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Results for “Atomic force microscopy (AFM)”

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PubMed2000

Atomic force microscopy-based detection of binding and cleavage site of matrix metalloproteinase on individual type II collagen helices.

Type II tropocollagen molecules were reacted with matrix metalloproteinase 8 (MMP-8) and the binding sites as well as the cleavage site of MMP-8 were detected on individual molecules using atomic force microscopy (AFM). Approximately 300-nm-long coiled-coil tropocollagen molecules were straightened and immobilized on an atomically flat surface for detection by AFM. The direct visualization of individual collagen molecules revealed heterogeneous characteristics of MMP-8:collagen complexes. We observed that there existed multiple MMP-8 nonspecific binding sites on the collagen molecules, but cleavage always took place at a unique site. When collagen molecules, straightened and immobilized on the surface, were reacted with MMP-8, a site of cleavage appeared as a gap in stretched molecules. This is the first report to visually show direct collagenase:collagen interactions using AFM. The described AFM-based analysis has potential as a protein analysis tool for understanding a complex mechanism of enzyme:substrate interactions.

Animals↗

PubMed2006

Characteristic features of Bacillus cereus cell surfaces with biosorption of Pb(II) ions by AFM and FT-IR.

In order to elucidate the potential mechanisms involved in the biosorption of metal ions, atomic force microscopy (AFM) and Fourier transform infrared (FT-IR) spectroscopy were used to characterize the interaction between Pb2+ and Bacillus cereus. AFM imaging of the biomass surfaces exposed to different concentrations of lead ions solution showed a major morphological change occurred after Pb2+ biosorption. The FT-IR spectra indicated the binding characteristics of the lead ions involved the carboxyl, hydroxyl and amino groups in the biomass. Equilibrium biosorption experiments of Pb2+ were carried out to investigate the effects of pH values and the initial metal concentrations. The experimental isotherm data were then modeled using Langmuir, Freundlich, and Redlich-Peterson isotherm equations. As a result, the Redlich-Peterson model yielded the best fit of experimental data. Kinetics experiments showed the biosorption was a rapid process and the pseudo-second-order model was successfully applied to predict the rate constant of biosorption.

Adsorption↗

PubMed2003

The extracellular matrix of the cuticle of Gordius panigettensis (Gordioiidae, Nematomorpha): observations by TEM, SEM and AFM.

The cuticle of Gordius panigettensis (Sciacchitano, 1955) was studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The cuticle is composed of 30-50 compact layers. The number of the layers is higher in the central part of the animal's body and decreases at the extremities. Each layer is composed of parallel tightly packed fibres approximately 640 nm in diameter and of indefinite length. The fibres run strictly parallel within each layer, while in adjoining layers they run at a variable angle from 45 degrees in the central body to 90 degrees in the extremities. Each fibre shows a barely detectable filamentous inner structure and is enveloped in a thin highly regular net formed by hexagonal meshes. Our results suggested that these fibres should be proteinaceous although non-collagenous. Thinner radial fibres run among the large fibres and across all the layers and span the whole thickness of the cuticle from the epithelial layer located deep underneath the large fibres up to the epicuticle on the external surface of the animal.

Animals↗

PubMedDate not supplied

AFM lithography of aluminum for fabrication of nanomechanical systems.

Nanolithography by local anodic oxidation of surfaces using atomic force microscopy (AFM) has proven to be more reproducible when using dynamic, non-contact mode. Hereby, the tip/sample interaction forces are reduced dramatically compared to contact mode, and thus tip wear is greatly reduced. Anodic oxidation of Al can be used for fabricating nanomechanical systems, by using the Al oxide as a highly selective dry etching mask. In our experiments, areas as large as 2 micro m x 3 micro m have been oxidized repeatedly without any sign of tip-wear. Furthermore, line widths down to 10nm have been routinely obtained, by optimization of AFM parameters, such as tip/sample distance, voltage and scan speed. Finally, AFM oxidation experiments have been performed on CMOS processed chips, demonstrating the first steps of fabricating fully functional nanomechanical devices.

Journal Article↗

PubMed2006

Immobilization and AFM of single 4 x 6-mer tarantula hemocyanin molecules.

The high molecular mass respiratory protein of the tarantula Eurypelma californicum, a 4 x 6-mer hemocyanin, was investigated by atomic force microscopy (AFM). Various substrates and methods were evaluated for immobilization of individual hemocyanin molecules on a solid surface. Samples were imaged after physisorption on mica and self-assembled monolayers, and after chemisorption on Au(111) and N-hydroxy-succinimide (NHS) functionalized surfaces. AFM measurements were carried out preferable in solution and contact mode, but also in Tapping mode and on air-dried samples. Adsorption of the protein on mica followed by drying and carrying out the measurements in Tapping mode gave the best results. In the AFM images the four hexamers of the native 4 x 6-mer hemocyanin have been defined. The results were compared with independent available structural data and represent a validation case for this technique applied for the first time on such giant and complex molecules. As observable in images taken by transmission electron microscopy and also proposed from SAXS data, 4 x 6-mers could be found where the half-molecules are tilted against each other. This study is a step in resolving conformational heterogeneities, involved in oxygen binding of hemocyanins, at the single-molecule level by AFM.

Air↗

PubMed2000

Direct characterization of the physicochemical properties of fungal spores using functionalized AFM probes.

A new method is described for characterizing the physicochemical properties of native microbial cells by using atomic force microscopy (AFM) with chemically functionalized probes. Adhesion forces were measured, under deionized water, between probes and model substrata functionalized with alkanethiol self-assembled monolayers terminated with OH and CH(3) groups. These were found to be 6 +/- 2 nN (n = 1024), 0.9 +/- 0.4 nN, and approximately 0 nN, for CH(3)/CH(3), CH(3)/OH, and OH/OH surfaces, respectively, and were not significantly influenced by changes of ionic strength (0.1 M NaCl versus deionized water). This shows that functionalized probes are very sensitive to changes of surface hydrophobicity. Using OH- and CH(3)-terminated probes, patterns of rodlets, approximately 10 nm in diameter, were visualized, under physiological conditions, at the surface of spores of Phanerochaete chrysosporium. Multiple (1024) force-distance curves recorded over 500 x 500-nm areas at the spore surface, either in deionized water or in 0.1 M NaCl solutions, always showed no adhesion for both OH- and CH(3)-terminated probes. Control experiments indicated that the lack of adhesion is not due to transfer of cellular material onto the probe, but to the hydrophilic nature of the spore surface.

Cell Adhesion↗

PubMed2006

Hydration-dehydration of adsorbed protein films studied by AFM and QCM-D.

The hydration-dehydration process of an adsorbed human serum albumin film has been studied using atomic force microscopy (AFM) and a quartz crystal microbalance (QCM). All measurements were performed with identically prepared protein films deposited on highly hydrophilic substrates. Both techniques are shown to be suitable for following in situ the kinetics of protein hydration, and for providing quantitative values of the adsorbed adlayer mass. The results obtained by the two methods have been compared and combined to study changes of physical properties of the films in terms of viscosity, shear, Young's modulus, density and film thickness. These properties were found to be reversible during hydration-dehydration cycles.

Adsorption↗

PubMed2004

A nano-view of West Nile virus-induced cellular changes during infection.

BACKGROUND: Microscopic imaging of viruses and their interactions with and effects on host cells are frequently held back by limitations of the microscope's resolution or the invasive nature of the sample preparation procedures. It is also difficult to have a technique that would allow simultaneous imaging of both surface and sub-surface on the same cell. This has hampered endeavours to elucidate virus-host interactions. Atomic Force Microscopy (AFM), which is commonly used in the physical sciences, is now becoming a good correlative form of microscopy used to complement existing optical, confocal and electron microscopy for biological applications RESULTS: In this study, the West Nile (Sarafend) virus-infected Vero cell model was used. The atomic force microscope was found to be useful in producing high resolution images of virus-host events with minimal sample processing requirements. The AFM was able to image the budding of the West Nile (Sarafend) virus at the infected cell surface. Proliferation of the filopodia and thickening of clusters of actin filaments accompanied West Nile virus replication. CONCLUSIONS: The study shows that the AFM is useful for virus-host interaction studies. The technique provides morphological information on both the virus and the host cell during the infection stages.

Journal Article↗

PubMed2005

Metal-oxide-Semiconductor capacitors fabricated by layer-by-layer nanoassembly and microfabrication.

Metal oxide semiconductor (MOS) capacitors were fabricated by electrostatic layer-by-layer self-assembly (LbL-SA) combined with a modified lift-off technique. The MOS capacitors were built on both n-type and p-type silicon substrates. The numbers of silicon dioxide (SiO2) nanoparticle layers were varied to characterize the electrical performance of MOS capacitors. Unlike the conventional process, LbL-SA allows us to deposit the thin films for a semiconductor device with a lower temperature, lower cost, and shorter processing time. The stability of the silica insulation layers was also investigated. Atomic force microscopy (AFM) served to monitor the film quality of the self-assembled thin films.

Metals↗

PubMed2000

Atomic force microscopy in structural biology: from the subcellular to the submolecular.

Atomic force microscopy (AFM) is capable of generating images within ranges of resolution that are of particular interest in biology. Although atomic resolution may not be possible with biological samples, a great deal of information can still be obtained from images that provide structures at a slightly lower level of resolution. The submolecular resolution images of bacteriorhodopsin and the chaperonin GroES, which revealed, respectively, individual loops and beta-turns, confirmed and complemented other structural investigations, while the molecular-level features in images of membrane-bound VacA, a cytotoxin from Helicobacter pylori, immediately suggested the possibility, subsequently proven, of channel-forming ability. A series of images with macromolecular resolution directly provided details on the mechanisms by which RNA polymerase nonspecifically translocates along DNA, and images with subcellular resolving power of erythrocytic cellular membranes showed, with unambiguous clarity, linear arrays of molecular complexes. In this review, we will describe some of the most biologically relevant findings that have been obtained with AFM within ranges of resolution from the submolecular to the molecular, and from the macromolecular to the subcellular. Furthermore, we will describe some of the sample conditions and imaging environments that are likely important to achieve a particular level of resolution.

Bacterial Proteins↗

PubMedDate not supplied

Annealing and melting behavior of poly(l-lactic acid) single crystals as revealed by in situ atomic force microscopy.

In situ annealing and melting of folded-chain single crystals of poly(l-lactic acid) (PLLA) was examined by temperature-controlled atomic force microscopy (AFM). Prominent changes in the crystal appearance during annealing could be followed in real time by the AFM at temperatures above the original crystallization temperature. Thickening of the crystal edges could be occasionally observed, and this indicates that the crystal edges are less perfect than the central, well-ordered regions. At higher annealing temperatures, melting of the unthickened part started. The melting of the unthickened region progressed from the boundaries of the thickened portion normal to the growth face, rather than to the folding surfaces. In addition, it is suggested that melting also initiates at defective or distorted sites in the crystal as revealed by transmission electron microscopy (TEM) and AFM.

Biocompatible Materials↗

PubMed2004

Viscoelasticity of gelatin surfaces probed by AFM noise analysis.

The viscoelastic properties of surfaces of swollen gelatin were investigated by analyzing the Brownian motion of an atomic force microscopy (AFM) cantilever in contact with the gel surface. A micron-sized glass sphere attached to the AFM cantilever is used as the dynamic probe. When the sphere approaches the gelatin surface, there is a static repulsive force without a jump into contact. The cantilever's Brownian movement is monitored in parallel, providing access to the dynamic sphere-surface interaction as quantified by the dynamic spring constant, kappa, and the drag coefficient, xi. Gelatin is used as a model substance for a variety of other soft surfaces, where the stiffness of the gel can be varied via the solvent quality, the bloom number, and the pH. The modulus derived from the static force-distance curve is in the kPa range, consistent with the literature. However, the dynamic spring constant as derived from the Brownian motion is much larger than the static differential spring constant dF/dz. On retraction, one observes a rather strong adhesion hysteresis. The strength of the bridge (as given by the dynamic spring constant and the drag coefficient) is very small.

Gelatin↗

PubMed2006

AFM study of the effect of metronidazole on surface structures of sulfate-reducing bacteria.

The effect of metronidazole (ME) on sulfate-reducing bacteria (SRB) was studied by atomic force microscopy (AFM) in this paper. Topography images of SRB cell show that after exposure to ME individual cell shape is sharply modified. Topography and phase images of SRB cell wall show that after exposure to ME not only the roughness of the cell wall increases but also the physical performance of SRB surface is changed to be uniform. AFM frictional loops show that after exposure to ME, SRB surface friction is increased remarkably.

Anti-Infective Agents↗

PubMed2006

Size distribution of pressure-decomposed casein micelles studied by dynamic light scattering and AFM.

Reversible and irreversible states of pressure-dissociated casein micelles were studied by in situ light scattering techniques and ex situ atomic force microscopy. AFM experiments performed at ambient pressure reveal heterogeneities across the micelle, suggesting a sub-structure on a 20 nm scale. At pressures between 50 and 250 MPa, the native micelles disintegrate into small fragments on the scale of the observed sub-structure. At pressures above 300 MPa the micelles fully decompose into their monomeric constituents. After pressure release two discrete populations of casein aggregates are observed, depending on the applied initial pressure: Between 160 and 240 MPa stable micelles with diameters near 100 nm without detectable sub-structures are formed. Casein micelles exposed to pressures above 280 MPa re-associate at ambient pressure yielding mini-micelles with diameters near 25 nm. The implications concerning structural models are discussed.

Biophysical Phenomena↗

PubMed2006

Structural investigations on native collagen type I fibrils using AFM.

This study was carried out to determine the elastic properties of single collagen type I fibrils with the use of atomic force microscopy (AFM). Native collagen fibrils were formed by self-assembly in vitro characterized with the AFM. To confirm the inner assembly of the collagen fibrils, the AFM was used as a microdissection tool. Native collagen type I fibrils were dissected and the inner core uncovered. To determine the elastic properties of collagen fibrils the tip of the AFM was used as a nanoindentor by recording force-displacement curves. Measurements were done on the outer shell and in the core of the fibril. The structural investigations revealed the banding of the shell also in the core of native collagen fibrils. Nanoindentation experiments showed the same Young's modulus on the shell as well as in the core of the investigated native collagen fibrils. In addition, the measurements indicate a higher adhesion in the core of the collagen fibrils compared to the shell.

Collagen Type I↗

PubMed1999

Atomic force microscopy to study the degranulation in rat peritoneal mast cells after activation.

We have studied the degranulation in rat peritoneal mast cells by atomic force microscopy (AFM). Although AFM has advantages close to electron microscopy (EM) in spatial resolution, visualization of surface topography in peritoneal mast cells has not been reported yet. In the present paper we have succeeded in visualizing the degranulation process of rat peritoneal mast cells by AFM. AFM images showed that secretory granules were about 1 microm in diameter and that they were densely packed in the cytoplasm surrounding the nucleus. After stimulation with compound 48/80 (5 microg/ml), the packed granules began to loosen through formation of irregular cracks, as if polymerization of actin filaments was changed; 3 min after stimulation with compound 48/80 at 37 degrees C, the packed granules were completely loosened and the nucleus was no longer covered by the granules. In addition, AFM images showed clear fringes at the edges of the spreading cells. The fringes were 30-50 nm in height and there existed many small matrixes which were secreted to the outside of the cell. The matrixes were about 80 nm in diameter and 40 nm in height. These kinds of secretory matrixes were observed here first by AFM.

Animals↗

PubMed2005

Phase contrast and DIC illumination for AFM hybrids.

High-resolution optical microscopy is an essential pre-requisite for life science force microscopy, particularly for applications in cell biology and medicine. Identification and validation of cells is typically established with techniques like phase contrast microscopy or differential interference contrast microscopy. The option to select or monitor individual cells online with such light microscopy techniques while performing atomic force microscopy (AFM) measurements is therefore extremely beneficial. Here, we report two conceptually different strategies to implement these light microscopy techniques in a fully functional AFM head at the ultimate resolution of the Abbe diffraction limit.

Epithelial Cells↗

PubMedDate not supplied

AFM observation of band-like cellulose assemblies produced by Acetobacter xylinum.

We succeeded in estimating the thickness of band-like cellulose assemblies by combined use of atomic force microscopy (AFM) and transmission electron microscopy. The thickness of "dense" band-like cellulose assemblies was estimated at 20-30 nm from their AFM height profiles, which was several times greater than that of "coarse" band-like and ribbonlike cellulose assemblies. On the basis of these results, the folded- chain model previously proposed was discussed, and a different organization of TC subunits was suggested for the "dense" band-like cellulose assembly.

Cellulose↗