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PubMed2003

Atomic force microscopy study of living diatoms in ambient conditions.

We present the first in vivo study of diatoms using atomic force microscopy (AFM). Three chain-forming, benthic freshwater species -Eunotia sudetica, Navicula seminulum and a yet unidentified species - are directly imaged while growing on glass slides. Using the AFM, we imaged the topography of the diatom frustules at the nanometre range scale and we determined the thickness of the organic case enveloping the siliceous skeleton of the cell (10 nm). Imaging proved to be stable for several hours, thereby offering the possibility to study long-term dynamic changes, such as biomineralization or cell movement, as they occur. We also focused on the natural adhesives produced by these unicellular organisms to adhere to other cells or the substratum. Most man-made adhesives fail in wet conditions, owing to chemical modification of the adhesive or its substrate. Diatoms produce adhesives that are extremely strong and robust both in fresh- and in seawater environments. Our phase-imaging and force-pulling experiments reveal the characteristics of these natural adhesives that might be of use in designing man-made analogues that function in wet environments. Engineering stable underwater adhesives currently poses a major technical challenge.

Adhesiveness↗
PubMed1998

The mechanical stability of immunoglobulin and fibronectin III domains in the muscle protein titin measured by atomic force microscopy.

The domains of the giant muscle protein titin (connectin) provide interaction sites for other sarcomeric proteins and fulfill mechanical functions. In this paper we compare the unfolding forces of defined regions of different titin isoforms by single-molecule force spectroscopy. Constructs comprising six to eight immunoglobulin (Ig) domains located in the mechanically active I-band part of titin are compared to those containing fibronectin III (Fn3) and Ig domains from the A-band part. The high spatial resolution of the atomic force microscope allows us to detect differences in length as low as a few amino acids. Thus constructs of different lengths may be used as molecular rulers for structural comparisons with other modular proteins. The unfolding forces range between 150 and 300 pN and differ systematically between the constructs. Fn3 domains in titin exhibit 20% lower unfolding forces than Ig domains. Fn3 domains from tenascin, however, unfold at forces only half those of titin Fn3 domains. This indicates that the tightly folded titin domains are designed to maintain their structural integrity, even under the influence of stretching forces. Hence, at physiological forces, unfolding is unlikely unless the forces are applied for a long time (longer than minutes).

Biomechanical Phenomena↗
PubMed2004

Chemical treatment of mica for atomic force microscopy can affect biological sample conformation.

An important aspect in the preparation of substrate materials to use in atomic force microscopy lies in the question of interactions introduced by treatments designed to immobilize the sample over the substrate. Here we used a mica substrate that was chemically modified with cationic nickel to immobilize actin filaments (F-actin). Chemical modification could be followed quantitatively by measuring the interaction force between the scanning tip and the mica surface. This approach allowed us to observe polymeric F-actin in a structure that resembles an actin gel. It also improved sample throughput and conferred sample stability as well as repeatability from run to run.

Actin Cytoskeleton↗
PubMed1996

Freeze-substitution as a preparative technique for immunoelectronmicroscopy: evaluation by atomic force microscopy.

Cryofixation followed by freeze substitution in osmium tetroxide was evaluated as a method for preparing biological specimens for immunoelectronmicroscopy. Samples were rapidly frozen by impact onto a sapphire block cooled with liquid nitrogen, substituted at -80 degrees C in acetone containing osmium tetroxide, and embedded in epoxy resin. With this protocol, excellent ultrastructure can be combined with localization of antigens that otherwise would be inactivated by the osmium, but labeling may need to be enhanced by chemically etching the sections prior to staining. The effects of etching on various structures in the sections were investigated by examining the sections with atomic force microscopy, an approach that yields three-dimensional views of the surface of the section. A considerable part of the section was removed or collapsed by the etching, and these effects occurred differentially in several components of the tissue and with different etching protocols. Nevertheless, the results suggest that the partial removal of the plastic by etching of freeze-substituted tissue can be explored as a method for exposing fine biological structures for observation with atomic force microscopy.

Animals↗
PubMed2003

Atomic force microscopy analysis and confocal Raman microimaging of coated pellets.

Polymer-coated pellets with different coating thicknesses have been studied regarding coating morphology and drug release properties with atomic force microscopy (AFM) and confocal Raman microscopy. The results were compared with those from scanning electron microscopy (SEM) and drug release profiles, which have been measured previously for these systems and found to vary depending on coating thickness. Results from AFM studies indicated that these pellets differ in the amount of crystalline material on the surface of the coating. The amount was found to be highest on the pellet with the thinnest coating. Confocal Raman microscopy studies confirmed that the active component (remoxipride hydrochloride monohydrate) is present at or close to the surface and that the amount is higher for the thinnest coating. AFM studies in aqueous media showed that the crystalline material on the surface was almost instantaneously dissolved and released into the liquid. AFM has proven to be a powerful tool in the study of the surface of dry formulations and in the study of the controlled release mechanism of a pharmaceutical in a liquid cell. The method can be combined with Raman, giving the added possibility to identify the chemical composition in selected small areas of the coating surface.

Crystallization↗
PubMed2006

Phase structure and enzymatic degradation of poly(L-lactide)/atactic poly(3-hydroxybutyrate) blends: an atomic force microscopy study.

Phase structures and enzymatic degradation of poly(l-lactide) (PLLA)/atactic poly(3-hydroxybutyrate) (ata-PHB) blends with different compositions were characterized by using atomic force microscopy (AFM). Differential scanning calorimetry (DSC) thermograms of PLLA/ata-PHB blends with different compositions showed two glass transition temperatures, indicating that the PLLA/ata-PHB blends are immiscible in the melt. Surface morphologies of the thin films for PLLA/ata-PHB blends were determined by AFM. Phase separated morphology was recognized from the AFM topography and phase images. The domain size of the components was dependent on the blend ratio. Enzymatic degradation of the PLLA/ata-PHB blends was performed by using both PHB depolymerase and proteinase K. Either PLLA or ata-PHB domains were eroded depending on the kinds of enzyme. Surface morphologies after enzymatic degradation have revealed the phase structure along the depth direction. Enzymatic adsorption of PHB depolymerase was examined on the surface of PLLA/ata-PHB blends. The enzyme molecules were found on both domains of the binary blends. The larger number of enzyme molecules was found on the PLLA domains relative to those on the ata-PHB domains, suggesting the higher affinity of the enzyme against PLLA domain.

Adsorption↗
PubMed1998

Imaging of the surface of living cells by low-force contact-mode atomic force microscopy.

The membrane surface of living CV-1 kidney cells in culture was imaged by contact-mode atomic force microscopy using scanning forces in the piconewton range. A simple procedure was developed for imaging of the cell surface with forces as low as 20-50 pN, i.e., two orders of magnitude below those commonly used for cell imaging. Under these conditions, the indentation of the cells by the tip could be reduced to less than l0 nm, even at the cell center, which gave access to the topographic image of the cell surface. This surface appeared heterogeneous with very few villosities and revealed, only in distinct areas, the submembrane cytoskeleton. At intermediate magnifications, corresponding to 20-5 microm scan sizes, the surface topography likely reflected the organization of submembrane and intracellular structures on which the plasma membrane lay. By decreasing the scan size, a lateral resolution better than 20 nm was routinely obtained for the cell surface, and a lateral resolution better than 10 nm was obtained occasionally. The cell surface appeared granular, with packed particles, likely corresponding to proteins or protein-lipid complexes, between approximately 5 and 30 nm xy size.

Animals↗
PubMed1998

Direct detection of domains in phospholipid bilayers by grazing incidence diffraction of neutrons and atomic force microscopy.

The geometry of domains in phospholipid bilayers of binary (1:1) mixtures of synthetic lecithins with a difference in chain length of four methylene groups has been studied by two independent, direct and complementary methods. Grazing incidence diffraction of neutrons provided gel domain sizes of less than 10 nm in both the gel and the coexistence phase of the mixture, while no domains were detected for the fluid phase. For the coexistence region, the neutron data suggest that domains grow in number rather than in size with decreasing temperature. Atomic force microscopy was used to study gel phase size and shape of the domains. The domains imaged by atomic force microscopy exhibit a rather irregular shape with an average size of 10 nm, thus confirming the neutron results for this phase. The good agreement between atomic force microscopy and neutron results, despite the completely different nature of their observables, has potential for the future development of refined models for the interpretation of neutron data from heterogeneous membranes in terms of regularly spaced and spatially extended scatterers.

Calorimetry, Differential Scanning↗
PubMed1997

Tertiary structure of the hepatic cell protein fibrinogen in fluid revealed by atomic force microscopy.

Fibrinogen participates in important cellular physiological processes, such as cell adhesion and blood clotting. Although the primary and secondary structures of fibrinogen are known, its tertiary structure is yet to be determined. In attempts to understand the tertiary structure of this important hydrated cellular and plasma membrane protein, the present study using atomic force microscopy was carried out. The techniques presented in this manuscript may also be applicable to enhance the imaging of live cells as well as their subcellular components. The authors have imaged fibrinogen by Tapping Mode atomic force microscopy in fluid. Purified human fibrinogen, together with 15-nm colloidal gold particles serving as an internal calibration standard, were adhered to a poly-L-lysine substrate on freshly cleaved mica. Atomic force microscopy images were obtained using oxide-sharpened silicon nitride probes, either unaltered or with an electron beam deposited extended tip. Although various structures were observed, the predominant forms consisted of a bi- or trinodular slightly curved linear shape. Approximately 300 of these structures were observed with six different tips (1 unaltered and 5 electron beam deposited) and their lengths and heights were analyzed. The mean length of the fibrinogen molecules was 65.8 nm and the mean height was 3.4 nm. The quantitative measurements were little influenced by the shape of the tip, whereas the sharper electron beam deposited tips seemed to produce qualitatively superior images.

Fibrinogen↗
PubMed1997

Paired helical filaments are twisted ribbons composed of two parallel and aligned components: image reconstruction and modeling of filament structure using atomic force microscopy.

To study the structure of Alzheimer paired helical filaments (PHF) we examined isolated detergent-insoluble PHF using atomic force microscopy with image reconstruction. The reconstructed AFM images of Alzheimer PHF most closely resembled ribbon-like helices with thin edges. The presence of a conspicuous furrow in the PHF midline indicated that PHF were composed of two distinctive strands. Our present conception of the overall configuration of PHF is consistent with that proposed by Crowther and Wischik in 1985 but includes an essential component of the prevailing model: the presence of two strands. Thus, our new model of PHF structure, based on atomic force microscopy-derived data, indicates that the true structure of PHF is actually a hybrid of the prevailing PHF model and a thin helical ribbon.

Alzheimer Disease↗
PubMed2006

Bitumen morphologies by phase-detection atomic force microscopy.

Summary Bitumen is a complex mixture of hydrocarbons for which microstructural knowledge is incomplete. In an effort to detail this microstructure, 13 bitumens were analysed by phase-detection atomic force microscopy. Based on morphology, the bitumens could be classified into three distinct groups. One group showed fine domains down to 0.1 microm, another showed domains of about 1 microm, and a third group showed up to four different domains or phases of different sizes and shapes. No correlation was found between the atomic force microscopy morphology and the composition based on asphaltenes, polar aromatics, naphthene aromatics and saturates. A high correlation was found between the area of the 'bee-like' structures and the vanadium and nickel content in bitumen, and between the atomic force microscopy groups and the average size of molecular planes made of fused aromatics. The morphology and the molecular arrangements in bitumen thus appear to be partly governed by the molecular planes and the polarity defined by metallic cations.

Journal Article↗
PubMed1999

Surface fine structure of treated dentine investigated with tapping mode atomic force microscopy (TMAFM).

OBJECTIVES: The objective of this study was to investigate the surface morphology and roughness of human dentine treated with different dentine conditioning agents with tapping mode atomic force microscopy (TMAFM). METHODS: Dentine samples from third permanent molar teeth were prepared as flat disks. The samples were treated with different conditioning agents for 15-30 s: aqueous phosphoric acid and those supplied with three commercial adhesive products. The surface morphology and roughness was measured with tapping mode atomic force microscopy, a highly resolving method which minimises unwanted side effects of conventional AFM. RESULTS: The tubules, the areas between the tubules and the entire surface showed variations in surface morphology and roughness, depending on the conditioning agent used. These differences might affect the bonding properties of the agents in dentine/polymer systems. CONCLUSIONS: The surface structure, such as the tubules diameters and depths, the local height differences of the surface between the tubules and the overall mean surface roughness Ra varies between samples treated with different conditioning agents. All conditioning agents, however, led to exposed and open tubules. CLINICAL RELEVANCE: The direct observation of effects of conditioning agents on dentine is important in order to judge their performance under clinically relevant conditions-free of desiccation artefacts. A vast number of conditioning agents is available today. The differences in their actual effects on dentine surfaces, however, are less than clear to many users.

Acid Etching, Dental↗
PubMed2002

Visualization of complexes of Hoechst 33258 and DNA duplexes in solution by atomic force microscopy.

Tertiary structure changes in DNA duplexes, induced by Hoechst 33258 binding, have been examined by the use of atomic force microscopy. Besides minor groove binding, which is an established mode of binding for this drug, Hoechst 33258 has now been found to show another binding mode, which causes an unwinding of the duplex. When the drug concentration is as high as 0.5 microg/ml, the Hoechst 33258 molecule seems to function as a clamp for two DNA chains and forms a condensate. The condensate was found to have a toroidal shape. By surveying more than 100 microscopic images of such condensates formed in I microg/ml drug solution, a mechanism of toroidal condensate formation has been proposed.

Bisbenzimidazole↗
PubMed2000

Surface finishes on stainless steel reduce bacterial attachment and early biofilm formation: scanning electron and atomic force microscopy study.

Three common finishing treatments of stainless steel that are used for equipment during poultry processing were tested for resistance to bacterial contamination. Methods were developed to measure attached bacteria and to identify factors that make surface finishes susceptible or resistant to bacterial attachment and biofilm formation. Samples of the treated surfaces (sand-blasted, sanded, and electropolished) were exposed to natural bacterial populations from chicken carcass rinses to allow growth of bacteria and development of biofilms on the surfaces. The kinetics of bacterial growth during surface exposure was followed by UV-visible spectrophotometry, and counts of bacteria and early biofilm formation were measured following scanning electron microscopy (SEM). The surface morphology of the samples was analyzed by atomic force microscopy (AFM) with samples from each of the batches of treatments used in the SEM studies. Relative differences in the surface morphology, including fractal dimensions, Z ranges, roughness, and other measurements corresponded by treatment with the differences in reduction of bacterial counts shown by SEM. The surface types varied in affinity for bacteria, and both physical and electrochemical treatments improved resistance of stainless steel to bacterial attachment. Electropolished stainless steel was the least rough surface and showed significantly fewer bacterial cells and beginning biofilm formations than the other treated surfaces. Food safety could be improved if bacterial populations could be reduced during processing by increasing the use of materials that are resistant to bacterial contamination. These findings will aid equipment manufacturers and processors in selecting materials and finishes that are most resistant to bacteria and biofilm formation.

Animals↗
PubMed2005

Imaging DNA molecules on mica surface by atomic force microscopy in air and in liquid.

DNA molecules immobilized on mica surface by various methods have been observed by atomic force microscopy both in air and in liquid. Divalent cations and 3-aminopropyltriethoxysilane (APTES) modified mica surface have been used to immobilize the DNA molecules. Optimal DNA and divalent cations concentration for AFM imaging are presented. Among the different methods of modifying mica surface with APTES, the water solution modifying method appears to get the best results. When using high DNA concentration for AFM imaging, DNA networks can be formed. A simple method to extend long DNA molecules is demonstrated. The optimal imaging conditions and AFM operating techniques are discussed. Different DNA immobilizing methods have been compared and evaluated.

Air↗