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At least 1,009 records · Page 56Linked to original sources
PubMed2004

Structure of beta-casein layers at the air/solution interface: atomic force microscopy studies of transferred layers.

We report the nanoscale structural changes associated with the interfacial gelation of adsorbed beta-casein layers as a function of aging time. Adsorbed layers were transferred to solid supports and imaged by atomic force microscopy. The aging of the layer was accompanied by the formation of distinct disk-shaped protein nanoparticles ( approximately 20 nm in diameter). Under conditions where a gelled layer was expected (from previous interfacial rheology experiments), we observed ordering of the particles and the formation of elongated aggregates or linear rows. Brewster angle microscopy images were also obtained during the adsorption and gelation processes and during the degradation of the protein layer following addition of the surfactant sodium dodecyl sulfate (SDS). If SDS was added prior to interfacial protein gelation, the layer developed a foamlike morphology consistent with a fluid interfacial protein layer. However, if SDS was added after gelation, the protein layer was observed to fracture, consistent with the behavior of a solid phase.

Air↗
PubMedDate not supplied

Hybridisation of short DNA molecules investigated with in situ atomic force microscopy.

By introducing the complementary DNA (cDNA) strand to a molecular layer of short single stranded DNA (ssDNA), immobilised on a gold surface, we have investigated hybridisation between the two DNA strands through the technique of in situ atomic force microscopy (AFM). Before introduction of cDNA, the ssDNA molecular layer was modulated with the spacer molecule mercaptohexanol (MCH), which makes the ssDNA molecules more accessible for hybridisation. With in situ AFM, we have monitored the formation of a smooth, mixed molecular layer containing ssDNA and MCH. Furthermore, the hybridisation between the two DNA strands has been studied. Introduction of the cDNA strand resulted in an increase in smoothness and thickness of the molecular layer. Both the increase in order and thickness of the molecular layer can be expected if hybridisation occurs, since double stranded DNA molecules have a more rigid and elongated structure than ssDNA molecules.

Biosensing Techniques↗
PubMed1993

Subfibrillar structure of type I collagen observed by atomic force microscopy.

We have imaged native rat tail and reconstituted bovine dermal type I collagen by atomic force microscopy, obtaining a level of detail comparable to that obtained on the same samples by transmission electron microscopy. The characteristic 60-70 nm D periodicity consists of ridges exhibiting high tip-sample adhesion alternating with 5-15-nm-deep grooves having low adhesion. We also observe an intraperiod or "minor" band consisting of 1-nm-deep grooves, and "microfibrils" arranged parallel to or inclined approximately 5 degrees to the fibril axis. In air collagen fibrils exhibit negligible compression under the forces exerted by the tip. When immersed in water the subfibrillar features disappear and the fibrils become softer, compressing by 5% of their height under an 11-nN force. Material on the surface of the sample sometimes accumulates on the atomic force microscope tip; contrary to expectation such tip contamination can improve as well as reduce resolution.

Animals↗
PubMed2004

Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second harmonic generation and atomic force microscopy.

Orientation and organization of two amphiphilic push-pull chromophores mixed with two phospholipids (dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine) in Langmuir-Blodgett (LB) monolayers are investigated by second harmonic generation. The LB monolayers have also been characterized by atomic force microscopy and UV-vis spectroscopy. The effective molecular orientations and hyperpolarizabilities of the chromophores are studied as a function of the phospholipid concentrations. The experimental results are discussed within the frame of a model of orientational distribution of the chromophores which gives the orientational mean angle and bounds on the orientational disorder. The mean orientation of the chromophores is found to be within 45-55 degrees whereas their hyperpolarizability coefficients, measured with respect to quartz, are estimated to be in the range (0.3-0.7) x 10(-27) esu taking account of the maximal orientational disorder.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1996

The nanometer-scale structure of amyloid-beta visualized by atomic force microscopy.

Amyloid-beta (A beta) is the major protein component of neuritic plaques found in Alzheimer's disease. Evidence suggests that the physical aggregation state of A beta directly influences neurotoxicity and specific cellular biochemical events. Atomic force microscopy (AFM) is used to investigate the three-dimensional structure of aggregated A beta and characterize aggregate/fibril size, structure, and distribution. Aggregates are characterized by fibril length and packing densities. The packing densities correspond to the differential thickness of fiber aggregates along a zeta axis (fiber height above the x-y imaging surface). Densely packed aggregates ( > or = 100 nm thick) were observed. At the edges of these densely packed regions and in dispersed regions, three types of A beta fibrils were observed. These were classified by fibril thickness into three size ranges: 2-3 nm thick, 4-6 nm thick, and 8-12 nm thick. Some of the two thicker classes of fibrils exhibited pronounced axial periodicity. Substructural features observed included fibril branching or annealing and a height periodicity which varied with fibril thickness. When identical samples were visualized with AFM and electron microscopy (EM) the thicker fibrils (4-6 nm and 8-12 nm thick) had similar morphology. In comparison, the densely packed regions of approximately > or = 100 nm thickness observed by AFM were difficult to resolve by EM. The small, 2- to 3-nm-thick, fibrils were not observed by EM even though they were routinely imaged by AFM. These studies demonstrate that AFM imaging of A beta fibrils can, for the first time, resolve nanometer-scale, zeta-axis, surface-height (thickness) fibril features. Concurrent x-y surface scans of fibrils reveal the surface submicrometer structure and organization of aggregated A beta. Thus, when AFM imaging of A beta is combined with, and correlated to, careful studies of cellular A beta toxicity it may be possible to relate certain A beta structural features to cellular neurotoxicity.

Alzheimer Disease↗
PubMed2006

DNA strand arrangement within the SfiI-DNA complex: atomic force microscopy analysis.

The SfiI restriction enzyme binds to DNA as a tetramer holding two usually distant DNA recognition sites together before cleavage of the four DNA strands. To elucidate structural properties of the SfiI-DNA complex, atomic force microscopy (AFM) imaging of the complexes under noncleaving conditions (Ca2+ instead of Mg2+ in the reaction buffer) was performed. Intramolecular complexes formed by protein interaction between two binding sites in one DNA molecule (cis interaction) as well as complexes formed by the interaction of two sites in different molecules (trans interaction) were analyzed. Complexes were identified unambiguously by the presence of a tall spherical blob at the DNA intersections. To characterize the path of DNA within the complex, the angles between the DNA helices in the proximity of the complex were systematically analyzed. All the data show clear-cut bimodal distributions centered around peak values corresponding to 60 degrees and 120 degrees. To unambiguously distinguish between the crossed and bent models for the DNA orientation within the complex, DNA molecules with different arm lengths flanking the SfiI binding site were designed. The analysis of the AFM images for complexes of this type led to the conclusion that the DNA recognition sites within the complex are crossed. The angles of 60 degrees or 120 degrees between the DNA helices correspond to a complex in which one of the helices is flipped with respect to the orientation of the other. Complexes formed by five different recognition sequences (5'-GGCCNNNNNGGCC-3'), with different central base pairs, were also analyzed. Our results showed that complexes containing the two possible orientations of the helices were formed almost equally. This suggests no preferential orientation of the DNA cognate site within the complex, suggesting that the central part of the DNA binding site does not form strong sequence specific contacts with the protein.

Base Sequence↗
PubMed2005

Interactions between saporin, a ribosome-inactivating protein, and DNA: a study by atomic force microscopy.

Saporins are enzymes belonging to the PNAG class (polynucleotide: adenosine glycosidase), plant enzymes commonly known as ribosome-inactivating proteins (RIP), as a result of their property of irreversibly damaging eukaryotic ribosomes. Direct imaging with tapping-mode atomic force microscopy (AFM) has been used to study pGEM-4Z plasmid DNA binding to the saporin-SO6 (isoform from Saponaria officinalis seeds). Saporin wrapped the plasmidic DNA, and distribution of the enzyme molecules along the DNA chain was markedly variable; plasmid digested with saporin-SO6 appeared fragmented or topologically modified. The supercoiled DNA strands were cleaved, giving rise to a linearized form and to relaxed forms. Electrophoretic analysis of the effect of standard preparations of saporin-SO6 on pGEM-4S confirmed the presence of DNA strand-cleaving activity.

DNA↗
PubMed2004

Assessing the flexibility of intermediate filaments by atomic force microscopy.

Eukaryotic cells contain three cytoskeletal filament systems that exhibit very distinct assembly properties, supramolecular architectures, dynamic behaviour and mechanical properties. Microtubules and microfilaments are relatively stiff polar structures whose assembly is modulated by the state of hydrolysis of the bound nucleotide. In contrast, intermediate filaments (IFs) are more flexible apolar structures assembled from a approximately 45 nm long coiled-coil dimer as the elementary building block. The differences in flexibility that exist among the three filament systems have been described qualitatively by comparing electron micrographs of negatively stained dehydrated filaments and by directly measuring the persistence length of F-actin filaments (approximately 3-10 microm) and microtubules (approximately 1-8 mm) by various physical methods. However, quantitative data on the persistence length of IFs are still missing. Toward this goal, we have carried out atomic force microscopy (AFM) in physiological buffer to characterise the morphology of individual vimentin IFs adsorbed to different solid supports. In addition, we compared these images with those obtained by transmission electron microscopy (TEM) of negatively stained dehydrated filaments. For each support, we could accurately measure the apparent persistence length of the filaments, yielding values ranging between 0.3 microm and 1 microm. Making simple assumptions concerning the adsorption mechanism, we could estimate the persistence length of an IF in a dilute solution to be approximately 1 microm, indicating that the lower measured values reflect constraints induced by the adsorption process of the filaments on the corresponding support. Based on our knowledge of the structural organisation and mechanical properties of IFs, we reason that the lower persistence length of IFs compared to that of F-actin filaments is caused by the presence of flexible linker regions within the coiled-coil dimer and by postulating the occurrence of axial slipping between dimers within IFs.

Actin Cytoskeleton↗
PubMed2000

Atomic force microscopy of DNA molecules stretched by spin-coating technique.

We have developed an effective approach to stretching DNA molecules with the flow of fluid generated by spin coating. Well-stretched A DNA molecules were observed using atomic force microscopy. Substrate properties sensitively affected the stretching behavior of DNA. Our experimental findings revealed that a mica surface treated with crystal violet, a cationic dye molecule, is suitable to the spin-coating procedure for stretching DNA. Moreover, compared with relaxed DNA, we observed reduced height of the stretched DNA, which was attributed mainly to elongation force applied to the DNA molecules from the fluid flow and strong adhesion force between DNA and the substrate. This simple and effective method for preparing stretched DNA could be useful in physically mapping genomic DNA in a high throughput.

Aluminum Silicates↗
PubMed2002

Structure and dynamics of the fusion pores in live GH-secreting cells revealed using atomic force microscopy.

Earlier studies in live pancreatic acinar cells identified new cellular structures at the cell plasma membrane called 'pits' and 'depressions', where membrane-bound secretory vesicles dock and fuse to release vesicular contents. In the current study, using atomic force microscopy we identify similar structures at the plasma membrane of GH-secreting cells of the pituitary and implicate their involvement in hormone release. Pits containing 100-200 nm in diameter depressions or fusion pores were identified in resting GH-secreting cells. Following stimulation of secretion the size of depression enlarged and gold-tagged GH antibody were found to bind to the pit structures in the stimulated GH cells. This study documents for the first time the presence of these structures and their involvement in secretions in a neuroendocrine cell.

Actins↗
PubMed1993

New approach for atomic force microscopy of membrane proteins. The imaging of cholera toxin.

We demonstrate that supported synthetic phospholipid bilayers, which are stabilized by lateral cross-linking in both leaflets, can be used for specimen preparation for atomic force microscopy of purified membrane proteins with high stability and excellent reproducibility under water or low-salt buffer. A bilayer containing 1,2-dipentacosa-10,12-diynoyl-phosphatidylcholine and 20 mol % ganglioside (GM1) was transferred onto the surface of mica from a Langmuir trough. Cholera toxin, both the B-subunit and the complete molecular randomly bound to the gangliosides, were imaged by atomic force microscopy in solution with a resolution of better than 2 nm. The pentameric structure of the B-subunit oligomers was well resolved. This result indicates that, with this preparation procedure, other membrane proteins may be studied at intermediate to high resolution under physiologically relevant conditions without the need for crystallization.

Cholera Toxin↗
PubMed2003

A combined atomic force microscopy and molecular dynamics simulation study on a plastocyanin mutant chemisorbed on a gold surface.

A mutant of copper plastocyanin, covalently bound to an Au (111) surface through an engineered disulfide bridge, was investigated in aqueous medium by atomic force microscopy (AFM) and molecular dynamics (MD) simulations. Tapping-mode AFM images revealed adsorption of single molecules which are homogeneously distributed over the substrate and strongly bound to gold and display uniform lateral size. A statistical analysis of the height of the macromolecules on the gold substrate evidenced a distribution around a mean value consistent with that expected from the crystallographic data and with a relatively large standard deviation. A 10-ns classical MD simulation of mutated plastocyanin, hydrated by a layer of water, covalently bound to a gold surface by one or two sulfur atoms, was performed. The simulations indicate that the bound protein retains, in both cases, its overall tertiary structure during the dynamic evolution. Moreover, the macro-molecule can assume different orientations with respect to the gold substrate, which give rise to a distribution of heights on the gold substrate. Experimental and MD simulation results are compared and discussed in connection with the topological and dynamical properties of the protein system.

Adsorption↗
PubMed2005

Ultra-high resolution imaging of DNA and nucleosomes using non-contact atomic force microscopy.

Visualisation of nano-scale biomolecules aids understanding and development in molecular biology and nanotechnology. Detailed structure of nucleosomes adsorbed to mica has been captured in the absence of chemical-anchoring techniques, demonstrating the usefulness of non-contact atomic force microscopy (NC-AFM) for ultra-high resolution biomolecular imaging. NC-AFM offers significant advantages in terms of resolution, speed and ease of sample preparation when compared to techniques such as cryo-electron microscopy and X-ray crystallography. In the absence of chemical modification, detailed structure of DNA deposited on a gold substrate was observed for the first time using NC-AFM, opening up possibilities for investigating the electrical properties of unmodified DNA.

Aluminum Silicates↗
PubMed2004

Analysis of environmental particles by atomic force microscopy, scanning and transmission electron microscopy.

Due to their large specific surface and their abundance, micro and nano particles play an important role in the transport of micropollutants in the environment. Natural particles are usually composed of a mixture of inorganic amorphous or crystalline material (mainly FeOOH, Fe(x)Oy, Mn(x)Oy and clays) and organic material (humics and polysaccharides). They all tend to occur as very small particles (1-1,000 nm in diameter). Most natural amorphous particles are unstable and tend to transform with time towards more crystalline forms, either by aging or possibly, by dissolution and re-crystallization. Such transformations affect the fate of sorbed micropollutants and the scavenging properties are therefore changed. As these entities are sensitive to dehydration (aggregation, changes in the morphology), it is highly important to observe their morphology in their natural environment and understand their composition at the scale of the individual particles. Also for the understanding and optimization of water treatment technologies, the knowledge of the occurrence and behavior of nano-particles is of high importance. Some of the possible particle analysis methods are presented: aggregation processes, biomineralization, bacterial adhesion, biofilms in freshwaters, ferrihydrite as heavy metals remover from storm water. These examples demonstrate the capabilities and focus of the microscopes. Atomic Force Microscopy (AFM) allows to analyze the particles in their own environment, meaning in air or in the water. Thus, native aspects of particles can be observed. As well, forces of interactions between particles or between particles and other surfaces such as membranes will be highly valuable data. Scanning Electron Microscopy (SEM) and for higher lateral resolution, Transmission Electron Microscopy (TEM) allow measurement of the morphology and composition. Especially, TEM coupled with Electron Energy Loss Spectroscopy (TEM-EELS) is a powerful technique for elemental analysis. Finally, general guidelines for the effective use of microscopic techniques are provided.

Bacteria↗
PubMedDate not supplied

Structural characterization of self-assembled polypeptide films on titanium and glass surfaces by atomic force microscopy.

Chemically modified biomaterial surfaces (titanium and glass) covered with polyelectrolyte self-assembled films formed by the alternating adsorption of cationic poly-L-lysine (PLL) and anionic poly-L-glutamic acid (PGA) were structurally characterized by atomic force microscopy. Complementary information concerning the thickness and layer-by-layer growth of the films was provided by optical waveguide light-mode spectroscopy. The frequently used ex situ and the rarely used in situ build-up methods were compared. Important aspects of the industrial applicability of these films, their stability in time, and possible differences in their morphology were investigated. The films revealed a granular pattern, with grain diameters of 270 +/- 87 nm for glass (up to 8 bilayers) and 303 +/- 89 nm for titanium (up to 10 bilayers), independently of the build-up procedure. Both surfaces displayed a rehydration capability, the titanium surface exhibiting a better stability in time. The high roughness values observed at acidic or basic pH are related to the degree of ionization of PGA and PLL.

Adsorption↗
PubMed2000

The mechanism of cellulase action on cotton fibers: evidence from atomic force microscopy.

Two cellulases from Trichoderma reesei--an exoglucanase, CBH I, and an endoglucanase, EG II--alone and in combination were incubated with cotton fibers. The effects of the cellulases on the surfaces of the cotton fibers were examined by atomic force microscopy. At high magnification, the physical effects on the fibers caused by the two types of enzymes were considerably different. Treatment with CBH I resulted in the appearance of distinct pathways or tracks along the length of the macrofibril. Treatment with EG II appeared to cause peeling and smoothing of the fiber surface. In combination, their effect was observed to be greatest when both enzymes were present simultaneously. When fibers smoothed by treatment with EG II were treated subsequently with CBH I, further evidence of path way formation caused by the action of CBH I along the fibers was observed. Incubation with a cellulase from Thermotoga maritima that lacks a cellulose binding domain had no effect on the surface of cotton fibers. These images provide the first physical evidence of differences in the effect of cellulase components action on the surface of cotton fibers and provide evidence for the movement or tracking of CBH I along the fibers. The first AFM image of CBH I molecules are presented.

Cellulase↗
PubMed1998

Fractal analysis of pharmaceutical particles by atomic force microscopy.

PURPOSE: Reliable methods are needed to characterize the surface roughness of pharmaceutical solid particles for quality control and for finding the correlations with other properties. In this study, we used fractal analysis to describe the surface roughness. METHODS: Atomic force microscopy (AFM) was used to obtain three-dimensional surface profiles. The variation method was used to calculate fractal dimensions. We have measured fractal dimensions of four granule samples, four powders, and two freeze-dried powders. RESULTS: A computer-program was written to implement the variation method. The implementation was verified using the model surfaces generated by fractional Brownian motion. The fractal dimensions of most particles and granules were between 2.1 and 2.2, and were independent of the scan size we measured. The freeze-dried samples, however showed wide variation in the values of fractal dimension, which were dependent on the scan size. As scan size increased, the fractal dimension also increased up to 2.5. CONCLUSIONS: Fractal analysis can be used to describe surface roughness of pharmaceutical particles. The variation method allows calculation of reliable fractal dimensions of surface profiles obtained by AFM. Careful analysis is required for the estimation of fractal dimension, since the estimates are dependent on the algorithm and the digitized model size (i.e., number of data points of the measured surface profile) used. The fractal dimension of pharmaceutical materials is also a function of the observation scale i.e., the scan size) used in the profile measurement. The multi-fractal features and the scale-dependency of fractal dimension result from the artificial processes controlling the surface morphology.

Algorithms↗
PubMed1999

Atomic force microscopy: a powerful tool to observe biomolecules at work.

Atomic force microscopes (AFMs) move a sharp tip attached to a soft cantilever in a TV-raster-like pattern over a surface and record deflections of the tip that correspond to the surface topography. When operated in physiological solutions, an AFM allows biomolecules to be observed in their native environment. Progress in instrumentation, sample-preparation methods and recording conditions has provided images of biomolecules and their assemblies that reveal submolecular details. In addition, the AFM allows conformational changes to be observed directly. This article discusses these points and illustrates them with some pertinent examples.

Microscopy, Atomic Force↗