Advances in science and technology during the past decade have greatly enhanced the level of the structural investigation of macromolecules – peptides and proteins. Biological mass spectrometry has become one of the most precise and sensitive techniques in peptide and protein analysis. However, increasing demands of biotechnological applications require further progress to be made. In the present thesis the development and improvement of peptide and protein characterization methods and techniques based on ion-electron and ion-photon reactions in electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry are described. The focus is on the development of the electron capture dissociation method, recently discovered by the group of professor McLafferty, into a high rate, efficient tandem mass spectrometrical technique. The rate and reliability of the electron capture dissociation technique were greatly increased by implementation of low-energy pencil electron beam injection systems based on indirectly heated dispenser cathodes. Further implementation of a hollow electron beam injection system combined, in a single experimental configuration, two rapid fragmentation techniques, high rate electron capture dissociation and infrared multiphoton dissociation. Simultaneous and consecutive irradiations of trapped ions with electrons and photons extended the possibilities for ion activation/dissociation reaction schemes and lead to improved peptide and protein characterization. Using these improvements, high rate electron capture dissociation was employed in time-limited experiments, such as liquid chromatography–tandem mass spectrometry and capillary electrophoresis-tandem mass spectrometry. The analytical applications of the developed techniques have been demonstrated in top-down sequencing of peptides and proteins up to 29 kDa, improved sequencing of peptides with multiple disulfide bridges and secondary fragmentation (w-ion formation), as well as extended characterization of peptide mixtures separated by liquid chromatography and capillary electrophoresis. For instance, the dissociation of peptides resulting from enzymatic digestion of proteins provided complementary structural information on peptides and proteins, as well as their post-translational modifications.

Le collagène est une protéine ubiquitaire qui joue un rôle central dans l'architecture et la tenue mécanique des tissus conjonctifs et est impliqué dans de nombreuses pathologies. Synthétisé sous forme de triples hélices, le collagène s'auto-assemble en fibrilles in vivo et in vitro pour former des réseaux tridimensionnels. La microscopie multiphoton basée sur la génération de seconde harmonique (SHG) est une technique très spécifique, permettant de visualiser, sans marquage, le collagène en profondeur dans des tissus, avec une résolution sub-micrométrique. Ce travail de thèse vise à développer des approches quantitatives en imagerie SHG du collagène, tant à l'échelle fibrillaire que tissulaire. Nous avons montré que la microscopie SHG permet de sonder la dynamique de formation du réseau collagénique jusqu'à l'échelle d'une fibrille unique. En outre, nous avons caractérisé la structuration de gels collagéniques contrôlée par ajout de nanoparticules de silice fonctionnalisées. Nous avons ensuite réalisé de l'imagerie corrélative électronique/SHG sur ces gels pour mesurer la sensibilité de notre microscope et calibrer la réponse d'une fibrille en fonction de son diamètre. De plus, nous avons pu évaluer l'hyperpolarisabilité d'une molécule et valider le modèle additif utilisé pour calculer la réponse d'une fibrille. Enfin, nous avons développé une analyse d'images spécifique permettant de quantifier l'organisation d'un tissu collagénique à l'échelle fibrillaire, dans le but d'explorer la relation fonction/structure d'un tissu. Ceci a été validé en étudiant la modification des propriétés biomécaniques de souris génétiquement modifiées modèle du syndrome d'Ehlers-Danlos.

Orientador: Carlos Lenz Cesar / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-11T10:58:51Z (GMT). No. of bitstreams: 1 Thomaz_AndreAlexandrede_M.pdf: 10062018 bytes, checksum: 1e19c55cb5a4e709c2015e2d90f3ac13 (MD5) Previous issue date: 2007 / Resumo: A pesquisa em fotônica biomedica está claramente tomando a direção do entendimento de processos biológicos a nível celular. A resolução necessária para atingir esse objetivo requer praticamente ferramentas fotônicas. Contudo, uma integração de diferentes ferramentes fotônicas e uma aproximação funcional serão necessárias para acessar os processos biomecânicos e bioquímicos celulares. Deste modo nós podemos observar eventos bioquímicos disparados mecanicamente ou eventos mecânicos disparados bioquimicamente, ou até mesmo observar simultâneamente eventos biomecânicos e bioquímicos disparados por outros meios, entre outros, eletricamente. Uma das grandes vantagens das ferramentas fotônicas é a sua facilidade de integração. Nós desenvolvemos uma ferramenta integrada incorporando pinça óptica simples com Microscopia Confocal "Single-photon" e Multifóton. O sistema consegue realizar microscopias de fluorescência excitada pela absorção de dois fótons e geração de segundo harmônico em conjunto com manipulações ópticas. Medidas de força, elasticidade e viscosidade de membranes esticadas podem ser monitoradas em tempo real pelas microscopias confocais, bem como protozoários capturados opticamente, como, por exemplo, Trypanosoma cruzi. Nós mostraremos vários exemplos do uso de tal ferramenta integrada e seu potencial para observar processos mecânicos e bioquímicos a nível celular / Abstract: The research in biomedical photonics is clearly evolving in the direction of the understanding of biological processes at the cell level. The spatial resolution to accomplish this task practically requires photonics tools. However, an integration of different photonic tools and a multimodal and functional approach will be necessary to access the mechanical and biochemical cell processes. This way we can observe mechanicaly triggered biochemical events or biochemicaly triggered mechanical events, or even observe simultaneously mechanical and biochemical events triggered by other means, e.g. electricaly. One great advantage of the photonic tools is its easiness for integration. Therefore, we developed such integrated tool by incorporating single Optical Tweezers with Confocal Single and Multiphoton Microscopies. This system can perform 2-photon excited fluorescence and Second Harmonic Generation microscopies together with optical manipulations. Force, elasticity and viscosity measurements of stretched membranes can be followed by real time confocal microscopies. Also opticaly trapped living protozoas, such as Trypanosoma cruzi. Integration with CARS microscopy is under way. We will show several examples of the use of such integrated instrument and its potential to observe mechanical and biochemical processes at cell level / Mestrado / Física / Mestre em Física

Spinal cord injury (SCI) triggers several lipid alterations in nervous tissue. It is characterized by extensive demyelination and the inflammatory response leads to accumulation of activated microglia/macrophages, which often transform into foam cells by accumulation of lipid droplets after engulfment of the damaged myelin sheaths. Using an experimental rat model, Raman microspectroscopy was applied to retrieve the modifications of the lipid distribution following SCI. Coherent anti-Stokes Raman scattering (CARS) and endogenous two-photon fluorescence (TPEF) microscopies were used for the detection of lipid-laden inflammatory cells. The Raman mapping of CH2 deformation mode intensity at 1440 cm−1 retrieved the lipid-depleted injury core. Preserved white matter and inflammatory regions with myelin fragmentation and foam cells were localized by specifically addressing the distribution of esterified lipids, i.e., by mapping the intensity of the carbonyl Raman band at 1743 cm−1, and were in agreement with CARS/TPEF microscopy. Principal component analysis revealed that the inflammatory regions are notably rich in saturated fatty acids. Therefore, Raman spectroscopy enabled to specifically detect inflammation after SCI and myelin degradation products.

This dissertation investigates two long standing issues in nonlinear optics: complete characterization of the ultrafast dynamics of simple molecules, and the potential of a two-photon laser using a bulk semiconductor gain medium. Within the Born-Oppenheimer approximation, nonlinear refraction in molecular liquids and gases can arise from both bound-electronic and nuclear origins. Knowledge of the magnitudes, temporal dynamics, polarization and spectral dependences of each of these mechanisms is important for many applications including filamentation, white-light continuum generation, all-optical switching, and nonlinear spectroscopy. In this work the nonlinear dynamics of molecules are investigated in both liquid and gas phase with the recently developed beam deflection technique which measures nonlinear refraction directly in the time domain. Thanks to the utility of the beam deflection technique we are able to completely determine the third-order response function of one of the most important molecular liquids in nonlinear optics, carbon disulfide. This allows the prediction of essentially any nonlinear refraction or two-photon absorption experiment on CS2. Measurements conducted on air (N2 and O2) and gaseous CS2 reveal coherent rotational revivals in the degree of alignment of the ensemble at a period that depends on its moment of inertia. This allows measurement of the rotational and centrifugal distortion constants of the isolated molecules. Additionally, the rotational contribution to the beam deflection measurement can be eliminated thanks to the particular polarization dependence of the mechanism. At a specific polarization, the dominant remaining contribution is due to the bound-electrons. Thus both the bound-electronic nonlinear refractive index of air, and second hyperpolarizability of isolated CS2 molecules, are measured directly. The later agrees well with liquid CS2 measurements, where local field effects are significant. The second major portion of this dissertation addresses the possibility of using bulk semiconductors as a two-photon gain medium. A two-photon laser has been a goal of nonlinear optics since shortly after the original laser*s development. In this case, two-photons are emitted from a single electronic transition rather than only one. This processes is known as two-photon gain (2PG). Semiconductors have large two-photon absorption coefficients, which are enhanced by ~2 orders of magnitude when using photons of very different energies, e.g., ћωa≈10ћωb. This enhancement should translate into large 2PG coefficients as well, given the inverse relationship between absorption and gain. Here, we experimentally demonstrate both degenerate and nondegenerate 2PG in optically excited bulk GaAs via pump-probe experiments. This constitutes, to my knowledge, the first report of nondegenerate two-photon gain. Competition between 2PG and competing processes, namely intervalence band and nondegenerate three-photon absorption (ND-3PA), in both cases are theoretically analyzed. Experimental measurements of ND-3PA agree with this analysis and show that it is enhanced much more than ND-2PG. It is found for both degenerate and nondegenerate photon pairs that the losses dominate the two-photon gain, preventing the possibility of a two-photon semiconductor laser.

<p dir="ltr">This thesis describes advancements in both chemical imaging and optical manipulation methodologies for their application in tandem monitoring and control of biochemical processes. We developed a fast acquisition multimodal nonlinear imaging platform based on pulse-picking to minimize photoperturbation to the sample during imaging. By frequency doubling the imaging source, through acousto-optic modulation and simple comparator circuitry, we developed a comprehensive platform that uses chemical specific signals generated during imaging to control the pixel location for laser activation for reaction control. This feedback loop allows for advanced decision logic on a pixel by pixel basis.</p>

No description available.

This project considers the co-rotating twin screw extrusion of a confectionery paste comprising powdered proteins, sugars, water and fats. As is the case with many food industry products, this process has been developed experimentally with little quantitative understanding of how variations in processing conditions influence the formation of the extrudate. A variety of techniques have therefore been developed to characterise and quantify the dispersive mixing, distributive mixing and rheological flow properties of this complex, multiphase, viscoelastic, unstable material. These techniques have then been utilised in a pilot plant extruder study of the mechanics of mixing and paste formation during extrusion, considering the influence of both processing conditions and screw profile. The internal evolution of paste microstructure has been successfully tracked along the length of screw profile using dead-stop extractions of the screws. A rigorous off-line assessment of shear yield strength behaviour using cone penetrometry has shown the use of conventional off-line rheometers to be unviable due to rapid post extrusion hardening. This highlighted the need for an in-line rheological measurement technique for continuous extrusion analysis where the extruded material is severely time dependent and not extractable. In pursuit of this, a novel arrangement of bender elements is proposed and trialled, to rapidly characterise material parameters of viscoelastic pastes. A second technique looking to extend the application of shear wave interface reflection to multiphase pastes is also trialled. A novel analysis of thermogravimetric data (TGA) has generated a viable index of distributive mixing, suitable for use on complex multi-component materials where thermal decomposition temperatures of the components are not well defined. Quantitative image analysis of pastes using scanning electron microscopy (SEM), optical microscopy protein staining and a novel application of multiphoton microscopy (MPM) have been used to visualise paste microstructure and quantify dispersive mixing. From the pilot plant extruder study, the application of these techniques was successful in mapping the evolution of paste mixing and the resulting microstructure, as well as identifying key differences between pastes mixed by twin screw extrusion and batch mixing.

Die Entwicklung Freier-Elektronen-Laser und einer neuen Generation von Strahlungsquellen erlaubt die Realisierung hoher Intensitäten und kurzer Pulsdauern. Im Regime niedriger Laserintensitäten war bisher die Dipolnäherung recht erfolgreich bei der Beschreibung der durch die Licht-Materie-Wechselwirkung erzeugten Dynamik, wodurch viele experimentell beobachtete Resultate reproduziert werden konnten. Bei den durch die neuen Strahlungsqullen erzeugten bisher unerreichten Intensitäten und Rönten-Wellenlängen kann die Dipolnäherung allerdings zusammenbrechen. Höhere Multipol-Wechselwirkungen, die mit dem Strahlungsdruck assoziiert werden, sollten dann erwartungsgemäß wichtig zur genauen Beschreibung der Wechselwirkungsdynamiken werden. In dieser Arbeit wird eine Methode zur Lösung der nichtrelativistischen zeitabhängigen Schrödingergleichung zur Beschreibung von Systemen mit einem einzelnen aktiven Elektron, das mit einem Laserfeld wechselwirkt, über die Dipolnäherung hinausgehend erweitert. Dabei wird sowohl die Taylor- als auch die Rayleight-Multipolentwicklung des Retardierungsterms ebener Wellen verwendet. Es wird erwartet, dass die Berücksichtigung höherer Ordnungen der Multipolwechselwirkung zu einer erhöhten Genauigkeit und Richtigkeit der Resultate führen. Weiterhin wird gezeigt, dass die Rayleigh-Multipolentwicklung für gleiche Laserparameter genauer ist und schneller zur Konvergenz der numerischen Rechnung führt. Die nicht-Dipoleffekte spiegeln is sowohl in den differentiellen als auch den totalen Ionisierungswahrscheinlichkeiten in Form von erhöhten Ionisierungsausbeuten, verzerrten ATI Strukturen und einer Asymmetrie in der Photoelektronenwinkelverteilung in der Polarisations und Propagationsrichtung wider. Es wird beobachtet, dass die nicht-Dipoleffekte mit der Intensität, Wellenlänge und Pulsdauer zunehmen. Es werden Ergebnisse sowohl für das Wasserstoffatom als auch das Heliumatom gezeigt. / The development of free-electron lasers and new generation light sources is enabling the realisation of high intensities and short pulse durations. In the weak-field intensity regime, the electric dipole approximation has been quite successful in describing the light-matter interaction dynamics reproducing many of the experimentally observed features. But at the unprecedented intensities and x-ray wavelengths produced by the new light sources, the electric dipole approximation is likely to break down. The role of higher multipole-order terms in the interaction Hamiltonian, associated with the radiation pressure, is then expected to become important in the accurate description of the interaction dynamics. This study extends the solution of the non-relativistic time dependent Schrödinger equation for a single active electron system interacting with short intense laser pulses beyond the standard dipole approximation. This is realized using both the Taylor and the Rayleigh plane-wave multipole expansion series of the spatial retardation term. The inclusion of higher multipole-order terms of the interaction is expected to increase the validity and accuracy of the calculated observables relative to the experimental measurements. In addition, it is shown that for equivalent laser parameters the Rayleigh multipole expansion series is more accurate and efficient in numerical convergence. The investigated non-dipole effects manifest in both differential and total ionization probabilities in form of the increased ion yields, the distorted above-threshold-ionization structure, and asymmetry of the photoelectron angular distribution in both polarization and propagation directions. The non-dipole effects are seen to increase with intensity, wavelength, and pulse duration. The results for hydrogen as well as helium atom are presented in this study.

Nous étudions de façon expérimentale les mécanismes d'excitation et de relaxation électroniques, sous irradiation par impulsion laser (UV et IR) ultracourte (60 fs - 1 ps), dans les matériaux diélectriques à large bande interdite. Les régimes explorés vont de l'ionisation sans modification permanente (quelques TW/cm²) jusqu'au régime ablatif (dizaines de TW/cm²). L'objectif de ce travail à caractère fondamental est la compréhension du processus d'endommagement laser dans nos conditions d'irradiation. D'abord, la connaissance de la densité électronique du matériau irradié aux différents stades d'excitation permet la quantification de cette interaction. La technique d'interférométrie résolue en temps est utilisée pour mesurer de façon directe cette quantité d'excitation. Cette mesure, effectuée au seuil de claquage pour différentes durées d'impulsion remet en question l'utilisation de densité d'excitation critique comme critère universel d'endommagement. Un nouveau critère, lié à l'énergie échangée est proposé. D'autre part, l'utilisation d'un schéma expérimental à deux impulsions " pompes " a permis la distinction des mécanismes d'excitation intervenant à l'échelle temporelle de l'ordre de la durée des impulsions utilisées. Nos résultats indiquent des comportements différents selon les matériaux utilisés. L'existence d'une avalanche électronique est observée dans certains matériaux (SiO2, NaCl) alors que ceci n'est pas le cas pour d'autres (Al2O3, MgO). Ces différences seront discutées en détail. Ensuite, nous mesurons le spectre en énergie des électrons excités par une technique complémentaire : la spectroscopie de photoémission. Ces résultats ont permis d'une part de montrer l'existence d'un effet croisé entre deux impulsions " pompes " qui se traduit par une augmentation de l'énergie des photoélectrons et d'autre part la mesure des temps caractéristiques de relaxation des électrons selon leur énergie cinétique. Enfin, une étude morphologique des cratères résultants de l'ablation a été effectuée et ce pour différents paramètres d'irradiation avec une seule impulsion (nombre de tirs, énergie et durée de l'impulsion) ainsi que pour le cas de l'association de deux impulsions en fonction de leur délai.