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PubMed2004

Hydrodynamic effects in fast AFM single-molecule force measurements.

Atomic force microscopy (AFM) allows the critical forces that unfold single proteins and rupture individual receptor-ligand bonds to be measured. To derive the shape of the energy landscape, the dynamic strength of the system is probed at different force loading rates. This is usually achieved by varying the pulling speed between a few nm/s and a few microm/s, although for a more complete investigation of the kinetic properties higher speeds are desirable. Above 10 microm/s, the hydrodynamic drag force acting on the AFM cantilever reaches the same order of magnitude as the molecular forces. This has limited the maximum pulling speed in AFM single-molecule force spectroscopy experiments. Here, we present an approach for considering these hydrodynamic effects, thereby allowing a correct evaluation of AFM force measurements recorded over an extended range of pulling speeds (and thus loading rates). To support and illustrate our theoretical considerations, we experimentally evaluated the mechanical unfolding of a multi-domain protein recorded at 30 microm/s pulling speed.

Algorithms↗

PubMed2006

Influence of DNA condensation state on transfection efficiency in DNA/polymer complexes: an AFM and DLS comparative study.

Atomic force microscopy (AFM) is used to describe the formation process of polymer/DNA complexes. Two main objectives of this research are presented. The first one is to apply AFM as an effective tool to analyse DNA molecules and different polycation/DNA complexes in order to evaluate their degree of condensation (size and shape). The other one is to search for a relationship between the condensation state of DNA and its transfection efficiency. In this study, linear methacrylate based polymers and globular SuperFect polymers are used in order to induce DNA condensation. Ternary complexes, composed of methacrylate based polymers and polyethylene glycol (PEG)-based copolymers, are also investigated. AFM allows us to confirm good condensation conditions and relate them (or not) to transfection efficiencies. These AFM results (obtained after drying in air) are compared with measurements deduced from Dynamic Light Scattering (DLS) experiments performed in water. This comparison allowed us to identify the structural modifications resulting from deposition on the mica surface.

Animals↗

PubMed1999

Combined ultramicrotomy for AFM and TEM using a novel sample holder.

A new sample holder that allows combined microtomy for atomic force microscopy (AFM) and transmission electron microscopy (TEM) is described. The main feature of this sample holder is a small central part holding the sample. This central part fits into the head of an atomic force microscope. AFM measurements can be performed with a sample mounted in this central part of the sample holder. This makes the alignment of a microtomed bulk sample unnecessary, and offers the opportunity of an easy and fast combined sample preparation for AFM and TEM.

Microscopy, Atomic Force↗

PubMed2004

Elastic properties of single amylose chains in water: a quantum mechanical and AFM study.

Recent single-molecule atomic force microscopy (AFM) experiments have revealed that some polysaccharides display large deviations from force-extension relationships of other polymers which typically behave as simple entropic springs. However, the mechanism of these deviations has not been fully elucidated. Here we report the use of novel quantum mechanical methodologies, the divide-and-conquer linear scaling approach and the self-consistent charge density functional-based tight binding (SCC-DFTB) method, to unravel the mechanism of the extensibility of the polysaccharide amylose, which in water displays particularly large deviations from the simple entropic elasticity. We studied the deformations of maltose, a building block of amylose, both in a vacuum and in solution. To simulate the deformations in solution, the TIP3P molecular mechanical model is used to model the solvent water, and the SCC-DFTB method is used to model the solute. The interactions between the solute and water are treated by the combined quantum mechanical and molecular mechanical approach. We find that water significantly affects the mechanical properties of maltose. Furthermore, we performed two nanosecond-scale steered molecular dynamics simulations for single amylose chains composed of 10 glucopyranose rings in solution. Our SCC-DFTB/MM simulations reproduce the experimentally measured force-extension curve, and we find that the force-induced chair-to-boat transitions of glucopyranose rings are responsible for the characteristic plateau in the force-extension curve of amylose. In addition, we performed single-molecule AFM measurements on carboxymethyl amylose, and we found that, in contrast to the results of an earlier work by others, these side groups do not significantly affect amylose elasticity. By combining our experimental and modeling results, we conclude that the nonentropic elastic behavior of amylose is governed by the mechanics of pyranose rings themselves and their force-induced conformational transitions.

Amylose↗

PubMed2000

Individual plasma proteins detected on rough biomaterials by phase imaging AFM.

In the past several years, atomic force microscopy (AFM) has provided topographic images of adsorbed plasma proteins in situ at unprecedented resolution. Imaging has been limited to adsorbed protein on relatively smooth model substrates such as mica, graphite, or self-assembled monolayers on which the small height of the protein can be observed from the background. The inherent roughness of biomaterial surfaces has prevented observation of adsorbed proteins in topographic images. We report imaging isolated fibrinogen molecules adsorbed on National Heart Lung and Blood Institute (NHLBI) reference materials polydimethylsiloxane and low-density polyethylene in situ using phase imaging AFM. Fibrinogen, a plasma protein important for blood coagulation and platelet aggregation, was adsorbed from dilute solution onto reference biomaterial surfaces at sub-monolayer coverage. Tapping mode AFM was used to image the samples. For polydimethylsiloxane, the lateral size of the surface features is much greater than the dimensions of proteins. This allowed adsorbed proteins to be observed in topographic images. The phase imaging signal of tapping mode AFM provides information on differences in material properties of the surface, and was used to distinguish individual protein molecules from the underlying polymer surface. On the low-density polyethylene surface, characteristic topographical features are of the same magnitude as the protein molecules, so that protein cannot be distinguished from the surface using topographic images. However, phase images were used to successfully locate and characterize the distribution of the protein. Phase imaging was not able to distinguish fibrinogen adsorbed onto expanded polytetrafluoroethylene. The utility and limitations of the phase imaging technique for characterizing protein adsorption to rough surfaces is discussed.

Adsorption↗

PubMed1999

Evidence for nonrandom behavior in 208-12 subsaturated nucleosomal array populations analyzed by AFM.

Atomic force microscopy was used to determine the population distributions in reconstituted, subsaturated 208-12 nucleosomal arrays. The features found in these distributions vary with the average nucleosome loading per template molecule (n(av)): at n(av) < 4, the distributions show a single peak whose breadth is equal to that expected for a random loading process; at n(av) = 4-8, the distributions are broader than random distributions and are complex; i.e., they contain multiple peaks and/or shoulders. Moreover, the peaks/shoulders typically occur at two nucleosome intervals, i.e., 2, 4, 6 or 3, 5, 7 nucleosomes. This two-nucleosome periodicity is statistically significant. The precise cause for such discrete features within the distributions is unknown, but at least these features would seem to indicate some pairwise preference in nucleosome occupation at these loading levels. In these intermediate-level (n(av) = 4-8) distributions, the major peak contains a larger fraction of the total templates than a random nucleosome loading process would produce. This feature indicates that at these intermediate population levels there is some tendency for correlated nucleosome loading among the templates. Hyperacetylated nucleosomal arrays show only subtle differences in their population distributions compared to nonacetylated arrays and demonstrate the above features. AFM allows one to study unfixed chromatin arrays; we find that nucleosomes on the 208-12 template demonstrate significant lability when they are not glutaraldehyde-fixed.

Acetylation↗

PubMed1998

Evaluation of Surface Ionization Parameters from AFM Data.

A new application of atomic force microscopy (AFM) for evaluation of surface ionization parameters on the basis of a charge-regulation model is demonstrated. Interactions between symmetrical silicon nitride and silica surfaces have been measured in aqueous electrolytic solutions at different pHs and separations from a few Debye lengths down to zero. The obtained experimental data are reported and interpreted on a quantitative level in the framework of Derjaguin's approximation; the computational procedure used for this purpose is outlined. Surface charge density, surface potential, and species concentrations are calculated as functions of pH and separation, unveiling the limits of applicability of classical constant-charge and constant-potential approximations. The integrity of the results on the charge properties of interacting surfaces is discussed in reference to electrokinetic and titration data, potential problems caused by the existence of non-DLVO forces and surface irregularities being highlighted. Copyright 1998 Academic Press.

Journal Article↗

PubMed1997

Examination of contact lens surfaces by Atomic Force Microscope (AFM).

PURPOSE: We evaluated the use of Atomic Force Microscopy (AFM) in examining the surfaces of unused and worn hydrogel contact lenses under natural, fully hydrated conditions. METHODS: Using the AFM contact mode, we examined hydrogel lenses (Acuvue, Surevue, NewVues, CSI Clarity, SeeQuence) that were hydrated. RESULTS: Surface morphologies characteristics of each lens type and wear history were readily observed. The surfaces of worn lenses showed evidence of abrasion and altered morphology. These changes varied with type of contact lens and conditions of use and by site on the lens. CONCLUSIONS: AFM is a very powerful tool for high resolution examination of hydrated contact lens surface structure. The method avoids artifacts due to dehydration and coating which can occur even with low voltage Scanning Electron Microscopy. Significant differences in contact lens surface morphology were observed before and after wear. These observations may be of importance in helping develop improved new lens polymers and ocular solutions.

Contact Lenses, Hydrophilic↗

PubMed2001

Internal structure of the starch granule revealed by AFM.

Atomic force microscopy images of sectioned native corn starch granules show evidence of the well-known radial organisation of the starch macromolecules, with the less-ordered hilum region near to the centre. Native granules show blocks 400-500 nm in size that span the growth rings. Lintnerised starch granules, where a mild acid hydrolysis has been used to remove the amorphous and less crystalline parts of the granule, clearly show smaller 'blocklets' within the rings approximately 10-30 nm in size. This level of organisation within the growth rings corresponds to the blocklet or superhelix structures that have been proposed in the literature for the association or clustering of amylopectin helices. Mechanical property imaging techniques have provided enhanced contrast to view this morphology, and shown the deformability of the starch structure under contact mode imaging conditions.

Amylopectin↗

PubMed2004

Structural calorimetry of main transition of supported DMPC bilayers by temperature-controlled AFM.

Atomic force microscopy at high temperature resolution (DeltaT < or approximately 0.1 K) provided a quantitative structural calorimetry of the transition from the fluid (Lalpha)- to the gel (Pbeta')-phase of supported dimyristoylphosphatidylcholine bilayers. Besides a determination of the main transition temperature (T0) and the van't Hoff transition enthalpy (DeltaHvH), the structural analysis in the nm-scale at T close to T0 of the ripple phase allowed an experimental estimation of the area of cooperative units from small lipid domains. Thereby, the corresponding transition enthalpy (DeltaH) of single molecules could be determined. The lipid organization and the corresponding parameters T0 and DeltaHvH (DeltaH) were modulated by heptanol or external Ca2+ and compared with physiological findings. The size of the cooperative unit was not significantly affected by the presence of 1 mM heptanol. The observed linear relationship of DeltaHvH and T0 was discussed in terms of a change in heat capacity.

Calcium↗

PubMed1994

Structures of large T antigen at the origin of SV40 DNA replication by atomic force microscopy.

For inorganic crystals such as calcite (CaCO3), Atomic Force Microscopy (AFM) has provided surface structure at atomic resolution (Ohnesorge and Binnig, 1993). As part of a broad effort to obtain high resolution for an individual protein or protein assembly (Binnig et al., 1986; Rugar and Hansma, 1990; Radmacher et al., 1992), we applied AFM to study the ATP-dependent double hexamer of SV40 large T antigen, which assembles around the viral origin of DNA replication. Multimeric mass has been determined in two-dimensional projected images by Scanning Transmission Electron Microscopy (STEM) (Mastrangelo et al., 1989). By AFM, if the DNA-protein preparation has been stained positively by uranyl acetate, the contour at the junction between hexamers is visible as a cleft, 2-4 nm deep. The cleft, whether determined as a fraction of height by AFM or as a fraction of mass thickness by STEM, is of comparable magnitude. On either side of the cleft, hexamers attain a maximum height of 13-16 nm. Monomers found in the absence of ATP show heights of 5-7 nm. Taken together, the z coordinates provide a surface profile of complete and partial replication assemblies consistent with the spatial distribution of recognition pentanucleotides on the DNA, and they contribute direct geometrical evidence for a ring-like hexamer structure.

Antigens, Viral, Tumor↗

PubMed2006

Optical and structural properties of sputter-deposited nanocrystalline Cu2O films: effect of sputtering gas.

We report the effect of the atomic mass of the sputtering gas (He, Ne, Ar, Kr, and Xe) on the structure and optical properties of nanocrystalline cuprous oxide (Cu2O) thin films deposited by dc magnetron sputtering. The crystal structure and surface morphology were studied by X-ray diffraction (XRD) and atomic force microscopy (AFM) respectively. We find that the atomic mass of the sputtering gas significantly affects the primary crystallite size as well as the surface morphology and texture. Optical reflectance and transmission measurements show that the nanocrystalline thin films are transparent over most of the visible region. The HOMO-LUMO gap obtained from optical absorption spectra show a size-dependent quantum shift with respect to the bulk band gap reported for Cu2O (2.1 eV).

Copper↗

PubMed2000

The use of atomic force microscopy for the observation of corneal epithelium surface.

PURPOSE: To evaluate the feasibility of imaging normal corneal epithelium by means of atomic force microscopy (AFM). METHODS: Twelve normal corneas from six albino rabbits were examined using a commercial atomic force microscope. Six corneas were examined in balanced salt solution after fixation in glutaraldehyde 2.5% and six without any fixation. Rectangular silicon nitride cantilevers with a spring constant of 10 to 20 mN/m were used. The measured forces after imaging were less than 100 pN. All reported images were made with 512x512-pixel definition with typical scan rates ranging from 1 to 5 Hz. RESULTS: High-quality images of corneal epithelium surface were obtained from fixed and unfixed specimens in magnifications ranging from x2000 to x2,000,000. Imaging of fixed specimens was always easier. In unfixed specimens fuzzy images were very common, probably because of the presence of the cell glycocalyx. AFM revealed the typical polygonal corneal epithelial cells. The cell surface was covered by microprojections; at cell borders the microprojections were arranged in two characteristic parallel rows. Craterlike formations were revealed in several specimens. The microprojections' morphology and their surface details were revealed using magnifications up to x2,000,000. Three-dimensional representation of the images facilitated better understanding of the surface topography. Measurements in horizontal and vertical plane were made using the section analysis tool. CONCLUSIONS: In this work the AFM parameters appropriate for corneal epithelium imaging in physiological medium were defined. AFM represents a new powerful tool for corneal epithelium imaging, and its application in this field warrants further investigation.

Animals↗

PubMed2004

Mapping of enzyme activity by detection of enzymatic products during AFM imaging with integrated SECM-AFM probes.

With the integration of submicro- and nanoelectrodes into atomic force microscopy (AFM) probes using microfabrication techniques, an elegant approach combining scanning electrochemical microscopy (SECM) with AFM has recently been introduced. Simultaneous contact mode imaging of a micropatterned sample with immobilized enzyme spots and imaging of enzyme activity is shown. In contrast to force spectroscopy the conversion of an enzymatic byproduct is directly detected during AFM imaging and correlated to the activity of the enzyme.

Electrochemistry↗

PubMed2004

Insertion of GPI-anchored alkaline phosphatase into supported membranes: a combined AFM and fluorescence microscopy study.

A new method based on combined atomic force microscopy (AFM) and fluorescence microscopy observations, is proposed to visualize the insertion of glycosylphosphatidyl inositol (GPI) anchored alkaline phosphatase from buffer solutions into supported phospholipid bilayers. The technique involves the use of 27 nm diameter fluorescent latex beads covalently coupled to the amine groups of proteins. Fluorescence microscopy allows the estimation of the relative protein coverage into the membrane and also introduces a height amplification for the detection of protein/bead complexes with the AFM. The coupling of the beads with the amine groups is not specific; this new and simple approach opens up new ways to investigate proteins into supported membrane systems.

Alkaline Phosphatase↗

PubMed2003

Modeling of adhesion in tablet compression--I. Atomic force microscopy and molecular simulation.

Adhesion problems during tablet manufacturing have been observed to be dependent on many formulation and process factors including the run time on the tablet press. Consequently, problems due to sticking may only become apparent towards the end of the development process when a prolonged run on the tablet press is attempted for the first time. It would be beneficial to predict in a relative sense if a formulation or new chemical entity has the potential for adhesion problems early in the development process. It was hypothesized that favorable intermolecular interaction between the drug molecules and the punch face is the first step or criterion in the adhesion process. Therefore, the rank order of adhesion during tablet compression should follow the rank order of these energies of interaction. The adhesion phenomenon was investigated using molecular simulations and contact mode atomic force microscopy (AFM). Three model compounds were chosen from a family of "profen" compounds. Silicon nitride AFM tips were modified by coating a 20-nm iron layer on the surfaces by sputter coating. Profen flat surfaces were made by melting and recrystallization. The modified AFM probe and each profen surface were immersed in the corresponding profen saturated water during force measurements using AFM. The work of adhesion between iron and ibuprofen, ketoprofen, and flurbiprofen in vacuum were determined to be -184.1, -2469.3, -17.3 mJ. m(-2), respectively. The rank order of the work of adhesion between iron and profen compounds decreased in the order: ketoprofen > ibuprofen > flurbiprofen. The rank order of interaction between the drug molecules and the iron superlattice as predicted by molecular simulation using Cerius(2) is in agreement with the AFM measurements. It has been demonstrated that Atomic Force Microscopy is a powerful tool in studying the adhesion phenomena between organic drug compounds and metal surface. The study has provided insight into the adhesion problems occurring during tablet compression and a direction for continued study.

Adhesiveness↗

PubMed2000

Total internal reflection microscopy and atomic force microscopy (TIRFM-AFM) to study stress transduction mechanisms in endothelial cells.

The cytoskeleton plays a key role in providing strength and structure to the cell. A force balance exists between the cytoskeleton and the extracellular matrix/substratum via the focal contact regions. The purpose of this study is to integrate atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM) data to determine the effect of localized force application over the cell surface on the cell's focal contacts size and position. TIRFM gives detailed information on the cell-substrate contact regions and AFM is a tool for elasticity measurements, force application, and topographic surface mapping of the cell. TIRFM data were calibrated by varying the intensity of the evanescent wave to change the interfacial angle at the glass-cell interface. The individual focal contact intensity was found to decrease with increasing interfacial angles from 66 degrees to 80 degrees as the depth of penetration varied from 150 to 66 nm. A measure of cellular mechanical properties was obtained by collecting a set of force curves over the entire cell using the Bioscope AFM. The nuclear region appears to be stiffer than the cell body. Preliminary results of the nanonewtons force application to the cell surface indicate that the cell-substrate contacts rearrange to offset the force. It is evident that the stress applied to the surface is transmitted to the cell-substrate contact region.

Adaptation, Physiological↗

PubMed2006

Structure of the rhodopsin dimer: a working model for G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) participate in virtually all physiological processes. They constitute the largest and most structurally conserved family of signaling molecules. Several class C GPCRs have been shown to exist as dimers in their active form and growing evidence indicates that many, if not all, class A receptors also form dimers and/or higher-order oligomers. High-resolution crystal structures are available only for the detergent-solubilized light receptor rhodopsin (Rho), the archetypal class A GPCR. In addition, Rho is the only GPCR for which the presumed higher-order oligomeric state has been demonstrated, by imaging native disk membranes using atomic force microscopy (AFM). Based on these data and the X-ray structure, an atomic model of Rho dimers has been proposed, a model that is currently scrutinized in various ways. AFM has also been used to measure the forces required to unfold single Rho molecules, thereby revealing which residues are responsible for Rho's stability. Recent functional analyses of fractions from solubilized disk membranes revealed that higher-order Rho oligomers are the most active species. These and other results have enhanced our understanding of GPCR structure and function.

Animals↗