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.     

Correlation between desorption force measured by atomic force microscopy and adsorption free energy measured by surface plasmon resonance spectroscopy for peptide-surface interactions

Surface plasmon resonance (SPR) spectroscopy is a useful technique for thermodynamically characterizing peptide-surface interactions; however, its usefulness is limited to the types of surfaces that can readily be formed as thin layers on the nanometer scale on metallic biosensor substrates. Atomic force microscopy (AFM), on the other hand, can be used with any microscopically flat surface, thus making it more versatile for studying peptide-surface interactions. AFM, however, has the drawback of data interpretation due to questions regarding peptide-to-probe-tip density. This problem could be overcome if results from a standardized AFM method could be correlated with SPR results for a similar set of peptide-surface interactions so that AFM studies using the standardized method could be extended to characterize peptide-surface interactions for surfaces that are not amenable for characterization by SPR. In this article, we present the development and application of an AFM method to measure adsorption forces for host-guest peptides sequence on surfaces consisting of alkanethiol self-assembled monolayers (SAMs) with different functionality. The results from these studies show that a linear correlation exists between these data and the adsorption free energy (ΔG(o)(ads)) values associated with a similar set of peptide-surface systems available from SPR measurements. These methods will be extremely useful to characterize thermodynamically the adsorption behavior for peptides on a much broader range of surfaces than can be used with SPR to provide information related to understanding protein adsorption behavior to these surfaces and to provide an experimental database that can be used for the evaluation, modification, and validation of force field parameters that are needed to represent protein adsorption behavior accurately for molecular simulations.     

Attractive bridging interactions in dense polymer brushes in good solvent measured by atomic force microscopy

Using an atomic force microscope (AFM), we have investigated the interaction forces exerted by latex particles bearing densely grafted polymer brushes consisting of poly(N,N-dimethylacrylamide) (PDMA), poly(methoxyethylacrylamide) (PMEA), poly(N-isopropylacrylamide) (PNIPAM), and PMEA-b-PNIPAM in aqueous media (good solvent). The brushes were prepared by controlled surface-initiated atom transfer radical polymerization, and the hydrodynamic thicknesses were measured by dynamic light scattering. The molecular weight (Mn), grafting density (sigma), and polydispersity (PDI) of the brushes were determined by gel permeation chromatography and multiangle laser light scattering after cleaving the polymer from the latex surface by hydrolysis. Force profiles of PDMA (0.017 nm(-2) < or = sigma < or = 0.17 nm-2) and PMEA (sigma = 0.054 nm-2) brushes were purely repulsive upon compression, with forces increasing with Mn and a, as expected, due to excluded volume interactions. At a sufficiently low grafting density (sigma = 0.012 nm-2), PDMA exhibited a long-range exponentially increasing attractive force followed by repulsion upon further compression. The long-range attractive force is believed to be due to bridging between the free chain ends and the AFM tip. The PNIPAM brush exhibited a bridging force at a grafting density of 0.037 nm(-2), a value lower than the sigma needed to induce bridging in the PDMA brush. Bridging was therefore found to depend on grafting density as well as on the nature of the monomer. The grafting densities of these polymers were larger than those typically associated with bridging. Bridging interactions were used to confirm the presence of PNIPAM in a block copolymer PMEA-b-PNIPAMA brush given that the original PMEA homopolymer brush produced a purely repulsive force. The attractive force was first detected in the block copolymer brush at a separation that increased with the length of the PNIPAM block.     

Time-dependent conformational changes in fibrinogen measured by atomic force microscopy

Tapping-mode atomic force microscopy was used to study the time-dependent changes in the structure of fibrinogen under aqueous conditions following adsorption on two model surfaces: hydrophobic graphite and hydrophilic mica. Fibrinogen was observed in the characteristic trinodular form, and the dimensions of the adsorbed molecules were consistent with previously reported values for these surfaces. On the basis of the differences in the relative heights of the D and the E domains, four orientation states were observed for fibrinogen adsorbed on both the surfaces. On graphite, the initial asymmetric orientation states disappeared with spreading over time. Some small lateral movements of the adsorbed proteins were observed on mica during repeated scanning, whereas no such movement was observed on graphite, indicating strong adhesion of fibrinogen to a hydrophobic surface. Spreading kinetics of fibrinogen on the two surfaces was determined by measuring the heights of the D and E domains over a time period of approximately 2 h. On graphite, the heights of both the D and E domains decreased with time to a lower plateau value of 1.0 nm. On mica, the heights of both the D and E domains showed an increase, rising to an upper plateau value of approximately 2.1 nm. The spreading of the D and E domains on graphite was analyzed using an 'exponential-decay-of-height' model. A spreading rate constant of approximately 4.7 x 10(-4) s(-1) was observed for the whole fibrinogen molecule adsorbed on graphite, corresponding to a free energy of unfolding of approximately 37 kT. Extrapolation of the exponential curve in the model to t = 0 yielded values of 2.3 and 2.2 nm for the heights of the D and the E domains at the time of contact with the hydrophobic graphite substrate, significantly less than their free solution diameters. A two-step spreading model is proposed to explain this observation.     

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.     

The dynamic nature of contact angles as measured by atomic force microscopy

Atomic force microscopy appears to be a useful tool for determining the contact angle for small particles. It is shown in this paper that the contact angle of a spherical polyethylene particle changes with the speed of the AFM piezoelectric translator. Such dynamic behavior of the contact angle and other uncertainties such as the position of the three-phase contact on the particle surface during bubble-particle interaction make it difficult to decide whether or not the AFM single-particle contact angle can be used to describe the hydrophobic state of the particle surface.     

Probing interactions between aggrecan and mica surface by the atomic force microscopy

Aggrecan is a bottlebrush shaped macromolecule found in the extracellular matrix of cartilage. The negatively charged glycosaminoglycan (GAG) chains attached to its protein backbone give aggrecan molecules a high charge density, which is essential for exerting high osmotic swelling pressure and resisting compression under external load. In solution, aggrecan assemblies are insensitive to the presence of calcium ions, and show distinct osmotic pressure versus concentration regimes. The aim of this study is to investigate the effect of ionic environment on the structure of aggrecan molecules adsorbed onto well‐controlled mica surfaces. The conformation of the aggrecan was visualized using Atomic Force Microscopy. On positively charged APS mica the GAG chains of the aggrecan molecules are distinguishable, and their average dimensions are practically unaffected by the presence of salt ions. With increasing aggrecan concentration they form clusters, and at higher concentrations they form a continuous monolayer of conforming molecules. On negatively charged mica, the extent of aggrecan adsorption varies with salt composition. Understanding aggrecan adsorption onto a charged surface provides insight into its interactions with bone and implant surfaces in the biological milieu. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2010     

Local morphological and dimensional changes of enzyme-degraded cellulose materials measured by atomic force microscopy

A study of the degradation effects of enzyme treatment on the dimensional changes of cellulose aggregate fibrils (CAFs) with dimensions of ∼100,000 × 3,000 × 300 nm from fully bleached kraft fiber was performed. CAFs were incubated with cellulase for up to 32 h. The insoluble CAFs fragments remaining after enzymatic hydrolysis were then subjected to variable relative humidity (RH). Each sample was imaged by an atomic force microscope (AFM) in tapping mode. The images were analyzed to determine the dimensional changes of the insoluble CAFs. Enzymatic hydrolysis continuously depolymerized the CAFs over 32 h, ultimately causing 20% of the CAFs to become soluble. Compared to initial dimensions of the reference CAFs with no enzymatic treatment, the dimensions of the enzyme treated CAFs were generally more responsive to humidity and exhibited an increased frequency of plastic deformations.     

Mechanical characterization of polymeric thin films by atomic force microscopy based techniques

Polymeric thin films have been awakening continuous and growing interest for application in nanotechnology. For such applications, the assessment of their (nano)mechanical properties is a key issue, since they may dramatically vary between the bulk and the thin film state, even for the same polymer. Therefore, techniques are required for the in situ characterization of mechanical properties of thin films that must be nondestructive or only minimally destructive. Also, they must also be able to probe nanometer-thick ultrathin films and layers and capable of imaging the mechanical properties of the sample with nanometer lateral resolution, since, for instance, at these scales blends or copolymers are not uniform, their phases being separated. Atomic force microscopy (AFM) has been proposed as a tool for the development of a number of techniques that match such requirements. In this review, we describe the state of the art of the main AFM-based methods for qualitative and quantitative single-point measurements and imaging of mechanical properties of polymeric thin films, illustrating their specific merits and limitations.     

Mechanistic aspects of protein/material interactions probed by atomic force microscopy

Physicochemical studies on the mechanisms of protein adsorption onto solid material surfaces have been extensively performed so far, mainly based on the analysis of factors such as the equilibrium adsorbed amount (adsorption isotherms), time-dependent change of adsorbed amount (adsorption kinetics), and conformational change of adsorbed protein. However, direct understanding of the strength of the molecular interaction between protein and the material surface has not been established yet. For this issue, the force measurement techniques of an atomic force microscope (AFM) using a protein-modified probe tip are recently becoming powerful tools to analyze the actual interaction forces between protein and material surfaces. In this mini review, we discuss the characteristics and interpretation of the AFM force-versus-distance curves (f–d curves) obtained with the protein-modified probe tip, and the relationship between the forces measured from the f–d curves and the driving forces in the natural process of protein adsorption. Relative degrees of each of the following contributions which determine the character of protein adsorption are clarified: (1) the intrinsic protein/material forces mediated by solvent, (2) the thermodynamic stability of protein/material adhesion interface, and (3) diffusion force of protein molecules. Within these driving forces, the latter two in particular are confirmed to play essential roles in determining the character of protein adsorption, based on the profiles of f–d curves.     

Interaction forces between metal-chelating lipid monolayers measured by colloidal probe atomic force microscopy

Surface forces between LB films of metal-chelating lipids in water have been studied using colloidal probe atomic force microscopy. The LB films of an amphiphile functionalized by the iminodiacetic acid group were prepared on hydrophobic glass substrates. The electric double layer repulsion operated between these LB film surfaces changed depending on pH reflecting the different protonation states of the iminodiacetic acid groups. The titration curve of the iminodiacetic acid monolayer was obtained from the force profiles. The Cu²⁺ complexation process was also monitored by measuring the force profiles at various Cu²⁺ ion concentrations.     

Dynamics of a disturbed sessile drop measured by atomic force microscopy (AFM)

A new method for studying the dynamics of a sessile drop by atomic force microscopy (AFM) is demonstrated. A hydrophobic microsphere (radius, r ～ 20-30 μm) is brought into contact with a small sessile water drop resting on a polytetrafluoroethylene (PTFE) surface. When the microsphere touches the liquid surface, the meniscus rises onto it because of capillary forces. Although the microsphere volume is 6 orders of magnitude smaller than the drop, it excites the normal resonance modes of the liquid interface. The sphere is pinned at the interface, whose small (<100 nm) oscillations are readily measured with AFM. Resonance oscillation frequencies were measured for drop volumes between 5 and 200 μL. The results for the two lowest normal modes are quantitatively consistent with continuum calculations for the natural frequency of hemispherical drops with no adjustable parameters. The method may enable sensitive measurements of volume, surface tension, and viscosity of small drops.     

Dynamic imaging of single DNA-protein interactions using atomic force microscopy

Atomic force microscopy (AFM) imaging of static DNA-protein complexes, in air and in liquid, can be used to directly obtain quantitative and qualitative information on the structure of different complexes. For example, DNA length, the location of preferential binding sites for proteins and bending of DNA as a result of the complexation can all be measured. Recording consecutive AFM images of DNA and protein molecules under conditions that they are still able to move and interact, or dynamic AFM imaging, however, can reveal information on the dynamic aspects of the interactions between these molecules. Here, an overview is given of the technical challenges that need to be considered for successful dynamic AFM imaging studies of individual DNA-protein interactions. Necessary technical improvements to the AFM set-up and the development of new sample preparation methods are described in this paper.     

Spring constants and adhesive properties of native bacterial biofilm cells measured by atomic force microscopy

Bacterial biofilms were imaged by atomic force microscopy (AFM), and their elasticity and adhesion to the AFM tip were determined from a series of tip extension and retraction cycles. Though the five bacterial strains studied included both Gram-negative and -positive bacteria and both environmental and laboratory strains, all formed simple biofilms on glass surfaces. Cellular spring constants, determined from the extension portion of the force cycle, varied between 0.16+/-0.01 and 0.41+/-0.01 N/m, where larger spring constants were measured for Gram-positive cells than for Gram-negative cells. The nonlinear regime in the extension curve depended upon the biomolecules on the cell surface: the extension curves for the smooth Gram-negative bacterial strains with the longest lipopolysaccharides on their surface had a larger nonlinear region than the rough bacterial strain with shorter lipopolysaccharides on the surface. Adhesive forces between the retracting silicon nitride tip and the cells varied between cell types in terms of the force components, the distance components, and the number of adhesion events. The Gram-negative cells' adhesion to the tip showed the longest distance components, sometimes more than 1 microm, whereas the shortest distance adhesion events were measured between the two Gram-positive cell types and the tip. Fixation of free-swimming planktonic cells by NHS and EDC perturbed both the elasticity and the adhesive properties of the cells. Here we consider the biochemical meaning of the measured physical properties of simple biofilms and implications to the colonization of surfaces in the first stages of biofilm formation.     

Kinetic study on the elastic change of vascular endothelial cells on collagen matrices by atomic force microscopy

The elasticity of vascular endothelial cells (HUVEC) was measured with an atomic force microscopy (AFM, Olympus), and analyzed by applying the Hertz model, and those data were compared with ones reported by us previously. The latter elasticity data were measured with AFM Instruments (Seiko) on the basis of the Young's modulus of gelatin gel, which was obtained from the measurement with a tensile tester. The elasticity of HUVEC was concluded to depend on the culture period. The elasticity of the cells cultured on type IV collagen for longer than 4 days led to average elasticity values higher than ca. 10(4)Pa. Moreover, the scattered values of elasticity decreased eminently in the AFM measurement of the cells at room temperature. A few cells, however, appeared to adhere long and tensely on matrix, which seems to one of reasons for relatively high elasticity in our previous works. The possibility of such high elasticity was considered to change in cell adhesion, anchoring on matrix during a long culture period.     

Changes in the surface structure of purple membrane upon illumination measured by atomic force microscopy

Bacteriorhodopsin (BR) patches with a diameter of 1 to 3 μm were investigated in their native state by atomic force microscopy (AFM) in buffer solution. The patches were immobilized deposited and investigated on mica in 150 mM KCl and 10 mM Tris-buffer at pH 8. Under this buffer condition they adsorb preferred with their extracellular side to the solid support mica. The structure of the two-dimensional light adapted crystals was resolved with an imaging force of about 100 pN up to a resolution of 13 Å. The topography of the surface gets smoother if an imaging force of 1000 pN was applied indicating that protruding structures are compressed. Upon illumination with white light, during imaging with a force of 200 pN, the surface structure of the BR lattice changed. The force- and light-induced structural changes were reversible.     

Quantification of particle-bubble interactions using atomic force microscopy: A review

The attachment of particles to bubbles in solution is of fundamental importance to several industrial processes, most notably in the process of froth flotation. During this process hydrophobic particles attach to air bubbles in solution, which allows them to be separated as froth at the surface. The addition of chemicals can help to modulate these interactions to increase the yield of the minerals of interest. Over the past decade the atomic force microscope (AFM) has been adapted for use in studying the forces involved in the attachment of single particles to bubbles in the laboratory. This allows the measurement of actual DLVO (Derjaguin, Landau, Vervey and Overbeek) forces and adhesive contacts to be measured under different conditions. In addition contact angles may be calculated from features of force versus distance curves. It is the purpose of this article to illustrate how the colloid probe technique can be used to make single particle-bubble interactions and to summarise the current literature describing such experiments.     

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.     

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.