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The crystallization of molecules from solution encompasses a number of key areas in science and technology, ranging from the purification and separation of industrial chemicals through to the arrangement of fragile biomacromolecules in ordered arrays suitable for structural analysis. However, as there are only a small number of techniques suitable for the study of such assemblies, many fundamental aspects governing the crystallization of molecules from solution are still poorly understood. In the studies presented here we have attempted to improve the understanding of this subject by investigating the crystallization of a series of molecules of both pharmaceutical and biological importance, using in situ Atomic Force Microscopy (AFM). A particular aim of the PhD was to develop the experimental protocols necessary to investigate macromolecular crystals known to exhibit poor diffraction properties, and subsequently to relate AFM data to these properties. The first study carried out concerned the crystallization and habit modification of a pharmaceutical excipient molecule, adipic acid. By using AFM we were able, for the first time, to directly observe the behavior of the dominant (100) face in both an air and liquid environment. A number of important observations were made including solute reorganization in air, etch pit formation and growth inhibition by the structurally related habit modifier, octanoic acid. We subsequently investigated various aspects of the crystallization of the model protein, lysozyme. The rate and mechanisms of growth of the (110) surface of the tetragonal crystal were observed using in situ AFM at a range of supersaturations, a study that uncovered a previously unreported mechanistic event. The (110) and (101) faces were then both investigated at higher resolution, revealing molecular resolution features corresponding directly to basic crystallographic data. The polymorphic characteristics displayed by many macromolecular crystals were then investigated in a short study concerning the growth and structure of the monoclinic form of the lysozyme crystal. The dominant (101) face of the crystal was investigated at both high and low protein/precipitant concentrations, allowing us to unambiguously distinguish between two crystalline forms of the same macromolecule. Finally, by utilizing the experimental techniques developed throughout the previous studies, we investigated a poorly diffracting crystal constructed from a protein found in Streptococcus pneumoniae, Response Regulator 02 receiver domain (RR02rec). By studying the surface of crystalline RR02rec with in situ AFM, we were able to uncover various features of the crystal lattice that may have contributed to the poor diffraction properties displayed by the crystal during previous X-ray studies. Besides revealing a range of new molecular scale details concerning the structure and growth of each of these crystal systems, these studies culminated in a successful attempt to relate direct microscopical observations of growth dynamics of a protein crystal system (RR02rec) to the limited results obtained from previous crystallographic studies performed on this protein. As an approach this offers considerable promise in identifying problems with certain crystals and, in conjunction with future advances in AFM technology, may offer information that could lead to the acceleration and enhancement of X-ray diffraction analyses.

With a proven ability to uncover fundamental biological processes, the atomic force microscope (AFM) represents one of the most valuable and versatile tools available to the biophysical sciences. We study the unsteady small-scale flows generated within the AFM by its sensing probe (a long thin cantilever), which have received relatively little attention to date, yet which are increasingly relevant in an age of microdevices. The early parts of this thesis investigate some canonical two-dimensional flows driven by oscillations of an infinite-length rigid cantilever. These prove amenable to analysis and enable us to investigate many of the important physical phenomena and compile a comprehensive collection of asymptotic expressions for the drag. The corresponding results lay out the influence of a nearby wall, geometry and oscillation frequency. The limitations of a two-dimensional approach are then explored through the development of a novel unsteady slender-body theory (USBT) for finite-length cylinders, an asymptotic treatment of which offers corrections to traditional resistive-force-theory (RFT) methods by accounting for geometric factors and flow inertia. These ideas are then extended to the study of thin rectangular plates. Two key parameters are identified which promote two-dimensionality in the flow, namely the frequency of oscillation and the proximity of a nearby boundary. We then examine flexible cylinders and plates by coupling the hydrodynamics to linearized elastic beam and plate equations, which simulate the hydrodynamically-damped high-speed deformable motion of the AFM's cantilever, when driven either externally or by Brownian motion. In the latter case, we adopt an approach which offers notable improvements over the most advanced method currently available to the AFM community.

Confocal scanning microscopy (CSM) is a much used and advantageous form of microscopy. Although CSM is superior to conventional microscopy in many respects, a major disadvantage is the complexity of the scanning process and the sometimes long time to perform the scan. In this thesis a novel non-scanning fluorescence confocal microscopy is investigated. The method uses a random time-varying speckle pattern to illuminate the specimen, recording a large number of independent full-field frames without the need for a scanning system. The recorded frames are then processed in a suitable way to give a confocal image. The goal of this research project is to confirm the effectiveness and practicality of speckle-illumination microscopy and to develop this proposal into a functioning microscope system. The issues to be addressed include modelling of the system performance, setting up experiments, computer control and image processing. This work makes the following contributions to knowledge: * The development of criteria for system performance evaluation * The development of methods for speckle processing, whereby the number of frames required for an image of acceptable quality can be reduced * The implementation of non-scanning fluorescence confocal microscopy based upon separate recording of the speckle patterns and the fluorescence frames, demonstrating the practicality and effectiveness of this method * The realisation of real-time image processing by optically addressed spatial light modulator, showing how this new form of optical arrangement may be used in practice The thesis is organised into three main segments. Chapters 1-2 review related work and introduce the concepts of fluorescence confocal microscopy. Chapters 3-5 discuss system modelling and present results of performance evaluation. Chapters 6-8 present experimental results based upon the separate recording scheme and the spatial light modulation scheme, draw conclusions and offer some speculative suggestions for future research.

Scanning near-field optical microscopy (SNOM) is a system that can image beyond the conventional diffraction limit. It does this by collecting the information contained within evanescent fields. This unique ability to image using evanescent fields also enables SNOM to directly measure the electric field distribution in waveguides, where light is guided by total internal reflection. When SNOM is used with a spectrally resolving detector, local temporal phenomena can be detected by analysing spectral interference in the spectra collected by the probe. This spectrally resolving configuration was used to directly measure inter-modal group velocity difference in a multimode ridge waveguide and, using the modes’ spatial profiles to experimentally determine the mode amplitude coefficient ratio. Such an ability to provide measurements on the local dispersion characteristics and relative modal amplitudes of guided light establishes SNOM as a route for investigating the conversion of current single mode photonic devices into multimode devices. The spectrally resolving SNOM system was also used to investigate the sources of temporal delays created by a quasi disordered scattering sample, which was based on John H. Conway’s pinwheel tiling. Whilst the measurements do not create a complete picture of the scattering phenomena in this work, suggestions for improvement are offered with the aim establishing spectrally resolving SNOM systems as tools for mapping localised temporal phenomena in disordered scattering systems.

De récentes études ont montré que les ondes électromagnétiques, proche d'une structure diffusante telle qu'une pointe de microscope à force atomique (AFM), peuvent être diffusées et détectées en champ lointain. Ainsi, la détection d'ondes de surface par microscopie optique en champ proche (SNOM) est une technique prometteuse dans le cadre des mesures thermiques aux petites échelles. Une telle technologie prend alors le nom de microscope TRSTM (Thermal Radiation Scanning Tunnelling Microscopy).Le travail de thèse présenté dans ce manuscrit se scinde en deux étapes. La première a trait à nos travaux expérimentaux basés sur le montage d'un dispositif TRSTM. Nous en décrivons les différentes composantes, ainsi que les difficultés rencontrées liées à son fonctionnement. En outre, divers outils numériques, destinés à détecter et extraire tout signal périodique utile, sont développés. La seconde étape se concentre sur nos travaux numériques. Nous y proposons un modèle de diffusion d'ondes électromagnétiques basé sur la FDTD (Finite-Difference Time-Domain) et la transformation champ proche/champ lointain. Ce modèle a été validé par l'étude de dipôles, puis de sphères dispersives à proximité d'un substrat diélectrique. Alors, un certain nombre de simulations de diffusion d'ondes évanescentes par une pointe, de diverses formes et de divers matériaux, proche d'une interface, est présenté. / Recent studies have shown that electromagnetic waves (in particular the thermal radiation), in the vicinity of a scattering object such as en atomic force microscope (AFM), can be scattered from near to far-field and thus detected. The detection of surface waves through scanning near-field optical microscopy (SNOM) is a promising technique for thermal measurement at small scales. Such technology is known as TRSTM. The thesis work presented in this manuscript is divided into tow part. The first one relates to our experimental work based on the development of a TRSTM device. Its various components are detailed, and the difficulties observed during its utilization are described. Furthermore, several digital tools, used to detect and extract any useful signal, are presented. The second part of our work focuses on the development of a numerical model based on the finite-difference time-domain (FDTD) and the near-field to far-field (NFTFF) transformation for the scattering of electromagnetic waves. This model has been validated by studying different cases of dipoles and dispersive spheres close to a dielectric substrate. Then, several simulations of scattering of evanescent waves by a tip, with various shapes and materials, near an interface, are presented.

In recent years, the confocal and two photon microscopes have become ubiquitous tools in life science laboratories. The reason for this is that both these systems can acquire three dimensional image data from biological specimens. Specifically, this is done by acquiring a series of two-dimensional images from a set of equally spaced planes within the specimen. The resulting image stack can be manipulated and displayed on a computer to reveal a wealth of information. These systems can also be used in time lapse studies to monitor the dynamical behaviour of specimens by recording a number of image stacks at a sequence of time points. The time resolution in this situation is, however, limited by the maximum speed at which each constituent image stack can be acquired. Various techniques have emerged to speed up image acquisition and in most practical implementations a single, in-focus, image can be acquired very quickly. However, the real bottleneck in three dimensional imaging is the process of refocusing the system to image different planes. This is commonly done by physically changing the distance between the specimen and imaging lens, which is a relatively slow process. It is clear with the ever-increasing need to image biologically relevant specimens quickly that the speed limitation imposed by the refocusing process must be overcome. This thesis concerns the acquisition of data from a range of specimen depths without requiring the specimen to be moved. A new technique is demonstrated for two photon microscopy that enables data from a whole range of specimen depths to be acquired simultaneously so that a single two dimensional scan records extended depth of field image data directly. This circumvents the need to acquire a full three dimensional image stack and hence leads to a significant improvement in the temporal resolution for acquiring such data by more than an order of magnitude. In the remainder of this thesis, a new microscope architecture is presented that enables scanning to be carried out in three dimensions at high speed without moving the objective lens or specimen. Aberrations introduced by the objective lens are compensated by the introduction of an equal and opposite aberration with a second lens within the system enabling diffraction limited performance over a large range of specimen depths. Focusing is achieved by moving a very small mirror, allowing axial scan rates of several kHz; an improvement of some two orders of magnitude. This approach is extremely general and can be applied to any form of optical microscope with the very great advantage that the specimen is not disturbed. This technique is developed theoretically and experimental results are shown that demonstrate its potential application to a broad range of sectioning methods in microscopy.

Le contexte de cette thèse est le recalage d'images endomicroscopiques. Le microendoscope multiphotonique fournit différentes trajectoires de balayage que nous considérons dans ce travail. Nous proposons d'abord une méthode de recalage non rigide dont l'estimation du mouvement est transformée en un problème d'appariement d'attributs dans le cadre des Log-Demons et d'ondelettes sur graphes. Nous étudions les ondelettes de graphe spectral (SGW) pour capturer les formes des images, en effet, la représentation des données sur les graphes est plus adaptée aux données avec des structures complexes. Nos expériences sur des images endomicroscopiques montrent que cette méthode est supérieure aux techniques de recalage d'images non rigides existantes. Nous proposons ensuite une nouvelle stratégie de recalage d'images pour les images endomicroscopiques acquises sur des grilles irrégulières. La transformée en ondelettes sur graphe est flexible et peut être appliquée à différents types de données, quelles que soient la densité de points et la complexité de la structure de données. Nous montrons également comment le cadre des Log-Demons peut être adapté à l'optimisation de la fonction objective définie pour les images acquises avec un échantillonnage irrégulier. / The context of this thesis is the image registration for endomicroscopic images. Multiphoton microendoscope provides different scanning trajectories which are considered in this work. First we propose a nonrigid registration method whose motion estimation is cast into a feature matching problem under the Log-Demons framework using Graph Wavelets. We investigate the Spectral Graph Wavelets (SGWs) to capture the shape feature of the images. The data representation on graphs is more adapted to data with complex structures. Our experiments on endomicroscopic images show that this method outperforms the existing nonrigid image registration techniques. We then propose a novel image registration strategy for endomicroscopic images acquired on irregular grids. The Graph Wavelet transform is flexible to apply on different types of data regardless of the data point densities and how complex the data structure is. We also show how the Log-Demons framework can be adapted to the optimization of the objective function defined for images with an irregular sampling.

Ce travail porte sur la description théorique de la microscopie à grille locale (SGM) et sur la résolution de modèles particuliers de contacts quantiques (QPC), analytiquement et numériquement. SGM est une technique expérimentale, qui mesure la conductance d'une nanostructure, lorsqu'une pointe de microscope a force atomique chargée balaye la surface, sans contacter cette dernière. Les images de SGM révèlent de nombreuses traits intéressants, tels que des lobes, des branches, des franges d'interférence et des motifs de damier. Aucune théorie généralement applicable, donnant une interprétation univoque, n’est disponible à ce jour. En utilisant la théorie de la diffusion de Lippman–Schwinger, nous avons développé une description perturbative de signal de SGM non invasive. Les expressions du premier et du second ordre ont été données, se rapportant aux régions de marche et de plateau de la courbe de conductance. Dans les systèmes invariants par renversement du temps (TRI), adaptés au premier plateau de conductance, les images SGM sont liées à la densité de charge à l`énergie de Fermi. Dans un système TRI, avec une symétrie spatiale centrale et de très larges contacts, les images sont aussi liées à la densité de courant, quelque soit le plateau. Nous présentons et discutons les images calculées pour deux modèles analytiques de QPC et les images obtenues numériquement avec la méthode des fonctions de Green récursives, reproduisant certains motifs observés expérimentalement, et pointant les difficultés fondamentales a se bien positionner sur le plateau de conductance. / This work is concerned with the theoretical description of the Scanning Gate Microscopy (SGM) in general and with solving particular models of the quantum point contact (QPC) nanostructure, analytically and numerically. SGM is an experimental technique, which measures the conductance of a nanostructure, while a charged AFM tip is scanned above its surface. It gives many interesting results, such as lobed and branched images, interference fringes and a chequerboard pattern. A generally applicable theory, allowing for unambiguous interpretation of the results, is still missing. Using the Lippman-Schwinger scattering theory, we have developed a perturbative description of non-invasive SGM signal. First and second order expressions are given, pertaining to the ramp- and plateau-regions of the conductance curve. The maps of time-reversal invariant (TRI) systems, tuned to the lowest conductance plateau, are related to the Fermi-energy charge density. In a TRI system with a four-fold spatial symmetry and very wide leads, the map is also related to the current density, on any plateau. We present and discuss the maps calculated for two analytically solvable models of the QPC and maps obtained numerically, with Recursive Green Function method, pointing to the experimental features they reproduce and to the fundamental difficulties in obtaining good plateau tuning which they reveal.

La microscopie SEEC (Surface Enhanced Ellipsometric Contrast) est une technique inventée au mans, il y a une dizaine d’années. Elle permet de visualiser des objets de taille nanoscopique entre polariseur et analyseur croisés en utilisant les propriétés non dépolarisantes de surfaces multicouches. Jusqu’au début de la thèse, seules des observations à l’air étaient possibles. Le but de cette thèse a consisté à adapter cette technique à l’observation in-situ d’objets en immersion dans l’eau.Pour cela, il a fallu inventer de nouvelles surfaces propres à ce nouveau milieu. Les calculs ont montrés que des surfaces fines d’or révélaient un bon contraste pour des objets de 1 nm en immersion dans l’eau. Expérimentalement, nous avons montré que pour exploiter au maximum le contraste SEEC, il est nécessaire de modifier l’éclairage. En parallèle de ces travaux expérimentaux, de nouveaux calculs ont montrés que l’utilisation d’épaisseurs encore plus fines permettait de visualiser ces objets avec un bon contraste et sans aucune modification de l’éclairage. Nous avons appelé cette nouvelle technique : la microscopie CONE. Nous avons découvert deux modes de mesure. Après avoir réalisé des fonctionnalisations homogènes et hétérogènes des surfaces d’or. Ces surfaces ont été utilisées en résonance plasmonique de surface (SPR) pour l’étude de fixation de protéine (adsorption et immobilisation) puis d’interaction protéine/protéine. Ces expériences ont ensuite servies de référence pour évaluer les microscopies SEEC et CONE. Par cela, nous avons prouvé que ces microscopies présentent de forts intérêts pour la détection in-situ de protéines avec un faible coût. / This thesis was supported by National Agency for Research with the project: ANR PNANO-07 SEEC. The Surface Enhanced Ellipsometric Contrast (SEEC) microscopy has invented in 2000 at Le Mans (France). This technique allows the visualization of nanoscopic object between crossed analyzer and polarizer. It’s possible if some special multilayer surfaces are used. There surfaces must have the particularity to not change the polarization of light during the reflection. Until the beginning of the project the SEEC microscopy was useful only for air observations. The goal of the thesis was to adapt this technique to observe on gold surfaces immerged in water and to compare the performance of the SEEC microscopy with Surface Plasmonique Resonance (SPR) in that configuration. The SPR is a biomolecular interaction study reference technique. SEEC microscopy lateral resolution was evaluate by fluorescence microscopy. Next, we realize two model experiments monitor in parallel by SEEC microscopy and by SPR: BSA immobilization and biotinylated IgG fixation by immobilized streptavidine. To compare measurements efficiently we did a huge preparation work (surface functionalizations and microfluidic) to have exactly same conditions in both techniques.Our results show SEEC microscopy cannot replace SPR for biomolecular interaction studies but it can be used as cheap immunological diagnostic technique. This work gives the path to follow on that direction.