La membrane plasmique constitue la première entité séparant la cellule de son environnement. A ce rôle de barrière s'ajoute celui de réguler la. Par conséquent, la membrane plasmique est une zone privilégiée pour le passage d'information. Cependant, son étude reste difficile, ne serait-ce que par l'extraordinaire complexité d'organisation de cet assemblage supramoléculaire.Mon projet de thèse vise à développer de nouvelles approches expérimentales pour explorer plus spécifiquement l'organisation et le rôle de la membrane plasmique d'une cellule dans les mécanismes de transduction de l'information. Deux axes ont été privilégiés : le premier, concerne la description de la dynamique d'organisation de la membrane ; le deuxième concerne l'inter-connectivité et la transmission du signal d'une cellule avec d'autres partenaires.Ce manuscrit se compose de plusieurs parties. Le premier chapitre introduira succinctement les questions biologiques. Dans le second chapitre, je présenterai des méthodes utilisées pour l'étude de la membrane. J'y présenterai aussi une série d'observation que j'ai réalisée sur la diffusion de l'EGFR. Le troisième chapitre sera consacré à la technique de corrélation croisée de fluorescence depuis le montage jusqu'à l'étude du modèle EGFR. Dans la quatrième partie, nous verrons comment les collaborations à l'interface biophysique ont permis des développements innovants sur un système de pinces optiques holographiques. J'y présenterai les applications de ce système à différent modèles d'intérêt biologique. Enfin, je conclurai ce document par une brève discussion autour des résultats obtenus aussi bien d'un point de vue méthodologique que biologique. / The plasma membrane separates the cell from its environment. But it is more than a barrier any cell has to communicate with the outside world. Therefore the plasma membrane plays a prime role in transferring and exchanging information. However, the biological study of the plasma membrane remains difficult due to the extraordinary complexity of it organization.My thesis is a part of an effort to develop new experimental approaches to explore more specifically the organization and the role of the plasma membrane in the signal transduction mechanisms. Two major aspects were followed: the first one concerns the description of the dynamics of membrane organization and of molecular interactions, the second concerns the inter-connectivity and signal transduction between a cell and other biological partners.This manuscript is composed of several parts. The first chapter briefly introduces the biological questions that I tried to answer. In the second chapter, I present the methods commonly used to study the membrane with a dynamic perspective. Additionally, I include a series of observations that I made on the EGF receptor diffusion. The third chapter is devoted to the fluorescence cross-correlation technique to study the assembly of the EGFR. In the fourth part, I demonstrate how scientific collaborations at the interface between biology and physics have led to the development of innovative solutions on a holographic optical tweezers system. I present applications of this system in different biological models. Finally, I conclude this thesis with a brief discussion about my technological and biological results.

"Thermal infrared (IR) imaging systems are widely used in medical, industrial, and defense applications. IR imaging systems utilize a lens to focus IR radiation onto a focal plane array (FPA) of IR detectors, which transduce the IR radiation from the scene into signals that can be further processed. In conventional IR imaging systems, electronic readout integrated circuitry (ROIC) is used to read out the information from the FPA, and computer signal processing allows for an IR image to be displayed on an electronic screen. However, the ROIC decreases the thermal isolation and sensitivity of the IR detectors in the FPA, and the computer processing and electronic display increase the cost, weight, and complexity of the IR imaging system. This thesis focuses on the development of an IR direct viewing system that does not require any ROIC, computer signal processing, or electronic display. This is accomplished through the use of microelectromechanical systems (MEMS) uncooled IR imaging detectors, which consist of arrays of bimaterial thermomechanical cantilever structures that tilt as a function of IR radiation from a scene. Other members of the WPI-ME/CHSLT group have previously shown that an interferometric optical readout mechanism based on digital holography and computer processing can eliminate the need for ROIC and be used to measure the nanometer scale tilt of the structures in a MEMS-based IR imaging system that was found to have a responsivity of 1.5 nm/K. However, these previously demonstrated results required significant computer processing and an electronic display. The hypothesis of the current work is that an optomechanical readout mechanism can be used to realize an IR direct viewer without the use of ROIC, computer signal processing, or an electronic display. Three optical readout mechanisms were identified for transducing the nanometer scale deformations of the MEMS structures in the FPA into a directly observable visible light image. Two of these, one using live holography and the other using Nomarski differential interference contrast (DIC), were based on interferometry, while the third, using reflectometry, was based on geometrical optics. The identified optical readout mechanisms were analytically evaluated based on the performance and perception of the human vision system (HVS), and preliminary experimental results were obtained using optical setups constructed for all three readout mechanisms. Based on the analytical and experimental investigations, reflectometry was selected as the most suitable readout mechanism for a direct viewer. A visible light camera was used with custom software to determine a temperature sensitivity of 137 mK for the reflectometry readout, and thermal images of scenes at human body temperature were demonstrated using limited computer processing. A false color, direct view, live IR imaging system was then demonstrated based on a two color reflectometry readout and the output was characterized with respect to the color differentiation sensitivity of the HVS. The system temperature sensitivity, based on the theoretical color differentiation sensitivity of a human observer, was found to be on the order of 10 K across a measuring range of roughly 400 °C, and objects with a temperature as low as approximately 150 °C were distinguishable. The advantages and limitations of the developed IR imaging system are identified and recommendations for further developments and future work are provided."

Binding-sites which facilitate the transport of substrates across membranes are ubiquitous in membrane proteins. To understand this fundamental process in cells, we build up a synthetic membrane system consisting of microfluidic channels and colloidal particles. Holographic optical tweezers are used to modulate the potential energy landscape in those channels. We show how to extract the underlying energy potential by analysing local transition probabilities. Our method is applicable both to equilibrium systems and non-equilibrium steady states. Our method offers improved robustness when dealing with fragmented trajectories or small ensembles of data compared to other established approaches, such as probability density function and splitting probability. Meanwhile, we utilise the intensity distribution of the optical traps generated by holographic optical tweezers to estimate energy landscapes featuring high energy barriers where transitions rarely occur. We use this newly developed experimental system to mimic the functionality of membrane protein transporters that are known to alternate their substrate-binding sites between the extracellular and cytosolic side of the membrane. We study particle transport through a channel coupled with an energy well that oscillates its position between the two entrances of the channel deterministically and stochastically. Optimised particle transport across the channel is obtained by adjusting the oscillation frequency. At the optimal oscillation frequency, the translocation rate of particles through the channel is a hundred times higher with respect to free diffusion across the channel. Our findings reveal the effect of time dependent potentials on particle transport across a channel. This work adds a new tool for the investigation of highly controlled membrane transport processes at the micron scale. Our results are relevant for improving our understanding of membrane transport especially for microfluidics application.

Current representation schemes for automatic text classification treat documents as syntactically unstructured collections of words (Bag-of-Words) or `concepts' (Bag-of-Concepts). Past attempts to encode syntactic structure have treated part-of-speech information as another word-like feature, but have been shown to be less effective than non-structural approaches. We propose a new representation scheme using Holographic Reduced Representations (HRRs) as a technique to encode both semantic and syntactic structure, though in very different ways. This method is unique in the literature in that it encodes the structure across all features of the document vector while preserving text semantics. Our method does not increase the dimensionality of the document vectors, allowing for efficient computation and storage. We present the results of various Support Vector Machine classification experiments that demonstrate the superiority of this method over Bag-of-Concepts representations and improvement over Bag-of-Words in certain classification contexts.

Current representation schemes for automatic text classification treat documents as syntactically unstructured collections of words (Bag-of-Words) or `concepts' (Bag-of-Concepts). Past attempts to encode syntactic structure have treated part-of-speech information as another word-like feature, but have been shown to be less effective than non-structural approaches. We propose a new representation scheme using Holographic Reduced Representations (HRRs) as a technique to encode both semantic and syntactic structure, though in very different ways. This method is unique in the literature in that it encodes the structure across all features of the document vector while preserving text semantics. Our method does not increase the dimensionality of the document vectors, allowing for efficient computation and storage. We present the results of various Support Vector Machine classification experiments that demonstrate the superiority of this method over Bag-of-Concepts representations and improvement over Bag-of-Words in certain classification contexts.

No description available.

Digital in-line holographic microscopy (DIHM) has been incorporated as an additional simultaneous in situ optical technique with ellipsomicroscopy for surface imaging and microscopy to study metastable pitting corrosion on stainless steel 316LVM in simulated biological solutions. DIHM adds microscopic volume imaging, allows one to detect local changes of the index of refraction in the vicinity of a pitting event, and allows one to track tracer particles and/or material ejected from the pitting sites. To improve the pitting corrosion resistance of stainless steel 316LVM, a simple surface treatment was tested and the aforementioned imaging techniques were used to verify that pitting occurred only on the wire face. Treatments consisted of polishing the samples to remove the passive layer, then immersing the wires in 90 C nanopure water for several hours. Treated wires show a marked increase in pitting corrosion resistance over untreated wires: the pit initiation potential increases by a minimum of 200 mV. Additional testing with scanning electron microscopy and energy dispersive X-ray spectroscopy indicate that the removal of sulphide inclusions from the surface is the most probable cause of this enhancement. To increase holographic reconstruction performance, Graphics Processing Units (GPUs) have been used; 4 Mpixel holograms are reconstructed using the dot product approximation of the Kirchhoff-Fresnel integral in 60 ms on a Tesla c1060 GPU. Errors in sizes and positions can easily be as large as 5 to 10 % for regions where the dot product approximation is not valid, so algorithms with fewer or no approximations are also required. Reconstructions for arbitrary holographic geometries using the full Kirchhoff-Fresnel integral take approximately 1 hour (compared to 1 week on a quad-core CPU), and reconstructions using convolution methods, in which the results of 256 reconstructions at 4096 x 4096 pixels in one plane are combined, take 17 s. This method is almost exact, with approximations only in the obliquity factor.

Lithium niobate (LiNbO3) and KTiOPO4 (KTP) are ferroelectric crystals of considerable interest in different fields of optics and optoelectronics. Due to its large values of the nonlinear optical, electro-optic (EO), piezoelectric and acousto-optical coefficients, LiNbO3 is widely used for laser frequency conversion using the quasiphase matching (QPM) approach where the sign of nonlinearity has been periodically modulated by electric field poling (EFP). In the microwave and telecommunication field LiNbO3 is used for surface acoustic devices and integrated optical modulators. KTP and its isomorphs, on the other hand, exhibit slightly lower nonlinear coefficients but have much higher photorefractive damage thresholds, so that it is mainly used in the fabrication of QPM devices for both UV, IR and visible light generation and in high power applications. This thesis focus on different key issues: (1) accurate characterization of specific optical properties of LiNbO3, which are of interest in nonlinear and EO applications; (2) in-situ visualization and characterization of domain reversal by EFP in LiNbO3 and KTP crystals for a through understanding of the ferroelectric domain switching; (3) fabrication of periodic surface structures at sub-micron scale in LiNbO for photonic applications. An interferometric method is used for accurate measurement of ordinary and extraordinary refractive indices in uniaxial crystals, which is of great interest in the proper design of QPM crystals. A digital holography (DH) based method is presented here for 2D characterization of the EO properties of LiNbO , which is considerably interesting in the applications where the proper design of the EO device requires a spatially resolved information about the EO behaviour and the existing pointwise techniques are not sufficient. A DH method for novel in-situ monitoring of domain reversal by EFP in both LiNbO3 and KTP, is also presented here. The technqiue could be used as a tool for high fidelity periodic domain engineering but also provides information about domain kinetics, internal field and crystals defects. 3 3 3 Finally this thesis presents novel results concerning nanoscale periodic surface structuring of congruent LiNbO3. Holographic lithography (HL) is used for sub-micron period resist patterning and electric overpoling for surface domain reversal. Surface structures are obtained by selective etching. Moiré effect is also used in the HL to fabricate complicated structures with multiple periods. The depth compatibility with waveguide implementation allows foreseeing possible applications of these structures for Bragg gratings or innovative photonic crystal devices, exploiting the additional nonlinear and EO properties typical of LiNbO3. / QC 20100824

Moving towards label-free technologies is essential for many clinical and research applications. Raman spectroscopy is a powerful tool in the field of biomedicine for label-free cell characterisation and disease diagnosis, owing to its high chemical specificity. However, Raman scattering is a relatively weak process and can require long acquisition times, thus hampering its integration to clinical technologies. Multimodal analysis is currently pushing the boundaries in biomedicine, obtaining more information than would be possible using a single mode and overcoming any limitations specific to a single technique. Digital holographic microscopy (DHM) is a rapid and label-free quantitative phase imaging modality, providing complementary information to Raman spectroscopy, and is thus an ideal candidate for combination in a multimodal system. Firstly, this thesis explores the use of wavelength modulated Raman spectroscopy (WMRS), for the classification of immune cell subsets. Following this a multimodal approach, combining Raman spectroscopy and DHM, is demonstrated, where each technique is considered individually and in combination. The complementary modalities provide a wealth of information (both chemical and morphological) for cell characterisation, which is a step towards achieving a label-free technology for the identification of human immune cells. The suitability of WMRS to discriminate between closely related neuronal cell types is also explored. Furthermore optical spectroscopic techniques are useful for the analysis of food and beverages. The use of Raman and fluorescence spectroscopy to successfully discriminate between various whisky and extra-virgin olive oil brands is demonstrated, which may aid the detection of counterfeit or adulterated samples. The use of a compact Raman device is utilised, demonstrating the potential for in-field analysis. Finally, monodisperse and highly spherical nanoparticles are synthesised. A short study demonstrates the potential for these nanoparticles to benefit the techniques of surface enhanced Raman spectroscopy and optical trapping, by way of minimising variability.

This work builds up on the greatest minds of Cambridge Holography: Adrian Cable, Edward Buckley, Jonathan Freeman, and Christoph Bay. Cable and Buckley, developed an OSPR algorithm which was the first to provide high-quality real-time hologram generation using general-purpose hardware while Freeman designed a method to correct arbitrary aberrations. As ingenious as the method was, the calculations were extensively lengthy. Addressing this issue, a variant of OSPR suited for correcting spatially-varying aberration is presented. The algorithm combines the approaches of Cable, Buckley and Freeman to provide real-time hologram generation while incorporating various corrections (aberration, distortion, and pixel shape envelope). A high-performance implementation on a mid-range GPU achieved hologram generation up to 12 fps. Following topic studied is an adaptive optical correction. This work attempts to construct a set of methods, forming an automated testbed for holographic projectors. Each model, after exiting the production line is placed on such testbed, having all of its imperfections characterized. Once calibrated, each model is able to display highest-quality image throughout its life-span. An application of this work to industry was carried in collaboration with Dr Phillip Hands (University of Edinburgh) and LumeJET. Three demonstrators are constructed intending for a cost-effective system for holographic lithography. They are characterized using the developed testbed. Using the supersampled Adaptive OSPR algorithm, the diffraction limit was surpassed 2.75 times allowing to increase the patterning area. This combines approaches of Cable, Buckley, Freeman and Bay to achieve a wide field-of-view and high pixel-count replay field using off-the-shelf components. This thesis is finished describing the work on 3D holography carried with Penteract28. It is shown that the 2D hologram in the presence of spatially-varying aberrations is mathematically equivalent to a 3D hologram. The same implementation of the algorithm can be used to provide real-time 3D hologram generation.