L’étude comparée des réactions de formation de paires d’ions et de simple photoionisation de la molécule NO2 induites par rayonnement synchrotron (RS) d’une part et par impulsions laser femtosecondes (fs) d’autre part, démontre le rôle remarquable de l’excitation résonante d’états de Rydberg dans la dynamique électronique et nucléaire induite. Trois réactions principales, la photoionisation non dissociative (NO2+ (X 1Σ+g) + e), la photoionisation dissociative (NO+ (X 1Σ+) + O(3P) + e) et la formation de paires d'ions, (NO+ (X 1Σ+) + O- (2P)), ont été caractérisées en utilisant la méthode des corrélations vectorielles ou spectroscopie en coïncidence des impulsions du photoélectron et des photoions, auprès des sources RS (SOLEIL, DESIRS) et lasers fs (CEA, Saclay), respectivement. Le diagramme de corrélation des énergies cinétiques électron-ion, première observable issue de ces mesures, met en évidence un partage de l’énergie en excès entre noyaux et électrons qui dépend fortement du mode d’excitation photonique. Les déviations significatives observées par rapport aux profils d’ionisation de type Franck Condon sont attribuées à des couplages vibroniques entre états excités NO2*, tels que ceux induits par une intersection conique. Les chemins réactionnels identifiés confirment le rôle de l’excitation des séries de Rydberg [R*(6a1)-1] et [R*(4b2)-1] intervenant comme états intermédiaires dans l’excitation multiphotonique ou dans le continuum d’ionisation exploré. Une étude complémentaire par spectroscopie à haute résolution des états [R*(6a1)-1] a été mise en œuvre (UBC, Vancouver).Pour une réaction de photoionisation dissociative (PID), l’observable la plus complète est la distribution angulaire des photoélectrons dans le référentiel lié à la vitesse de recul de l’ion fragment (RFPAD) déduite de la mesure de la corrélation vectorielle (Vi, Ve, P). Afin d’accéder aux éléments de matrice dipolaire décrivant la photoionisation de l’état électronique considéré, le formalisme développé en collaboration avec R. R. Lucchese (Texas A&M) décrivant la photoémission dans le référentiel moléculaire pour la simple PID d'une molécule linéaire par excitation à un photon, a été étendu à l'étude des réactions de PID par excitation multiphotonique d'une molécule polyatomique, telle que la molécule NO2 de symétrie C2v. L’analyse multivariée de la RFPAD multiphotonique proposée constitue une stratégie fructueuse en vue d’extraire l’information optimale sur la dynamique complexe de photoionisation et de réaliser une comparaison détaillée entre les résultats expérimentaux et les calculs de photoionisation des états excités de la molécule. / The comparative study of ion pair formation and simple photoionization of the NO2 molecule induced by synchrotron radiation (SR) on the one hand and by femtosecond (fs) pulses on the other hand reveals the remarkable role of Rydberg states in the induced electronic and nuclear dynamics. Three main reactions, namely (NO2+ (X 1Σ+g) + e) non dissociative photoionization, (NO+ (X 1Σ+) + O(3P) + e) dissociative photoionization and (NO+ (X 1Σ+) + O- (2P)) ion pair formation have been characterized using the vector correlation method, or photoion and photoelectron coincidence momentum spectroscopy, at SR sources (SOLEIL DESIRS) and at fs laser platforms (CEA, Saclay), respectively. The electron-ion kinetic energy correlation diagram, which is the first observable obtained from these measurements, highlights the excess energy sharing among nuclei and electrons, which strongly depends on the photon excitation mode. The observed remarkable deviations from Franck Condon ionization profiles are attributed to vibronic couplings such as those induced at a conical intersection. The identified reaction pathways confirm the role of the [R*(6a1)-1] and [R*(4b2)-1] Rydberg series excitation as stepping states in multiphoton excitation or in the explored ionization continua. A complementary study of high resolution spectroscopy of [R*(6a1)-1] Rydberg series has been performed (UBC, Vancouver). For a dissociative photoionization (DPI) process, the most complete observable is the photoelectron angular distribution in the reference frame attached to the recoil ion fragment velocity (RFPAD) deduced from the measured (Vi, Ve, P) vector correlation. In order to get access to the dipole matrix elements describing photoionization of the considered excited electronic state, the formalism developed in collaboration with R. R. Lucchese (Texas A&M) describing molecular frame photoemission for a DPI of a linear molecule by one-photon excitation has been extended to the study of DPI processes induced by multiphoton excitation for a polyatomic molecule, such as the NO2 molecule of C2v symmetry. The proposed multivariate analysis of the multiphoton RFPAD constitutes a successful strategy to extract the optimal information on the complex photoionization dynamics and to perform a detailed comparison between experimental results and calculations of photoionization of the molecular excited states.

We demonstrate experimentally that the multiphoton ionization rate in gallium arsenide depends on the alignment of the laser polarization with respect to the crystal axis. We show real-time modulation of 1900nm laser ionization rate, through viewing transmission, which mimics the symmetry of the semiconductor crystal. We propose that the modulation in the ionization rate arises because the varying reduced effective carrier mass, as predicted by Keldysh theory. We show direct comparison of the experimental transmission modulation depth with that predicted by Keldysh theory. This opens up a novel method for real-time non-invasive crystallography of crystalline materials.

A compact multi-photon microscope (MPM) was designed and developed with the use of low-cost mode-locked fiber lasers operating at 1040nm and 1560nm. The MPM was assembled in-house and the system aberration was investigated using the optical design software: Zemax. A novel characterization methodology based on 'nonlinear knife-edge' technique was also introduced to measure the axial, lateral resolution, and the field curvature of the multi-photon microscope's image plane. The field curvature was then post-corrected using data processing in MATLAB. A customized laser scanning software based on LabVIEW was developed for data acquisition, image display and controlling peripheral electronics. Finally, different modalities of multi-photon excitation such as second- and third harmonic generation, two- and three-photon fluorescence were utilized to study a wide variety of samples from cancerous cells to 2D-layered materials.

Malaria affects 200 million people annually, resulting in 584,000 - 1,238,000 deaths. The majority of these deaths occur in children, less than 5 years of age, in sub-Saharan Africa and are due to cerebral malaria (CM), a neuropathology induced primarily by the species Plasmodium (P.) falciparum. The pathogenesis of CM remains poorly understood and the mechanisms involved in acquired protection against the syndrome in malaria-endemic regions are undefined. Utilising the well characterised P. berghei ANKA experimental infection model of cerebral malaria (ECM), results presented in this thesis show that the development of ECM is associated with the accumulation and arrest of pathogenic CD8+ T cells within the perivascular spaces of the brain. Accumulation of activated CD8+ T cells, without arrest, was observed in the perivascular spaces of the brains of mice infected with the non-ECM causing P. berghei NK65 strain. These data show that the behaviour of intracerebral CD8+ T cells specifies their pathogenic function during malaria infection. The development of ECM was associated with extensive disruption to the BBB, which developed in the absence of extensive CD8+ T cell-dependent endothelial cell apoptosis. We modified the ECM model, establishing an infection-drug cure strategy, to investigate the immunological basis of parasite exposure-induced resistance to ECM development. Three rounds of infection-drug cure promoted resistance to ECM, which was associated with reduced intracerebral expression of genes involved in defence response, regulation of apoptosis, chemotaxis, CTL activity, antigen processing and presentation and cell adhesion, compared with ECM susceptible mice. Additionally, CD8+ T cell activation was suppressed in exposure-induced resistant mice and was associated with the antibody dependent expansion of a splenic plasmacytoid DC population, with a regulatory phenotype. The infection-induced protection against ECM was critically dependent upon secreted antibody production. A long standing problem in studying the immune response to malaria infection has been the inability to track parasite-specific CD4+ T cell responses. To address this, we generated and validated new transgenic P. berghei parasites expressing the model antigen, ovalbumin (OVA), either in the parasite cytoplasm or on the parasitophorous vacuole membrane (PVM). We found that cellular location and expression level of the antigen influence the induction and magnitude of parasite-specific T-cell responses. These parasites thus provide knowledge on the factors that influence the recognition of parasite antigens by the immune system and represent useful tools to study the development and function of antigen-specific T-cell responses during malaria infection. The results in this thesis improve our understanding of the events that lead to the development of CM, and the host immune responses that develop following parasite exposure to protect against it. The results should contribute towards the rational development of adjunctive therapies and effective vaccines for human CM.

Nonlinear optical microscopy (NLOM) utilizing femtosecond laser pulses is well suited for imaging living tissues. This work reports on the design and development of an optical fiber based multispectral NLOM developed around a laser generating broadband sub-10-fs pulses. An all-mirror dispersion-compensation setup is used to correct for quadratic and cubic phase distortions induced within the NLOM. Mouse tail tendon was used to characterize sub-10-fs pulses by interferometric autocorrelation. This is an effective method for characterizing dispersion from the optical system, immersion medium, and wet biological sample. The generation of very short autocorrelations demonstrates the ability to compensate for phase distortions within the imaging system and efficient second-harmonic upconversion of the ultrashort pulse spectrum within collagen. Reconstruction of ultrashort pulses at the focal plane of the objective allows the excitation of multiple fluorescent probes simultaneously. Multiple fluorescent probe excitation and spectral discrimination is demonstrated using mixtures of fluorescent dye solutions and an in-vitro angiogenesis model containing human umbilical vein endothelial cells (HUVEC’s) expressing multiple fluorescent proteins. Sub-10-fs pulses can be propagated through polarization-maintaining single mode fiber (PMF) for use in NLOM. We demonstrate delivery of near transform-limited, 1 nJ pulses from a Ti:Al2O3 oscillator via PMF to the NLOM focal plane while maintaining 120 nm of bandwidth. Negative group delay dispersion (GDD) introduced to pre-compensate normal dispersion of the optical fiber and microscope optics ensured linear pulse propagation through the PMF. Nonlinear excitation of multiple fluorophores simultaneously and polarization sensitive NLOM imaging using second harmonic generation in collagen was demonstrated using PMF delivered pulses. Two-photon excited fluorescence spectra and second harmonic images taken with and without the fiber indicates that the fiber based system is capable of generating optical signals that are within a factor of two to three of our traditional NLOM.

The nanoscale manipulation of soft-matter, such as biological tissues, in its native environment has promising applications in medicine to correct for defects (eg. eye cataracts) or to destroy malignant regions (eg. cancerous tumours). To achieve this we need the ability to first image and then do precise ablation with sub-micron resolution with the same setup. For this purpose, we designed and built a multiphoton microscope and tested it on goldfish gills and bovine cells. We then studied light-matter interaction on a hard polymer (PMMA) because the nature of ablation of soft-matter in its native environment is complex and not well understood. Ablation and modification thresholds for successive laser shots were obtained. The ablation craters revealed 3D nanostructures and polarization dependent orientation. The interaction also induced localized porosity in PMMA that can be controlled.

We demonstrate experimentally that the multiphoton ionization rate in gallium arsenide depends on the alignment of the laser polarization with respect to the crystal axis. We show real-time modulation of 1900nm laser ionization rate, through viewing transmission, which mimics the symmetry of the semiconductor crystal. We propose that the modulation in the ionization rate arises because the varying reduced effective carrier mass, as predicted by Keldysh theory. We show direct comparison of the experimental transmission modulation depth with that predicted by Keldysh theory. This opens up a novel method for real-time non-invasive crystallography of crystalline materials.

The nanoscale manipulation of soft-matter, such as biological tissues, in its native environment has promising applications in medicine to correct for defects (eg. eye cataracts) or to destroy malignant regions (eg. cancerous tumours). To achieve this we need the ability to first image and then do precise ablation with sub-micron resolution with the same setup. For this purpose, we designed and built a multiphoton microscope and tested it on goldfish gills and bovine cells. We then studied light-matter interaction on a hard polymer (PMMA) because the nature of ablation of soft-matter in its native environment is complex and not well understood. Ablation and modification thresholds for successive laser shots were obtained. The ablation craters revealed 3D nanostructures and polarization dependent orientation. The interaction also induced localized porosity in PMMA that can be controlled.

Nonlinear optical microscopy (NLOM) utilizing femtosecond laser pulses is well suited for imaging living tissues. This work reports on the design and development of an optical fiber based multispectral NLOM developed around a laser generating broadband sub-10-fs pulses. An all-mirror dispersion-compensation setup is used to correct for quadratic and cubic phase distortions induced within the NLOM. Mouse tail tendon was used to characterize sub-10-fs pulses by interferometric autocorrelation. This is an effective method for characterizing dispersion from the optical system, immersion medium, and wet biological sample. The generation of very short autocorrelations demonstrates the ability to compensate for phase distortions within the imaging system and efficient second-harmonic upconversion of the ultrashort pulse spectrum within collagen. Reconstruction of ultrashort pulses at the focal plane of the objective allows the excitation of multiple fluorescent probes simultaneously. Multiple fluorescent probe excitation and spectral discrimination is demonstrated using mixtures of fluorescent dye solutions and an in-vitro angiogenesis model containing human umbilical vein endothelial cells (HUVEC’s) expressing multiple fluorescent proteins. Sub-10-fs pulses can be propagated through polarization-maintaining single mode fiber (PMF) for use in NLOM. We demonstrate delivery of near transform-limited, 1 nJ pulses from a Ti:Al2O3 oscillator via PMF to the NLOM focal plane while maintaining 120 nm of bandwidth. Negative group delay dispersion (GDD) introduced to pre-compensate normal dispersion of the optical fiber and microscope optics ensured linear pulse propagation through the PMF. Nonlinear excitation of multiple fluorophores simultaneously and polarization sensitive NLOM imaging using second harmonic generation in collagen was demonstrated using PMF delivered pulses. Two-photon excited fluorescence spectra and second harmonic images taken with and without the fiber indicates that the fiber based system is capable of generating optical signals that are within a factor of two to three of our traditional NLOM.

Application of the nonlinear optical microscopy (NLOM) for investigation of biological samples has, to date, primarily focused upon the qualitative analysis of images. The general consensus is that the nonlinear optical (NLO) techniques provide enough bio- chemical information when compared to, for example, visible light microscopy. Herein, it is presented a detailed study where a set of tools for quantitative extraction of infor- mation from NLO images were developed and tested for the analysis of complex tissue assemblies. Two-photon excited autofluorescence (TPEF), second-harmonic generation (SHG), and coherent anti-Stokes Raman scattering (CARS) were used for the charac- terization of atherosclerotic plaques. Our NLO-based image analysis of animal arteries affected by atherosclerotic plaque accumulation revealed that images of the healthy regions of the artery can be readily distinguished by marked differences in morphology, due to a fluorescent signal generated from the presence of generally intact elastic layer. Regions affected by lesions were dominated by lipid-rich cells and collagen fibers; the elastic layer was disrupted and the presence of fluorescent particles were also detected. Next, the potential of using information extracted from NLO images lead us to the development of a new optical index for plaque burden (OIPB). Through the OIPB, it was possible to investigate and to classify the plaque severity regarding the already established and currently used definition during clinical analyses. Extrapolating to and anticipating future applications, several methods for extracting specific information from images acquired by each NLOM modality were developed and tested. Texture analysis, particle-specific features, fractal analysis and directionality of components within the images were successfully adapted and tailored to better extract relevant information from the NLO images. Even though the methods presented in this thesis were mostly tested in images from arterial plaques, there is strong evidence that all tools presented here are capable of tracking changes that occur in many medical conditions and applications.