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PubMed2005

Probing mechanical properties of living cells by atomic force microscopy with blunted pyramidal cantilever tips.

Atomic force microscopy (AFM) allows the acquisition of high-resolution images and the measurement of mechanical properties of living cells under physiological conditions. AFM cantilevers with blunted pyramidal tips are commonly used to obtain images of living cells. Measurement of mechanical properties with these tips requires a contact model that takes into account their blunted geometry. The aim of this work was to develop a contact model of a blunted pyramidal tip and to assess the suitability of pyramidal tips for probing mechanical properties of soft gels and living cells. We developed a contact model of a blunted pyramidal tip indenting an elastic half-space. We measured Young's modulus (E) and the complex shear modulus (G*= G' +i G" ) of agarose gels and A549 alveolar epithelial cells with pyramidal tips and compared them with those obtained with spherical tips. The gels exhibited an elastic behavior with almost coincident loading and unloading force curves and negligible values of G". E fell sharply with indentation up to approximately 300 nm , showing a linear regime for deeper indentations. A similar indentation dependence of E with twofold lower values at the linear regime was obtained with the spherical tip fitted with Hertz's model. The dependence of E on indentation in cells paralleled that found in gels. Cells exhibited viscoelastic behavior with G"/G' approximately 1/4 . Pyramidal tips commonly used for AFM imaging are suitable for probing mechanical properties of soft gels and living cells.

Biomechanical Phenomena↗
PubMed1999

Analysis of indentation: implications for measuring mechanical properties with atomic force microscopy.

Indentation using the atomic force microscope (AFM) has potential to measure detailed micromechanical properties of soft biological samples. However, interpretation of the results is complicated by the tapered shape of the AFM probe tip, and its small size relative to the depth of indentation. Finite element models (FEMs) were used to examine effects of indentation depth, tip geometry, and material nonlinearity and heterogeneity on the finite indentation response. Widely applied infinitesimal strain models agreed with FEM results for linear elastic materials, but yielded substantial errors in the estimated properties for nonlinear elastic materials. By accounting for the indenter geometry to compute an apparent elastic modulus as a function of indentation depth, nonlinearity and heterogeneity of material properties may be identified. Furthermore, combined finite indentation and biaxial stretch may reveal the specific functional form of the constitutive law--a requirement for quantitative estimates of material constants to be extracted from AFM indentation data.

Biomechanical Phenomena↗
PubMed2000

Imaging microtubules and kinesin decorated microtubules using tapping mode atomic force microscopy in fluids.

The atomic force microscope has been used to investigate microtubules and kinesin decorated microtubules in aqueous solution adsorbed onto a solid substrate. The netto negatively charged microtubules did not adsorb to negatively charged solid surfaces but to glass covalently coated with the highly positively charged silane trimethoxysilylpropyldiethylenetriamine (DETA) or a lipid bilayer of 1,2-dipalmitoyl-3-dimethylammoniumpropane. Using electron beam deposited tips for microtubules adsorbed on DETA, single protofilaments could be observed showing that the resolution is up to 5 nm. Under conditions where the silane coated surfaces are hydrophobic, microtubules opened, presumably at the seam, whose stability is lower than that of the bonds between the other protofilaments. This led to a "sheet" with a width of about 100 nm firmly attached to the surface. Microtubules decorated with a stoichiometric low amount of kinesin molecules in the presence of the non-hydrolyzable ATP-analog 5'-adenylylimidodiphosphate could also be adsorbed onto silane-coated glass. Imaging was very stable and the molecules did not show any scan-induced deformation even after hundreds of scans with a scan frequency of 100 Hz.

Animals↗
PubMed2000

Plasmodium falciparum-infected erythrocytes: qualitative and quantitative analyses of parasite-induced knobs by atomic force microscopy.

We used the combination of an atomic force microscope and a light microscope equipped with epifluorescence to serially image Plasmodium falciparum-infected erythrocytes. This procedure allowed us to determine unambiguously the presence and developmental stage of the malaria parasite as well as the number and size of knobs in singly, doubly, and triply infected erythrocytes. Knobs are not present during the ring stage of a malaria infection but a lesion resulting from invasion by a merozoite is clearly visible on the erythrocyte surface. This lesion is visible into the late trophozoite stage of infection. Knobs begin to form during the early trophozoite stage of infection and have a single-unit structure. Our data suggest the possibility that a two-unit structure of knobs, which was reported by Aikawa et al. (1996, Exp. Parasitol. 84, 339-343) using atomic force microscopy, appears to be a double-tipped image. The number of knobs per unit of host cell surface area is directly proportional to parasite number in both early and late trophozoite stages. These results indicate that knob formation by one parasite does not influence knob formation by other parasites in a multiply infected erythrocyte. In addition, knob volume is not influenced by either parasite stage or number at the late trophozoite stage, indicating that the number of component molecules per knob is constant throughout the parasite maturation process.

Animals↗
PubMed1996

Biological cryo atomic force microscopy: a brief review.

Despite many successes, atomic force microscopy (AFM) of biological specimens at room temperature is still severely limited by at least two factors: the softness and the thermal motion of flexible multi-domain/subunit molecules. Both problems can be overcome by imaging biological structures at cryogenic temperatures. Even though the instrumentation is considerably more complex and earlier attempts were largely unsuccessful, cryo-AFM has recently been demonstrated on a number of biological specimens, using an AFM operated in liquid nitrogen vapor under ambient pressure. In this brief review, both the method of instrumentation and the latest biological applications are discussed. Not only has the cryo-AFM attained high resolution on those specimens that could not be well imaged at room temperature, but it has also produced potentially important information on several specimens. These results firmly establish the cryo-AFM as a useful and versatile structural probe in biology with its own unique capabilities.

Animals↗
PubMed2006

Nuclear pore complex structure and plasticity revealed by electron and atomic force microscopy.

To study the ultrastructure of nuclear pore complexes (NPCs), a wide spectrum of different electron microscopy (EM) or atomic force microscopy (AFM) techniques can be employed. The combination of these methods can reveal new insights into the structural and functional organization of this important supramolecular machine through which nucleocytoplasmic transport occurs. Negative staining, quick freezing/freeze-drying/rotary metal shadowing, embedding and thin sectioning, cryoelectron microscopy and tomography, scanning electron microscopy, or combination with immunolabeling techniques are tools for collecting data and information about the three-dimensional structure and architecture of the NPCs. AFM enables investigation of the functional dynamics of native NPCs under physiological conditions.

Animals↗
PubMed2006

Pore size characterization of monolith for electrochromatography via atomic force microscopy studies in air and liquid phase.

This paper investigates the use of scanning electron microscopy (SEM) and atomic force microscopy (AFM) for the characterization of monoliths used in capillary electrochromatography (CEC) while focusing on the nature of the information available from both techniques. SEM imaging revealed a compact structure of non-porous micrometer sized particles homogeneously agglomerated. With a simple AFM methodology, we found by direct observation that the same material exhibits mesopores in the nanometer range while SEM showed non-porous surfaces. These results obtained by AFM clearly showed that micrometer sized particles shrank and micrometer sized pores increased in the monolith when wetted. Thus, AFM was capable of demonstrating the morphological differences between wet and dried monolithic materials that are not possible by other imaging methods at micrometer resolution.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2004

Topographic effects on adhesive force mapping of stretched DNA molecules by pulsed-force-mode atomic force microscopy.

Adhesive interaction between a tip and a sample surface was examined on a microscopic scale by pulsed-force-mode atomic force microscopy (PFM-AFM). The signal measured by monitoring pull-off force is influenced by various factors such as topography, elasticity, electrostatic charges, and adsorbed water on surfaces. Here, we focus on the topographic effects on the adhesive interaction. To clarify the topographic influence, the adhesive force measurement of a stretched DNA molecule with a smaller radius of curvature than that of a tip was carried out at low relative humidity (RH) with an alkanethiol-modified tip. The experimental conditions such as low RH and the use of the alkanethiol-modified tip were required to minimise the influence of water capillary force on hydrated DNA strands. The hydrophobic modification of a substrate surface was also important to minimise the adsorbed water effect. The DNA molecules were stretched on the substrate surfaces by an immobilisation process called a dynamic molecular combing method. The two-component vapour-phase surface modification with an alkylsilane mixed with a silane derivative containing an amino end group enhanced the DNA adsorption due to the electrostatic interaction. The experimental results for the topographic effects on the adhesive force mapping were reproducible.

Adhesiveness↗
PubMed2002

Investigating live and fixed epithelial and fibroblast cells by atomic force microscopy.

PURPOSE: To investigate the use of atomic force microscopy (AFM) to image live and fixed cell in culture. Rabbit corneal fibroblasts, Chang conjunctival cells, and transformed human corneal epithelial cells were chosen so that AFM parameters could be set for future use in toxicologic and pharmacologic studies of ocular cells. METHODS: Contact mode AFM was performed under air and in balanced salt solution (BSS) using live and fixed cells. All cell lines were imaged in the height mode for optimal resolution of cellular features. RESULTS: Images of fixed cells showed no discernible differences in surface features when visualized in air or under physiologic solution. Structural differences were observed, however, between fixed and live cells in BSS. Although the AFM technique provides high quality images of live cells under BSS, sub-membrane features of live cells are more well-defined compared to fixed cells. It was also possible to image live cells in air if imaging was completed within 10 minutes of removal of the cells from culture medium. Images of cytoskeletal features under air were similar to those obtained under BSS. CONCLUSIONS: The atomic force microscopy technique can be used to study cells and provide sub-cellular details at resolution equal to or in some situations better than the scanning electron microscopy technique. However, parameters for imaging have to be tailored for individual experimental goals.

Acetates↗
PubMed2002

Investigating live and fixed epithelial and fibroblast cells by atomic force microscopy.

PURPOSE: To investigate the use of atomic force microscopy (AFM) to image live and fixed cell in culture. Rabbit corneal fibroblasts, Chang conjunctival cells, and transformed human corneal epithelial cells were chosen so that AFM parameters could be set for future use in toxicologic and pharmacologic studies of ocular cells. METHODS: Contact mode AFM was performed under air and in balanced salt solution (BSS) using live and fixed cells. All cell lines were imaged in the height mode for optimal resolution of cellular features. RESULTS: Images of fixed cells showed no discernible differences in surface features when visualized in air or under physiologic solution. Structural differences were observed, however, between fixed and live cells in BSS. Although the AFM technique provides high quality images of live cells under BSS, sub-membrane features of live cells are more well-defined compared to fixed cells. It was also possible to image live cells in air if imaging was completed within 10 minutes of removal of the cells from culture medium. Images of cytoskeletal features under air were similar to those obtained under BSS. CONCLUSION: The atomic force microscopy technique can be used to study cells and provide sub-cellular details at resolution equal to or in some situations better than the scanning electron microscopy technique. However, parameters for imaging have to be tailored for individual experimental goals.

Acetates↗
PubMed2005

Atomic force microscopy measurement of the elastic properties of the kidney epithelial cells.

Direct interaction force measurements using atomic force microscopy (AFM) were carried out between a silicon nitride tip and renal epithelial cells (Madin-Darby Canine Kidney-MDCK and proximal tubular epithelial cells derived from pig kidneys, LLC-PK1). The approaching (extending) portion of the force/distance curves is considered, and repulsive forces in the long range of 2-3 microm were seen in both MDCK as well as LLC-PK1 cells growing under normal conditions. The repulsive force in the shorter distance range of 50-200 nm was also observed, when cells were damaged exposing the underlying basal membrane. LLC-PK1 cells were more prone to damage than the MDCK cells, hence short-range forces were common in the former and long-range forces in the latter cells. The functional dependence of repulsive force on the indentation depth changes, at small indentation depth the force increases linearly, while at larger indentations the force is a quadratic function of the distance, which is attributed to the elasticity of the membrane and the solid-like response of cells, respectively. The oxalate treatment of cells for 2-4 h gives rise to an increase in the elastic modulus of the cells.

Animals↗
PubMedDate not supplied

Force measurement for antigen-antibody interaction by atomic force microscopy using a photograft-polymer spacer.

To determine the intermolecular force on protein-protein interaction (PPI) by atomic force microscopy (AFM), a photograft-polymer spacer for protein molecules on both surfaces of the substrate and AFM probe tip was developed, and its effectiveness was assessed in a PPI model of a pair of human serum albumin (HSA) and its monoclonal antibody (anti-HSA). A carboxylated photoiniferter, N-(dithiocarboxy)sarcosine, was derivatized on both surfaces of the glass substrate and AFM probe tip, and subsequently water-soluble nonionic vinyl monomers, N,N-dimethylacrylamide (DMAAm), were graft-polymerized on them upon ultraviolet light irradiation. DMAAm-photograft-polymerized spacers with carboxyl groups at the growing chain end but with different chain lengths on both surfaces were prepared. The proteins were covalently bound to the carboxyl terminus of the photograft-polymer chain using a water-soluble condensation agent. The effects of the graft-spacer length on the profile of the force-distance curves and on the unbinding characteristics (unbinding force and unbinding distance) were examined in comparison with those in the case of the commercially available poly(ethylene glycol) (PEG) spacer. The frequency of the nonspecific adhesion force profile was markedly decreased with the use of the photograft spacers. Among the force curves detected, a high frequency of single-peak curves indicating the unbinding process of a single pair of proteins and a very low frequency of multiple-peak profiles were observed for the photograft spacers, regardless of the graft chain length, whereas a high frequency of no-force peaks was noted. These observations were in marked contrast with those for the PEG spacer. The force peak values determined ranged from 88 to 94 pN, irrespective of the type of spacer, while the standard deviation of force distribution observed for the photograft spacer was lower than that for the PEG spacer, indicating that the photograft spacers provide a higher accuracy of force determination.

Adhesiveness↗
PubMed2006

Direct force measurement of the interaction between liposome and the C2A domain of synaptotagmin I using atomic force microscopy.

The binding force between a liposome and the C2A domain of synaptotagmin I was determined by an atomic force microscopy (AFM). Liposomes were immobilized on the surface of the L1 sensor chip and the C2A domains, which recognize phosphatidylserine, were chemically conjugated onto a gold-coated cantilever tip. The average interaction force between the C2A domain and the liposome was 306 (+/-57) pN while the force between untreated cantilever and the liposome was 58 (+/-16) pN. This work helps understand the physicochemical interactions between proteins and lipid vesicles for the design of high affinity protein probes against the apoptotic cell surface.

Binding Sites↗
PubMed1999

Graphical method for force analysis: macromolecular mechanics with atomic force microscopy.

We present a graphical method for a unifying, quantitative analysis of molecular bonding-force measurements by atomic force microscopy (AFM). The method is applied to interpreting a range of phenomena commonly observed in the experimental AFM measurements of noncovalent, weak bonds between biological macromolecules. The analysis suggests an energy landscape underlying the intermolecular force and demonstrates that many observations, such as "snaps-on," "jumps-off," and hysteresis loops, are different manifestations of a double-well energy landscape. The analysis gives concrete definitions for the operationally defined "attractive" and "adhesive" forces in terms of molecular parameters. It is shown that these operationally defined quantities are usually functions of the experimental setup, such as the stiffness of the force probe and the rate of its movement. The analysis reveals a mechanical instability due to the multistate nature of molecular interactions and provides new insight into macromolecular viscosity. The graphical method can equally be applied to a quantitative analysis of multiple unfolding of subunits of the giant muscle protein titin under AFM.

Connectin↗
PubMed1996

Atomic Force Microscopy of Interfacial Protein Films

Atomic force microscopy (AFM) has been used to image interfacial films of bovine serum albumen and beta-casein produced at hexadecane/water and air/water interfaces, respectively. Planar oil/water and air/water interfaces have been used to model protein films such as may form in emulsions and foams. The protein films were picked up onto mica sheets and imaged under butanol. Both systems studied yielded homogeneous flat networks which could be imaged at molecular resolution and which demonstrate the potential for using AFM to probe interfacial networks.

Journal Article↗
PubMed2002

Atomic force microscopy and magnetic force microscopy study of model colloids.

Atomic force microscopy (AFM) is used to study the size, shape, and polydispersity of a variety of magnetic and nonmagnetic model colloids, previously imaged by transmission electron microscopy (TEM) only. Both height and phase images are analyzed and special attention is given to 3D morphology and softness of particles, as well as structures and presence of secondary components in the colloid, difficult to investigate with TEM. Several methods of tip characterization followed by deconvolution were applied in order to improve the accuracy of lateral diameter determination. In the case of magnetite particles dispersed in conventional ferrofluids, we explore both experimentally and theoretically the possibility of using magnetic force microscopy (MFM). We propose and discuss several models which allow to estimate the magnetic moment of a single domain superparamagnetic sphere using MFM, which cannot be done with other techniques; alternatively the tip magnetization can be determined.

Journal Article↗
PubMed2002

Monitoring high-temperature solid-solid phase transitions of HMX with atomic force microscopy.

Using high-temperature atomic force microscopy we have observed the solid-solid phase transition of octahydro-1,3,5,7,-tetrazocine (HMX) in real time. Massive surface reconstruction occurs during the first-order transition. The temperature induced increase in void space and surface roughness observed in the delta polymorph of HMX serve to increase the growth rate and volume of shock initiated hot spots and hence reaction sensitivity.

Journal Article↗