Robust strategies for automated AFM force curve analysis-II: adhesion-influenced indentation of soft, elastic materials

In the first of this two-part discourse on the extraction of elastic properties from atomic force microscopy (AFM) data, a scheme for automating the analysis of force-distance curves was introduced and experimentally validated for the Hertzian (i.e., linearly elastic and noninteractive probe-sample pairs) indentation of soft, inhomogeneous materials. In the presence of probe-sample adhesive interactions, which are common especially during retraction of the rigid tip from soft materials, the Hertzian models are no longer adequate. A number of theories (e.g., Johnson-Kendall-Roberts and Derjaguin-Muller-Toporov), covering the full range of sample compliance relative to adhesive force and tip radius, are available for analysis of such data. We incorporated Pietrement and Troyon's approximation (2000, "General Equations Describing Elastic Indentation Depth and Normal Contact Stiffness Versus Load," J. Colloid Interface Sci., 226(1), pp. 166-171) of the Maugis-Dugdale model into the automated procedure. The scheme developed for the processing of Hertzian data was extended to allow for adhesive contact by applying the Pietrement-Troyon equation. Retraction force-displacement data from the indentation of polyvinyl alcohol gels were processed using the customized software. Many of the retraction curves exhibited strong adhesive interactions that were absent in extension. We compared the values of Young's modulus extracted from the retraction data to the values obtained from the extension data and from macroscopic uniaxial compression tests. Application of adhesive contact models and the automated scheme to the retraction curves yielded average values of Young's modulus close to those obtained with Hertzian models for the extension curves. The Pietrement-Troyon equation provided a good fit to the data as indicated by small values of the mean-square error. The Maugis-Dugdale theory is capable of accurately modeling adhesive contact between a rigid spherical indenter and a soft, elastic sample. Pietrement and Troyon's empirical equation greatly simplifies the theory and renders it compatible with the general automation strategies that we developed for Hertzian analysis. Our comprehensive algorithm for automated extraction of Young's moduli from AFM indentation data has been expanded to recognize the presence of either adhesive or Hertzian behavior and apply the appropriate contact model.     

Nano-mechanical exploration of the surface and sub-surface of hydrated cells of Staphylococcus epidermidis

The surface of hydrated cells of Staphylococcus epidermidis has been probed using an atomic force microscope. While local force measurements over the surface of bacteria reveal a heterogeneous chemical surface, with heterogeneous mechanical properties, different kinds of force curves appear with high frequency, and are thought to provide information on features contributing strongly to the overall mechanical and surface behaviour of the cell. Force curves often present two different mechanical regimes, being the first one (outer) of about 48 nm thick, and presenting a local relative elasticity of about 0.08 N/m, which is about a third of the relative elasticity of the inner part of the cell wall, harder, with a relative elasticity of about 0.24 N/m, in water. Both regimes appears as straight lines in the force versus distance curves (the ‘corresponding’ stress–strain curves in contact mechanics), but hysteresis is observed between the approach and the retraction line in the inner regime, indicating a degree of viscoelasticity. No viscoelasticity is observed in the outer regime, however, which presents quite linear and juxtaposed approach-retraction lines. These kinds of force curves do not present measurable pull-off forces nor snap-in forces, which indicates an almost null interaction between tip and bacterial surface, which could be in agreement with the measured very high hydrophobicity of this strain. Another kind of force curve has been observed recurrently, showing peaks in the retraction curves. Adhesive pull-off forces were measured giving an average of about 2 nN. Interestingly, however, these force curves appear only when quite irregular and wavy retraction curves are present, from the very beginning of its trace (maximum indentation). This leads us to think that these pull-off forces measured by our AFM do not give information on surface forces-unbinding events at the surface of the bacteria, but could be related to events at the sub-surface of the cell surface. Oscillations seen in the retraction curve in the portion corresponding to the contact with the bacteria surface could be due to rupture phenomena within the multilayered cell wall architecture expected in Gram-positive bacteria as Staphylococcus epidermidis, which could result in local irreversible deformations of the cell surface. Imaging with a sharp tip in contact mode sometimes leads to surface damage. Force curves recorded over damaged parts of the cell surface showed a completely different behaviour, in many cases with two well-defined high-adhesion peaks, and also interestingly, with snap-in forces of about 0–2 nN, which seems to indicate a completely different electrical/hydrophobicity state only a few nanometers down from the surface. Similar indentation effects can occur in the contact of a bacterial cell with a solid surface, even when showing only atomic-molecular-scale roughness, thus interacting not only with the very surface of the cell, especially when soft layers are present in the outer. Our results highlight the importance of the cell surface mechanical properties and their interplay with purely surface properties when analyzing cell–material interaction, and show the AFM as a useful method for investigating this.     

Atomic force microscopy-based force spectroscopy--biological and biomedical applications

The use of atomic force microscopy (AFM) applied to biological systems to generate high resolution images is gaining a wider acceptance. However, the most remarkable advances are being achieved on the use of the AFM to measure inter- and intramolecular interaction forces with piconewton resolution, not only to demonstrate this ability but also actually to solve biological and biomedical relevant questions. Single-molecule force spectroscopy recognition studies enable the detection of specific interaction forces, based on the AFM sensitivity and the possibility of manipulating individual molecules. In this review, we describe the basic principles of this methodology and some of the practical aspects involved. The ability to measure interactions at the single-molecule level is illustrated by some relevant examples. A special focus is given to the study of the fibrinogen-erythrocyte binding and its relevance as a cardiovascular risk factor. An approach to the latter problem by single-molecule force spectroscopy allowed the molecular recognition, characterization, and partial identification of a previously unknown receptor for fibrinogen on human erythrocytes.     

Probing the interaction forces between hydrophobic peptides and supported lipid bilayers using AFM

Despite the vast body of literature that has accumulated on tilted peptides in the past decade, direct information on the forces that drive their interaction with lipid membranes is lacking. Here, we attempted to use atomic force microscopy (AFM) to explore the interaction forces between the Simian immunodeficiency virus peptide and phase-separated supported bilayers composed of various lipids, i.e. dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, dioleoylphosphatidic acid and dipalmitoylphosphatidylethanolamine. Histidine-tagged peptides were attached onto AFM tips terminated with nitrilotriacetate and tri(ethylene glycol) groups, an approach expected to ensure optimal exposure of the C-terminal hydrophobic domain. Force-distance curves recorded between peptide-tips and the different bilayer domains always showed a long-range repulsion upon approach and a lack of adhesion upon retraction, in marked contrast with the hydrophobic nature of the peptide. To explain this unexpected behaviour, we suggest a mechanism in which lipids are pulled out from the bilayer due to strong interactions with the peptide-tip, in agreement with the very low force needed to extract lipids from supported bilayers.     

An overview of the biophysical applications of atomic force microscopy

The potentialities of the atomic force microscopy (AFM) make it a tool of undeniable value for the study of biologically relevant samples. AFM is progressively becoming a usual benchtop technique. In average, more than one paper is published every day on AFM biological applications. This figure overcomes materials science applications, showing that 17 years after its invention, AFM has completely crossed the limits of its traditional areas of application. Its potential to image the structure of biomolecules or bio-surfaces with molecular or even sub-molecular resolution, study samples under physiological conditions (which allows to follow in situ the real time dynamics of some biological events), measure local chemical, physical and mechanical properties of a sample and manipulate single molecules should be emphasized.     

Atomic force microscopy-based molecular studies on the recognition of immunogenic chlorinated ovalbumin by macrophage receptors

This report presents simple and reliable approach developed to study the specific recognition events between chlorinated ovalbumin (OVA) and macrophages using atomic force microscopy (AFM). Thanks to the elimination of nonspecific adhesion, the interactions of the native and chlorinated OVA with a membrane of macrophages could be quantified using exclusively the so-called adhesion frequency (AF). The proposed system not only enabled the application of AFM-based force measurements for such poorly defined ligand-receptor pairs but also significantly improved both the acquisition and the processing of the data. The proteins were immobilized on the gold-coated AFM tips from the aqueous solutions containing charged thiol adsorbates. Such surface dilution of the proteins ensured the presence of single or just a few macromolecules at the tip-surface contact. The formation of negatively charged monolayer on the tip dramatically limited its nonspecific interactions with the macrophage surface. In such systems, AF was used as a measure of the recognition events even if the interaction forces varied significantly for sets of measurements. The system with the native OVA, a weak immunogen, showed only negligible AF compared with 85% measured for the immunogenic chlorinated OVA. The AF values varied with the tip-macrophage contact time and loading velocity. Blocking of the receptors by the chlorinated OVA was also confirmed. The developed approach can be also used to study other ligand-receptor interactions in poorly defined biological systems with intrinsically broad distribution of the rupture forces, thus opening new fields for AFM-based recognition on molecular level.     

Analysis of DNA interactions using single-molecule force spectroscopy

Protein–DNA interactions are involved in many biochemical pathways and determine the fate of the corresponding cell. Qualitative and quantitative investigations on these recognition and binding processes are of key importance for an improved understanding of biochemical processes and also for systems biology. This review article focusses on atomic force microscopy (AFM)-based single-molecule force spectroscopy and its application to the quantification of forces and binding mechanisms that lead to the formation of protein–DNA complexes. AFM and dynamic force spectroscopy are exciting tools that allow for quantitative analysis of biomolecular interactions. Besides an overview on the method and the most important immobilization approaches, the physical basics of the data evaluation is described. Recent applications of AFM-based force spectroscopy to investigate DNA intercalation, complexes involving DNA aptamers and peptide– and protein–DNA interactions are given.     

肌动蛋白的原子力显微镜研究

原子力显微镜 (AFM )是一种能够在生理条件下对生物大分子、活细胞表面以及细胞膜下结构进行在体或离体研究的强有力的新型工具 ,具有原子级的成像分辨率和纳牛顿级的力测定功能。目前原子力显微镜已被广泛地应用于生物大分子、超分子体系的结构解析、动力学过程观察 ,分子力学研究及细胞功能鉴定。原子力显微镜能够通过尖锐探针扫描待测样品表面 ,收集被测样品表面地貌坐标数据从而对单分子或细胞进行成像或操作 ,并能通过移动探针、记录探针与样品之间的作用力 ,对生物大分子 (蛋白质、核酸和多糖等 )的结构力学特性进行分析以获取分子构象、功能及其相互关系的有用信息。肌动蛋白是一种细胞内普遍存在 ,具有广泛、复杂生理功能的重要蛋白质 ,原子力显微镜的各项功能已广泛地用于肌动蛋白结构、功能及动力学研究。通过综述原子力显微镜在肌动蛋白研究中的应用 ,阐明了原子力显微镜在现代生命科学研究中的重要意义及巨大应用前景。     

Relative microelastic mapping of living cells by atomic force microscopy.

The spatial and temporal changes of the mechanical properties of living cells reflect complex underlying physiological processes. Following these changes should provide valuable insight into the biological importance of cellular mechanics and their regulation. The tip of an atomic force microscope (AFM) can be used to indent soft samples, and the force versus indentation measurement provides information about the local viscoelasticity. By collecting force-distance curves on a time scale where viscous contributions are small, the forces measured are dominated by the elastic properties of the sample. We have developed an experimental approach, using atomic force microscopy, called force integration to equal limits (FIEL) mapping, to produce robust, internally quantitative maps of relative elasticity. FIEL mapping has the advantage of essentially being independent of the tip-sample contact point and the cantilever spring constant. FIEL maps of living Madine-Darby canine kidney (MDCK) cells show that elasticity is uncoupled from topography and reveal a number of unexpected features. These results present a mode of high-resolution visualization in which the contrast is based on the mechanical properties of the sample.     

Staphylococcus aureus-Fibronectin Interactions with and without Fibronectin-Binding Proteins and Their Role in Adhesion and Desorption

Adhesion and residence-time-dependent desorption of two Staphylococcus aureus strains with and without fibronectin (Fn) binding proteins (FnBPs) on Fn-coated glass were compared under flow conditions. To obtain a better understanding of the role of Fn-FnBP binding, the adsorption enthalpies of Fn with staphylococcal cell surfaces were determined using isothermal titration calorimetry (ITC). Interaction forces between staphylococci and Fn coatings were measured using atomic force microscopy (AFM). The strain with FnBPs adhered faster and initially stronger to an Fn coating than the strain without FnBPs, and its Fn adsorption enthalpies were higher. The initial desorption was high for both strains but decreased substantially within 2 s. These time scales of staphylococcal bond ageing were confirmed by AFM adhesion force measurement. After exposure of either Fn coating or staphylococcal cell surfaces to bovine serum albumin (BSA), the adhesion of both strains to Fn coatings was reduced, suggesting that BSA suppresses not only nonspecific but also specific Fn-FnBP interactions. Adhesion forces and adsorption enthalpies were only slightly affected by BSA adsorption. This implies that under the mild contact conditions of convective diffusion in a flow chamber, adsorbed BSA prevents specific interactions but does allow forced Fn-FnBP binding during AFM or stirring in ITC. The bond strength energies calculated from retraction force-distance curves from AFM were orders of magnitude higher than those calculated from desorption data, confirming that a penetrating Fn-coated AFM tip probes multiple adhesins in the outermost cell surface that remain hidden during mild landing of an organism on an Fn-coated substratum, like that during convective diffusional flow.     

Disulfide bonds in the outer layer of keratin fibers confer higher mechanical rigidity: correlative nano-indentation and elasticity measurement with an AFM.

Nanomechanical properties of biological fibers are governed by the morphological features and chemically heterogeneous constituent subunits. However, very little experimental data exist for nanoscale correlation between heterogeneous subunits and their mechanical properties. We have used keratin-rich wool fibers as a model of composite biological fibers; a wool fiber is a simple two component cylindrical system consisting of a core cellular component surrounded by an outer cell layer and their ultrastructure and chemical composition are well-characterized. The core is 16-40 micrometer in diameter and rich in axially aligned keratin microfibrils. Outer cells have multiple laminar layers, 60-600 nm thick and distinctly rich in disulfide bonds. We used an atomic force microscope (AFM) to examine the nanomechanical properties of various structural components using complementary techniques of force-volume imaging and nano-indentation. AFM images of transverse sections of fibers were obtained in ambient environment, and the mechanical properties of several identified regions were examined. The outer cell layer showed a significantly higher mechanical stiffness than the internal cellular core region. Chemical reduction of disulfide bonds eliminated such dichotomy of mechanical strengths, indicating that the higher rigidity of the outer layer is attributed primarily to the presence of extensive disulfide bonding in the exo-cuticle. This is the first detailed correlative study of nano-indentation and regional elasticity measurements in composite biological systems, including mammalian biological fibers.     

Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or “tapping mode” AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming.PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials.The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials.     

Nanomechanical recognition of sphingomyelin-rich membrane domains by atomic force microscopy

Sphingomyelin (SM) is a reservoir of signaling lipids and forms specific lipid domains in biomembranes together with cholesterol. In this study, atomic force microscopy (AFM) and force measurement were applied to investigate the interaction of SM-binding protein toxin, lysenin, with N-palmitoyl-D-erythro-sphingosylphosphorylcholine (palmitoyl sphingomyelin, PSM) bilayer spread over a mica substrate, in an aqueous buffer solution. Lysenin molecules were grafted on a silicon nitride tip for AFM by siloxane-thiol-amide coupling. The bilayers were prepared by the Langmuir-Blodgett (LB)/Langmuir-Schaefer (LS) method. By repeating cycles of tip approach/retraction motion, single-molecular adhesion motions were observed on the force curve, characterized as "fishing curves". The addition of cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) did not alter the peak force but increased the peak extension. Mixtures of PSM/DOPC/cholesterol exhibited 2-dimensional two-phase domain separation. The characteristic fishing curves were observed exclusively in one of the phases, indicating the selective interaction of the lysenin tip to PSM-rich membrane domains. Our results indicate that the AFM tips conjugated with lysenin are useful to detect the surface distribution of SM-rich membrane domains as well as the nanomechanical properties of the domains.     

Frequency modulation atomic force microscopy reveals individual intermediates associated with each unfolded I27 titin domain

In this study, we apply a dynamic atomic force microscopy (AFM) technique, frequency modulation (FM) detection, to the mechanical unfolding of single titin I27 domains and make comparisons with measurements made using the AFM contact or static mode method. Static mode measurements revealed the well-known force transition occurring at 100-120 pN in the first unfolding peak, which was less clear, or more often absent, in the subsequent unfolding peaks. In contrast, some FM-AFM curves clearly resolved a force transition associated with each of the unfolding peaks irrespective of the number of observed unfolded domains. As expected for FM-AFM, the frequency shift response of the main unfolding peaks and their intermediates could only be detected when the oscillation amplitudes used were smaller than the interaction lengths being measured. It was also shown that the forces measured for the dynamical interaction of the FM-AFM technique were significantly lower than those measured using the static mode. This study highlights the potential for using dynamic AFM for investigating biological interactions, including protein unfolding and the detection of novel unfolding intermediates.     

Indentation and adhesive probing of a cell membrane with AFM: theoretical model and experiments

In probing adhesion and cell mechanics by atomic force microscopy (AFM), the mechanical properties of the membrane have an important if neglected role. Here we theoretically model the contact of an AFM tip with a cell membrane, where direct motivation and data are derived from a prototypical ligand-receptor adhesion experiment. An AFM tip is functionalized with a prototypical ligand, SIRPalpha, and then used to probe its native receptor on red cells, CD47. The interactions prove specific and typical in force, and also show in detachment, a sawtooth-shaped disruption process that can extend over hundreds of nm. The theoretical model here that accounts for both membrane indentation as well as membrane extension in tip retraction incorporates membrane tension and elasticity as well as AFM tip geometry and stochastic disruption. Importantly, indentation depth proves initially proportional to membrane tension and does not follow the standard Hertz model. Computations of detachment confirm nonperiodic disruption with membrane extensions of hundreds of nm set by membrane tension. Membrane mechanical properties thus clearly influence AFM probing of cells, including single molecule adhesion experiments.     

Robust strategies for automated AFM force curve analysis--I. Non-adhesive indentation of soft, inhomogeneous materials

The atomic force microscope (AFM) has found wide applicability as a nanoindentation tool to measure local elastic properties of soft materials. An automated approach to the processing of AFM indentation data, namely, the extraction of Young's modulus, is essential to realizing the high-throughput potential of the instrument as an elasticity probe for typical soft materials that exhibit inhomogeneity at microscopic scales. This paper focuses on Hertzian analysis techniques, which are applicable to linear elastic indentation. We compiled a series of synergistic strategies into an algorithm that overcomes many of the complications that have previously impeded efforts to automate the fitting of contact mechanics models to indentation data. AFM raster data sets containing up to 1024 individual force-displacement curves and macroscopic compression data were obtained from testing polyvinyl alcohol gels of known composition. Local elastic properties of tissue-engineered cartilage were also measured by the AFM. All AFM data sets were processed using customized software based on the algorithm, and the extracted values of Young's modulus were compared to those obtained by macroscopic testing. Accuracy of the technique was verified by the good agreement between values of Young's modulus obtained by AFM and by direct compression of the synthetic gels. Validation of robustness was achieved by successfully fitting the vastly different types of force curves generated from the indentation of tissue-engineered cartilage. For AFM indentation data that are amenable to Hertzian analysis, the method presented here minimizes subjectivity in preprocessing and allows for improved consistency and minimized user intervention. Automated, large-scale analysis of indentation data holds tremendous potential in bioengineering applications, such as high-resolution elasticity mapping of natural and artificial tissues.     

Direct force measurements between cellulose surfaces and colloidal silica particles

The interaction of cellulose layers with colloidal silica particles was investigated by direct force measurements with the atomic force microscope (AFM). Upon approach, repulsive forces were found between the negatively charged silica particles and the cellulose surface. The forces were interpreted quantitatively in terms of electrostatic interactions due to overlap of diffuse layers originating from negatively charged carboxylic groups on the cellulose surface. The diffuse layer charge density of cellulose was estimated to be 0.80 mC/m2 at pH 9.5 and 0.21 mC/m2 at pH 4. The forces upon retraction are characterized by molecular adhesion events, whereby individual cellulose chains desorb from the probe surface. The retraction profiles are dominated by well-defined force plateaus, which correspond to single-chain desorption forces of 35-42 pN. We surmise that adsorption of cellulose to probe surfaces is dominated by nonelectrostatic forces, probably originating from hydrogen bonding. Electrostatic contributions to desorption force could be detected only at high pH, where the silica surface is highly charged.     

What makes bacterial pathogens so sticky?

Pathogenic bacteria use a variety of cell surface adhesins to promote binding to host tissues and protein-coated biomaterials, as well as cell–cell aggregation. These cellular interactions represent the first essential step that leads to host colonization and infection. Atomic force microscopy (AFM) has greatly contributed to increase our understanding of the specific interactions at play during microbial adhesion, down to the single-molecule level. A key asset of AFM is that adhesive interactions are studied under mechanical force, which is highly relevant as surface-attached pathogens are often exposed to physical stresses in the human body. These studies have identified sophisticated binding mechanisms in adhesins, which represent promising new targets for antiadhesion therapy.     

Atomic force microscopy measurements. Measurements of binding strength between a single pair of molecules in physiological solutions

Atomic force microscopy (AFM) measurements of intermolecular binding strength between a single pair of complementary cell adhesion molecules in physiological solutions provided the first quantitative evidence for their cohesive function. This novel AFM based nanobiotechnology opens a molecular mechanic approach for studying structure to function related properties of any type of individual biological macromolecules. The presented example of Porifera cell adhesion glyconectin proteoglycans showed that homotypic carbohydrate to carbohydrate interactions between two primordial proteogylycans can hold the weight of 1600 cells. Thus, glyconectin type carbohydrates, as the most peripheral cell surface molecules of sponges (today’s simplest living Metazoa), are proposed to the primary cell adhesive molecules essential for the evolution of the multicellularity.