Microscopy techniques have been developed and refined over multiple decades, but innovation around single photon modalities has slowed. The advancement of the utility of information acquired, and minimum resolution available is seemingly reaching an asymptote. The fusion of light microscopy and well-studied nonlinear processes has broken through this barrier and enabled the collection of vast amounts of additional information beyond the topographical information relayed by traditional microscopes. Through nonlinear imaging modalities, chemical information can also be extracted from tissue. Nonlinear microscopy also can beat the resolution limit caused by diffraction, and offers up three-dimensional capabilities. The power of nonlinear imaging has been demonstrated by countless research groups, solidifying it as a major player in biomedical imaging. The value of a nonlinear imaging system could be enhanced if a reduction in size would permit the insertion into bodily cavities, as has been demonstrated by linear imaging endoscopes. The miniaturization of single photon imaging devices has led to significant advancements in diagnostics and treatment in the medical field. Much more information can be extracted from a patient if the tissue can be imaged in vivo, a capability that traditional, bulky, table top microscopes cannot offer. The development of new technologies in optics has enabled the miniaturization of many critical components of standard microscopes. It is possible to combine nonlinear techniques with these miniaturized elements into a portable, hand held microscope that can be applied to various facets of the biomedical field. The research demonstrated in this thesis is based on the selection, testing and assembly of several miniaturized optical components for use as a nonlinear imaging device. This thesis is the first demonstration of a fibre delivered, microelectromechanical systems mirror with miniaturized optics housed in a portable, hand held package. Specifically, it is designed for coherent anti-Stokes Raman scattering, second harmonic generation, and two-photon excitation fluorescence imaging. Depending on the modality being exploited, different chemical information can be extracted from the sample being imaged. This miniaturized microscope can be applied to diagnostics and treatments of spinal cord diseases and injuries, atherosclerosis research, cancer tumour identification and a plethora of other biomedical applications. The device that will be revealed in the upcoming text is validated by demonstrating all designed-for nonlinear modalities, and later will be used to perform serialized imaging of myelin of a single specimen over time.

No description available.

<p> </p> <p>High-speed flows are important for defense, national security, and transportation applications and generate harsh environments where simplifying assumptions such as the ideal gas law are not valid due to nonequilibrium and chemistry effects. These flows are difficult and expensive to replicate experimentally, so the development and improvement of high-speed vehicles often relies on high-fidelity computational fluid dynamics (CFD) models. The successful modeling of complicated phenomena, such as heat transfer in a turbulent boundary layer, relies on validation by experimental data taken with high spatiotemporal resolution, precision, and accuracy. Precise experimental measurement of temperature, an important thermodynamic property for CFD models, is difficult with physical probes which are typically slow and perturb the flow. Instead, hybrid femtosecond/picosecond (fs/ps) coherent anti-Stokes Raman scattering (CARS) allows for non-intrusive, spatially-resolved, collision-free thermometry at kHz repetition rates with high precision and accuracy. </p> <p>The goal of this thesis is to advance hybrid fs/ps CARS for extension to high-speed flows, with particular improvements to the spatial extent, probe characteristics, and precision of the technique. A novel method for multipoint measurements in a simple and effective optical arrangement is demonstrated, enabling single-shot and averaged measurements of temperature and O₂/N₂ concentration along a linear array of probe volumes. The generation of a variable-pulsewidth probe beam by a ps slicer, electro-optic modulator, fiber amplifier, and custom narrowband amplifier system is used for improved signal-to-noise ratios at low pressure. Simultaneous CARS thermometry and femtosecond laser electronic excitation tagging (FLEET) velocimetry are performed in the freestream of Mach 3 and Mach 4 nitrogen flows. These measurements reveal the need to quantify and establish the ultimate precision of the hybrid fs/ps CARS technique. Sources of uncertainty in hybrid fs/ps CARS thermometry are determined through a theoretical uncertainty analysis and the predicted precision of the technique is confirmed experimentally in room temperature nitrogen. Benchtop measurements in a supersonic nozzle are used to indicate spatial and temporal simultaneity between FLEET and CARS measurements and hybrid fs/ps CARS thermometry is performed in a high-speed, low temperature flow.</p>

La peau est un organe qui enveloppe le corps, elle est une barrière naturelle importante et efficace contre différents envahisseurs. Pour le traitement des maladies dermatologiques ainsi que dans l'industrie cosmétique, les applications topiques sur la peau sont largement utilisées. Ainsi beaucoup d'efforts ont été investis dans la recherche sur la peau visant à comprendre l'absorption moléculaire et les mécanismes rendant efficace la pénétration. Cependant, il reste difficile d'obtenir une visualisation 3D de haute résolution combinée à une information chimique- ment spécifique et quantitative dans la recherche sur la peau. La spectroscopie et la microscopie non-linéaire, incluant la fluorescence excitée à 2-photon (TPEF), la diffusion Raman spontanée, la diffusion Raman cohérente anti-Stokes (CARS) et la diffusion Raman stimulée (SRS), sont introduits dans ce travail pour l'identification sans ambiguïté de la morphologique de la peau et la détection de molécules appliquées de façon topique. Plusieurs méthodes quantitatives basées sur la spectroscopie et la microscopie non-linéaire sont proposées pour l'analyse chimique en3D sur la peau artificielle, ex vivo et in vivo sur la peau humaine. De plus, afin de s'adapter aux applications cliniques à venir, un design endoscopique est étudié pour permettre l'imagerie non-linéaires dans les endoscopes flexibles. / Skin is an organ that envelops the entire body, acts as a pivotal, efficient natural barrier to- wards various invaders. For the treatment of major dermatological diseases and in the cosmetic industry, topical applications on skin are widely used, thus many efforts in skin research have been aimed at understanding detailed molecular absorption and efficient penetration mechanisms. However, it remains difficult to obtain high-resolution visualization in 3D together with chemical selectivity and quantification in skin research. Nonlinear spectroscopy and microscopy, including two-photon excited fluorescence (TPEF), spontaneous Raman scattering, coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), are introduced in this work for unambiguous skin morphological identification and topical applied molecules detection. Sev- eral quantitative methods based on nonlinear spectroscopy and microscopy are designed for 3D chemical analysis in reconstructed skin, ex vivo and in vivo on human skin. Furthermore, to adapt to forthcoming clinical applications, an endoscopic design is investigated to bring nonlin- ear imaging in flexible endoscopes.

Nonlinear optical microscopy is a collection of very powerful imaging techniques. Linear optical microscopes probe the refractive index and absorption, which both stem from the first-order linear electric susceptibility. Especially in biological tissue, the variation in the refractive index is often small and the tissue is, in many cases, transparant. Nonlinear optical microscopes on the other hand probe the nonlinear higher-order susceptibilities, which can be chemically sensitive, leading to the capability to achieve label-free imaging. Nonlinear optical microscopes have been in development for more than thirty years and they are based on numerous nonlinear optical processes. The ones I will concentrate on in this thesis are second harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS), and stimulated Raman Scattering (SRS). The first technique is commonly used to image collagen as those molecules have a particularly large second-order nonlinear susceptibility due to their chiral structure. CARS and SRS on the other hand are often used because they resonantly target vibrational resonances in molecules, giving rise to the aforementioned label-free imaging. Deep understanding of the nonlinear imaging process is crucial to the interpretation of the images these techniques produce. Computational tools are exceptionally suited for this task as they allow studying the electromagnetic field anywhere in the sample as well as the far-field, and one can change any of the material properties to study their effect. One such tool is finite-difference time-domain (FDTD) that our group developed for nonlinear optical microscopy simulations. It is a direct discretization of Maxwell's equation. While computationally costly, it does allow any arbitrary shaped sample to be simulated. The sample can have frequency dependent refractive indexes, and also nonlinear media with third-order nonlinearities such as Kerr media and Raman-active media, but also second-order nonlinearities for SHG. The code is designed in such a way that it can run on thousands of CPUs on a wide variety of compute cluster which allows our group to obtain nanoscale resolution. Another computational tool I use is the free-space Green's function solution to the Helmholtz equation, which can be used to calculate the Hertz vector in the frequency domain, both in the near- and far-field, based on the induced nonlinear polarization. The electric field is then calculated from this Hertz vector. This technique is much faster then FDTD and also allows for arbitrary shapes of the nonlinear electric susceptibility in the sample. However, it assumes a homogeneous refractive index throughout the entire spatial domain and requires complete knowledge of the input beam or beams that induce the nonlinear polarization. In this thesis, I use these tools to study the image formation process of various nonlinear optical processes mentioned earlier. For example, I study the effect of an inhomogeneous refractive index on the images produced by these microscopes. In literature the index of refraction is almost always assumed to be homogeneous, because, as mentioned before, the inhomogeneity of the refractive index is often small. However, I show that these small differences in the index of refraction can have a significant effect on the measured far-field intensity signal. For example, in SRS and CARS images, the measured signal can increase by an order of magnitude depending on the index mismatch and structure of the sample. Additionally, significant shifts in perceived position occur. Even nonresonant nonlinear signals can be evoked purely through a mismatch in linear refractive index. Computational modelling can also help reveal additional detail. As SHG is a coherent process, subwavelength information can be inferred through the phase information. Our experimental collaborators built an interferometric SHG (I-SHG) microscope for exactly that purpose. We used this to image collagen fibrils, which are all aligned in a parallel fashion. However, because collagen fibrils have a chiral molecular structure, they can point either ``up'' or ``down''. Using my Green's function simulations of the SHG imaging process of collagen fibrils, I was able to predict the standard deviation in the measured phase and link it to the orientation of collagen fibrils in the focal spot of the probing laser beam, even though the diameters are far below the minimum resolvable capabilities of the microscope. We found that the ``upwards'' fibrils make up 46--53% of the sample. Even with a normal SHG microscope that does not measures phase, additional subresolution information is obtainable. With our collaborators we measured the ratio of the forward SHG intensity signal to that in the backward direction and with my simulations, we are able to link this to the fibril diameters in collagen tissue. Thus we inferred that the fibril diameter increases as a function of tissue depth. Furthermore, a computational technique called ptychography is able to retrieve phase information without an interferometric reference beam. Additionally, it increases resolution to the theoretical limit, independent of the laser focal spot size, and corrects for distortions in the input beam as well. I have developed this technique for use with nonlinear optical microscopy and was able to show it is a viable alternative to I-SHG by imaging simulated rat tail tendon at the diffraction limit while retrieving the orientation of the fibrils through the phase of the SHG signal. I also implemented the algorithm for CARS, where the phase information can be used to greatly increase the signal-to-noise ratio by reducing the nonresonant background radiation that results from competing nonlinear optical processes. I showed an example of this by imaging a simulated fibroblast cell where the CARS process was tuned to the lipid droplets inside of the cell. I am currently in talk with experimentalists to apply this theoretical technique to experiments as that would further demonstrate the impact of my work. Finally, keeping in theme with the collagen fibrils, I show that the ratio of the forward SHG signal to the backward signal, the F/B ratio, is affected by a mismatch in the refractive index for fibrils larger than 100nm. This measure is an indicator of fibril diameter and thus important for making qualitative predictions. Single fibrils are generally too small to be significantly affected by near-field effects, but the bigger fibrils can be. Fibrils in rat tail tendon have a distribution of fibrils diameters and the large fibrils occur infrequent. However, I found that the large fibrils are largely responsible for the forward as well as backward signal, thus refractive index mismatches still affect the F/B ratio significantly despite their infrequency. The F/B ratio for a collection of fibrils placed in a n=1.47 medium was found to be 31.8±0.7% higher than for those in a n=1.33 medium. Our experimental colleagues have done preliminary measurements on mouse tail tendon where they found an increase of 40±20%, in line with the value of 28.1±0.6% that I found for simulations with mouse tail tendon. In conclusion, the theoretical tools I have used in my thesis have provided me with the ability to study nonlinear optical image formation processes with a level of detail that would be near-impossible to do experimentally. I have used this ability to show how refractive index mismatches, such as those found in biological tissue, can significantly distort the far-field intensity signals. I have shown this for SRS and CARS where the far-field intensity signal appeared an order-of-magnitude larger compared to the same sample without a refractive index mismatch with the background medium. Additionally, shifts in the perceived position of the object under investigation were observed and I showed the presence of a nonresonant background signal in AM-SRS. Likewise I showed that in the SHG imaging of collagen fibrils significant changes in the F/B ratio can occur. All of these effects have important implications as these types of images as biomedical researches rely on the correct interpretation of nonlinear optical microscopy images for both research and diagnostics. Apart from showing the effect of a refractive index mismatch, I have also shown that computation modelling can be used to infer subwavelength features in SHG imaging experiments of collagen fibril such as fibril orientation and fibril diameter. These methods have the potential to aid medical researchers as changes in the structure of collagen are often an early indicator of diseases such as osteoarthritis. Finally, I showed that the ptychography algorithm I developed for nonlinear optical microscopy is able to retrieve phase information of the nonlinear electric susceptibility in SHG and CARS imaging while also enhancing the resolution and correcting for distortions in the input beams. I can also use much larger laser spot sizes than in conventional experiments without compromising the obtained resolution, thus fewer measurements are required. The technique is not limited to SHG and CARS either; it will work for other nonlinear optical processes as well. Experimental verification of nonlinear ptychography will be done soon. This technique has to potential to significantly improve current imaging techniques since access to the phase information allows one to observe additional information about the sample as we showed with the I-SHG microscope.

L’étude expérimentale des processus physico-chimiques de la combustion nécessite de disposer de diagnostics non-intrusifs. Le présent manuscrit reporte le développement du diagnostic laser de mesure de température DRASC (Diffusion Raman anti-Stokes Cohérente) en régime d’impulsions laser femtoseconde pour lequel la configuration à sonde à dérive de fréquence (CPP) a permis d’effectuer des mesures instantanées de température à 1kHz. Un travail à la fois théorique, numérique et expérimental a permis d’extraire la température des spectres DRASC instantanés acquis dans des mélanges air/argon (300-600K) et en flamme prémélangée CH4/Air avec une précision de l’ordre de 1% à 2100 K. La validité de ces résultats est obtenues par des confrontations numérique/expérimental pour différentes grandeurs d’influence. Cette étude permettra dans un proche futur d’appliquer le diagnostic DRASC fs CPP dans des flammes turbulentes représentatives d’écoulements réels observés en combustion aéronautique. / The experimental study of the physico-chemical processes of combustion requires the use of non-intrusive diagnostics. This manuscript reports the development of the CARS (Coherent Anti-Stokes Raman Scattering)) laser diagnostic in the femtosecond pulse regime for which the Chirped Pulse Probe (CPP) configuration enabled instantaneous measurements of temperature at 1kHz. A theoretical, numerical and experimental study allowed highlighting the possibility to measure temperature from the data processing of instantaneous DRASC spectra acquired in air/argon mixtures (300-600K) and in premixed flame CH4/Air with an accuracy of 1% at 2100 K. Validity of these results was obtained from numerical/experimental confrontations for different scalar parameters configurations. This study would enable in the near future the application of the CPP fs CARS diagnostic in turbulent flames representative of real flows observed in aeronautical combustion.

Broadband Coherent Anti-Stokes Raman scattering (B-CARS) is an advanced Raman spectroscopy technique used to investigate the vibrational properties of materials. B-CARS combines the spectral sensitivity of spontaneous Raman scattering with the enhanced signal intensity of coherent Raman techniques. While B-CARS has been successfully applied in biomedicine for ultra-fast imaging of biological tissue, its potential in solid-state physics remains largely unexplored. This work delves into the challenges and adaptations necessary to apply B-CARS to crystalline materials and shows its potential as a powerful tool for high-speed, hyperspectral investigations. The theoretical part of this work covers inelastic light-matter scattering fundamentals and the signal generation process of B-CARS, with special attention given to the so-called Non-Resonant Background (NRB). This sample-unspecific signal amplifies the B-CARS intensity but also distorts the shape and position of the measured spectral peaks. A reliable NRB correction becomes crucial to retrieve precise spectral parameters containing information on the investigated material's crystallographic structure, defect density, and stress distribution. The first results chapter presents a practical guideline for an optimized workflow of sample preparation, measurement procedure, and data analysis. The influences of sample surfaces, focus positioning, and polarization sensitivity are discussed. The successful NRB removal is achieved by adapting an algorithm initially designed for biomedical purposes. The second chapter involves a transnational Round Robin investigating the same set of materials using different experimental setups. The influences of laser source, detection range, and transmission vs. epi detection are explored to optimize the experimental parameters. This work showcases applications such as high-speed, hyperspectral imaging of ferroelectric domain walls in LiNbO3, demonstrating the potential of B-CARS in the cutting-edge field of domain wall engineering. Additionally, imaging and polarization-sensitive measurements are shown for MoO3 flakes, paving the way for B-CARS investigations of 2D materials. The final chapter presents advanced techniques, such as Three-Color CARS and Time-Delay CARS, applied to crystalline materials. Three-Color CARS is especially promising, as it enhances the signal intensity for low-frequency Raman modes, which are particularly interesting for solid-state physics compared to the usual large-shift modes investigated in biomedical research. Meanwhile, Time-Delay CARS is sensitive to relaxation processes of vibrational and NRB states, enabling experimental NRB removal and lifetime measurements. Additionally, a neural network-based NRB removal method is presented, eliminating the need for a prior NRB spectrum and offering rapid computation. In summary, this work demonstrates the successful implementation of B-CARS for crystalline materials and provides a comprehensive guideline for the optimal experimental setup, workflow, and data processing. The application of B-CARS for imaging bulk crystalline materials, ferroelectric domain walls, and 2D structures shows promising possibilities for future research.

Dual-broadband and dual-pump nanosecond CARS experiments were performed to investigate the pure rotational transitions of the nitric oxide molecule. The former experiment was initially utilized to determine the pure rotational structure while the latter focused on polarization suppression of the pure rotational transitions of nitric oxide. A polarization calculation and analysis were conducted on the rotational and vibrational transitions of nitrogen, and the pure rotational transitions of nitric oxide were subjected to a similar polarization scheme. The electronic transitions that arise due to the spin-split nature of nitric oxide ground electronic energy levels were suppressed by the polarization scheme in a similar manner to the rotational S branch transitions. Results have been compared with a spectral simulation developed by Dr. Lucht, and the theory is partially presented. Comparison between simulation and experimental data yielded favorable agreement for the pure rotational transitions of nitric oxide.

L'utilisation de techniques de spectroscopie cohérente comme la diffusion Raman anti-Stokes cohérente (DRASC) sont communément utilisées pour l'analyse quantitative de milieux réactifs.Dans le cadre des progrès récents des sources lasers et des détecteurs, ce travail de thèse propose une adaptation de la technique de mesure de température au régime des impulsions ultra-courtes (femtoseconde, picoseconde) à haute cadence.Le développement d'un dispositif expérimental de DRASC en régime hybride fs/ps utilisant un laser femtoseconde et un réseau de Bragg en volume a permis de générer une impulsion de sonde de 30 ps. Dans la configuration choisie pour ce travail, la résolution spectrale est de 0,7 cm-1, ce qui réprésente l'état de l'art. Ce dispositif est appliqué à la spectroscopie ro-vibrationnelle de plusieurs espèces moléculaires.Pour décrire l'interaction dans ce régime temporel, un modèle de simulation des spectres DRASC est implémenté. Cette modélisation prend notamment en compte le profil en amplitude et phase de la sonde ainsi que son retard par rapport à l'excitation femtoseconde. L'influence des collisions moléculaires est discutée dans le cadre du régime hybride.Les ajustements des spectres calculés sur des spectres expérimentaux enregistrés dans l'air ambiant et dans une flamme de prémélange CH4/air, permettent de valider la modélisation proposée dans ce travail. En particulier, la température est mesurée à froid et à chaud à pression atmosphérique, avec une précision comparable à l'état de l'art dans le régime hybride fs/ps.Ce travail permet d'obtenir des résultats probants, et de valider la pertinence du montage développé pour la thermométrie. Fort de ces résultats, des applications en chambre de combustion réelles sont envisagées à court terme à l'ONERA, notamment par l'extrapolation du modèle à haute pression. / Coherent spectroscopy such as Coherent Anti-Stokes Raman Scattering (CARS) is commonly used for the study of reactive media.Following the recent progress encountered in laser sources and detectors, this thesis work proposes an adaptation of the technique for temperature measurements that exploits the ultra-short pulse regime (femtosecond, picosecond) to operate at high repetition-rate.The developed hybrid fs/ps-CARS setup delivers a 30-ps probe pulse using a femtosecond laser source and a Volume Bragg Grating. The obtained spectral resolution is 0,7 cm-1, which represents, within this pulse generation framework, the state of the art. This setup is applied to ro-vibrational spectroscopy of several molecular species.The interaction in the hybrid regime is described through a CARS simulation model that has been implemented. The modelling takes into account both the amplitude and the phase profiles of the probe pulse, and its delay to the femtosecond excitation. Influence of molecular collisions in the hybrid regime is also discussed.This modelling has been validated by fitting calculated spectra on experimental spectra recorded in ambient air, and in a premixed CH4/air flame. Temperature has been then measured at atmospheric pressure in these media. The precision is similar to the hybrid fs/ps-CARS thermometry state of the art.This work validates our N2 thermometry approach based on both the developed CARS setup and simulation model. Short term perspectives of this work are measurements on real combustion chambers at ONERA, by extrapolating our simulation model at high pressure.

Orientador: Carlos Lenz César / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin / Made available in DSpace on 2018-09-01T03:43:43Z (GMT). No. of bitstreams: 1 Pelegati_VitorBianchin_D.pdf: 6822381 bytes, checksum: 86749ce34dc184aeb7ed4b4ee47d70b3 (MD5) Previous issue date: 2016 / Resumo: Técnicas de microscopias ópticas são as principais ferramentas capazes de observar células e tecidos biológicos em tempo real e com mínimo dano. Essa área foi revolucionada recentemente através das microscopias confocais de varredura a laser e as microscopias de óptica não linear, naturalmente confocais. Entre os processos não lineares temos, a fluorescência excitada por dois ou mais fótons, geração de segundo harmônico [Second Harmonic Generation - SHG] e terceiro harmônico [Third Harmonic Generation - THG]. SHG e THG são técnicas de óptica não linear coerentes, não necessitam de marcadores exógenos e permitem reconstrução de imagens em três dimensões com resolução espacial subcelular. As técnicas de fluorescência permitem visualizar estruturas específicas no espaço, mas não permitem discriminar as substâncias químicas nas estruturas celulares, e as técnicas de SHG e THG não possuem especificidade química. Espectroscopia Raman possui especificidade química através das propriedades vibracionais das moléculas e pode ser usada como mecanismo de contraste na aquisição de imagens. Comparada com a espectroscopia/microscopia infravermelho, a microscopia Raman traz a informação das vibrações moleculares do infravermelho para o visível, eliminando os problemas da baixa resolução espacial e opacidade das amostras. Entretanto a baixa sensibilidade dessa técnica implica em tempos de aquisição de imagens muito longos, da ordem de horas, inviabilizando acompanhar a dinâmica de processos celulares em tempo real. Como solução para essa baixa sensibilidade do espalhamento Raman espontâneo, surgiu a microscopia por espalhamento Raman Coerente anti-Stokes [Coherent Anti-Stokes Raman Scattering - CARS]. Comparado com Raman espontâneo, a microscopia CARS representa aumento de 4 a 5 ordens de grandeza na sensitividade da técnica, diminuindo os tempos de aquisição ao ponto de viabilizar a aquisição em taxas de vídeos (mais rápido do que 30 quadros por segundo) e estudos em tempo real. Essa tese é dedicada ao estudo experimental e teórico, assim como de algumas aplicações, das técnicas de óptica não linear, com destaque para processos de óptica não linear coerentes. Apresentamos de forma detalhada três sistemas experimentais para a aquisição de imagens de Raman coerente e um sistema integrado com várias técnicas de óptica não linear. Mostramos as primeiras imagens de CARS realizadas no Brasil. Além do CARS convencional, trabalhamos com outra técnica de CARS de ordem mais alta, o CARS cascata [cascade CARS - CCARS], e, no melhor do nosso conhecimento, apresentamos as primeiras imagens internacionais obtidas com essa metodologia. CCARS aumenta o contraste da técnica CARS, diminuindo o fundo não ressonante, um problema que aflige a comunidade científica dedicada ao uso dessa técnica. Além da diminuição do fundo não ressonante, a emissão do CCARS acontece em um comprimento de onda diferente de qualquer outro efeito não linear coerente, significando um acréscimo de complexidade mínimo para sua detecção quando comparado com o CARS. Por último mostramos algumas aplicações realizadas com o sistema experimental desenvolvido para integrar diversas modalidades ópticas em paralelo, especialmente da geração de harmônicos com a fluorescência excitada por dois fótons e suas variantes, como microscopia de tempo de vida de fluorescência (Fluorescence Lifetime Imaging ¿ FLIM) / Abstract: Optical microscopies techniques are the main tools capable of observing cell and biological tissues in real time and with minimum damage. This area have recently been revolutionized by confocal laser scanning microscopies and non-linear microscopies, naturally confocal. Among the non-linear process we have, the two or more photons excited fluorescence, second harmonic generation [SHG] and third harmonic generation [THG]. SHG and THG are coherent nonlinear techniques, they do not require exogenous markers and allow three dimension imaging reconstruction with subcellular resolution. The fluorescence techniques allow visualizing specific structures in space, but do not allow discriminating the chemical substances in cellular structures, SHG and THG techniques do not have chemical specificity. Raman spectroscopy has chemical specificity through the vibrational properties of the molecules and can be used as a contrast mechanism for imaging acquisition. Compared to infrared spectroscopy/microscopy, Raman microscopy brings information about molecular vibration from infrared to visible, eliminating the low resolution and sample opacity problems. However, this technique low sensibility implies in very long imaging acquisition times, order of hours, making it not viable for following cellular process dynamics in real time. As an answer for the spontaneous Raman scattering low sensibility, the coherent anti-Stokes Raman scattering [CARS] emerged. Compared to spontaneous Raman, CARS microscopy presents an increase of 4 to 5 orders of magnitude in the sensitivity of the technique, lowering the acquisition times to the point of making video acquisition (faster than 30 frames per second) and real time studies possible. This thesis is dedicated to the experimental and theoretical study, as well as some applications, of the non-linear techniques, with emphasis on coherent non-linear optical processes. We present in detailed form three experimental systems for the acquisition of coherent Raman images, and a system with the integration of various non-linear techniques. We show the first CARS images acquired in Brazil. In addition to conventional CARS, we worked with other higher order CARS technique, the cascade CARS [CCARS], and, in the best of our knowledge, we present the first international image acquired with this methodology. CCARS increases the contrast from CARS technique, decreasing the non-resonant background, a problem that afflicts the scientific community dedicated to the use of this technique. Besides the decrease of the non-resonant background, the CCARS emission occurs in a different wavelength from any other non-linear coherent effect, meaning a minimum complexity increase for its detection when compared with CARS. Finally we show some applications performed with the experimental system developed to integrate several optical modalities in parallel, especially the generation of harmonics with two photons excitation fluorescence and their variants such as Fluorescence Lifetime Imaging [FLIM] / Doutorado / Física / Doutor em Ciências / 830406/2010 / CAPES