Parallel nanometric 3D tracking of intracellular gold nanorods using multifocal two-photon microscopy.

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.

Measuring gravity with milligram levitated masses.

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.

Distinct defects in collagen microarchitecture underlie vessel-wall failure in advanced abdominal aneurysms and aneurysms in Marfan syndrome.

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.

Mechanical properties of the extracellular matrix of the aorta studied by enzymatic treatments.

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.

Size of the localized electron emission sites on a closed multiwalled carbon nanotube.

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.

Single-walled carbon nanotubes as scaffolds to concentrate DNA for the study of DNA-protein interactions.

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.

Top-down approach to fiber-top cantilevers.

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.

Making carbon nanotube electron sources of defined lengths and with closed caps.

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.

Quantitative force versus distance measurements in amplitude modulation AFM: a novel force inversion technique.

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.

Vibration isolation with high thermal conductance for a cryogen-free dilution refrigerator.

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.

Analysis of interactions of signaling proteins with phage-displayed ligands by fluorescence correlation spectroscopy.

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.

Quantitative comparison of different iron forms in the temporal cortex of Alzheimer patients and control subjects.

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.

Human-brain ferritin studied by muon spin rotation: a pilot study.

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.

A novel approach to quantify different iron forms in ex-vivo human brain tissue.

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.

Interlaboratory round robin on cantilever calibration for AFM force spectroscopy.

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.

Fabrication and characterization of polymer insulated carbon nanotube modified electrochemical nanoprobes.

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.

Mannan-binding lectin: structure, oligomerization, and flexibility studied by atomic force microscopy.

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.

Heterogeneous nucleation of three-dimensional protein nanocrystals.

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.

Electron emission from individual indium arsenide semiconductor nanowires.

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.