From lipid bilayers to synaptic vesicles: atomic force microscopy on lipid-based systems

The aim of this thesis was to apply the techniques of the atomic force microscope (AFM) to biological samples, namely lipid-based systems. To this end several systems with biological relevance based on self-assembly, such as a solid-supported membrane (SSM) based sensor for transport proteins, a bilayer of the natural lipid extract from an archaebacterium, and synaptic vesicles, were investigated by the AFM. For the characterization of transport proteins with SSM-sensors proteoliposomes are adsorbed that contain the analyte (transport protein). However the forces governing bilayer-bilayer interactions in solution should be repulsive under physiological conditions. I investigated the nature of the interaction forces with AFM force spectroscopy by mimicking the adsorbing proteoliposome with a cantilever tip, which was functionalized with charged alkane thiols. The nature of the interaction is indeed repulsive, but the lipid layers assemble in stacks on the SSM, which expose their unfavourable edges to the medium. I propose a model by which the proteoliposomes interact with these edges and fuse with the bilayer stacks, so forming a uniform layer on the SSM. Furthermore I characterized freestanding bilayers from a synthetic phospholipid with a phase transition at 41°C and from a natural lipid extract of the archaebacterium Methanococcus jannaschii. The synthetic lipid is in the gel-phase at room temperature and changes to the fluid phase when heated to 50°C. The bilayer of the lipid extract shows no phase transition when heated from room temperature to the growth temperature (~ 50°C) of the archeon. Synaptic vesicles are the containers of neurotransmitter in nerve cells and the synapsins are a family of extrinsic membrane proteins, that are associated with them, and believed to control the synaptic vesicle cycle. I used AFM imaging and force spectroscopy together with dynamic light scattering to investigate the influence of synapsin I on synaptic vesicles. To this end I used native, untreated synaptic vesicles and compared them to synapsin-depleted synaptic vesicles. Synapsin-depleted vesicles were larger in size and showed a higher tendency to aggregate compared to native vesicles, although their mechanical properties were alike. I also measured the aggregation kinetics of synaptic vesicles induced by synapsin I and found that the addition of synapsin I promotes a rapid aggregation of synaptic vesicles. The data indicate that synapsin I affects the stability and the aggregation state of synaptic vesicles, and confirm the physiological role of synapsins in the assembly and regulation of synaptic vesicle pools within nerve cells.

Atomic force microscopy of biomimetic systems

This thesis was driven by the ambition to create suitable model systems that mimic complex processes in nature, like intramolecular transitions, such as unfolding and refolding of proteins, or intermolecular interactions between different cell compo-nents. Novel biophysical approaches were adopted by employing atomic force mi-croscopy (AFM) as the main measurement technique due to its broad diversity. Thus, high-resolution imaging, adhesion measurements, and single-molecule force distance experiments were performed on the verge of the instrumental capabilities. As first objective, the interaction between plasma membrane and cytoskeleton, me-diated by the linker protein ezrin, was pursued. Therefore, the adsorption process and the lateral organization of ezrin on PIP2 containing solid-supported membranes were characterized and quantified as a fundament for the establishment of a biomimetic model system. As second component of the model system, actin filaments were coated on functionalized colloidal probes attached on cantilevers, serving as sensor elements. The zealous endeavor of creating this complex biomimetic system was rewarded by successful investigation of the activation process of ezrin. As a result, it can be stated that ezrin is activated by solely binding to PIP2 without any further stimulating agents. Additional cofactors may stabilize and prolong the active conformation but are not essentially required for triggering ezrin’s transformation into an active conformation. In the second project, single-molecule force distance experiments were performed on bis-loop tetra-urea calix[4]arene-catenanes with different loading rates (increase in force per second). These macromolecules were specifically designed to investigate the rupture and rejoining mechanism of hydrogen bonds under external load. The entangled loops of capsule-like molecules locked the unbound state of intramolecular hydrogen bonds mechanically, rendering a rebinding observable on the experimental time scale. In conjunction with Molecular Dynamics simulations, a three-well potential of the bond rupture process was established and all kinetically relevant parameters of the experiments were determined by means of Monte Carlo simulations and stochastic modeling. In summary, it can be stated that atomic force microscopy is an invaluable tool to scrutinize relevant processes in nature, such as investigating activation mechanisms in proteins, as shown by analysis of the interaction between F-actin and ezrin, as well as exploring fundamental properties of single hydrogen bonds that are of paramount interest for the complete understanding of complex supramolecular structures.

Gravity-based Local Clock Synchronization in Wireless Sensor Networks

Contention-based MAC protocols follow periodic listen/sleep cycles. These protocols face the problem of virtual clustering if different unsynchronized listen/sleep schedules occur in the network, which has been shown to happen in wireless sensor networks. To interconnect these virtual clusters, border nodes maintaining all respective listen/sleep schedules are required. However, this is a waste of energy, if locally a common schedule can be determined. We propose to achieve local synchronization with a mechanism that is similar to gravitation. Clusters represent the mass, whereas synchronization messages sent by each cluster represent the gravitation force of the according cluster. Due to the mutual attraction caused by the clusters, all clusters merge finally. The exchange of synchronization messages itself is not altered by LACAS. Accordingly, LACAS introduces no overhead. Only a not yet used property of synchronization mechanisms is exploited.

G-force measurements at Mount Nyiragongo

To evaluate the mechanical stress on the volcanic edifice that results from lava lake level variations, we deployed a self-recording, differential capacitance (MEMS Inertial Sensor STMicroelectronics LIS3LV02DQ), 3-axis X6-1A accelerometer (Gulf Coast Data Concepts, LLC) at a distance of ~100m from the center of the Nyiragongo lava lake on freshly erupted lava flows. The device range was used in high (12-bit) resolution mode, which corresponds to a sensitivity of about 1 mg. The device was set to high-sensitivity mode with four additional bits to improve resolution, yet with a much lower signal-noise ratio. Once in position, the accelerometer continuously recorded data for three-day periods in June 2010. The system was oriented so that the X- and Y-axes form a plain parallel to the lava lake. During data collection, we did not attempt to calibrate the precision of the angle because relative G-force measurements were required instead of absolute G-force measurements. To distinguish the tiny accelerations caused by temperature differentials of the atmosphere, from the forces caused by magma movements, the temperature of the X6-1A device was continuously recorded. Temperature variations were corrected for by applying a de-correlation method to the recorded signal. Data was collected at 20 Hz, regrouped into batches that cover 1 hour per observation and associated with one averaged temperature measurement. This method was reproducible because diurnal temperature variations were the main cause for heating and cooling.

Novel system for bite-force sensing and monitoring based on magnetic eear field communication

Intraoral devices for bite-force sensing have several applications in odontology and maxillofacial surgery, as bite-force measurements provide additional information to help understand the characteristics of bruxism disorders and can also be of help for the evaluation of post-surgical evolution and for comparison of alternative treatments. A new system for measuring human bite forces is proposed in this work. This system has future applications for the monitoring of bruxism events and as a complement for its conventional diagnosis. Bruxism is a pathology consisting of grinding or tight clenching of the upper and lower teeth, which leads to several problems such as lesions to the teeth, headaches, orofacial pain and important disorders of the temporomandibular joint. The prototype uses a magnetic field communication scheme similar to low-frequency radio frequency identification (RFID) technology (NFC). The reader generates a low-frequency magnetic field that is used as the information carrier and powers the sensor. The system is notable because it uses an intra-mouth passive sensor and an external interrogator, which remotely records and processes information regarding a patient?s dental activity. This permits a quantitative assessment of bite-force, without requiring intra-mouth batteries, and can provide supplementary information to polysomnographic recordings, current most adequate early diagnostic method, so as to initiate corrective actions before irreversible dental wear appears. In addition to describing the system?s operational principles and the manufacture of personalized prototypes, this report will also demonstrate the feasibility of the system and results from the first in vitro and in vivo trials.

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.

Detection and localization of individual antibody-antigen recognition events by atomic force microscopy.

A methodology has been developed for the study of molecular recognition at the level of single events and for the localization of sites on biosurfaces, in combining force microscopy with molecular recognition by specific ligands. For this goal, a sensor was designed by covalently linking an antibody (anti-human serum albumin, polyclonal) via a flexible spacer to the tip of a force microscope. This sensor permitted detection of single antibody-antigen recognition events by force signals of unique shape with an unbinding force of 244 +/- 22 pN. Analysis revealed that observed unbinding forces originate from the dissociation of individual Fab fragments from a human serum albumin molecule. The two Fab fragments of the antibody were found to bind independently and with equal probability. The flexible linkage provided the antibody with a 6-nm dynamical reach for binding, rendering binding probability high, 0.5 for encounter times of 60 ms. This permitted fast and reliable detection of antigenic sites during lateral scans with a positional accuracy of 1.5 nm. It is indicated that this methodology has promise for characterizing rate constants and kinetics of molecular recognition complexes and for molecular mapping of biosurfaces such as membranes.

Disposable electrochromic polyaniline sensor based on a redox response using a conventional camera: A first approach to handheld analysis

We present a disposable optical sensor for Ascorbic Acid (AA). It uses a polyaniline based electrochromic sensing film that undergoes a color change when exposed to solutions of ascorbic acid at pH 3.0. The color is monitored by a conventional digital camera working with the hue (H) color coordinate. The electrochromic film was deposited on an Indium Tin Oxide (ITO) electrode by cyclic voltammetry and then characterized by atomic force microscopy, electrochemical and spectroscopic techniques. An estimation of the initial rate of H, as ΔH/Δt, is used as the analytical parameter and resulted in the following logarithmic relationship: ΔH/Δt = 0.029 log[AA] + 0.14, with a limit of detection of 17 μM. The relative standard deviation when using the same membrane 5 times was 7.4% for the blank, and 2.6% (for n = 3) on exposure to ascorbic acid in 160 μM concentration. The sensor is disposable and its applicability to pharmaceutical analysis was demonstrated. This configuration can be extended for future handheld configurations.

Operator's, organizational, and intermediate (direct support and general support) maintenance manual (including repair parts and special tools list) : sensor, capacitance proximity DT-548()/FSS-9 (V) FSN 6350-228-2606.

"May 1974."

Operator's, organizational, and intermediate (direct support and general support) maintenance manual (including repair parts and special tools list) : sensor, magnetic weapon DT-547()/FSS-9(V) FSN 6350-228-2590.

"May 1974."

Organizational concepts for the sensor-to-shooter world : the impact of real-time information on airpower targeting /

"May 1997."

Development of a carbon dioxide partial pressure sensor

Photocopy.

Force sensing for measuring human body movement

The research developed in this thesis explores the sensing and inference of human movement in a dynamic way, as opposed to conventional measurement systems, that are only concerned with discrete evaluations of stimuli in sequential time. Typically, conventional approaches are used to infer the dynamic movement of the body; such as vision and motion tracking devices, with either a human diagnosis or complex image processing algorithm to classify the movement. This research is therefore the first of its kind to attempt and provide a movement classifying algorithm through the use of minimal sensing points, with the application for this novel system, to classify human movement during a golf swing. There are two main categories of force sensing. Firstly, array-type systems consisting of many sensing elements, and are the most commonly researched and commercially available. Secondly, reduced force sensing element systems (RFSES) also known as distributive systems have only been recently exploited in the academic world. The fundamental difference between these systems is that array systems handle the data captured from each sensor as unique outputs and suffer the effects of resolution. The effect of resolution, is the error in the load position measurement between sensing elements, as the output is quantized in terms of position. This can be compared to a reduced sensor element system that maximises that data received through the coupling of data from a distribution of sensing points to describe the output in discrete time. Also this can be extended to a coupling of transients in the time domain to describe an activity or dynamic movement. It is the RFSES that is to be examined and exploited in the commercial sector due to its advantages over array-based approaches such as reduced design, computational complexity and cost.

Graphene-assisted microfiber for optical-power-based temperature sensor

Combined the large evanescent field of microfiber with the high thermal conductivity of graphene, a sensitive all-fiber temperature sensor based on graphene-assisted micro fiber is proposed and experimentally demonstrated. Microfiber can be easily attached with graphene due to the electrostatic 6 force, resulting in an effective interaction between graphene and the evanescent field of microfiber. The change of the ambient temperature has a great influence on the conductivity of graphene, leading to the variation of the effective refractive index of microfiber. Consequently, the optical power transmission will be changed. The temperature sensitivity of 0.1018 dB/°C in the heating process and 0.1052 dB/°C in the cooling process as well as a high resolution of 0.0098 °C is obtained in the experiment. The scheme may have great potential in sensing fields owing to the advantages of high sensitivity, compact size, and low cost.

Performance analysis of polymer optical fibre based Fabry-Perot sensor formed by two uniform Bragg gratings

The stress sensitivity of polymer optical fibre (POF) based Fabry-Perot sensors formed by two uniform Bragg gratings with finite dimensions is investigated. POF has received high interest in recent years due to its different material properties compared to its silica counterpart. Biocompatibility, a higher failure strain and the highly elastic nature of POF are some of the main advantages. The much lower Young’s modulus of polymer materials compared to silica offers enhanced stress sensitivity to POF based sensors which renders them great candidates for acoustic wave receivers and any kind of force detection. The main drawback in POF technology is perhaps the high fibre loss. In a lossless fibre the sensitivity of an interferometer is proportional to its cavity length. However, the presence of the attenuation along the optical path can significantly reduce the finesse of the Fabry-Perot interferometer and it can negatively affect its sensitivity at some point. The reflectivity of the two gratings used to form the interferometer can be also reduced as the fibre loss increases. In this work, a numerical model is developed to study the performance of POF based Fabry-Perot sensors formed by two uniform Bragg gratings with finite dimensions. Various optical and physical properties are considered such as grating physical length, grating effective length which indicates the point where the light is effectively reflected, refractive index modulation of the grating, cavity length of the interferometer, attenuation and operating wavelength. Using this model, we are able to identify the regimes in which the PMMA based sensor offer enhanced stress sensitivity compared to silica based one.