Mechanical manifestations of rare atomic jumps in dynamic force microscopy.

The resonance frequency and the excitation amplitude of a silicon cantilever have been measured as a function of distance to a cleaved KBr(001) surface with a low-temperature scanning force microscope (SFM) in ultrahigh vacuum. We identify two regimes of tip-sample distances. Above a site-dependent critical tip-sample distance reproducible data with low noise and no interaction-induced energy dissipation are measured. In this regime reproducible SFM images can be recorded. At closer tip-sample distances, above two distinct atomic sites, the frequency values jump between two limiting curves on a timescale of tens of milliseconds. Furthermore, additional energy dissipation occurs wherever jumps are observed. We attribute both phenomena to rarely occurring changes in the tip apex configuration which are affected by short-range interactions with the sample. Their respective magnitudes are related to each other. A specific candidate two-level system is also proposed.

Rapid and label-free nanomechanical detection of biomarker transcripts in human RNA.

The availability of entire genome sequences has triggered the development of microarrays for clinical diagnostics that measure the expression levels of specific genes. Methods that involve labelling can achieve picomolar detection sensitivity, but they are costly, labour-intensive and time-consuming. Moreover, target amplification or biochemical labelling can influence the original signal. We have improved the biosensitivity of label-free cantilever-array sensors by orders of magnitude to detect mRNA biomarker candidates in total cellular RNA. Differential gene expression of the gene 1-8U, a potential marker for cancer progression or viral infections, has been observed in a complex background. The measurements provide results within minutes at the picomolar level without target amplification, and are sensitive to base mismatches. This qualifies the technology as a rapid method to validate biomarkers that reveal disease risk, disease progression or therapy response. We foresee cantilever arrays being used as a tool to evaluate treatment response efficacy for personalized medical diagnostics.

Energy level alignment at metal-octaethylporphyrin interfaces.

We studied the electronic structure of copper-octaethylporphyrin (CuEOP) adsorbed on three metal surfaces--Ag(001), Ag(111), and Cu(111)--by means of ultraviolet photoelectron spectroscopy (UPS). The adsorption-induced work function shifts saturate roughly beyond two monolayers. The saturation values are substrate dependent, negative, and range from -1.30 to -0.85 eV. This shift is larger than that for tetraphenylporphyrins. The two highest occupied molecular orbitals (HOMO and HOMO-1) of the organic are clearly resolved in the UPS spectra. The origin of the negative work function shift is discussed.

Study of the mechanical properties of myomesin proteins using dynamic force spectroscopy.

Myomesin is the most prominent structural component of the sarcomeric M-Band that is expressed in mammalian heart and skeletal muscles. Like titin, this protein is an intracellular member of the Ig-fibronectin superfamily, which has a flexible filamentous structure and which is largely composed of two types of domain that are similar to immunoglobulin (Ig)-like and fibronectin type III (FNIII) domains. Several myomesin isoforms have been identified, and their expression patterns are highly regulated both spatially and temporally. Particularly, alternative splicing in the central part of the molecule gives rise to an isoform, EH (embryonic heart)-myomesin, containing a serine and proline-rich insertion with no well-defined secondary structure, the EH segment. EH-myomesin represents the major myomesin isoform at embryonic stages of mammalian heart and is rapidly down-regulated around birth, but it is re-expressed in the heart of patients suffering from dilated cardio-myopathy. Here, in order to facilitate a better understanding of the physiological, and possibly pathological, functions of myomesin proteins, we explore the mechanical stability, elasticity and force-driven structural changes of human myomesin's sub-molecular segments using single-molecule force spectroscopy and protein engineering. We find that human myomesin molecules are composed of modules (Ig and FNIII), that are designed to withstand force and we demonstrate that the human cardiac EH segment functions like an additional elastic stretch in the middle part of the EH-myomesin and behaves like a random coil. Consequently myomesin isoforms (proteins with or without the EH segment) have different elastic properties, the EH-myomesin being the more compliant one. These findings imply that the compliance of the M-band increases with the amount of EH-myomesin it contains. So, we provide the evidence that not only titin but also other sarcomeric proteins have complicated visco-elastic properties depending on the contractile parameters in different muscle types.

Octyl-decorated Fréchet-type dendrons: a general motif for visualisation of static and dynamic behaviour using scanning tunnelling microscopy?

A detailed STM study of monolayers of 3,5-bis[(3,5-bisoctyloxyphenyl)methyloxy]benzaldehyde and 3,5-bis[(3,5-bisoctyloxyphenyl)methyloxy]benzyl alcohol adsorbed on graphite is presented. Very highly resolved scanning tunnelling microscopy images are observed at room temperature in air allowing the analysis of the conformation of the adsorbed molecules. These long-chain alkyl-decorated Fréchet-type dendrons are a powerful assembly motif and initially form a pattern based on trimeric units, assembled into hexagonal host structures with a pseudo-unit cell of seven molecules, one of which remains highly mobile. Over time, the supramolecular ordering changes from a trimeric into a dimeric pattern. The chirality arising from the adsorption onto a surface of the dendrons is discussed.

Sublattice identification in scanning force microscopy on alkali halide surfaces.

We propose and apply to the KBr(001) surface a new procedure for species recognition in scanning force microscopy (SFM) of ionic crystal surfaces which show a high symmetry of the charge arrangement. The method is based on a comparison between atomistic simulations and site-specific frequency versus distance measurements. First, by taking the difference of force-distance curves extracted at a few judiciously chosen surface sites we eliminate site-independent long-range forces. The obtained short-range force differences are then compared with calculated ones assuming plausible tip apex models. This procedure allows for the first time identification of the tip apex polarity and of the positive and negative sublattices in SFM images of the (001) cleavage surface of an ionic crystal with the rock salt structure.

Quantitative measurement of short-range chemical bonding forces.

We report direct force measurements of the formation of a chemical bond. The experiments were performed using a low-temperature atomic force microscope, a silicon tip, and a silicon (111) 7x7 surface. The measured site-dependent attractive short-range force, which attains a maximum value of 2.1 nanonewtons, is in good agreement with first-principles calculations of an incipient covalent bond in an analogous model system. The resolution was sufficient to distinguish differences in the interaction potential between inequivalent adatoms, demonstrating the ability of atomic force microscopy to provide quantitative, atomic-scale information on surface chemical reactivity.

Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates.

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level, predicting relative off-rates, potentially of great value for combinatorial chemistry and biology.

Dynamic force spectroscopy of single DNA molecules.

To explore the analytic relevance of unbinding force measurements between complementary DNA strands with an atomic force microscope, we measured the forces to mechanically separate a single DNA duplex under physiological conditions by pulling at the opposite 5'-ends as a function of the loading rate (dynamic force spectroscopy). We investigated DNA duplexes with 10, 20, and 30 base pairs with loading rates in the range of 16-4,000 pN/s. Depending on the loading rate and sequence length, the unbinding forces of single duplexes varied from 20 to 50 pN. These unbinding forces are found to scale with the logarithm of the loading rate, which is interpreted in terms of a single energy barrier along the mechanical separation path. The parameters describing the energy landscape, i.e. , the distance of the energy barrier to the minimum energy along the separation path and the logarithm of the thermal dissociation rate, are found to be proportional to the number of base pairs of the DNA duplex. These single molecule results allow a quantitative comparison with data from thermodynamic ensemble measurements and a discussion of the analytic applications of unbinding force measurements for DNA.

Plasmon transmissivity and reflectivity of narrow grooves in a silver film.

Surface plasmon (SP) reflectivity and transmissivity of narrow grooves in silver films are studied. The SP source is the probe of a scanning near-field optical microscope. Locally detected leakage radiation from the SP provides detailed information on the paths of SP propagation, in particular the influence of perturbations. Global detection provides representative average data on the SP properties of a given metal film and its structures. A groove of 200 nm width, for instance, reflects/transmits about 15%/80% of 'blue-green' SP radiation at normal incidence.

Specific antigen/antibody interactions measured by force microscopy.

Molecular recognition between biotinylated bovine serum albumin and polyclonal, biotin-directed IG antibodies has been measured directly under various buffer conditions using an atomic force microscope (AFM). It was found that even highly structured molecules such as IgG antibodies preserve their specific affinity to their antigens when probed with an AFM in the force mode. We could measure the rupture force between individual antibody-antigen complexes. The potential and limitations of this new approach for the measurement of individual antigen/antibody interactions and some possible applications are discussed.

Binding strength between cell adhesion proteoglycans measured by atomic force microscopy.

Measurement of binding forces intrinsic to adhesion molecules is necessary to assess their contribution to the maintenance of the anatomical integrity of multicellular organisms. Atomic force microscopy was used to measure the binding strength between cell adhesion proteoglycans from a marine sponge. Under physiological conditions, the adhesive force between two cell adhesion molecules was found to be up to 400 piconewtons. Thus a single pair of molecules could hold the weight of 1600 cells. High intermolecular binding forces are likely to form the basis for the integrity of the multicellular sponge organism.

Sled-type motion on the nanometer scale: determination of dissipation and cohesive energies of c60.

The tribological properties of C(60) on the mesoscopic scale were investigated with a scanning force microscope, which allowed simultaneous measurements of normal and lateral forces under ultrahigh-vacuum conditions. Islands of C(60), deposited on NaCl(001), could be moved by the action of the probing tip in a controlled way. Different modes of motion, such as translation and rotation, were observed. An extremely small dissipation energy of about 0.25 millielectron volt per molecule and a cohesive energy of 1.5 electron volts were determined in these nanometer-scale experiments. The corresponding shear strength of 0.05 to 0.1 megapascal was smaller by one order of magnitude than typical values of boundary lubricants. For C(60) on graphite, disruption of the islands was observed and collective motion of the islands could not be achieved. These results could find use in the field of nanotechnology; for example, C(60) islands could be developed into a sled-type transport system on the nanometer scale.

Creation of liquid crystal waveguides with scanning force microscopy.

The rubbing of a polymer layer, a commonly applied process, leads to an anisotropic surface morphology, aligning liquid crystal molecules. Scanning force microscopy can be used to intentionally create areas with a similar anisotropy by operating the instrument at loads in the range of 10(-7) to 10(-5) newtons. These areas have an orientation effect on liquid crystals indistinguishable from the rubbing process, which allows a systematic investigation of the orientation properties of an alignment layer as a function of its nanometer-scale morphology. Refractive index patterns can be tailored with this method by scratching a suitable area, as demonstrated by fabrication of an optical waveguide 6 micrometers wide and 5 millimeters long.

Topographic and magnetic-sensitive scanning tunneling microscope study of magnetite.

The topographic and magnetic surface structure of a natural single crystal of magnetite (Fe(3)0(4)), a common mineral, has been studied from the submicrometer scale down to the atomic scale with a scanning tunneling microscope having nonmagnetic tungsten as well as ferromagnetic iron probe tips. Several different (001) crystal planes were imaged to atomic resolution with both kinds of tips. A selective imaging of the octahedrally coordinated Fe B-sites in the Fe-O planes, and even a selective imaging of the different magnetic ions Fe(2+) and Fe(3+), has been achieved, demonstrating for the first time that magnetic imaging can be realized at the atomic level.