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Gravity differs from all other known fundamental forces because it is best described as a curvature of space-time. For that reason, it remains resistant to unifications with quantum theory. Gravitational interaction is fundamentally weak and becomes prominent only at macroscopic scales. This means, we do not know what happens to gravity in the microscopic regime where quantum effects dominate and whether quantum coherent effects of gravity become apparent. Levitated mechanical systems of mesoscopic size offer a probe of gravity, while still allowing quantum control over their motional state. This regime opens the possibility of table-top testing of quantum superposition and entanglement in gravitating systems. Here, we show gravitational coupling between a levitated submillimeter-scale magnetic particle inside a type I superconducting trap and kilogram source masses, placed approximately half a meter away. Our results extend gravity measurements to low gravitational forces of attonewton and underline the importance of levitated mechanical sensors.

We present the design and implementation of a mechanical low-pass filter vibration isolation used to reduce the vibrational noise in a cryogen-free dilution refrigerator operated at 10 mK, intended for scanning probe techniques. We discuss the design guidelines necessary to meet the competing requirements of having a low mechanical stiffness in combination with a high thermal conductance. We demonstrate the effectiveness of our approach by measuring the vibrational noise levels of an ultrasoft mechanical resonator positioned above a superconducting quantum interference device. Starting from a cryostat base temperature of 8 mK, the vibration isolation can be cooled to 10.5 mK, with a cooling power of 113 µW at 100 mK. We use the low vibrations and low temperature to demonstrate an effective cantilever temperature of less than 20 mK. This results in a force sensitivity of less than 500 zN/Hz and an integrated frequency noise as low as 0.4 mHz in a 1 Hz measurement bandwidth.

We present a quantitative study of different molecular iron forms found in the temporal cortex of Alzheimer (AD) patients. Applying the methodology we developed in our previous work, we quantify the concentrations of non-heme Fe(III) by Electron Paramagnetic Resonance (EPR), magnetite/maghemite and ferrihydrite by SQUID magnetometry, together with the MRI transverse relaxation rate [Formula: see text], to obtain a systematic view of molecular iron in the temporal cortex. Significantly higher values of [Formula: see text], a larger concentration of ferrihydrite, and a larger magnetic moment of magnetite/maghemite particles are found in the brain of AD patients. Moreover, we found correlations between the concentration of the iron detected by EPR, the concentration of the ferrihydrite mineral and the average iron loading of ferritin. We discuss these findings in the framework of iron dis-homeostasis, which has been proposed to occur in the brain of AD patients.

Muon spin rotation is employed to investigate the spin dynamics of ferritin proteins isolated from the brain of an Alzheimer's disease (AD) patient and of a healthy control, using a sample of horse-spleen ferritin as a reference. A model based on the Néel theory of superparamagnetism is developed in order to interpret the spin relaxation rate of the muons stopped by the core of the protein. Using this model, our preliminary observations show that ferritins from the healthy control are filled with a mineral compatible with ferrihydrite, while ferritins from the AD patient contain a crystalline phase with a larger magnetocrystalline anisotropy, possibly compatible with magnetite or maghemite.

Within the last three decades Scanning Probe Microscopy has been developed to a powerful tool for measuring surfaces and their properties on an atomic scale such that users can be found nowadays not only in academia but also in industry. This development is still pushed further by researchers, who continuously exploit new possibilities of this technique, as well as companies that focus mainly on the usability. However, although imaging has become significantly easier, the time required for a safe approach (without unwanted tip-sample contact) can be very time consuming, especially if the microscope is not equipped or suited for the observation of the tip-sample distance with an additional optical microscope. Here we show that the measurement of the absolute tip-sample capacitance provides an ideal solution for a fast and reliable pre-approach. The absolute tip-sample capacitance shows a generic behavior as a function of the distance, even though we measured it on several completely different setups. Insight into this behavior is gained via an analytical and computational analysis, from which two additional advantages arise: the capacitance measurement can be applied for observing, analyzing, and fine-tuning of the approach motor, as well as for the determination of the (effective) tip radius. The latter provides important information about the sharpness of the measured tip and can be used not only to characterize new (freshly etched) tips but also for the determination of the degradation after a tip-sample contact/crash., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The nondestructive imaging of subsurface structures on the nanometer scale has been a long-standing desire in both science and industry. A few impressive images were published so far that demonstrate the general feasibility by combining ultrasound with an atomic force microscope. From different excitation schemes, heterodyne force microscopy seems to be the most promising candidate delivering the highest contrast and resolution. However, the physical contrast mechanism is unknown, thereby preventing any quantitative analysis of samples. Here we show that friction at material boundaries within the sample is responsible for the contrast formation. This result is obtained by performing a full quantitative analysis, in which we compare our experimentally observed contrasts with simulations and calculations. Surprisingly, we can rule out all other generally believed responsible mechanisms, like Rayleigh scattering, sample (visco)elasticity, damping of the ultrasonic tip motion, and ultrasound attenuation. Our analytical description paves the way for quantitative subsurface-AFM imaging.

We calculate the change of the properties of a resonator, when coupled to a semiclassical spin by means of the magnetic field. Starting with the Lagrangian of the complete system, we provide an analytical expression for the linear response function for the motion in the case of a mechanical resonator and the current for the case of an electromagnetic resonator, thereby considering the influence of the resonator on the spin and vice versa. This analysis shows that the resonance frequency and effective dissipation factor can change significantly due to the relaxation times of the spin. We first derive this for a system consisting of a spin and mechanical resonator and thereafter apply the same calculations to an electromagnetic resonator. Moreover, the applicability of the method is generalized to a resonator coupled to two-level systems and more, providing a key to understand some of the problems of two-level systems in quantum devices., Competing Interests: The authors declare no competing financial interests.

We propose a novel combination of methods to study the physical properties of ferric ions and iron-oxide nanoparticles in post-mortem human brain, based on the combination of Electron Paramagnetic Resonance (EPR) and SQUID magnetometry. By means of EPR, we derive the concentration of the low molecular weight iron pool, as well as the product of its electron spin relaxation times. Additionally, by SQUID magnetometry we identify iron mineralization products ascribable to a magnetite/maghemite phase and a ferrihydrite (ferritin) phase. We further derive the concentration of magnetite/maghemite and of ferritin nanoparticles. To test out the new combined methodology, we studied brain tissue of an Alzheimer's patient and a healthy control. Finally, we estimate that the size of the magnetite/maghemite nanoparticles, whose magnetic moments are blocked at room temperature, exceeds 40-50 nm, which is not compatible with the ferritin protein, the core of which is typically 6-8 nm. We believe that this methodology could be beneficial in the study of neurodegenerative diseases such as Alzheimer's Disease which are characterized by abnormal iron accumulation in the brain.

Collapse models predict a tiny violation of energy conservation, as a consequence of the spontaneous collapse of the wave function. This property allows us to set experimental bounds on their parameters. We consider an ultrasoft magnetically tipped nanocantilever cooled to millikelvin temperature. The thermal noise of the cantilever fundamental mode has been accurately estimated in the range 0.03-1 K, and any other excess noise is found to be negligible within the experimental uncertainty. From the measured data and the cantilever geometry, we estimate the upper bound on the continuous spontaneous localization collapse rate in a wide range of the correlation length r_{C}. Our upper bound improves significantly previous constraints for r_{C}>10^{-6}  m, and partially excludes the enhanced collapse rate suggested by Adler. We discuss future improvements.

The application of ultrasound in an Atomic Force Microscope (AFM) gives access to subsurface information. However, no commercially AFM exists that is equipped with this technique. The main problems are the electronic crosstalk in the AFM setup and the insufficiently strong excitation of the cantilever at ultrasonic (MHz) frequencies. In this paper, we describe the development of an add-on that provides a solution to these problems by using a special piezo element with a lowest resonance frequency of 2.5 MHz and by separating the electronic connection for this high frequency piezo element from all other connections. In this sense, we support researches with the possibility to perform subsurface measurements with their existing AFMs and hopefully pave also the way for the development of a commercial AFM that is capable of imaging subsurface features with nanometer resolution.

We present a new method to analyse simultaneous Topography and RECognition Atomic Force Microscopy data such that it becomes possible to measure single molecule binding rates of surface bound proteins. We have validated this method on a model system comprising a S-layer surface modified with Strep-tagII for binding sites and strep-tactin bound to an Atomic Force Microscope tip through a flexible Poly-Ethylene-Glycol linker. At larger distances, the binding rate is limited by the linker, which limits the diffusion of the strep-tactin molecule, but at lateral distances below 3 nm, the binding rate is solely determined by the intrinsic molecular characteristics and the surface geometry and chemistry of the system. In this regime, Kon as determined from single molecule TREC data is in agreement with Kon determined using traditional biochemical methods., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Pulse tube refrigerators are becoming more common, because they are cost efficient and demand less handling than conventional (wet) refrigerators. However, a downside of a pulse tube system is the vibration level at the cold-head, which is in most designs several micrometers. We implemented vibration isolation techniques which significantly reduced vibration levels at the experiment. These optimizations were necessary for the vibration sensitive magnetic resonance force microscopy experiments at milli-kelvin temperatures for which the cryostat is intended. With these modifications we show atomic resolution scanning tunneling microscopy on graphite. This is promising for scanning probe microscopy applications at very low temperatures.

Experiments in Heterodyne Force Microscopy (HFM) show the possibility to image deeply buried nanoparticles below a surface. However, the contrast mechanism and the motion of the cantilever, which detects the subsurface signal, are not yet understood. We present a numerical study of the cantilever motion in different HFM modes using realistic tip-sample interactions. The results provide information on the sensitivity to the heterodyne signal. The parameters in our calculations are chosen as closely as possible to the situation in real experiments to enable (future) comparisons based on our predictions. In HFM both the tip and the sample are excited at slightly different ultrasonic frequencies such that a difference frequency is generated that can contain subsurface information. We calculate the amplitude and phase of the difference frequency generated by the motion of the cantilever. The amplitude shows a local maximum in the attractive Van-der-Waals regime and an even higher plateau in the repulsive regime. The phase shifts 180° or 90°, depending on the mode of operation. Finally, we observe oscillations in both the amplitude and the phase of the difference frequency, which are caused by a shift of the resonance frequency of the cantilever and an involved transient behavior., (© 2013 Elsevier B.V. All rights reserved.)

Applying heterodyne force microscopy (HFM), it has been impressively demonstrated that it is possible to obtain subsurface information: 20 nm large gold nanoparticles that were buried 500 nm deep have been imaged. It is the heterodyne signal that contains the subsurface information. We elucidate, both theoretically and experimentally, the sensitivity to the heterodyne signal as a function of the tip-sample distance. This is crucial information for experiments as the distance, and therefore the sensitivity, is tunable. We show that the amplitude of the heterodyne signal has a local maximum in the attractive part of the tip-sample interaction, before it surprisingly reaches an even higher plateau, when the tip-sample interaction is repulsive. This can only be explained by a non-decreasing amplitude of the ultrasonic motion of the tip, although it is in full contact with the surface. We confirm this counterintuitive tip behavior experimentally even on a hard surface like silicon.

We report a novel technique for long-term parallel three dimensional (3D)-tracking of gold nanorods in live cells with nanometer resolution. Gold nanorods feature a strong plasmon-enhanced two-photon luminescence, can be easily functionalized, and have been shown to be nontoxic. These properties make gold nanorods very suitable for in vivo two-photon luminescence microscopy. By rapid multifocal scanning, we combine the advantages of 3D molecular tracking methods using wide-field imaging with the advantages of two-photon microscopy. Isolated gold nanorods can be localized with a resolution of 4 nm in the xy-plane and 8 nm in the z-direction. The polarization-dependence of the two-photon luminescence signal can be used to resolve the angular orientation, even when two gold nanorods are separated by less than the diffraction limit. Individual nanorods in live U2OS cells could be followed in 3 dimensions for over 30 min, with a photon noise limited accuracy, and a time resolution of 50 ms in 2D and 500 ms in 3D.

Microparticles, also known as microvesicles, found in blood plasma, urine, and most other body fluids, may serve as valuable biomarkers of diseases such as cardiovascular diseases, systemic inflammatory disease, thrombosis, and cancer. Unfortunately, the detection and quantification of microparticles are hampered by the microscopic size of these particles and their relatively low abundance in blood plasma. The use of a combination of microfluidics and atomic force microscopy to detect microparticles in blood plasma circumvents both problems. In this study, capture of a specific subset of microparticles directly from blood plasma on antibody-coated mica surface is demonstrated. The described method excludes isolation and washing steps to prepare microparticles, improves the detection sensitivity, and yields the size distribution of the captured particles. The majority of the captured particles have a size ranging from 30 to 90 nm, which is in good agreement with prior results obtained with microparticles immediately isolated from fresh plasma. Furthermore, the qualitative shape of the size distribution of microparticles is shown not to be affected by high-speed centrifugation or the use of the microfluidic circuit, demonstrating the relative stable nature of microparticles ex vivo.

Genomic DNA in bacteria exists in a condensed state, which exhibits different biochemical and biophysical properties from a dilute solution. DNA was concentrated on streptavidin-covered single-walled carbon nanotubes (Strep-SWNTs) through biotin-streptavidin interactions. We reasoned that confining DNA within a defined space through mechanical constraints, rather than by manipulating buffer conditions, would more closely resemble physiological conditions. By ensuring a high streptavidin loading on SWNTs of about 1 streptavidin tetramer per 4 nm of SWNT, we were able to achieve dense DNA binding. DNA is bound to Strep-SWNTs at a tunable density and up to as high as 0.5 mg mL(-1) in solution and 29 mg mL(-1) on a 2D surface. This platform allows us to observe the aggregation behavior of DNA at high concentrations and the counteracting effects of HU protein (a histone-like protein from Escherichia coli strain U93) on the DNA aggregates. This provides an in vitro model for studying DNA-DNA and DNA-protein interactions at a high DNA concentration., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The microarchitecture of different components of the extracellular matrix (ECM) is crucial to our understanding of the properties of a tissue. In the study presented here, we used a top-down approach to understand how the interplay among different fibers determines the mechanical properties of real tissues. By selectively removing different elements of the arterial wall, we were able to measure the contribution of the different constituents of the ECM to the mechanical properties of the whole tissue. Changes in the network structure were imaged with the use of two-photon microscopy. We used an atomic force microscope to measure changes in the mechanical properties by performing nanoindentation experiments. We show that although the removal of a key element of the ECM reduced the local stiffness by up to 50 times, the remaining tissue still formed a coherent network. We also show how this method can be extended to study the effects of cells on real tissues. This new (to our knowledge) way of studying the ECM will not only help physicists gain a better understanding of biopolymers, it will be a valuable tool for biomedical researchers studying processes such as wound healing and cervix ripening., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Recent experiments in the field of subsurface atomic force microscopy have demonstrated that it is possible to nondestructively image micro- and even nanoparticles that are embedded significantly deep within the bulk of a sample. In order to get insights into the contrast formation mechanism, we performed a finite element analysis and an analytical study, in which we calculated the amplitude and phase variation on the surface of an ultrasound wave that has traveled through the sample. Our calculations were performed as closely as possible to the situation in the experiments to enable a (future) comparison based on our predictions. We show that Rayleigh scattering of acoustic waves accounts for the measured contrast and we verify the characteristic Rayleigh dependences. The numerical results show that the contrast is independent of the depth at which a particle is buried, whereas the analytical study reveals a 1/depth dependence. In addition, we find a large deviation in the width of the particle in the contrast at the surface when applying the numerical or the analytical calculation respectively. These results indicate the importance of both the reflections of sound waves at the sample interfaces and bulk damping, as both are treated differently in our two models.

We have measured the size of the localized electron emission sites on multiwalled carbon nanotubes (MWNTs) with caps closed by a fullerenelike structure. MWNTs were individually mounted on tungsten support tips and imaged with a field emission microscope (FEM). The magnification of the FEM was calibrated using electron ray tracing and verified by comparing transmission electron microscope images. The FEM image was also tested for effects of the lateral energy spread. We found ring-shaped emission areas with three flattened sides, of a radius of 1.7±0.3 nm, and separated by 5±1 nm., (© 2012 American Physical Society)

Magnetic resonance force microscopy (MRFM) is a powerful technique to detect a small number of spins that relies on force detection by an ultrasoft magnetically tipped cantilever and selective magnetic resonance manipulation of the spins. MRFM would greatly benefit from ultralow temperature operation, because of lower thermomechanical noise and increased thermal spin polarization. Here we demonstrate MRFM operation at temperatures as low as 30 mK, thanks to a recently developed superconducting quantum interference device (SQUID)-based cantilever detection technique, which avoids cantilever overheating. In our experiment, we detect dangling bond paramagnetic centres on a silicon surface down to millikelvin temperatures. Fluctuations of such defects are supposedly linked to 1/f magnetic noise and decoherence in SQUIDs, as well as in several superconducting and single spin qubits. We find evidence that spin diffusion has a key role in the low-temperature spin dynamics.

Single-molecule force spectroscopy studies performed by Atomic Force Microscopes (AFMs) strongly rely on accurately determined cantilever spring constants. Hence, to calibrate cantilevers, a reliable calibration protocol is essential. Although the thermal noise method and the direct Sader method are frequently used for cantilever calibration, there is no consensus on the optimal calibration of soft and V-shaped cantilevers, especially those used in force spectroscopy. Therefore, in this study we aimed at establishing a commonly accepted approach to accurately calibrate compliant and V-shaped cantilevers. In a round robin experiment involving eight different laboratories we compared the thermal noise and the Sader method on ten commercial and custom-built AFMs. We found that spring constants of both rectangular and V-shaped cantilevers can accurately be determined with both methods, although the Sader method proved to be superior. Furthermore, we observed that simultaneous application of both methods on an AFM proved an accurate consistency check of the instrument and thus provides optimal and highly reproducible calibration. To illustrate the importance of optimal calibration, we show that for biological force spectroscopy studies, an erroneously calibrated cantilever can significantly affect the derived (bio)physical parameters. Taken together, our findings demonstrated that with the pre-established protocol described reliable spring constants can be obtained for different types of cantilevers., (Copyright © 2011. Published by Elsevier B.V.)

Taking inspiration from conventional top-down micromachining techniques, we have fabricated a low mass gold fiber-top cantilever via align-and-shine photolithography. The cantilever is characterized by measuring its resonance frequency and mechanical quality factor. Our results show that the device grants mass sensitivity comparable to that reported for similar standard cantilevers. This proof-of-concept paves the way to series production of highly sensitive fiber-top devices for remote detection of biochemical substances., (© 2011 Optical Society of America)

A method is reported to make an electron source consisting of an individual multi-walled carbon nanotube (MWNT) mounted on a tungsten support tip, and cut to length using localized electron beam irradiation in a scanning electron microscope. The apex of the MWNT was transformed into a closed cap with at least one fullerene-like layer via an annealing process involving simultaneous heating and the extraction of an emission current of ∼ 1 mA. The electron emission occurred at localized emission sites. The electron emission showed Fowler-Nordheim behavior, was highly stable with time, and exhibited a low energy spread. The structure of the caps of two MWNTs was studied with transmission electron microscopy before and after the cap closure.

Scanning probe microscopy is a frequently used nanometer-scale surface investigation technique. Unfortunately, its applicability is limited by the relatively low image acquisition speed, typically seconds to minutes per image. Higher imaging speeds are desirable for rapid inspection of samples and for the study of a range of dynamic surface processes, such as catalysis and crystal growth. We have designed a new high-speed scanning probe microscope (SPM) based on micro-electro mechanical systems (MEMS). MEMS are small, typically micrometer size devices that can be designed to perform the scanning motion required in an SPM system. These devices can be optimized to have high resonance frequencies (up to the MHz range) and have very low mass (10(-11)kg). Therefore, MEMS can perform fast scanning motion without exciting resonances in the mechanical loop of the SPM, and hence scan the surface without causing the image distortion from which conventional piezo scanners suffer. We have designed a MEMS z-scanner which we have integrated in commercial AFM (atomic force microscope) and STM (scanning tunneling microscope) setups. We show the first successful AFM experiments.

Electrochemical nanoprobes were fabricated from polymer insulated multiwalled carbon nanotube modified tapping mode atomic force microscope probes. An electrochemically active length of carbon nanotube was exposed by laser ablation of the insulating polymer. Characterization of these probes is done by cyclic voltammetry of ferrocenemethanol in an aqueous solution and by finite element analysis. The fabricated nanoelectrodes were found to be stable and yielded an interfacial electron transfer rate constant (k(0)) of 1.073 +/- 0.36 cm s(-1) for ferrocenemethanol.

The high speed performance of a scanning probe microscope (SPM) is improved if a microelectromechanical systems (MEMS) device is employed for the out-of-plane scanning motion. We have carried out experiments with MEMS high-speed z-scanners (189 kHz fundamental resonance frequency) in both atomic force microscope and scanning tunneling microscope modes. The experiments show that with the current MEMS z-scanner, lateral tip speeds of 5 mm/s can be achieved with full feedback on surfaces with significant roughness. The improvement in scan speed, obtained with MEMS scanners, increases the possibilities for SPM observations of dynamic processes. Even higher speed MEMS scanners with fundamental resonance frequencies in excess of a megahertz are currently under development.

Background: Microparticles (MPs) are small vesicles released from cells of different origin, bearing surface antigens from parental cells. Elevated numbers of blood MPs have been reported in (cardio)vascular disorders and cancer. Most of these MPs are derived from platelets., Objectives: To investigate whether atomic force microscopy (AFM) can be used to detect platelet-derived MPs and to define their size distribution., Methods: Blood MPs isolated from seven blood donors and three cancer patients were immobilized on a modified mica surface coated with an antibody against CD41 prior to AFM imaging. AFM was performed in liquid-tapping mode to detect CD41-positive MPs. In parallel, numbers of CD41-positive MPs were measured using flow cytometry. Mouse IgG1 isotype control was used as a negative control., Results: AFM topography measurements of the number of CD41-positive MPs were reproducible (coefficient of variation=16%). Assuming a spherical shape of unbound MPs, the calculated diameter of CD41-positive MPs (dsph) ranged from 10 to 475 nm (mean: 67.5+/-26.5 nm) and from 5 to 204 nm (mean: 51.4+/-14.9 nm) in blood donors and cancer patients, respectively. Numbers of CD41-positive MPs were 1000-fold higher than those measured by flow cytometry (3-702x10(9) L(-1) plasma vs. 11-626x10(6) L(-1) plasma). After filtration of isolated MPs through a 0.22-microm filter, CD41-positive MPs were still detectable in the filtrate by AFM (mean dsph: 37.2+/-11.6 nm), but not by flow cytometry., Conclusions: AFM provides a novel method for the sensitive detection of defined subsets of MPs in the nanosize range, far below the lower limit of what can be measured by conventional flow cytometry.

A compact, two-stage nanomanipulator was designed and built for use inside a scanning electron microscope. It consists of a fine stage employing piezostacks that provide a 15 microm range in three dimensions and a coarse stage based on commercially available stick-slip motors. Besides the fabrication of enhanced probes for scanning probe microscopy and the enhancement of electron field emitters, other novel manipulation processes were developed, such as locating, picking up, and positioning small nanostructures with an accuracy of approximately 10 nm. In combination with in situ I-V experiments, welding, and etching, this results in a multipurpose nanofactory, enabling a new range of experiments.

An aneurysm of the aorta is a common pathology characterized by segmental weakening of the artery. Although it is generally accepted that the vessel-wall weakening is caused by an impaired collagen metabolism, a clear association has been demonstrated only for rare syndromes such as the vascular type Ehlers-Danlos syndrome. Here we show that vessel-wall failure in growing aneurysms of patients who have aortic abdominal aneurysm (AAA) or Marfan syndrome is not related to a collagen defect at the molecular level. On the contrary our findings indicate similar (Marfan) or even higher collagen concentrations (AAA) and increased collagen cross-linking in the aneurysms. Using 3D confocal imaging we show that the two conditions are associated with profound defects in collagen microarchitecture. Reconstructions of normal vessel wall show that adventitial collagen fibers are organized in a loose braiding of collagen ribbons. These ribbons encage the vessel, allowing the vessel to dilate easily but preventing overstretching. AAA and aneurysms in Marfan syndrome show dramatically altered collagen architectures with loss of the collagen knitting. Evaluations of the functional characteristics by atomic force microscopy showed that the wall has lost its ability to stretch easily and revealed a second defect: although vascular collagen in normal aortic wall behaves as a coherent network, in AAA and Marfan tissues it does not. As result, mechanical forces loaded on individual fibers are not distributed over the tissue. These studies demonstrate that the mechanical properties of tissue are strongly influenced by collagen microarchitecture and that perturbations in the collagen networks may lead to mechanical failure.

Mannan-binding lectin (MBL) is the archetypical pathogen recognition molecule of the innate immune defense. Upon binding to microorganisms, reactions leading to the destruction of the offender ensue. MBL is an oligomer of structural subunits each composed of three identical polypeptides. We used atomic force microscopy to reveal tertiary and quaternary structures of MBL. The images in both air and buffer show a quaternary structure best described as "sertiform", that is, a hub from which the subunits fan out. The dimensions conform to those calculated from primary and secondary structures. The subunits associate with a preferred angle of 40 degrees between them. This angle is stable with respect to the degree of oligomerization for MBL of four subunits or more. Due to an interruption in the collagenous sequence, the arms of the subunits are expected to form a kink. We find that approximately 30% of the subunits are kinked and the kink angle distributed, quite broadly, around 145 degrees . The conformation and flexibility of the MBL molecule that we observe differ distinctly from the popular view of a "bouquet-like" configuration as that found for related members of the complement system such as C1q. This structural information will further the understanding of the specific functioning of the MBL pathway of complement activation.

Magnetic resonance force microscopy (MRFM) makes use of the spectroscopic nature of magnetic resonance to add unambiguous elemental selectivity to scanning probe microscopy. We show isotopic selectivity of MRFM for three nuclei, (1)H, (31)P, and (13)C, in organic materials. We also detect a roughly 1 nm thick layer of naturally occurring adsorbates on a gold surface by measuring the magnetic resonance signal of the hydrogen contained in the layer. Finally, we detect the signal from hydrogen present on a carbon nanotube and use it to perform a three-dimensional magnetic resonance image of the 10 nm diameter object.

A new method for extracting quantitative data from amplitude modulation dynamic force-distance measurements is developed. The method is based on the harmonic oscillator model of vibrating atomic force microscope cantilevers, and is capable of extracting both the conservative and dissipative parts of the tip-sample interaction from a measurement of oscillation amplitude and phase as a function of distance. Numerical simulations are used to demonstrate the validity of the method. Further proof of the accuracy of this method is provided by a measurement of electrostatic forces between an AFM tip and a graphite sample.

Fluorescent correlation spectroscopy (FCS) was used to measure binding affinities of ligands to ligates that are expressed by phage-display technology. Using this method we have quantified the binding of the 14-3-3 signaling protein to artificial peptide ligand. As a ligand we used the R18 artificial peptide expressed as a fusion in the cpIII coat protein that is present in 3 to 5 copies in an M13 phage. Comparisons of binding affinities were made with free R18 ligands using FCS. The result showed a relatively high binding affinity for the phage-displayed R18 peptide compared with binding to free fluorescently labeled R18. Quantification was supported by titration of the phage numbers using atomic force microscopy (AFM). AFM was shown to accurately determine phage numbers in solution as a good alternative for electron microscopy. It was shown to give reliable data that correlated perfectly with those of the viable phage numbers determined by classical bacterial infection studies. In conclusion, a very fast and sensitive method for the selection of new peptide ligands or ligates based on a quantitative assay in solution has been developed.

Nucleation is the rate-limiting step in protein crystallization. Introducing heterogeneous substrates may in some cases lower the energy barrier for nucleation and thereby facilitate crystal growth. To date, the mechanism of heterogeneous protein nucleation remains poorly understood. In this study, the nucleating properties of fragments of human hair in crystallization experiments have been investigated. The four proteins that were tested, lysozyme, glucose isomerase, a polysaccharide-specific Fab fragment and potato serine protease inhibitor, nucleated preferentially on the hair surface. Macrocrystals and showers of tiny crystals of a few hundred nanometres thickness were obtained also under conditions that did not produce crystals in the absence of the nucleating agent. Cryo-electron diffraction showed that the nanocrystals diffracted to at least 4 A resolution. The mechanism of heterogeneous nucleation was studied using confocal fluorescent microscopy which demonstrated that the protein is concentrated on the nucleating surface. A substantial accumulation of protein was observed on the sharp edges of the hair's cuticles, explaining the strong nucleating activity of the surface.

A procedure was developed to mount individual semiconductor indium arsenide nanowires onto tungsten support tips to serve as electron field-emission sources. The electron emission properties of the single nanowires were precisely determined by measuring the emission pattern, current-voltage curve, and the energy spectrum of the emitted electron beam. The two investigated nanowires showed stable, Fowler-Nordheim-like emission behavior and a small energy spread. Their morphology was characterized afterward using transmission electron microscopy. The experimentally derived field enhancement factor corresponded to the one calculated using the basic structural information. The observed emission behavior contrasts the often unstable emission and large energy spread found for semiconductor emitters and supports the concept of Fermi-level pinning in indium arsenide nanowires. Indium arsenide nanowires may thus present a new type of semiconductor electron sources.

The conductivity of two photosynthetic protein-pigment complexes, a light harvesting 2 complex and a reaction center, was measured with an atomic force microscope capable of performing electrical measurements. Current-voltage measurements were performed on complexes embedded in their natural environment. Embedding the complexes in lipid bilayers made it possible to discuss the different conduction behaviors of the two complexes in light of their atomic structure.

The main function of the transmembrane light-harvesting complexes in photosynthetic organisms is the absorption of a light quantum and its subsequent rapid transfer to a reaction center where a charge separation occurs. A combination of freeze-thaw and dialysis methods were used to reconstitute the detergent-solubilized Light Harvesting 2 complex (LH2) of the purple bacterium Rhodopseudomonas acidophila strain 10050 into preformed egg phosphatidylcholine liposomes, without the need for extra chemical agents. The LH2-containing liposomes opened up to a flat bilayer, which were imaged with tapping and contact mode atomic force microscopy under ambient and physiological conditions, respectively. The LH2 complexes were packed in quasicrystalline domains. The endoplasmic and periplasmic sides of the LH2 complexes could be distinguished by the difference in height of the protrusions from the lipid bilayer. The results indicate that the complexes entered in intact liposomes. In addition, it was observed that the most hydrophilic side, the periplasmic, enters first in the membrane. In contact mode the molecular structure of the periplasmic side of the transmembrane pigment-protein complex was observed. Using Föster's theory for describing the distance dependent energy transfer, we estimate the dipole strength for energy transfer between two neighboring LH2s, based on the architecture of the imaged unit cell.

Carbon nanotubes can act as electron sources with very rigid structures, making them particularly interesting for use as point electron sources in high-resolution electron-beam instruments. Promising results have been reported with respect to some important requirements for such applications: a stable emitted current and a long lifetime. Two parameters of an electron source affect the resolution of these instruments: the energy spread of the emitted electrons and a parameter called the reduced brightness, which depends on the angular current density and the virtual source size. Several authors have measured a low energy spread associated with electron emission. Here we measure the reduced brightness, and find a value that is more than a factor of ten larger than provided by state-of-the-art electron sources in electron microscopes. In addition, we show that an individual multi-walled carbon nanotube emits most current into a single narrow beam. On the basis of these results, we expect that carbon nanotube electron sources will lead to a significant improvement in the performance of high-resolution electron-beam instruments.

The FIN1 gene from the yeast Saccharomyces cerevisiae encodes a basic protein with putative coiled-coil regions. Here we show that in large-budded cells a green fluorescent protein-Fin1 fusion protein is visible as a filament between the two spindle pole bodies. In resting cells the protein is undetectable, and in small-budded cells it is localized in the nucleus. During late mitosis it localizes on the spindle pole bodies. Filaments of cyano fluorescent protein-tagged Fin1 colocalize with filaments of green fluorescent protein-tagged Tub1 only in large-budded cells. By electron and atomic force microscopy we showed that purified recombinant Fin1p self-assembles into filaments with a diameter of approximately 10 nm. Our results indicate that the Fin1 protein forms a cell cycle-specific filament, additional to the microtubules, between the spindle pole bodies of dividing yeast cells.

A double quantum dot device is a tunable two-level system for electronic energy states. A dc electron current was used to directly measure the rates for elastic and inelastic transitions between the two levels. For inelastic transitions, energy is exchanged with bosonic degrees of freedom in the environment. The inelastic transition rates are well described by the Einstein coefficients, relating absorption with stimulated and spontaneous emission. The most effectively coupled bosons in the specific environment of the semiconductor device used here were acoustic phonons. The experiments demonstrate the importance of vacuum fluctuations in the environment for quantum dot devices and potential design constraints for their use for preparing long-lived quantum states.

A tunable Kondo effect has been realized in small quantum dots. A dot can be switched from a Kondo system to a non-Kondo system as the number of electrons on the dot is changed from odd to even. The Kondo temperature can be tuned by means of a gate voltage as a single-particle energy state nears the Fermi energy. Measurements of the temperature and magnetic field dependence of a Coulomb-blockaded dot show good agreement with predictions of both equilibrium and nonequilibrium Kondo effects.

Studies of the ground and excited states in semiconductor quantum dots containing 1 to 12 electrons showed that the quantum numbers of the states in the excitation spectra can be identified and compared with exact calculations. A magnetic field induces transitions between the ground and excited states. These transitions were analyzed in terms of crossings between single-particle states, singlet-triplet transitions, spin polarization, and Hund's rule. These impurity-free quantum dots allow "atomic physics" experiments to be performed in magnetic field regimes not accessible for atoms.