused a stable, exponentially decaying oscillatory force.     

Unbinding of the streptavidin-biotin complex by atomic force microscopy: a hybrid simulation study

A hybrid molecular simulation technique, which combines molecular dynamics and continuum mechanics, was used to study the single-molecule unbinding force of a streptavidin-biotin complex. The hybrid method enables atomistic simulations of unbinding events at the millisecond time scale of atomic force microscopy (AFM) experiments. The logarithmic relationship between the unbinding force of the streptavidin-biotin complex and the loading rate (the product of cantilever spring constant and pulling velocity) in AFM experiments was confirmed by hybrid simulations. The unbinding forces, cantilever and tip positions, locations of energy barriers, and unbinding pathway were analyzed. Hybrid simulation results from this work not only interpret unbinding AFM experiments but also provide detailed molecular information not available in AFM experiments.