Contrast Formation in Kelvin Probe Force Microscopy of Single π-Conjugated Molecules

We report the contrast formation in the local contact potential difference (LCPD) measured by Kelvin probe force microscopy (KPFM) on single charge-transfer complexes (CTCs) on a NaCl bilayer on Cu(111). At different tip heights, we found quantitatively different LCPD contrasts that characterize different properties of the molecule. In the small distance regime, the tip penetrates the electron density of the molecule, and the contrast is related to the size and topography of the electron shell of the molecule. For larger distances, the LCPD contrast corresponds to the electrostatic field above the molecule. However, in the medium-distance regime, that is, for tip heights similar to the size of the molecule, the nonspherical distribution of π- and σ-electrons often conceals the effect of the partial charges within the molecule. Only for large distances does the LCPD map converge toward the simple field of a dipole for a polar molecule.

Can we infer plant facilitation from remote sensing? a test across global drylands

Facilitation is a major force shaping the structure and diversity of plant communities in terrestrial ecosystems. Detecting positive plant–plant interactions relies on the combination of field experimentation and the demonstration of spatial association between neighboring plants. This has often restricted the study of facilitation to particular sites, limiting the development of systematic assessments of facilitation over regional and global scales. Here we explore whether the frequency of plant spatial associations detected from high-resolution remotely sensed images can be used to infer plant facilitation at the community level in drylands around the globe. We correlated the information from remotely sensed images freely available through Google Earth with detailed field assessments, and used a simple individual-based model to generate patch-size distributions using different assumptions about the type and strength of plant–plant interactions. Most of the patterns found from the remotely sensed images were more right skewed than the patterns from the null model simulating a random distribution. This suggests that the plants in the studied drylands show stronger spatial clustering than expected by chance. We found that positive plant co-occurrence, as measured in the field, was significantly related to the skewness of vegetation patch-size distribution measured using Google Earth images. Our findings suggest that the relative frequency of facilitation may be inferred from spatial pattern signals measured from remotely sensed images, since facilitation often determines positive co-occurrence among neighboring plants. They pave the road for a systematic global assessment of the role of facilitation in terrestrial ecosystems. Read More: http://www.esajournals.org/doi/10.1890/14-2358.1

Fatal steamer accident; blunt force injuries and drowning in post-mortem MSCT and MRI.

Steamer accidents, through contact with the bucket wheel, are very seldom today. No publication of such a kind of fatal accident could be found in literature. We present the case of a fatal steamer accident, in which the findings of a blunt traumatization of a person by the ship was completely documented by post-mortem combined multi-slice computed tomography (MSCT) and magnetic resonance imaging (MRI) examinations. A rupture of the aorta was detected using both radiological methods without use of radiopaque material. Radiological examination revealed a comminuted fracture of the thorax vertebrae at the same level as the aortic rupture. Injuries of the soft tissues of the back, caused by the bucket wheel of the steamer, were also diagnosed. In addition to the signs of blunt force trauma the findings of drowning such as an over inflation of the lungs, fluid in the stomach and duodenum were revealed. Furthermore, algological analysis detected diatoms in the lung tissue and blood from the left heart. Therefore, the cause of death was considered being a combination of fatal hemorrhage, caused by the aortic rupture, and drowning. We conclude that virtual autopsy using combined post-mortem MSCT and MRI is a useful tool for documentation, visualisation and analysis of the findings of blunt force trauma and drowning with a large potential in forensic medicine.

Force-controlled patch clamp of beating cardiac cells.

From its invention in the 1970s, the patch clamp technique is the gold standard in electrophysiology research and drug screening because it is the only tool enabling accurate investigation of voltage-gated ion channels, which are responsible for action potentials. Because of its key role in drug screening, innovation efforts are being made to reduce its complexity toward more automated systems. While some of these new approaches are being adopted in pharmaceutical companies, conventional patch-clamp remains unmatched in fundamental research due to its versatility. Here, we merged the patch clamp and atomic force microscope (AFM) techniques, thus equipping the patch-clamp with the sensitive AFM force control. This was possible using the FluidFM, a force-controlled nanopipette based on microchanneled AFM cantilevers. First, the compatibility of the system with patch-clamp electronics and its ability to record the activity of voltage-gated ion channels in whole-cell configuration was demonstrated with sodium (NaV1.5) channels. Second, we showed the feasibility of simultaneous recording of membrane current and force development during contraction of isolated cardiomyocytes. Force feedback allowed for a gentle and stable contact between AFM tip and cell membrane enabling serial patch clamping and injection without apparent cell damage.

SrGAP2-Dependent Integration of Membrane Geometry and Slit-Robo-Repulsive Cues Regulates Fibroblast Contact Inhibition of Locomotion

Migrating fibroblasts undergo contact inhibition of locomotion (CIL), a process that was discovered five decades ago and still is not fully understood at the molecular level. We identify the Slit2-Robo4-srGAP2 signaling network as a key regulator of CIL in fibroblasts. CIL involves highly dynamic contact protrusions with a specialized actin cytoskeleton that stochastically explore cell-cell overlaps between colliding fibroblasts. A membrane curvature-sensing F-BAR domain pre-localizes srGAP2 to protruding edges and terminates their extension phase in response to cell collision. A FRET-based biosensor reveals that Rac1 activity is focused in a band at the tip of contact protrusions, in contrast to the broad activation gradient in contact-free protrusions. SrGAP2 specifically controls the duration of Rac1 activity in contact protrusions, but not in contact-free protrusions. We propose that srGAP2 integrates cell edge curvature and Slit-Robo-mediated repulsive cues to fine-tune Rac1 activation dynamics in contact protrusions to spatiotemporally coordinate CIL.

A low-mass impact sensor for high-speed firmness sensing of fruits.

A low-mass impact sensor for high-speed firmness sensing of fruits was built and tested. Results of tests with a rubber ball indicated that the impact measurement was not sensitive to the distance between the impactor and the impacting surface of the sample within the range of 8 to 23 mm, and was not sensitive to how the sample was held. Tests with kiwifruits and peaches show good correlation between firmness readings obtained with the impact sensor and those obtained with the penetrometer. The best correlation was between the slope of the impact curve (at mid-point) and the force-deformation firmness. Preliminary test showed that the sensor could sense fruit firmness at a speed of 5 fruits/s.

New version of a laboratory impact device for firmness sensing of fruits.

Results of previous studies conducted by different researchers have shown that impact techniques can be used to evaluate firmness (Delwiche et al., 1989; Delwiche et al.;1996; Jaren et al., 1992; Ruiz Altisent et al., 1996). To impact the fruit with a small spherical impactor of known mass and radius of curvature and measure the acceleration of the impactor is a technique described by Chen et al. (1985) and used by several researchers for sensing fruit firmness (Jaren et al., 1992; Correa et al.; 1992). The advantages of this method vs. a force sensor that measures the force as a function of time is that the measured impact-acceleration response is independent of the fruit mass and is less sensitive to the variation in the radius of curvature of the fruit (Chen et al., 1996). Ruiz Altisent et al. (1993) developed and used a 50 g impactor with a 19 mm diameter spherical tip, dropping from different height for fruits (apples, pears, avocados, melons, peaches ...). Another impact device for firmness sensing of fruits was developed by Chen and Ruiz Altisent (1996). They designed and fabricated an experimental low-mass impact sensor for high-speed sensing of fruit firmness. The impactor consisted of a semi-spherical impacting tip attached to the end (near the centre of percussion) of a pivoting arm. Impact is done by swinging the impactor to collide with the fruit. It has been implemented for on-line use. In both devices a small accelerometer is mounted behind the impacting tip. Lateral impactor and vertical impactor have been used in laboratory and the results from non-destructive impact tests have contributed to standardise methods to measure fruit firmness: Barreiro (1992) compared impact parameters and results of Magness-Taylor penetration tests for apples, pears, apricots [and peaches; Agulheiro (1994) studied the behaviour of the impact parameters during seven weeks of cold storage of two melon varieties; Ortiz (1998) used low energy impact and NIR procedures to segregate non crispy, non firm and soft peaches. Steinmetz (1996) compared various non-destructive firmness sensors, based on sound, impact and micro-deformation.

Estudio de la adherencia de armaduras corroídas y su influencia en la capacidad resistente de elementos de hormigón armado

La verificación de la seguridad estructural, tanto de estructuras que permitan un cierto grado de deterioro en su dimensionado como de estructuras existentes deterioradas, necesita disponer de modelos de resistencia que tengan en cuenta los efectos del deterioro. En el caso de la corrosión de las armaduras en las estructuras de hormigón armado, la resistencia depende de múltiples factores tales como la sección del acero corroído, el diagrama tensión-deformación del acero corroído, la adherencia hormigón-acero corroído, la fisuración o desprendimiento del hormigón debido a la expansión de los productos de corrosión. En este sentido, la transferencia de las fuerzas a través de la superficie de contacto entre el hormigón y el acero, la adherencia, es uno de los aspectos más importantes a considerar y es la base del comportamiento del hormigón armado como elemento estructural. La adherencia debe asegurar el anclaje de las armaduras y transmitir la tensión tangencial que aparece en las mismas como consecuencia de la variación de las solicitaciones a lo largo de un elemento estructural. Como consecuencia de la corrosión de las armaduras, el desarrollo de la adherencia se altera y, por tanto, la transferencia de la tensión longitudinal. Esta Tesis Doctoral aborda el comportamiento en estado límite último de la adherencia en el hormigón estructural con armaduras corroídas. El objetivo principal es la obtención de un modelo suficientemente realista y fiable para la evaluación de la adherencia con armaduras corroídas en el marco de la verificación de la seguridad estructural de elementos de hormigón armado con armaduras corroídas. Para ello se ha llevado a cabo un programa experimental de ensayos tipo pull-out excéntricos, con diferentes probetas, unas sin corrosión y otras sometidas tanto a procesos de corrosión natural como a procesos de corrosión acelerada, con diferentes grados de deterioro. Este tipo de ensayo de adherencia representa de forma realista y fiable realista los esfuerzos de adherencia en la zona de anclaje. Por otra parte, para la realización de estos ensayos se ha puesto a punto, además del procedimiento de ensayo, un sistema de adquisición de datos entre los que se incluye el empleo de sensores de tipo fibra óptica con redes de Bragg embebidos en la armadura para determinar los parámetros representativos de la adherencia en el hormigón estructural con armaduras corroídas. Por otra parte, la recopilación de los datos de los estudios de adherencia con armaduras corroídas procedentes de la literatura científica, además de los resultados de la presente investigación, junto con la identificación de las variables relevantes en el comportamiento de la adherencia con armaduras sanas y corroídas ha servido para la obtención de una formulación realista y fiable para la evaluación conjunta de la adherencia con armaduras sanas y corroídas a partir de modelos de regresión múltiple. La formulación propuesta ha sido validada mediante criterios estadísticos y comparada con otras formulaciones propuestas en la literatura científica. Además se ha realizado un análisis de las variables influyentes de la formulación propuesta. También se ha obtenido un modelo numérico simple y eficiente, validado con alguno de los ensayos realizados en esta tesis, para simular la adherencia con armaduras sanas y corroídas. Finalmente, se presenta un procedimiento para realizar la evaluación de vigas deterioradas por corrosión mediante el método de los campos de tensiones que incluye la evaluación de la adherencia mediante la formulación sugerida en esta Tesis Doctoral. Las conclusiones alcanzadas en este trabajo han permitido evaluar la adherencia con armaduras corroídas de forma realista y fiable. Asimismo, se ha podido incluir la evaluación de la adherencia en el marco de la verificación de la seguridad estructural en elementos de hormigón armado deteriorados por corrosión. ABSTRACT Structural safety verification of both structures allowing a certain degree of deterioration in design and deteriorated existing structures needs strength models that factor in the effects of deterioration. In case of corrosion of steel bars in reinforced concrete structures, the resistance depends on many things such as the remaining cross-section of the corroded reinforcement bars, the stress-strain diagrams of steel, the concrete-reinforcement bond and corrosion-induced concrete cracking or spalling. Accordingly, the force transfer through the contact surface between concrete and reinforcement, bond, is one of the most important aspects to consider and it is the basis of the structural performance of reinforced concrete. Bond must assure anchorage of reinforcement and transmit shear stresses as a consequence of the different stresses along a structural element As a consequence of corrosion, the bond development may be affected and hence the transfer of longitudinal stresses. This PhD Thesis deals with ultimate limit state bond behaviour in structural concrete with corrode steel bars. The main objective is to obtain a realistic and reliable model for the assessment of bond within the context of structural safety verifications of reinforced concrete members with corroded steel bars. In that context, an experimental programme of eccentric pull-out tests were conducted, with different specimens, ones without corrosion and others subjected to accelerated or natural corrosion with different corrosion degrees. This type of bond test reproduces in a realistic and reliable way bond stresses in the anchorage zone. Moreover, for conducting these tests it was necessary to develop both a test procedure and also a data acquisition system including the use of an embedded fibre-optic sensing system with fibre Bragg grating sensors to obtain the representative parameters of bond strength in structural concrete with corroded steel bars. Furthermore, the compilation of data from bond studies with corroded steel bars from scientific literature, including tests conducted in the present study, along with the identification of the relevant variables influencing bond behaviour for both corroded and non-corroded steel bars was used to obtain a realistic and reliable formulation for bond assessment in corroded and non-corroded steel bars by multiple linear regression analysis. The proposed formulation was validated with a number of statistical criteria and compared to other models from scientific literature. Moreover, an analysis of the influencing variables of the proposed formulation has been performed. Also, a simplified and efficient numerical model has been obtained and validated with several tests performed in this PhD Thesis for simulating the bond in corroded and non-corroded steel bars. Finally, a proposal for the assessment of corrosion-damaged beams with stress field models including bond assessment with the proposed formulation is presented. The conclusions raised in this work have allowed a realistic and reliable bond assessment in corroded steel bars. Furthermore, bond assessment has been included within the context of structural safety verifications in corrosion-damaged reinforced concrete elements.

Development and fabrication of optical biosensors based on biophotonic sensing cells (BICELLs)

El objetivo de esta tesis es el desarrollo y caracterización de biosensores ópticos sin marcado basados en celdas sensoras biofotónicas (BICELLs). Éstas son un nuevo concepto de biosensor desarrollado por el grupo de investigación y consiste en la combinación de técnicas de interrogación vertical, junto a estructuras fotónicas producidas usando métodos de micro- y nanofabricación. Varias conclusiones son extraídas de este trabajo. La primera, que se ha definido una BICELL estándar basada en interferómetros Fabry-Perot (FP). Se ha demostrado su capacidad para la comparación de rendimiento entre BICELLs estructuradas y para la realización de inmunoensayos de bajo coste. Se han estudiado diferentes técnicas de fabricación disponibles para la producción de BICELLs. Se determinó que la litografía de contacto a nivel de oblea produce estructuras de bajo coste, reproducibles y de alta calidad. La resolución alcanzada ha sido de 700 nm. El estudio de la respuesta a inmunoensayos de las BICELLs producidas se ha desarrollado en este trabajo. Se estudió la influencia de BICELLs basadas en diferentes geometrías y tamaños. De aquí resulta un nuevo enfoque para predecir el comportamiento de respuesta para la detección biológica de cualquier biosensor óptico estructurado, relacionando su superficie efectiva y su sensibilidad óptica. También se demostró una técnica novedosa y de bajo coste para la caracterización experimental de la sensibilidad óptica, basada en el depósito de películas ultradelgadas. Finalmente, se ha demostrado el uso de BICELLs desarrolladas en esta tesis, en la detección de aplicaciones reales, tales como hormonas, virus y proteínas. ABSTRACT The objective of this thesis is the development and characterization of optical label-free biosensors based on Bio-Photonic sensing Cells (BICELLs). BICELL is a novel biosensor concept developed by the research group, and it consists of a combination of vertical interrogation optical techniques and photonic structures produced by using micro- and nano-fabrication methods. Several main conclusions are extracted from this work. Firstly, a standard BICELL is defined based on FP interferometers, which demonstrated its capacity for accomplishing performance comparisons among different structured BICELLs, as well as to achieve low-cost immunoassays. Different available fabrication techniques were studied for BICELL manufacturing. It is found that contact lithography at wafer scale produce cost-effective, reproducible and high quality structures. The resolution achieved was 700 nm. Study on the response of developed BICELLs to immunoassays is performed within this work. It is therefore studied the influence of BICELLs based on different geometries and sizes in the immunoassay, which resulted in a new approach to predict the biosensing behaviour of any structured optical biosensor relating to its effective surface and optical sensitivity. Also, it is demonstrated a novel and low-cost characterization technique of the experimental optical sensitivity, based on ultrathin-film deposition. Finally, it is also demonstrated the capability of using the developed BICELLs in this thesis for real applications detection of hormones, virus and proteins.

VHDL-based system design of a cognitive sensorimotor loop (CSL) for haptic Human-Machine Interaction (HMI)

This document is a summary of the Bachelor thesis titled “VHDL-Based System Design of a Cognitive Sensorimotor Loop (CSL) for Haptic Human-Machine Interaction (HMI)” written by Pablo de Miguel Morales, Electronics Engineering student at the Universidad Politécnica de Madrid (UPM Madrid, Spain) during an Erasmus+ Exchange Program at the Beuth Hochschule für Technik (BHT Berlin, Germany). The tutor of this project is Dr. Prof. Hild. This project has been developed inside the Neurobotics Research Laboratory (NRL) in close collaboration with Benjamin Panreck, a member of the NRL, and another exchange student from the UPM Pablo Gabriel Lezcano. For a deeper comprehension of the content of the thesis, a deeper look in the document is needed as well as the viewing of the videos and the VHDL design. In the growing field of automation, a large amount of workforce is dedicated to improve, adapt and design motor controllers for a wide variety of applications. In the specific field of robotics or other machinery designed to interact with humans or their environment, new needs and technological solutions are often being discovered due to the existing, relatively unexplored new scenario it is. The project consisted of three main parts: Two VHDL-based systems and one short experiment on the haptic perception. Both VHDL systems are based on a Cognitive Sensorimotor Loop (CSL) which is a control loop designed by the NRL and mainly developed by Dr. Prof. Hild. The CSL is a control loop whose main characteristic is the fact that it does not use any external sensor to measure the speed or position of the motor but the motor itself. The motor always generates a voltage that is proportional to its angular speed so it does not need calibration. This method is energy efficient and simplifies control loops in complex systems. The first system, named CSL Stay In Touch (SIT), consists in a one DC motor system controller by a FPGA Board (Zynq ZYBO 7000) whose aim is to keep contact with any external object that touches its Sensing Platform in both directions. Apart from the main behavior, three features (Search Mode, Inertia Mode and Return Mode) have been designed to enhance the haptic interaction experience. Additionally, a VGA-Screen is also controlled by the FPGA Board for the monitoring of the whole system. This system has been completely developed, tested and improved; analyzing its timing and consumption properties. The second system, named CSL Fingerlike Mechanism (FM), consists in a fingerlike mechanical system controlled by two DC motors (Each controlling one part of the finger). The behavior is similar to the first system but in a more complex structure. This system was optional and not part of the original objectives of the thesis and it could not be properly finished and tested due to the lack of time. The haptic perception experiment was an experiment conducted to have an insight into the complexity of human haptic perception in order to implement this knowledge into technological applications. The experiment consisted in testing the capability of the subjects to recognize different objects and shapes while being blindfolded and with their ears covered. Two groups were done, one had full haptic perception while the other had to explore the environment with a plastic piece attached to their finger to create a haptic handicap. The conclusion of the thesis was that a haptic system based only on a CSL-based system is not enough to retrieve valuable information from the environment and that other sensors are needed (temperature, pressure, etc.) but that a CSL-based system is very useful to control the force applied by the system to interact with haptic sensible surfaces such as skin or tactile screens. RESUMEN. Este documento es un resumen del proyecto fin de grado titulado “VHDL-Based System Design of a Cognitive Sensorimotor Loop (CSL) for Haptic Human-Machine Interaction (HMI)” escrito por Pablo de Miguel, estudiante de Ingeniería Electrónica de Comunicaciones en la Universidad Politécnica de Madrid (UPM Madrid, España) durante un programa de intercambio Erasmus+ en la Beuth Hochschule für Technik (BHT Berlin, Alemania). El tutor de este proyecto ha sido Dr. Prof. Hild. Este proyecto se ha desarrollado dentro del Neurorobotics Research Laboratory (NRL) en estrecha colaboración con Benjamin Panreck (un miembro del NRL) y con Pablo Lezcano (Otro estudiante de intercambio de la UPM). Para una comprensión completa del trabajo es necesaria una lectura detenida de todo el documento y el visionado de los videos y análisis del diseño VHDL incluidos en el CD adjunto. En el creciente sector de la automatización, una gran cantidad de esfuerzo está dedicada a mejorar, adaptar y diseñar controladores de motor para un gran rango de aplicaciones. En el campo específico de la robótica u otra maquinaria diseñada para interactuar con los humanos o con su entorno, nuevas necesidades y soluciones tecnológicas se siguen desarrollado debido al relativamente inexplorado y nuevo escenario que supone. El proyecto consta de tres partes principales: Dos sistemas basados en VHDL y un pequeño experimento sobre la percepción háptica. Ambos sistemas VHDL están basados en el Cognitive Sesnorimotor Loop (CSL) que es un lazo de control creado por el NRL y cuyo desarrollador principal ha sido Dr. Prof. Hild. El CSL es un lazo de control cuya principal característica es la ausencia de sensores externos para medir la velocidad o la posición del motor, usando el propio motor como sensor. El motor siempre genera un voltaje proporcional a su velocidad angular de modo que no es necesaria calibración. Este método es eficiente en términos energéticos y simplifica los lazos de control en sistemas complejos. El primer sistema, llamado CSL Stay In Touch (SIT), consiste en un sistema formado por un motor DC controlado por una FPGA Board (Zynq ZYBO 7000) cuyo objetivo es mantener contacto con cualquier objeto externo que toque su plataforma sensible en ambas direcciones. Aparte del funcionamiento básico, tres modos (Search Mode, Inertia Mode y Return Mode) han sido diseñados para mejorar la interacción. Adicionalmente, se ha diseñado el control a través de la FPGA Board de una pantalla VGA para la monitorización de todo el sistema. El sistema ha sido totalmente desarrollado, testeado y mejorado; analizando su propiedades de timing y consumo energético. El segundo sistema, llamado CSL Fingerlike Mechanism (FM), consiste en un mecanismo similar a un dedo controlado por dos motores DC (Cada uno controlando una falange). Su comportamiento es similar al del primer sistema pero con una estructura más compleja. Este sistema no formaba parte de los objetivos iniciales del proyecto y por lo tanto era opcional. No pudo ser plenamente desarrollado debido a la falta de tiempo. El experimento de percepción háptica fue diseñado para profundizar en la percepción háptica humana con el objetivo de aplicar este conocimiento en aplicaciones tecnológicas. El experimento consistía en testear la capacidad de los sujetos para reconocer diferentes objetos, formas y texturas en condiciones de privación del sentido del oído y la vista. Se crearon dos grupos, en uno los sujetos tenían plena percepción háptica mientras que en el otro debían interactuar con los objetos a través de una pieza de plástico para generar un hándicap háptico. La conclusión del proyecto fue que un sistema háptico basado solo en sistemas CSL no es suficiente para recopilar información valiosa del entorno y que debe hacer uso de otros sensores (temperatura, presión, etc.). En cambio, un sistema basado en CSL es idóneo para el control de la fuerza aplicada por el sistema durante la interacción con superficies hápticas sensibles tales como la piel o pantallas táctiles.

In situ atomic force microscopy study of Alzheimer’s β-amyloid peptide on different substrates: New insights into mechanism of β-sheet formation

We have applied in situ atomic force microscopy to directly observe the aggregation of Alzheimer’s β-amyloid peptide (Aβ) in contact with two model solid surfaces: hydrophilic mica and hydrophobic graphite. The time course of aggregation was followed by continuous imaging of surfaces remaining in contact with 10–500 μM solutions of Aβ in PBS (pH 7.4). Visualization of fragile nanoscale aggregates of Aβ was made possible by the application of a tapping mode of imaging, which minimizes the lateral forces between the probe tip and the sample. The size and the shape of Aβ aggregates, as well as the kinetics of their formation, exhibited pronounced dependence on the physicochemical nature of the surface. On hydrophilic mica, Aβ formed particulate, pseudomicellar aggregates, which at higher Aβ concentration had the tendency to form linear assemblies, reminiscent of protofibrillar species described recently in the literature. In contrast, on hydrophobic graphite Aβ formed uniform, elongated sheets. The dimensions of those sheets were consistent with the dimensions of β-sheets with extended peptide chains perpendicular to the long axis of the aggregate. The sheets of Aβ were oriented along three directions at 120° to each other, resembling the crystallographic symmetry of a graphite surface. Such substrate-templated self-assembly may be the distinguishing feature of β-sheets in comparison with α-helices. These studies show that in situ atomic force microscopy enables direct assessment of amyloid aggregation in physiological fluids and suggest that Aβ fibril formation may be driven by interactions at the interface of aqueous solutions and hydrophobic substrates, as occurs in membranes and lipoprotein particles in vivo.

Controlled unzipping of a bacterial surface layer with atomic force microscopy

We have combined high-resolution atomic force microscopy (AFM) imaging and force spectroscopy to gain insight into the interaction forces between the individual protomers of the hexagonally packed intermediate (HPI) layer of Deinococcus radiodurans. After imaging the HPI layer, the AFM stylus was attached to individual protomers by enforced stylus-sample contact to allow force spectroscopy experiments. Imaging of the HPI layer after recording force-extension curves allowed adhesion forces to be correlated with structural alterations. By using this approach, individual protomers of the HPI layer were found to be removed at pulling forces of ≈300 pN. Furthermore, it was possible to sequentially unzip entire bacterial pores formed by six HPI protomers. The combination of high-resolution AFM imaging of individual proteins with the determination of their intramolecular forces is a method of studying the mechanical stability of supramolecular structures at the level of single molecules.

A molecular basis for nitric oxide sensing by soluble guanylate cyclase

Nitric oxide (NO) functions as a signaling agent by activation of the soluble isoform of guanylate cyclase (sGC), a heterodimeric hemoprotein. NO binds to the heme of sGC and triggers formation of cGMP from GTP. Here we report direct kinetic measurements of the multistep binding of NO to sGC and correlate these presteady state events with activation of enzyme catalysis. NO binds to sGC to form a six-coordinate, nonactivated, intermediate (kon > 1.4 × 108 M−1⋅s−1 at 4°C). Subsequent release of the axial histidine heme ligand is shown to be the molecular step responsible for activation of the enzyme. The rate at which this step proceeds also depends on NO concentration (k = 2.4 × 105 M−1⋅s−1 at 4°C), thus identifying a novel mode of regulation by NO. NO binding to the isolated heme domain of sGC was also rapid (k = 7.1 ± 2 × 108 M−1⋅s−1 at 4°C); however, no intermediate was observed. The data show that sGC acts as an extremely fast, specific, and highly efficient trap for NO and that cleavage of the iron-histidine bond provides the driving force for activation of sGC. In addition, the kinetic data indicate that transport or stabilization of NO is not necessary for effective signal transmission.

Imaging ROMK1 inwardly rectifying ATP-sensitive K+ channel protein using atomic force microscopy.

The inwardly rectifying K+ channel ROMK1 has been implicated as being significant in K+ secretion in the distal nephron. ROMK1 has been shown by immunocytochemistry to be expressed in relevant nephron segments. The development of the atomic force microscope has made possible the production of high resolution images of small particles, including a variety of biological macromolecules. Recently, a fusion protein of glutathione S-transferase (GST) and ROMK1 (ROMK1-GST) has been used to produce a polyclonal antibody for immunolocalization of ROMK1. We have used atomic force microscopy to examine ROMK1-GST and the native ROMK1 polypeptide cleaved from GST. Imaging was conducted with the proteins in physiological solutions attached to mica. ROMK1-GST appears in images as a particle composed of two units of similar size. Analyses of images indicate that the two units have volumes of approximately 118 nm3, which is close to the theoretical volume of a globular protein of approximately 65 kDa (the molecular mass of ROMK1-GST). Native GST exists as a dimer, and the images obtained here are consistent with the ROMK1-GST fusion protein's existence as a heterodimer. In experiments on ROMK1 in aqueous solution, single molecules appear to aggregate, but contact to the mica was maintained. Addition of ATP to the solution produced a change in height of the aggregates. This change (which was reversible) suggests that ATP induces a structural change in the ROMK1 protein. The data show that atomic force microscopy is a useful tool for examination of purified protein molecules under near-physiological conditions, and furthermore, that structural alterations in the proteins may be continuously investigated.

3D Visual Data-Driven Spatiotemporal Deformations for Non-Rigid Object Grasping Using Robot Hands

Sensing techniques are important for solving problems of uncertainty inherent to intelligent grasping tasks. The main goal here is to present a visual sensing system based on range imaging technology for robot manipulation of non-rigid objects. Our proposal provides a suitable visual perception system of complex grasping tasks to support a robot controller when other sensor systems, such as tactile and force, are not able to obtain useful data relevant to the grasping manipulation task. In particular, a new visual approach based on RGBD data was implemented to help a robot controller carry out intelligent manipulation tasks with flexible objects. The proposed method supervises the interaction between the grasped object and the robot hand in order to avoid poor contact between the fingertips and an object when there is neither force nor pressure data. This new approach is also used to measure changes to the shape of an object’s surfaces and so allows us to find deformations caused by inappropriate pressure being applied by the hand’s fingers. Test was carried out for grasping tasks involving several flexible household objects with a multi-fingered robot hand working in real time. Our approach generates pulses from the deformation detection method and sends an event message to the robot controller when surface deformation is detected. In comparison with other methods, the obtained results reveal that our visual pipeline does not use deformations models of objects and materials, as well as the approach works well both planar and 3D household objects in real time. In addition, our method does not depend on the pose of the robot hand because the location of the reference system is computed from a recognition process of a pattern located place at the robot forearm. The presented experiments demonstrate that the proposed method accomplishes a good monitoring of grasping task with several objects and different grasping configurations in indoor environments.