Histology has long recognized the intimate link between structure and function. Over centuries histologists have utilized an assortment of optical microscopy techniques to elucidate functional attributes of tissues through investigating tissue architecture. This thesis includes developments in the field of nonlinear optical microscopy for use in histology and pathology. The main contributions focused on the study of fibrillar collagen in the extracellular matrix (ECM) with polarization-dependent second harmonic generation (P-SHG) microscopy and the study of harmonophore-stained cellular nuclei with third harmonic generation (THG) microscopy. The P-SHG microscopy technique, “polarization-in, polarization-out” (PIPO), was developed to accurately determine the second-order polarization properties of thin tissue sections. The polarization instrumentation was implemented into a nonlinear optical microscope and a custom fitting algorithm extracted ratios of the second-order nonlinear susceptibility elements at every pixel of an obtained image. Hierarchical organization, at every level of structure, can contribute significantly to the macroscopic second-order polarization properties of fibrillar collagen in the ECM and quantifiable differences between the various tissue architectures were observed. A framework was developed, based on the collagen hierarchical organization, to interpret the submicron polarization properties of various tissues. Complimentary to the P-SHG study of connective tissue, the structure of hematoxylin and eosin (H&E) stained nuclei was revealed by THG microscopy. Imaging the 3D organization of nuclei was possible using the inherent optical sectioning provided by nonlinear microscopy. The origin of THG was investigated with spectrally- and temporally-resolved measurements, as well as the THG ratio method. A rather complex situation involving multiple dye complexes was revealed. The structure of dye aggregates was investigated with THG PIPO microscopy. The techniques of PIPO and harmonophore-stained harmonic generation microscopy show great potential for ultimately furthering understanding of tissue structure and function. H&E stained tissue investigations with THG microscopy has applications as a tool for cancer diagnostics. PIPO can elucidate the symmetry and organization of materials beyond tissues, including starch, nanowires, and protein crystals. In pathology, the developed collagen framework has strong implications, as collagen is recognized as playing a more active role in a number of diseases including idiopathic pulmonary fibrosis, wound repair, and tumour development and progression.

Histology has long recognized the intimate link between structure and function. Over centuries histologists have utilized an assortment of optical microscopy techniques to elucidate functional attributes of tissues through investigating tissue architecture. This thesis includes developments in the field of nonlinear optical microscopy for use in histology and pathology. The main contributions focused on the study of fibrillar collagen in the extracellular matrix (ECM) with polarization-dependent second harmonic generation (P-SHG) microscopy and the study of harmonophore-stained cellular nuclei with third harmonic generation (THG) microscopy. The P-SHG microscopy technique, “polarization-in, polarization-out” (PIPO), was developed to accurately determine the second-order polarization properties of thin tissue sections. The polarization instrumentation was implemented into a nonlinear optical microscope and a custom fitting algorithm extracted ratios of the second-order nonlinear susceptibility elements at every pixel of an obtained image. Hierarchical organization, at every level of structure, can contribute significantly to the macroscopic second-order polarization properties of fibrillar collagen in the ECM and quantifiable differences between the various tissue architectures were observed. A framework was developed, based on the collagen hierarchical organization, to interpret the submicron polarization properties of various tissues. Complimentary to the P-SHG study of connective tissue, the structure of hematoxylin and eosin (H&E) stained nuclei was revealed by THG microscopy. Imaging the 3D organization of nuclei was possible using the inherent optical sectioning provided by nonlinear microscopy. The origin of THG was investigated with spectrally- and temporally-resolved measurements, as well as the THG ratio method. A rather complex situation involving multiple dye complexes was revealed. The structure of dye aggregates was investigated with THG PIPO microscopy. The techniques of PIPO and harmonophore-stained harmonic generation microscopy show great potential for ultimately furthering understanding of tissue structure and function. H&E stained tissue investigations with THG microscopy has applications as a tool for cancer diagnostics. PIPO can elucidate the symmetry and organization of materials beyond tissues, including starch, nanowires, and protein crystals. In pathology, the developed collagen framework has strong implications, as collagen is recognized as playing a more active role in a number of diseases including idiopathic pulmonary fibrosis, wound repair, and tumour development and progression.

[pt] Neste trabalho apresenta-se a implementação de um sistema de medição de pulsos ópticos curtos gerados por lasers semicondutores. Através de uma técnica indireta de medida, que usa a radiação de segundo harmônico gerada internamente nesses lasers, a duração dos pulsos curtos de luz é estimada. São apresentadas as principais considerações teóricas e experimentais envolvidas com a emissão de segundo harmônico em diodos lasers e são discutidas as principais limitações da técnica. Mostra-se que embora a determinação exata da duração dos pulsos exija medidas adicionais, é possível inferir com boa resolução temporal a duração de pulsos ópticos gerados nos regimes de chaveamento de ganho e mode-locking. / [en] In this work the implementation of a measurement system of short optical pulses generated by semiconductor lasers is presented. This indirect method estimates the pulse duration using the internally generated second harmonic radiation from these lasers. The main theoretical and experimental considerations related with the second harmonic emission are also presented. Although the precise pulse duration requires aditional measurements it is possible to estimate the duration of optical pulses generated by gain-switching and mode-loching regimes with good time-resolution.

Les nanocristaux à structure non-centrosymétrique présentent des propriétés optiques non linéaires prometteuses pour une utilisation en tant que marqueurs optiques en imagerie biomédicale, avec un intérêt significatif en termes de suivi sur de longues durées et de profondeur de pénétration dans les tissus biologiques. Le développement de ces marqueurs nécessite la détermination de leurs efficacités optiques non linéaires afin de pouvoir sélectionner les nanocristaux les plus prometteurs. Pour cela, la technique de diffusion Hyper-Rayleigh a été adaptée à la caractérisation de suspensions de nanoparticules (BaTiO3, KNbO3, KTP, LiNbO3 et ZnO, BiFeO3) pour lesquelles l’influence de paramètres comme la taille, la concentration ou l’état d’agrégation a été analysée et discutée. Les nanocristaux de BiFeO3 possèdent une efficacité optique non linéaire largement supérieure aux autres particules, démontrant leur potentiel pour la réalisation de nano-sondes optiques particulièrement performantes. Des mesures résolues en polarisation ont également été mises en œuvre pour déterminer les coefficients optiques non linéaires indépendants des particules étudiées. Dans ce cadre, une étude a permis de mettre en évidence l’influence de la forme des nanocristaux sur cette réponse. / Non-centrosymetric nanocrystals show promising nonlinear optical properties for being used as optical labels in bio-imaging applications, with significant interest for observations of long duration and for penetration depth into biological tissues. The development of such biomarkers requires the determination of their nonlinear optical properties to select the best potential markers. In this thesis, Hyper-Rayleigh Scattering (HRS) technique is used to determine nonlinear efficiencies of several nanocystals (BaTiO3, KNbO3, KTP, LiNbO3, ZnO and BiFeO3). These ensemble measurements have been performed on nanocrystals suspensions, for which the influence of parameters such as size, concentration and aggregation state was discussed. BiFeO3nanocrystals offer the best nonlinear optical efficiency compared to other particles, showing their potential as efficient optical biomarkers. Polarisation-resolved measurements have also been performed to retrieve individual coefficients of the nonlinear tensor of the investigated materials and influent parameters such as nanocrystals shape have been identified.

O diagnóstico preciso das características morfológicas e metabólicas de um tecido e/ou órgão com a finalidade de identificar alterações patológicas, ou avaliar um determinado tratamento, é de grande importância nas áreas de biologia e medicina. Uma excelente alternativa para este diagnóstico, e que permite uma visualização com resolução celular, são imagens de microscopia óptica. Tradicionalmente, analisam-se as características celulares através de processos histológicos; contudo, mais recentemente essa mesma análise tornou-se possível em tecidos sem a necessidade deste preparo histológico. Fenômenos de óptica não-linear, como a fluorescência devido à absorção de dois fótons e a geração de segundo harmônico, são exemplos de processos que podem ser realizados sem preparo histológico com o objetivo de se obter imagens microscópicas em diferentes profundidades com resolução celular. Este projeto teve por objetivo desenvolver um microscópio óptico de varredura a laser baseado em processos ópticos não lineares, para adquirir imagens de tecidos e órgãos, nas condições in vitro, in vivo e ex vivo. O microscópio óptico montado é composto por: um laser de pulsos ultracurtos sintonizável (Ti:Safira), um sistema de varredura espacial (dois espelhos conectados a galvanômetros e conjugados por dois espelhos esféricos, para varredura lateral, e uma plataforma piezoelétrica para varredura axial), uma lente objetiva (20X, abertura numérica de 1,0, imersão em água e distância funcional de 2,0 mm) e um sistema de aquisição e controle. A resolução lateral obtida foi de (0,8±0,1) μm e axial de (4,4±1,5) μm, suficiente para a realização de imagens com resolução subcelular de tecidos biológicos. Imagens de fluorescência e por geração de segundo harmônico foram obtidas com sucesso a partir de tecido ex vivo de pele e fígado de rato, pele de porco e de membrana corioalantóica. Estas imagens revelaram aspectos tidos como relevantes na análise morfo-histopatológica – como estruturas nucleares e de membrana, e a presença de colágeno, e com vantagens como coleta de informação vinda de diferentes camadas do tecido. A montagem desse sistema apresenta potencial para contribuir em estudos em diagnóstico e tratamento de lesões sejam feitos de modo que, no futuro, essa análise resulte em diagnósticos mais precisos e tratamentos mais efetivos. / Accurate diagnosis of the morphological and metabolic conditions of a tissue and/or an organ is essential to define the presence of pathological changes, and to evaluate the response during a number of treatments. The use of optical techniques for biological tissue imaging is an excellent alternative for this purpose. Such techniques allow non-invasive diagnostic procedures, with cellular resolution, and usually provide almost instantaneous response. The use of nonlinear optical techniques such as fluorescence promoted by two-photon absorption is one example of optical technique in which we obtain images of living tissue with spatial resolution at cellular level. The purpose of this study is the assembly and characterization of a custom-made non-linear microscope. This microscope allows customized adjustment for in vitro, in vivo and ex vivo imaging of biological samples. The excitation is done using a tunable femtosecond Ti:Sapphire laser. Two galvanometer mirrors conjugated by two spherical mirrors are used for the lateral scan and for the axial scan a piezoeletric stage is utilized. The light is focused in tissue by an 20X objective lens, in water immersion, numerical aperture of 1.0, and working distance of 2.0 mm. The lateral resolution obtained was (0.8 ± 0.1) μm and (4.4 ± 1.5) μm for axial resolution, which is sufficient for images with sub-cellular resolution to be achieved in biological tissues. Fluorescence and second harmonic generation images were performed using epithelial and hepatic tissue. Those images revealed aspects considered relevant in morpho-histopathology – such as nuclear and citoplasm membrane structures, and the presence of collagen. By means of the microscope it is possible to have images in different depths of tissues with sub-cellular resolution. The assembly of such an equipment shall represent a potential contribution to diagnostics and lesion treatment fields, so that it may result in more precise detection of diseases and more effective treatments in the future.

Filmes ultrafinos de polímeros que contém azocromóforos são importantes para muitas aplicações como armazenamento óptico, formação de grades de relevo superficiais, alinhamento de camadas de cristal líquido e dispositivos ópticos com propriedades não-lineares. Dentre as muitas técnicas de fabricação de filmes orgânicos ultrafinos, a técnica de Automontagem eletrostática camada a camada (Layer-by-Layer, LBL) é muito atrativa devido a sua simplicidade e versatilidade, permitindo o controle na espessura e na composição dos filmes em escalas nanométricas. Contudo, a completa caracterização estrutural de tais filmes enfrenta dificuldades devido à falta de técnicas experimentais apropriadas. Utilizamos a Geração de Segundo Harmônico (SHG) para estudar a orientação molecular de filmes automontados de um polieletrólito catiônico (PAH - Poli(alilamina hidroclorada)) e um polieletrólito aniônico contendo azocromóforos como grupos laterais (Ma-co-DR13) sobre substrato de vidro. O sinal de SHG é proporcional à susceptibilidade não-linear de segunda ordem do filme, que por sua vez depende da distribuição orientacional dos azocromóforos nesse filme. Os resultados indicam que existem uma orientação preferencial dos azocromóforos, que leva a uma não-linearidade óptica significativa. Entretanto, a intensidade do sinal e a anisotropia não são homogêneas por toda a amostra, indicando a presença de domínios orientacionais, que é verificado por meio da Microscopia a Ângulo de Brewster (BAM). O sinal médio de SHG não aumenta com a espessura do filme, indicando que a ordem orientacional das sucessivas bicamadas são independentes. Analisando o sinal de SHG em função das polarizações de saída e entrada, alguns parâmetros da distribuição orientacional dos azocromóforos podem ser deduzidos. Ajustando as medidas de SHG a uma distribuição modelo concluímos que os cromóforos possuem uma certa distribuição angular com um ângulo médio em relação à superfície do plano de aproximadamente 40° e uma pequena anisotropia ao longo do plano do filme. Utilizamos também o Método da Máxima Entropia (MEM) para determinar a distribuição mais larga possível compatível com nossos dados experimentais e comparar então com a distribuição modelo obtida através dos procedimentos de ajuste. / Ultrathin films of polymers containing azochromophores are important for many applications such as optical data storage, formation of surface relief gratings, liquid crystal alignment layers and non-linear optical devices. Among several techniques for fabricating organic thin films, the Layer-By-Layer electrostatic self-assembly (LBL) is very attractive due to its simplicity and versatility, allowing one to control film thickness and composition in the nanometer scale. However, thorough structural characterization of such films is often difficult due to lack of appropriate experimental techniques. We have used optical second-harmonic generation (SHG) to study the molecular orientation of Layer-by-Layer films of a cationic polyelectrolyte ((PAH - Poly(allylamine hydrochloride)) and a anionic polyelectrolyte containing azochromophores with azo side groups (PAH/Ma-co-DR13) on a glass substrate. The SHG signal is proportional to the second-order nonlinear susceptibility of the film, which in turn depends on the orientational distribution of the azo chromophores in the film. The results indicate that there is a preferential orientation of the azo chromophores in the film, leading to a significant optical nonlinearity. However, both the signal strength and its anisotropy are not homogeneous throughout the sample, indicating the presence of orientational domains. This is verified through Brewster Angle Microscopy (BAM). The average SHG signal does not increase with film thickness, indicating that the orientational order of successive bilayers are independent. Analyzing the SHG signal as a function of the input and output polarizations, a few parameters of the azochromophore orientational distribution can be deduced. Fitting the SHG signal to a simple model distribution, we have concluded that the chromophores have an angular distribution with a mean tilt from the surface plane of approximately 41° and a slight in-plane anisotropy. We have also used the Maximum-Entropy Method (MEM) to determine the widest orientational distribution compatible with our data and compared it to the model distribution obtained by the fitting procedure.

Orientador: Flavio Caldas da Cruz / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-14T07:51:57Z (GMT). No. of bitstreams: 1 NunezPortela_Mayerlin_M.pdf: 5161095 bytes, checksum: e6a7c2d2eb88d6f484ae5211642dbec1 (MD5) Previous issue date: 2009 / Resumo: Os relógios atômicos são usados na atualidade em um grande número de aplicações científicas e tecnológicas que vão desde experimentos de relatividade e determinação de constantes fundamentais, até sistema de navegação (GPS) e telecomunicações. A proxima geração de relógios atômicos de alta precisão estará baseada em transições ópticas de átomos frios. Neste trabalho foi desenvolvido um laser de estado sólido de Nd:YLF bombeado por um laser de diodo e duplicado em frequência em 657 nm. Este sistema é proposto como oscilador local em um relógio atômico óptico baseado em átomos de cálcio. Comparado com os lasers de diodo, este apresenta uma potência maior no vermelho, um ruído de frequência e amplitude menores e a possibilidade de transferência remota usando obras ópticas no comprimento de onda fundamental de 1314 nm. Duplicação em frequência intra-cavidade é feita usando um cristal de BiBO, com superfícies anti-refletoras, e com um casamento de fase crítico tipo I à temperatura ambiente. Uma potência de 270 mW na saída do vermelho foi obtida para uma potência de bombeamento de 11.6 W. / Abstract: Atomic clocks are used today in a number of scientific and technological applications, ranging from tests of relativity, or variations of fundamental constants, to the use in navigation and telecommunication. The next generation of such high precision devices will be based on optical transitions of suitable laser cooled and trapped atoms. In this work we describe a frequency-doubled, diode-pumped solid-state Nd:YLF ring laser emitting at 657 nm, proposed as a local oscillator in an optical atomic clock based on laser cooled and trapped calcium atoms. Compared to diode lasers, its main advantages include higher power, less intrinsic frequency noise, and the possibility of remote transfer in optical fibers using the fundamental light at 1314 nm. Frequency doubling is performed inside the cavity using a 10 mm long AR-coated BiBO crystal, under type I, critical phase-matching at room temperature. Red output power of 270 mW was achieved for 11.6 W of pumping power. / Mestrado / Física Atômica e Molecular / Mestre em Física

Orientadores: Konradin Metze, Carlos Lenz Cesar / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T10:46:07Z (GMT). No. of bitstreams: 1 Vieira-Damiani_Gislaine_D.pdf: 4044155 bytes, checksum: 7a862a3866981d1827845318ce37b121 (MD5) Previous issue date: 2012 / Resumo: A hipertensão arterial sistêmica (HAS) bem como o envelhecimento provoca mudanças na estrutura dos grandes vasos sanguíneos - aorta e seus ramos - propiciando o desenvolvimento de processos degenerativos que são a causa de diversas doenças. O uso de ferramentas fotônicas na aquisição de imagens, associado a recursos matemáticos para a interpretação delas representa um avanço para as análises histopatológicas, pois permitem a visualização e compreensão de pequenas estruturas que antes eram impossíveis de serem observadas. O objetivo desse trabalho foi associar estas duas tecnologias (ferramentas fotônicas e recursos matemáticos) e com isso criar uma metodologia para a análise simultânea de fibras elásticas e colágenas na aorta. Para tanto utilizamos aorta ascendente de 72 pacientes, sendo 22 normotensos, 38 portadores de HAS e 12 aortas de dissecção. As lâminas coradas com hematoxilina eosina foram examinadas no microscópio multifoton, com dois fótons: laser de argônio para fluorescência da eosina, corante de fibras elásticas e Ti:safira para SHG, sinal gerado por moléculas de colágeno. A distribuição e organização das fibras elásticas e colágenas foram analisadas pelas seguintes variáveis: morfometria geométrica, derivadas da matriz de co-ocorrência de Haralick, Transformada de Fourier e fluorescência ótica integrada. Usando estes descritores da textura associados a fractais, observamos que a geração do SHG é dependente não só da presença do colágeno como também do arranjo destas fibras. Observamos ainda que em indivíduos normotensos, quando comparados aos portadores de HAS, ocorre uma diminuição na distribuição do sinal SHG ao longo da espessura da camada média partindo da íntima em sentido à adventícia. Dessa maneira concluímos que os maiores distúrbios das fibras elásticas, nos indivíduos normais ocorrem na transição do terço interno para o médio, enquanto que nos portadores de HAS eles estão distribuídos em toda a espessura da aorta. Além disso, estes estudos nos permitiram verificar que a dissecção da aorta ocorre entre dois reforços de colágeno, uma vez que este fenômeno foi constatado entre dois picos de SHG / Abstract: The arterial hypertension as well as aging induces changes in the structure of large blood vessels - aorta and its branches - leading to development of degenerative processes which are the cause of many diseases. The use of photonics tools for image acquisition, associated to mathematical resources for interpretation of them represents an advance in histopathological analysis, because it allows the visualization and understanding of small structures that were impossible to be observed before. The main objective of this study was to associate both technologies (photonics tool and mathematical resources) to create a new methodology to evaluate, simultaneously, elastic and collagen fibers in aorta. For this we've used autopsies of ascending aortas from 72 patients, being 22 samples from normotensives individuals, 38 from HAS patients and 12 aortas from dissection. HE-stained paraffin sections from ascending aortas were analyzed by multifoton microscopy, with 2 types of photons: Two-photon excited fluorescence (TPEF) for elastin and Ti:safira for SHG to analyze collagen fibers. The distribution and organization of elastic and collagen fibers were analyzed by the following variables: geometric morphometric, derived from the co-occurrence matrix of Haralick, Fourier Transform and Fluorescence optics integrated. Using these texture descriptors associated to analysis of fractals, we've observed that SHG generation is not only dependent on the presence of collagen but on the arrangement of these fibers as well. We also observed that in normotensives individuals, if compared to HAS patients, occurs a decrease in the SHG intensity along the medial thickness from intimate in direction to adventitia. Thus we conclude that the major disorders of elastic fibers in normal subjects occur in the transition from the third layer to the middle, while in HAS individuals these disorders are distributed throughout the thickness of the aorta. Furthermore, this study has allowed us to verify that the aortic dissection has occurred between two peaks of SHG, since this phenomenon was observed between two ribs collagen / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutora em Fisiopatologia Médica

Cette thèse a pour but de déterminer les meilleures conditions possibles pour réaliser une génération de seconde harmonique (SHG) à partir d'un fondamental dans la bande C télécom (autour de 1.55 JJm) dans des microdisques de phosphure de gallium intégrés sur silicium. Il s'inscrit dans les efforts actuels de développement de circuits photoniques intégrés en étudiant une proposition technique pour tirer parti des effets non linéaires en optique. A partir d'une résolution approchée de l'équation de propagation des ondes électromagnétiques comportant un terme source d'origine non linéaire, il a permis d'aboutir à deux "cartes" indiquant, pour l'une, les conditions nécessaires à la SHG, pour l'autre, l'efficacité théorique attendue suivant les paramètres retenus. S'appuyant sur l'expertise du laboratoire Folon en matière d'intégration monolithique de GaP sur Si, une étude de l'influence sur la SHG de certains défauts de cristallisation a été menée et a conduit à la proposition de deux nouvelles techniques d'accord de phase dans les disques de GaP épitaxiés sur Si. D'un point de vue expérimental, ce travail de thèse a permis, d'une part, de doter le laboratoire d'un banc de fabrication de fibres effilées (tapers) et fibres effilées gaufrées (dimpled tapers) et, d'autre part, d'un banc de caractérisation optique des microdisques adapté à l'étude de la SHG. / The goal of this thesis is to determine the best conditions to manage the second harmonic generation (SHG) from afundamental in the telecom C-band (around 1.55 (Jm) in gallium phosphide microdisks integrated on silicon. Findingtechnical solutions to take advantage of the non linear effects in these devices is a contribution to the actual efforts madeto develop integrated photonics circuits.In this work, the approximate resolution of the electromagnetic wave propagation equation completed by a nonlinearsource led to two "charts" indicating, for one, the necessary conditions for SHG, and for the other, the conversionefficiency theoretically allowed according to the chosen parameters. Thanks to the Foton laboratory expertise inmonolithical integration of GaP on Si, the influence on the SHG of specific structural defects was studied. This has led tothe proposal of two new phase matching techniques in GaP/Si microdisks.From an experimental point of view, this work led to the fabrication of two setups, the fi rst one, to make tapered opticalfibres and dimpled tapers, the second one, to make optical characterizations of microdisks and to study SHG in these systems.

Ce travail s’inscrit dans le contexte des microscopies optiques à très haute résolution. Nous proposons un nouveau concept de sonde active pour la microscopie optique en champ proche (SNOM), exploitant les effets de génération de second harmonique (SHG) de molécules. L’idée développée vise à s'affranchir de l’une des principales limitations des sondes actives fluorescentes réalisées jusqu'à présent : l'accrochage des sondes à l'extrémité de la pointe SNOM, étape toujours délicate et souvent peu fiable. Pour ce faire, nous avons mis en œuvre une technique qui consiste à utiliser la localisation du champ électrique au sein d’une jonction pointe métallique-substrat conducteur immergée dans une solution de molécules non-linéaires dipolaires. L’interaction champ-molécules entraine l’orientation locale un nano-volume de ces molécules dont l’excitation par un laser permet ensuite la génération d’un signal de second harmonique. Après avoir validé ce concept dit de « nano-EFISHG » (Electric Field Induced SHG) nous avons conçu un nouveau banc expérimental, dédié à l'imagerie de second harmonique haute résolution : celui-ci a permis d'obtenir les premières images présentant un contraste de second harmonique sur un échantillon structuré à l'échelle micronique.Nous avons ensuite travaillé à l’optimisation de la résolution de l’expérience mise en place : nous avons notamment démontré la possibilité de tirer parti d’effets d’exaltation locale du champ électromagnétique se produisant à l'extrémité de pointes ou de nano-objets métalliques. L’extrapolation des résultats obtenus montre que de telles exaltations devraient permettre d’atteindre des résolutions de l’ordre de 50 nm. / This work was achieved within the context of high resolution optical microscopy. We propose a new concept of active probe for near-field optical microscopy (SNOM), exploiting the effect of second harmonic generation (SHG) of molecules. The idea intends to avoid one of the main limitations of currently developed fluorescent active probes: the anchoring of the probes at the end of a SNOM tip which is a very delicate and often unreliable step. The technique implemented here consists in using the electric field localization in a metallic tip – conducting substrate junction immersed in a solution containing dipolar non-linear molecules. The interaction between the molecules and the electric field gives rise to the local orientation of a nano-volume of these molecules whose excitation by a laser allows generation of a second harmonic signal.After validation of this concept named as “nanoEFISHG” (Electric Field Induced SHG) we have designed a new experimental setup, dedicated to high resolution second harmonic imaging. Successful implementation of this setup has leaded to the recording of the first images presenting a second harmonic contrast on a sample structured at the micronic scale. Next step has consisted in working towards optimization of the experimental resolution: we have especially study the possibility of taking advantages of local field enhancement effects occurring at metallic nano-structures or sharp tip’s apex. The extrapolation of the obtained results shows that such effects should allow to reach resolutions about 50 nm.