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PubMed2003

DNA height in scanning force microscopy.

The measured height of DNA molecules adsorbed on a mica substrate by scanning probe microscopy is always less than the theoretical diameter. In this paper we show that, when imaged in ambient conditions, the molecules are usually immersed in the salt layer used to adsorb them to the substrate. This layer distorts the measurement of DNA height and is the main source of error but not the only one. We have performed different experiments to study this problem using two scanning force techniques: non-contact tapping mode in air and jumping mode in aqueous solution, where the dehydration phenomena is minimized. Height measurements of DNA in air using tapping mode reveal a height of 0.7+/-0.2nm. This value increases up to 1.5+/-0.2nm when the salt layer, in which the molecules are embedded, is removed. Jumping experiments in water give a value of 1.4+/-0.3nm when the maximum applied force is 300pN and 1.8+/-0.2nm at very low forces, which confirms the removal of the salt layer. Still, in all our experiments, the measured height of the DNA is less than the theoretical value. Our results show that although the salt layer present is important, some sample deformation due to either the loading force of the tip or the interaction with the substrate is also present.

Adsorption↗
PubMed1995

Human dentine as a hydrogel.

The pores (tubules) of human dentine in 0.02-cm planoparallel sections of newly extracted permanent teeth were investigated. By the conventional scanning electron microscopy these pores appear empty, but by the newly developed scanning-probe microscopy the presence of a complex matrix could be established. By measuring the transport of neutral myoglobin by diffusion alone and diffusion+bulk flow, the area of dentine occupied by the matrix was calculated to be 1.9 +/- 0.9% and 2.3 +/- 0.5%, respectively. The hydraulic conductivity was surprisingly small, 1.35 +/- 0.55 x 10(-7) ml/(s.cm2 dentine) at a pressure difference of 0.1 kPa across a 1-cm thick section. This suggests a hydrogel with a relatively dense network, the width of meshes estimated at 2 x 30 nm. In line with this concept, enzymatic degradation of the organic matter increased the hydraulic conductivity 3000 times. By studying the transport of negatively charged myoglobin, the matrix was calculated to carry 18 mEq/l of positive charges. Due to the consequent attraction of small, negative ions and thence of water, the pressure within the matrix would be about 1.33 kPa, a force which will act to immobilize the water in the channels. The concept of a hydrogel in the dentine tubules was also supported by the finding that shielding the charges with bathing media of high ionic strength reduced the hydraulic conductivity.

Adolescent↗
PubMed1975

Scanning electron microscopy and electron probe microanalysis of calcified alpha-elastin coacervates.

The uptake of calcium and phosphorus from a serum calcification medium by coacervated alpha-elastin was studied by electron probe microanalysis and scanning electron microscopy. Calcium and phosphorus were bound by the coacervate in ratios similar to that of hydroxyapatite. A significant difference was observed in the secondary electron image of the coacercules from the serum during calcification of the coacervate.

Animals↗
PubMed2000

Formation of three-dimensional protein-lipid aggregates in monolayer films induced by surfactant protein B.

This study focuses on the structural organization of surfactant protein B (SP-B) containing lipid monolayers. The artificial system is composed of the saturated phospholipids dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) in a molar ratio of 4:1 with 0.2 mol% SP-B. The different "squeeze-out" structures of SP-B were visualized by scanning probe microscopy and compared with structures formed by SP-C. Particularly, the morphology and material properties of mixed monolayers containing 0.2 mol% SP-B in a wide pressure range of 10 to 54 mN/m were investigated revealing that filamentous domain boundaries occur at intermediate surface pressure (15-30 mN/m), while disc-like protrusions prevail at elevated pressure (50-54 mN/m). In contrast, SP-C containing lipid monolayers exhibit large flat protrusions composed of stacked bilayers in the plateau region (app. 52 mN/m) of the pressure-area isotherm. By using different scanning probe techniques (lateral force microscopy, force modulation, phase imaging) it was shown that SP-B is dissolved in the liquid expanded rather than in the liquid condensed phase of the monolayer. Although artificial, the investigation of this system contributes to further understanding of the function of lung surfactant in the alveolus.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Covalent anchoring of proteins onto gold-directed NHS-terminated self-assembled monolayers in aqueous buffers: SFM images of clathrin cages and triskelia.

N-Hydroxysuccinimide-terminated self-assembled monolayers with linear (CH2)10 chains were prepared on ultraflat Au(111) surfaces from dithiobis(succinimidylundecanoate). These monolayers, which are covalently chemisorbed to gold via thiolate bonds, form a highly reactive amino-group specific carpet at the liquid-solid interface. Proteins bind to it covalently in aqueous buffers under mild conditions; this provides a (general) procedure for protein immobilization for scanning probe microscopy. Using this technique, we have obtained what we believe are the first scanning force microscopy images of clathrin cages and of their in situ disassembly, yielding typical triskelia under non-denaturing conditions.

Animals↗
PubMed1993

Analysis of biomedical polymer surfaces: polyurethanes and plasma-deposited thin films.

The surface characterization of biomaterials is important for understanding the biological reactivity of surfaces and for monitoring surface reproducibility and contamination. Electron spectroscopy for chemical analysis (ESCA), secondary ion mass spectrometry (SIMS), contact-angle methods, vibrational spectroscopic methods, and scanning probe microscopies are briefly reviewed. Examples are presented using these methods to characterize RF plasma-deposited surfaces based upon acetone and oxygen for cell culture and Biomer¿ surfaces.

Biocompatible Materials↗
PubMed2003

The birth and evolution of surface science: child of the union of science and technology.

This article is an account of the birth and evolution of surface science as an interdisciplinary research area. Surface science emanated from the confluence of concepts and tools in physics and chemistry with technological innovations that made it possible to determine the structure and properties of surfaces and interfaces and the dynamics of chemical reactions at surfaces. The combination in the 1960s and 1970s of ultra-high-vacuum (i.e., P < 10(-7) Pascal or 10(-9) Torr) technology with the recognition that electrons in the energy range from 50 to 500 eV exhibited inelastic collision mean free paths of the order of a few angstroms fostered an explosion of activity. The results were a reformulation of the theory of electron solid scattering, the nearly universal use of electron spectroscopies for surface characterization, the rise of surface science as an independent interdisciplinary research area, and the emergence of the American Vacuum Society (AVS) as a major international scientific society. The rise of microelectronics in the 1970s and 1980s resulted in huge increases in computational power. These increases enabled more complex experiments and the utilization of density functional theory for the quantitative prediction of surface structure and dynamics. Development of scanning-probe microscopies in the 1990s led to atomic-resolution images of macroscopic surfaces and interfaces as well as videos of atoms moving about on surfaces during growth and diffusion. Scanning probes have since brought solid-liquid interfaces into the realm of atomic-level surface science, expanding its scope to more complex systems, including fragile biological materials and processes.

Journal Article↗
PubMed2002

Chiral recognition in dimerization of adsorbed cysteine observed by scanning tunnelling microscopy.

Stereochemistry plays a central role in controlling molecular recognition and interaction: the chemical and biological properties of molecules depend not only on the nature of their constituent atoms but also on how these atoms are positioned in space. Chiral specificity is consequently fundamental in chemical biology and pharmacology and has accordingly been widely studied. Advances in scanning probe microscopies now make it possible to probe chiral phenomena at surfaces at the molecular level. These methods have been used to determine the chirality of adsorbed molecules, and to provide direct evidence for chiral discrimination in molecular interactions and the spontaneous resolution of adsorbates into extended enantiomerically pure overlayers. Here we report scanning tunnelling microscopy studies of cysteine adsorbed to a (110) gold surface, which show that molecular pairs formed from a racemic mixture of this naturally occurring amino acid are exclusively homochiral, and that their binding to the gold surface is associated with local surface restructuring. Density-functional theory calculations indicate that the chiral specificity of the dimer formation process is driven by the optimization of three bonds on each cysteine molecule. These findings thus provide a clear molecular-level illustration of the well known three-point contact model for chiral recognition in a simple bimolecular system.

Cysteine↗
PubMed2003

Simultaneous in situ total internal reflectance fluorescence/atomic force microscopy studies of DPPC/dPOPC microdomains in supported planar lipid bilayers.

In situ simultaneous total internal reflectance fluorescence and in situ scanning probe microscopy performed on a phase-segregated supported planar lipid bilayer enabled direct in situ real-time correlated topographical and fluorescence images of nanometer-sized gel and fluid-phase lipid domains, presaging future in situ studies of membrane protein assemblies by single molecule imaging.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1996

Atomic force microscopy of DNA, nucleoproteins and cellular complexes: the use of functionalized substrates.

Progress towards rapid and simple characterization of biomolecular samples by scanning probe microscopy is impeded mainly by limitations of the current approach to sample preparation. We are working on approaches based on chemical functionalization of mica. Treatment of mica with aminopropyltriethoxy silane (APTES) makes the surface positively charged (AP-mica) and able to hold DNA in place for imaging, even in water. We have shown that AP-mica is an appropriate substrate for numerous nucleoprotein complexes as well. The AFM images of the complex of DNA with RecA protein are stable and indicate a structural periodicity for this filament. AP-mica holds strongly such large DNA complexes as kinetoplast DNA (kDNA) and is an appropriate substrate for their imaging with AFM. We have further develop this approach for making hydrophobic substrates. Silylation of mica surface with hexamethyldisilazane (Me-mica) allowed us to get AFM images of chlorosomes, an antenna complex isolated from green photosynthetic bacteria. Me-mica may be converted into a positively charged substrate after treatment with water solutions of tetraethylammonium bromide or cetyltrimethylammonium bromide. These activated surfaces show high activity towards binding the DNA molecules.

Aluminum Silicates↗
PubMed2004

Carbon nanotube scanning probe for imaging in aqueous environment.

Carbon nanotubes (CNTs) used as a probe for scanning probe microscopy has become one of the many potential usages of CNTs that is finding real applications in scientific research and industrial communities. It has been proposed that the unique mechanical buckling properties of the CNT would lessen the imaging force exerted on the sample and, thus, make CNT scanning probes ideal for imaging soft materials, including biological samples in liquid environments. The hydrophobic nature of the CNT graphitic sidewall is clearly chemically incompatible with the aqueous solution requirements in some biological imaging applications. In this paper, we present electron micrograph results demonstrating the instability of CNT scanning probes when submerged in aqueous solution. Moreover, we also introduce a novel approach to resolve this chemical incompatibility problem. By coating the CNT probe with ethylenediamine, thus rendering the CNT probe less hydrophobic, we demonstrate the liquid imaging capability of treated CNT probes. Experimental data for imaging in aqueous solutions are presented, which include an ultrathin Ir film and DNA molecules on a mica surface.

Ethylenediamines↗
PubMed2001

Thermal imaging of composites.

Active thermal imaging techniques and their applications to composite materials are reviewed. The techniques included are transient thermography, scanning thermal microscopy and scanning thermal probe microscopy. The factors that affect the images produced by both pulsed and periodic forms of active heating are considered. For pulsed heating, experimental and numerical modelling results for carbon fibre-reinforced plastic are used to show how the resolution of subsurface features depends on their size and depth and on the anisotropy in thermal materials properties common in such composites. For periodic heating, thermal wave characteristics are introduced to show how the resolution of subsurface features also depends on modulation frequency and focal spot radius. Examples are given of the applications of scanning thermal microscopy and scanning thermal probe microscopy that illustrate the potential of these techniques for the imaging of composite materials.

Journal Article↗
PubMed2003

Monolayer assemblies of a de novo designed 4-alpha-helix bundle carboprotein and its sulfur anchor fragment on Au(111) surfaces addressed by voltammetry and in situ scanning tunneling microscopy.

Mapping and control of proteins and oligonucleotides on metallic and nonmetallic surfaces are important in many respects. Electrochemical techniques based on single-crystal electrodes and scanning probe microscopies directly in aqueous solution (in situ SPM) have recently opened perspectives for such mapping at a resolution that approaches the single-molecule level. De novo design of model proteins has evolved in parallel and holds promise for testing and controlling protein folding and for new tailored protein structural motifs. In this report we combine these two strategies. We present a scheme for the synthesis of a new 4-alpha-helix bundle carboprotein built on a galactopyranoside derivative with a thiol anchor aglycon suitable for surface immobilization on gold. The carboprotein with thiol anchor in monomeric and dimeric (disulfide) form, the thiol anchor alone, and a sulfur-free 4-alpha-helix bundle carboprotein without thiol anchor have been prepared and investigated for comparison. Cyclic and differential pulse voltammetry (DPV) of the proteins show desorption peaks around -750 mV (SCE), whereas the thiol anchor desorption peak is at -685 mV. The peaks are by far the highest for thiol monomeric 4-alpha-helix bundle carboprotein and the thiol anchor. This pattern is supported by capacitance data. The DPV and capacitance data for the thiolated 4-alpha-helix bundle carboproteins and the thiol anchor hold a strong Faradaic reductive desorption component as supported by X-ray photoelectron spectroscopy. The desorption peak of the sulfur-free 4-alpha-helix bundle carboprotein, however, also points to a capacitive component. In situ scanning tunneling microscopy (in situ STM) of the thiol anchor discloses an adlayer with small domains and single molecules ordered in pin-striped supramolecular structures. In situ STM of thiolated 4-alpha-helix bundle carboprotein monomer shows a dense monolayer in a broad potential range on the positive side of the desorption potential. The coverage decreases close to this potential and single-molecule structures become apparent. The in situ STM contrast is also strengthened, indicative of a new redox-based tunneling mechanism. The data overall suggest that single-molecule mapping of natural and synthetic proteins on well-characterized surfaces by electrochemistry and in situ STM is within reach.

Amino Acid Sequence↗
PubMed2001

AFM imaging in solution of protein-DNA complexes formed on DNA anchored to a gold surface.

A chemical procedure for anchoring DNA molecules to gold surfaces was used to facilitate the imaging of DNA and DNA-protein complexes in buffer solution by tapping mode atomic force microscopy (TMAFM). For preparing flat gold surfaces, a novel approach was employed by evaporating small amounts of gold onto freshly cleaved mica to give flat films that were stable under aqueous buffer conditions. The thickness of the investigated films ranged from 1 to 10 nm. For typical films of 4-6 nm, which were stable under aqueous buffer conditions, the root mean square (RMS) roughness ranged between 0.25 and 0.5 nm, as measured by atomic force microscopy (AFM). This roughness is comparable to that of obtained by the template stripped gold (TSG) technique, which is widely used in scanning probe microscopy but involves more preparation steps. In order to visualize DNA and DNA-protein complexes by TMAFM, the DNA was chemisorbed to the gold surface through a linker carrying a terminal thiol group at the 5'-end of each of the DNA strands. The modified DNA fragments were bound to the gold films and imaged in buffer solution, while unmodified DNA could not be visualized. Since the DNA was not dried during the process, it can be assumed that its native conformation was retained. This mode of anchoring did not prevent interaction with proteins, as confirmed by the observation that the topology of a complex formed by adding the protein to a surface-anchored DNA was the same as that obtained by anchoring a pre-formed complex to the gold surface. We attribute this observation to the fact that the DNA is anchored to the gold surfaces only through its ends, therefore the DNA-support interaction is minimized but imaging is still possible.

Bacterial Proteins↗
PubMed2003

EC STM investigations of corrosion due to chloride solutions on thin CrN coatings.

The focus of the investigations presented is to evaluate local alterations caused by chloride ions affecting thin, magnetron-sputtered CrN layers. Scanning-probe microscopy and analysis techniques are used for this estimation. Thin CrN layers were deposited by reactive magnetron sputtering. They were investigated in electrochemical scanning tunnelling microscopy (EC STM) by cyclic voltammetry in 1 mol L(-1) NaCl. Simultaneously, the surface topography changes were recorded with STM. Above 100 mV the anodic oxidation leads to formation of chromium(III) hydroxide and at sample potentials above 350 mV oxidation of Cr(OH)(2) and Cr(OH)(3) towards chromium(VI) as a soluble chromate starts. Transpassive dissolution of the coating takes place above 900 mV. Yellow colour of the electrolyte is a visible sign for the formation of chromium(VI). Changes of the surface topography indicate the formation of surface layers at anodic potentials. At cathodic potentials increase in current is measured due to the reduction of chromium(III) hydroxide to divalent chromium and metallic chromium. Roughness of surface topography increases.Follow-up explorations with scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), atomic-force microscopy (AFM), scanning tunnelling microscopy/scanning tunnelling spectroscopy (STM/STS) and X-ray photoelectron spectroscopy (XPS) not only evidence the formation of various chromium oxides, but also indicate the existence of chromium hydroxide.

Journal Article↗
PubMed2002

Self-sensing piezoresistive cantilever and its magnetic force microscopy applications.

A newly developed Si self-sensing piezoresistive cantilever is presented. Si piezoresistive cantilevers for scanning microscopy are fabricated by Si micro-machining technique. The sensitivity of the piezoresistive cantilever is comparable to the current laser detecting system. Topographic images are successfully obtained with the piezoresistive cantilever and some comparisons are made with the laser detecting system. Furthermore, the magnetic film (Co-Cr-Pt) is coated on the tip of the piezoresistive cantilever for magnetic force microscopy (MFM) application. The magnetic images are successfully obtained with the self-sensing MFM piezoresistive cantilever. The self-sensing piezoresistive cantilevers have been successfully applied in scanning probe microscopy and MFM.

Journal Article↗
PubMed2004

Near-field infrared imaging and spectroscopy of a thin film polystyrene/poly(ethyl acrylate) blend.

The application of broadband, near-field infrared microscopy to the characterization of the mesoscale structure of a thin film polymer blend is described. Key features of this instrument, which couples the nanoscale spatial resolution of scanning probe microscopy with the chemical specificity of vibrational spectroscopy, include broad tunability and bandwidth, parallel spectral detection for high image acquisition rates, and infrared-transparent aperture probes. Nearfield spectral transmission images of a thin film of polystyrene/poly(ethyl acrylate) acquired in the C-H stretching region are reported. An assessment of the relative importance of transmission image contrast mechanisms is a significant aim of this work. Analysis of the near-field infrared spectra indicates that the image contrast in the C-H stretching region is largely due to near-field coupling and/or scattering effects. Identification and differentiation of the operative contrast mechanisms on the basis of their relative dependence on wavelength is discussed. Analysis of the contrast attributed to absorption is consistent with the chemical morphology of this sample derived from previous chemical modification/atomic force microscopy studies.

Journal Article↗
PubMed2002

Monolayers of a de novo designed 4-alpha-helix bundle carboprotein and partial structures on Au(111)-surfaces.

Mapping of structure and function of proteins adsorbed on solid surfaces is important in many contexts. Electrochemical techniques based on single-crystal metal surfaces and in situ scanning probe microscopies (SPM) have recently opened new perspectives for mapping at the single-molecule level. De novo design of model proteins has evolved in parallel and holds promise for test and control of protein folding and for new tailored protein structural motifs. These two strategies are combined in the present report. We present a synthetic scheme for a new 4-alpha-helix bundle carboprotein built on a galactopyranoside derivative with a thiol anchor aglycon suitable for surface immobilization on gold. The galactopyranoside with thiol anchor and the thiol anchor alone were prepared for comparison. Voltammetry of the three molecules on Au(111) showed reductive desorption peaks caused by monolayer adsorption via thiolate-Au bonding. In situ STM of the thiol anchor disclosed an ordered adlayer with clear domains and molecular features. This holds promise, broadly for single-molecule voltammetry and the SPM and scanning tunnelling microscopy (STM) of natural and synthetic proteins.

Electrochemistry↗