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Results for “Atomic force microscopy (AFM)”

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PubMed1998

Imaging two-dimensional arrays of soluble proteins by atomic force microscopy in contact mode using a sharp supertip.

A sharp tip with high aspect ratio is required for imaging biological macromolecules by atomic force microscopy (AFM). A tip with the end radius of curvature less than 3 nm has been reproducibly fabricated by means of electron beam deposition (EBD) in a field-emission scanning electron microscope. Two-dimensional protein arrays of ferritin and catalase, prepared at air/water interface and transferred onto silicon wafer, could be imaged both in air and in water by AFM using this sharp EBD-tip in contact mode. The negative staining preparation conventionally used in the transmission electron microscopy of protein was applied and shown to be quite effective in fixing the protein arrays for the AFM imaging in air. Individual molecules of ferritin and catalase were visible in the two-dimensional arrays. Also, imaging in water of these protein arrays presented molecular images clearer than in air, due probably to the absence of the adhesion force and the resulting weak lateral force during scanning. These images convince us of the capability of this supertip for AFM studies of biological molecules under aqueous conditions.

Air↗

PubMed2004

SNOM and AFM microscopy techniques to study the effect of non-ionizing radiation on the morphological and biochemical properties of human keratinocytes cell line (HaCaT).

In this study we have employed atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM) techniques to study the effect of the interaction between human keratinocytes (HaCaT) and electromagnetic fields at low frequency. HaCaT cells were exposed to a sinusoidal magnetic field at a density of 50 Hz, 1 mT. AFM analysis revealed modification in shape and morphology in exposed cells with an increase in the areas of adhesion between cells. This latter finding was confirmed by SNOM indirect immunofluorescence analysis performed with a fluorescent antibody against the adhesion marker beta4 integrin, which revealed an increase of beta4 integrin segregation in the cell membrane of 50-Hz exposed cells, suggesting that a higher percentage of these cells shows a modified pattern of this adhesion marker.

Cell Adhesion↗

PubMed1995

Scanning probe microscopy in microbiology.

Scanning probe microscopy (SPM) is emerging as an important alternative to electron microscopy as a technique for analyzing submicron details on biological surfaces. Microbiological specimens such as viruses, bacteriophages, and ordered bacterial surface layers and membranes have played an important role in the development of scanning tunnelling microscopy (STM) and atomic force microscopy (AFM) in cellular and molecular biology. Early STM studies involving metal-coated bacteriophage T4 polyheads, Methanospirillum hungatei, and Deinococcus radiodurans HPI layer clearly demonstrated that resolution was comparable to TEM on similarly prepared specimens and only limited by metal graininess. However, except for thin films or layers, novel biological information has been difficult to obtain since imaging native surfaces of such biomaterials as proteins or nucleic acids by STM proved to be unreliable. With the development of atomic force microscopes, which allow imaging of similar native structures, SPM applications have widened to include straightforward surface structure analysis, analysis of surface elastic and inelastic properties, bonding force measurements between molecules, and micro-manipulations of such individual molecules as DNA. AFM images have progressed from relatively crude representations of specimen topography to nanometer scale representations of native hydrated surfaces. It appears from the study of microbiological specimens that direct visualization of dynamic molecular events or processes may soon become a reality.

Bacteria↗

PubMed2006

Controlling gold atom penetration through alkanethiolate self-assembled monolayers on Au{111} by adjusting terminal group intermolecular interactions.

The penetration behavior of thermally evaporated Au on S(CH(2))(15)CH(3), S(CH(2))(15)CO(2)CH(3), S(CH(2))(15)CO(2)H, K-modified S(CH(2))(15)CO(2)CH(3), and K-modified S(CH(2))(15)CO(2)H self-assembled monolayers (SAM) on Au substrates is investigated. Gold is a particularly interesting metal since vapor-deposited Au atoms are known to pass through alkanethiolate SAMs on Au{111} substrates at room temperature. Here we show that it is possible to control Au penetration by adjusting the interactions between terminal groups. It is found that Au atoms evenly penetrate into the CH(3) and CO(2)CH(3) films, forming smooth buried layers below the organic thin films. For the CO(2)H film, although Au atoms can still penetrate through it, filaments and mushroomlike clusters form due to H-bonding between film molecules. In the case of the K-modified CO(2)CH(3) or CO(2)H films, however, most Au atoms form islands at the vacuum interface. These results suggest that van der Waals forces and H-bonds are not strong enough to block Au from going through but that ionic interactions are able to block Au penetration. The measurements were performed primarily using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and atomic force microscopy (AFM). The combination of these highly complementary probes provides a very useful strategy for the study of metal atom behavior on SAMs.

Journal Article↗

PubMed2000

Single-molecular AFM probing of specific DNA sequencing using RecA-promoted homologous pairing and strand exchange.

The specific sequence in a linearlized double-stranded DNA target has been identified at a single-molecular level by atomic force microscopy (AFM). This was accomplished using RecA-coated, single-stranded DNA probes which were paired with a specific complementary DNA sequence in a linear double-stranded DNA target by strand-exchange reaction at a homologous sequence site with target DNA. The sites of interaction between the nucleoprotein filaments and the double-stranded DNA targets were directly visualized by AFM in solution containing 4 mM magnesium acetate. Measurements of the position of RecA-coated probes paired to individual target DNA showed that DNA probes specifically paired at their corresponding homologous target sequences. Strand exchange promoted by RecA and the visualization by AFM provided a rapid and efficient way to identify homologous sequence on a single-molecule target DNA.

Base Sequence↗

PubMed2006

Localized attraction correlates with bacterial adhesion to glass and metal oxide substrata.

Bacterial adhesion to surfaces does not always proceed according to theoretical expectations. Discrepancies are often attributed to surface heterogeneities that provide localized, favorable sites for bacterial attachment. The presence of these favorable deposition sites for bacteria, however, has never been directly measured. Atomic force microscopy (AFM) was used to quantify the distribution of attractive sites on clean substrata. Surfaces of silica and three different metal oxides mapped by adhesion force with regular or colloidal AFM tips showed a heterogeneous distribution of adhesion forces. Adhesion forces were normally distributed based on a colloid probe, but regular tips revealed a proportionately larger number of relatively more adhesive sites. No correlation was found between the average adhesion force (tip or colloid) and macroscopic adhesion tests using five strains of bacteria. However, when AFM tip results were compared to bacterial adhesion data on the basis of only the stickiest sites (the 5% of sites with the largest adhesion force), there was a good correlation of AFM data with adhesion data. These results demonstrate for the first time how overall bacterial adhesion to a surface effectively correlates with a relatively small fraction of highly adhesive sites rather than averaged adhesion force as detected using AFM.

Adhesiveness↗

PubMedDate not supplied

Quantification of cell adhesion force with AFM: distribution of vitronectin receptors on a living MC3T3-E1 cell.

Distribution of vitronectin (VN) receptors on a living murine osteoblastic cell was successfully measured by atomic force microscopy (AFM). First, the distribution of the integrin beta(5) subunit which constitutes a part of the VN receptor on the cell was confirmed by conventional immunohistochemistry after fixing the cell. To visualize the distribution of the receptor on a living cell by an independent and potentially a more quantitative method, the AFM was used with a microbead attached to the cantilever tip to increase the area of contact and VN was immobilized on the microbead. Force measurements were then performed over a large area of a living murine osteoblastic cell using the microbead covered with VN.

Animals↗

PubMed2005

Force probe molecular dynamics simulations.

Many proteins are molecular nano-machines, which perform their biological function via well-coordinated structural transitions. Often, these motions occur on much slower time scales than those accessible to conventional molecular dynamics techniques, which are limited to submicrosecond time scales by current computer technology. This is also true for ligand binding and unbinding reactions. Force probe simulations (or steered molecular dynamics) provide a powerful means to overcome this limitation, and thus to get insight into the atomistic mechanisms that underlie biological functions such as ligand binding. This chapter provides a basic introduction into this method. It further sketches a simple nonequilibrium statistical mechanics treatment that shows how to relate the results of force probe simulations to atomic force microscopy (AFM) or optical tweezer experiments. As an example, enforced unbinding simulations of streptavidin/biotin complexes are detailed.

Biomechanical Phenomena↗

PubMed2003

Surface trapping and AFM detection of DNA topological intermediates generated from an oxidative chemical nuclease.

Direct observation of DNA topological intermediates generated from a 'chemical nuclease' treatment has been made by atomic force microscopy (AFM). The intermediates were trapped at the mica-water interface and imaging was carried out in the dynamic force mode. Complete conversion from supercoiled circular state to relaxed circular/linear state has been observed over a time scale of 8 min. Implication of such studies in complementing gel electrophoresis data has been predicted.

Aluminum Silicates↗

PubMed1998

A new system for the measurement of electrogenicity produced by ion pumps using a thin polymer film: examination of wild type bacteriorhodopsin and the D96N mutant over a wide pH range.

We developed a new assay system for the measurement of capacitive electric currents generated by ion pumps using the thin polymer film 'Lumirror' (Toray Co., Japan). This system enables us to examine the electrogenicity of ion pumps over a wide range of experimental conditions with high reproducibility due to the mechanical and chemical stability, the high electric resistance and the high electric capacitance of the thin polymer film. Using this method, we examined the photoelectric response of wild type bacteriorhodopsin and its D96N mutant over a wide pH range (2.8-10.0). The results were explained in terms of the affinities of the proton binding sites for translocated protons. A possibility that the direction of the proton transfer from the Schiff base was influenced by the protonation/deprotonation state of the surrounding proton binding sites was suggested. We also found that this film can be used as a substrate for atomic force microscopy (AFM) samples and hence the active purple membrane was observed with AFM.

Bacteriorhodopsins↗

PubMed2006

Deformation and nano-rheology of red blood cells: an AFM investigation.

Interaction forces, deformation and nano-rheology of individual red blood cells in physiologically relevant solution conditions have been determined by colloid probe atomic force microscopy (AFM). On approach of the physically immobilised cell and silica glass spherical probe surfaces, deformation of the red blood cell was observed in the force curves. At low levels of deformation, spring constants were determined in the range 3-6 m Nm(-1), whereas for higher levels of deformation, the forces increase non-linearly and on retraction, significant force curve hysteresis is observed (i.e. lower forces upon retraction). The extent of force curve hysteresis was dependent on both the drive velocity and loading force, typical of a viscoelastic system. The response of the red blood cell has been described by viscoelastic theory, where the short and long time scale elastic moduli and relaxation times are determined, i.e. the cell's nano-rheological properties elucidated. In addition to a time independent elastic modulus of 4.0 x 10(3)Nm(-2) at low levels of deformation, time-dependent elastic moduli ranges are observed (3.5 x 10(4) to 5.5 x 10(4)Nm(-2) at intermediate levels of deformation and 1.5 x 10(5) to 3.0 x 10(5)Nm(-2) at higher levels of deformation). That is, one elastic and more than one viscoelastic response to the red blood cell deformation is evident, which is considered to reflect the cellular structure.

Cells, Immobilized↗

PubMed2003

Polysaccharide properties probed with atomic force microscopy.

In recent years, polysaccharides have been extensively studied using atomic force microscopy (AFM). Owing to its high lateral and vertical resolutions and ability to measure interaction forces in liquids at pico- or nano-Newton level, the AFM is an excellent tool for characterizing biopolymers. The first imaging studies showed the morphology of polysaccharides, but gradually more quantitative image analysis techniques were developed as the AFM grew easier to use in aqueous liquids and in non-contact modes. Recently, AFM has been used to stretch polysaccharides and characterize their physicochemical properties by application of appropriate polymer stretching models, using a technique called single-molecule force spectroscopy. From application of such models as the wormlike chain, freely jointed chain, extensible-freely jointed chain, etc., properties such as the contour length, persistence length and segment elasticity or spring constant can be calculated for polysaccharides. The adhesion between polysaccharides and surfaces has been quantified with AFM, and this application is particularly useful for studying polysaccharides on microbial and other types of cells, because their adhesion is controlled by biopolymer characteristics. This review presents a synthesis of the theory and techniques currently in use to probe the physicochemical properties of polysaccharides with AFM.

Animals↗

PubMed2004

QCM-D on mica for parallel QCM-D-AFM studies.

Quartz crystal microbalance with dissipation monitoring (QCM-D) has developed into a recognized method to study adsorption processes in liquid, such as the formation of supported lipid bilayers and protein adsorption. However, the large intrinsic roughness of currently used gold-coated or silica-coated QCM-D sensors limits parallel structural characterization by atomic force microscopy (AFM). We present a method for coating QCM-D sensors with thin mica sheets operating in liquid with high stability and sensitivity. We define criteria to objectively assess the reliability of the QCM-D measurements and demonstrate that the mica-coated sensors can be used to follow the formation of supported lipid membranes and subsequent protein adsorption. This method allows combining QCM-D and AFM investigations on identical supports, providing detailed physicochemical and structural characterization of model membranes.

Aluminum Silicates↗

PubMed2001

Exploitation of reactivity and selectivity in cellulose functionalization using unconventional media for the design of products showing new superstructures.

A variety of new cellulose solvents was investigated toward their potential as media for the functionalization of the polyglucane. Thus, mixtures of dimethyl sulfoxide (DMSO)/tetrabutylammonium fluoride trihydrate (TBAF), N-methylmorpholine-N-oxide (NMMNO)/DMSO, melts of LiClO(4).3H(2)O, and aqueous solutions of Ni(tren)(OH)(2) [tren = tris(2-aminoethyl)amine] were applied as reaction media. In case of the new solvent, DMSO/TBAF its usefulness for derivatization reactions including the etherification with sodium monochloroacetate and the acylation with vinyl esters of carbonic acids was studied. The structural features of the products were analyzed by means of (1)H NMR spectroscopy (after depolymerization or peresterification), (13)C NMR spectroscopy, and HPLC after complete hydrolytic chain degradation. The results were compared with those obtained from derivatives prepared using the solvent N,N-dimethylacetamide (DMAc)/LiCl and conventional, heterogeneous synthesis. It can be shown that in case of carboxymethylation reactions the reaction medium applied has a drastic influence both on the course of reaction and on the structural features of the products. A highly efficient tool was found to be atomic force microscopy (AFM), showing remarkable differences in the superstructures of the differentially synthesized derivatives.

Acetylation↗

PubMed2004

In situ AFM studies of astrocyte-secreted apolipoprotein E- and J-containing lipoproteins.

The three-dimensional shapes and sizes of plasma lipoproteins and astrocyte-secreted lipoproteins (ASLPs) were characterized with the aid of in situ atomic force microscopy (AFM), which has the unique ability to study three-dimensional nanostructures under physiological conditions. Apolipoprotein E (apoE) and apolipoprotein J (apoJ) are the two most abundant apolipoproteins produced in the central nervous system (CNS). This study revealed that ASLPs containing apoE3, apoE4, or apoJ significantly differ from high density lipoprotein particles, thought to be their closest analogs in plasma, in aggregation properties, size, and shape. ASLPs were found to be significantly flatter and smaller than their plasma counterparts. Plasma lipoproteins were able to form ordered arrays on a mica surface at high concentration, but ASLPs did not. Rather, they formed amorphous aggregates at similar concentrations. Comprehensive quantitative characterization of particle size and shape was facilitated by two advances in AFM image analysis: (1) automated analysis through image-recognition algorithms, and (2) correction for the finite size of the AFM probe based on geometric modeling. This study and the developed AFM methodologies open the way to further in situ AFM studies of the lipoproteins in general and more specifically of CNS lipoproteins.

Animals↗

PubMed2004

Atomic force microscopy imaging of hair: correlations between surface potential and wetting at the nanometer scale.

We report investigations of hair surface potential under wetting at the nanometric scale by atomic force microscopy (AFM). Surface potential imaging was used to characterize the electrostatic properties of the hair samples. We found that the surface potential noticeably increases along the edges of the cuticles. These results are correlated with wetting behavior of different liquids performed using AFM in noncontact mode.

Hair↗

PubMed2005

Immobilization and condensation of DNA with 3-aminopropyltriethoxysilane studied by atomic force microscopy.

We used different methods to modify a mica surface with 3-aminopropyltriethoxysilane (APTES), and then used it as substrate to immobilize DNA for atomic force microscopy (AFM) observation. The evaporation method and solution modifying method were investigated and evaluated. The solution modifying method was found to be relatively simple and effective. Using an APTES solution-modified mica surface, DNA immobilization appeared more reproducible and it could be imaged in liquid. The mixed solution of APTES and DNA was dropped directly onto the mica surface for AFM imaging. We found that DNA can condense in APTES water solutions. Toroids, rods and intermediate structures of condensation were captured by AFM.

DNA↗

PubMed2004

A torsional resonance mode AFM for in-plane tip surface interactions.

Changing the method of tip/sample interaction leads to contact, tapping and other dynamic imaging modes in atomic force microscopy (AFM) feedback controls. A common characteristic of these feedback controls is that the primary control signals are based on flexural deflection of the cantilever probes, statically or dynamically. We introduce a new AFM mode using the torsional resonance amplitude (or phase) to control the feedback loop and maintain the tip/surface relative position through lateral interaction. The torsional resonance mode (TRmode ) provides complementary information to tapping mode for surface imaging and studies. The nature of tip/surface interaction of the TRmode facilitates phase measurements to resolve the in-plane anisotropy of materials as well as measurements of dynamic friction at nanometer scale. TRmode can image surfaces interleaved with TappingMode with the same probe and in the same area. In this way we are able to probe samples dynamically in both vertical and lateral dimensions with high sensitivity to local mechanical and tribological properties. The benefit of TRmode has been proven in studies of water adsorption on HOPG surface steps. TR phase data yields approximately 20 times stronger contrast than tapping phase at step edges, revealing detailed structures that cannot be resolved in tapping mode imaging. The effect of sample rotation relative to the torsional oscillation axis of the cantilever on TR phase contrast has been observed. Tip wear studies of TRmode demonstrated that the interaction forces between tip and sample could be controlled for minimum tip damage by the feedback loop.

Journal Article↗