Thesis (PhD (Physics))--Stellenbosch University, 2008. / The advent of high intensity short pulse lasers has opened the door to investigating buried solid-solid interfaces through the technique of optical second harmonic generation (SHG). This has led to extensive study of technologically important systems such as the Si/SiO2 interface. In this study, SHG is employed to study the interface between highly boron doped p+-type Si and its native oxide layer (SiO2). Previous studies from this laboratory have extensively investigated the photo-induced charge transfer process across the Si/SiO2 interface in the case of undoped natively oxidized Si by means of SHG, with initial SHG measurements being performed on boron doped p+-type Si. The natively oxidized p+-type Si/SiO2 sample was placed on a computer controlled positioning system which allowed for translation of the sample and rotation around the azimuth. The laser system employed was characterized in terms of spectral composition, pulse duration, pulse repetition rate, spatial pro le and pulse energy in order to ensure quantitative measurements. The SHG signal generated from the sample interface was recorded in re ection. Under the applied irradiation conditions, defects are created at the interface by the near infra red (NIR) femtosecond radiation from the laser. These defects are then populated via multi-photon processes by electrons and to a lesser extent holes. The charge transfer across the interface induces an interfacial electric eld. This photo-induced electric eld is in addition to the built-in interfacial electric eld caused by positive ionization of naturally occurring interfacial defects due to the strong doping of the bulk Si. It is this interfacial electric eld, consisting of the built-in doping induced eld and the photo-induced electron and hole elds, that is probed by SHG. The SHG signal is strongly dependent on the magnitude of this interfacial electric eld as the electric eld induced second harmonic (EFISH) signal dominates all other contributions to the observed SHG signal in the case of the Si/SiO2 system. The temporal evolution of the SHG signal is recorded for di erent intensities from virgin as well as the pre-irradiated samples. This yields information about the time scales on which the charge separation occurs as well as the in- uence of existing photo-induced trap sites on the charge separation process, since the strength of the SHG signal is an indirect measure of the interfacial electric eld strength. The angular dependence of the SHG signal (SH rotational anisotropy measurements) for both the initial signal (when the doping induced electric eld dominates) and the saturated signal (when the electron induced electric eld dominates) is measured. Both these measurements show a four fold symmetry but with a relative 45 phase shift between them. This iii is taken as con rmation of the reversal of the interfacial electric eld direction. The initial SHG signal as a function of intensity is also recorded for di erent incident wavelengths. The variation in the non-quadratic dependence of the initial SHG signal on the incident intensity is attributed to a resonant enhancement of two-photon absorption and subsequent screening of the interfacial electric eld by charge carriers. The measurement performed and the results obtained contribute to the understanding of the photo-induced charge separation process across buried solid-solid interfaces, speci cally as it applies to the important Si/SiO2 interface.

Articular cartilage has been imaged using the following multi-photon modalities: Second Harmonic Generation (SHG), Two-photon Fluorescence (TPF) and Coherent Anti-Stokes Raman Scattering (CARS). A simple epi detection microscope was constructed for SHG and TPF imaging in the early stages of this research. Later the imaging was transferred to a new microscope system which allowed simultaneous forwards and epi detection and combined CARS imaging with TPF and SHG. Multiphoton spectroscopic studies were conducted on both intact tissue samples and the major components of the extracellular matrix, in order to identify sources of TPF. Fluorescence was detected from type II collagen, elastin and samples of purified collagen and elastin crosslinks. Age related glycation crosslinks of collagen may be a significant source of TPF. No fluorescence was detected from proteoglycans. In intact, unfixed healthy articular cartilage the cells were observed via CARS, surrounded in their pericellular matrix which is characterised by an increase in TPF. The collagen of the extra cellular matrix showed up clearly in the SHG images. Diseased cartilage was also imaged revealing microscopic lesion at the articular surface in early osteoarthritis and highly fibrous collagen structures and cell clusters in more advanced degeneration. In young healthy cartilage a network of elastin fibres were found lying parallel to the articular surface in the most superficial 50μm of the tissue. Regional variations in these fibres were also investigated. The fibres appeared mainly long and straight suggesting that they may be under tension, further work is needed to identify whether they have a mechanical function. The polarization sensitivity of the SHG from collagen has been investigated for both cartilage and tendon. In the most superficial tissue these measurements can be used directly to determine the collagen fibre orientation. However at increasing depths the effects of biattenuation and birefringence must be considered. Healthy cartilage has a characteristic pattern of polarization sensitivity with depth and this changes at lesions indicating a disruption of the normal collagen architecture. The methods developed in this thesis demonstrate the use of non-linear microscopy to visualise the structure of the extracellular matrix and cells in intact unstained tissue. They should also be appropriate in many areas of cell and matrix biology.

Les phénomènes de diffusion cohérente non-linéaire ont été récemment proposés en alternatives à la fluorescence comme processus de marquage en microscopie multiphotonique. Les matériaux couramment appliqués dans ce contexte buttent toutefois sur une limite inférieure en taille déterminée par le seuil de détection de signaux faibles en optique non-linéaire. Aucun des efforts récents en détection en génération de second-harmonique (GSH), qui est le processus non-linéaire d’ordre le plus bas, n’a permis de descendre à ce jour au-dessous d’une barrière en taille de 40nm même en ayant recours aux techniques de détection les plus sensibles telles que le comptage de photons uniques. Les nanoparticules (NPs) restent ainsi dans la famille des nano-diffuseurs de “grande“ taille. Il apparaît toutefois possible de déplacer de façon significative cette limite inférieure vers les plus petites tailles en substituant aux isolants diélectriques ou aux semi-conducteurs à grands gaps des particules quantiques (PQs) à base de semi-conducteurs à gaps directs.Dans ce travail, un nouveau type de nanosondes hautement non-linéaires a été conçu et développé de façon à franchir cette barrière de taille minimale pour atteindre l’échelle de nanoparticules uniques. Nous considérons ainsi l’excitation résonnante à deux photons de nanoparticules quantiques individuelles à base de CdTe (de la famille des “zinc-blendes”) d’un diamètre d’environ 12.5nm, qui fournissent une émission cohérente efficace par GSH jusqu’à hauteur de 105 comptages de photons par seconde. Elles présentent de plus l’avantage d’une remarquable sensibilité à l’orientation de leur réseau cristallin octupolaire.De plus, il a été démontré que les effets de confinement quantique déterminent fortement les caractéristiques de la susceptibilité non-linéaire du second-ordre χ(2). La caractérisation quantitative du χ(2) des PQs, en particulier leur dispersion spectrale et leur dépendance en taille est menée par spectroscopie de particules uniques ainsi qu’en moyenne d’ensemble par diffusion Hyper-Rayleigh (HRS). Nous fournissons en particulier la preuve que sous certaines conditions, le χ(2) de structures à base de semi-conducteurs en mode de confinement quantique peut très largement dépasser sa valeur en milieu massif. De plus, un nouveau type de PQs hybridant des semi-conducteurs en géométries de type “bâtonnet sur sphère” (BS) a été développé sur la base de composantes cristallines de symétries différentes, afin d’augmenter leur non-linéarité quadratique effective, tout en maintenant leur taille dans un régime proche d’un fort confinement quantique. Le nouveau tenseur hybride complexe χ(2) est analysé en terme d’interférence des susceptibilités constitutives, en prenant en compte les différentes formes et symétries associées aux composantes octupolaires et dipolaires.Il en résulte pour de telles structures une exaltation significative du χ(2), qui excède celle des PQs à constituant unique compte tenu du couplage entre matériaux non-linéaires et d’un temps de décohérence plus long, que nous attribuons à un effet de separation de charge photo-induit. / Nonlinear coherent scattering phenomena from single nanoparticles have been recently proposed as alternative processes for fluorescence in multiphoton microscopy staining. Commonly applied nanoscale materials, however, have reached a certain limit in size dependent detection efficiency of weak nonlinear optical signals. None of the recent efforts in detection of second-harmonic generation (SHG), the lowest order nonlinear process, have been able to cross a ~40 nm size barrier for nanoparticles (NPs), thus remaining at the level of “large” nanoscatterers, even when resorting to the most sensitive detection techniques such as single-photon counting technology. As we realize now, this size limitation can be significantly lowered when replacing dielectric insulators or wide gap semiconductors by direct-gap semiconducting quantum dots (QDs). Herein, a new type of highly nonlinear nanoprobes is engineered in order to surpass above mentioned size barrier at the single nanoparticle scale. We consider two-photon resonant excitation in individual zinc-blende CdTe QDs of about 12.5 nm diameter, which provide efficient coherent SHG radiation, as high as 105 Hz, furthermore exhibiting remarkable sensitivity to spatial orientation of their octupolar crystalline lattice. Moreover, quantum confinement effects have been found to strongly contribute to the second-order nonlinear optical susceptibility χ(2) features. Quantitative characterization of the χ(2) of QDs by way of their spectral dispersion and size dependence is therefore undertaken by single particle spectroscopy and ensemble Hyper-Rayleigh Scattering (HRS) studies. We prove that under appropriate conditions, χ(2) of quantum confined semiconducting structures can significantly exceed that of bulk. Furthermore, a novel type of semiconducting hybrid rod-on-dot (RD) QDs is developed by building up on crystalline moieties of different symmetries, in order to increase their effective quadratic nonlinearity while maintaining their size close to a strong quantum confinement regime. The new complex hybrid χ(2) tensor is analyzed by interfering the susceptibilities from each component, considering different shape and point group symmetries associated to octupolar and dipolar crystalline structures. Significant SHG enhancement is consequently observed, exceeding that of mono-compound QDs, due to a coupling between two nonlinear materials and slower decoherence, which we attribute to the induced spatial charge separation upon photoexcitation.

Optical imaging methods have the potential to detect ovarian cancer at an early, curable stage. Optical imaging has the disadvantage that high resolution techniques require access to the tissue of interest, but miniature endoscopes that traverse the natural orifice of the reproductive tract, or access the ovaries and fallopian tubes through a small incision in the vagina wall, can provide a minimally-invasive solution. We have imaged both rodent and human ovaries and fallopian tubes with a variety of endoscope-compatible modalities. The recent development of fiber-coupled femtosecond lasers will enable endoscopic multiphoton microscopy (MPM). We demonstrated two-and three-photon excited fluorescence (2PEF, 3PEF), and second-and third-harmonic generation microscopy (SHG, THG) in human ovarian and fallopian tube tissue. A study was undertaken to understand the mechanisms of contrast in these images. Six patients (normal, cystadenoma, and ovarian adenocarcinoma) provided ovarian and fallopian tube biopsies. The tissue was imaged with three-dimensional optical coherence tomography, multiphoton microscopy, and frozen for histological sectioning. Tissue sections were stained with hematoxylin and eosin, Masson's trichrome, and Sudan black. Approximately 1 mu m resolution images were obtained with an excitation source at 1550 nm. 2PEF signal was absent. SHG signal was mainly from collagen. 3PEF and THG signal came from a variety of sources, including a strong signal from fatty connective tissue and red blood cells. Adenocarcinoma was characterized by loss of SHG signal, whereas cystic abnormalities showed strong SHG. There was limited overlap of two-and three-photon signals, suggesting that three-photon imaging can provide additional information for early diagnosis of ovarian cancer.

La conception de molécules pour l’optique non linéaires (ONL) est devenue un centre de recherche de pointe pour les télécommunications, les technologies de l'information et le stockage de données optiques. Les molécules dipolaires substituées par des groupes donneur-accepteur ont été les chromophores les plus étudiés pour l’ONL. Cependant les molécules dipolaires diverses limitations telles que leur transparence optique, leur faible stabilité thermique et leur tendance à adopter un alignement antiparallèle à l'état solide. Récemment, une nouvelle classe de matériaux est apparue basé sur des symétries octupolaire qui ne possède pas de dipôle permanent, pour les applications ONL. Au niveau structural, la structure générique idéale pour des molécules tridimensionnelles avec une distribution de charges octupolaire est un cube avec des charges opposées alternées à chaque angle. À ce jour, aucune molécule représentant le cube vrai (déformé) avec huit charges alternées aux sommets et délocalisation des charges complètes entre les plans supérieurs et inférieurs n’a été décrite. Dans le cadre de cette thèse, des complexes de lanthanides III à partir de phthalocyanines de type ABAB présentant en alternance des groupes donneurs et accepteurs d'électrons ont été synthétisés représentant le premier réel octupole. Ces structures ont été caractérisé par UV-NIR, X-Ray et présentent les plus élevés hyperpolarisabilité quadratique jamais enregistré pour des molécules octupolaires. En outre, ce travail a été étendu à divers type de double-decker de lanthanides homoleptiques non-octupolaire basé sur des phthalocyanines AB3, A4, B4, T4. Il a été observé que ces complexes présentent aussi des mesures d’hyperpolarisabilité quadratique élevés, mais inferieur a celles trouvé pour la série de complexes Ln (ABAB)2 octupolaire, comme prévu. / The design of nonlinear optical (NLO) molecules has become a focus of current research in telecommunications, information technologies and optical data storage. Donor-acceptor substituted dipolar molecules have been the most investigated NLO chromophores. Dipolar molecules, however, have several limitations such as low optical transparency, low thermal stability and their strong tendency to adopt anti-parallel packing in the solid state. Recently, a new class of materials based on octupolar symmetries, which lack permanent dipole moments, has been proposed for NLO applications. At a structural level, it can be shown that the basic template for 3D octupolar molecules comes to a cube with alternating charges at the corners such as donor and acceptor substituent. Despite all the various structures reported, it is worth noting that no molecules actually representing the “real” octupolar cube have been obtained so far. In this thesis, we showed that the real octupolar cube can be demonstrated by lanthanide III complexes based on ABAB type phthalocyanine featuring alternating electron donor and electron acceptor groups. These structures are characterized by UV-NIR, X-Ray and exhibit highest quadratic hyperpolarizability ever reported for an octupolar molecule. Moreover, this work was extended to nonoctupolar lanthanide homoleptic double-decker complexes based on AB3, A4, B4, T4 type phthalocyanines. It was observed that these molecules present a quite large quadratic hyperpolarizability too, but smaller than the one obtained for the Ln(ABAB)2 series, as expected. / Doğrusal olmayan optic (NLO) özellik gösteren moleküllerin tasarımı telekomünikasyon, bilgi teknolojileri ve optic very depolama alanlarında güncel bir araştırma konusudur. Alıcı-verici grup bağlı iki kutuplu moleküller en çok araştırılmış NLO malzemeleridirler. Ancak, iki kutuplu moleküllerin düşük optic şeffaflık, düşük termal kararlılık ve katı halde parallel olmayan yığılmaları gibi çeşitli sınırlamaları vardır. Son zamanlarda, kalıcı dipol momentleri olmayan octupolar simetriye dayalı, yeni bir malzeme sınıfı NLO uygulamaları için ileri sürülmüştür. Yapısal olarak bu 3D octupolar moleküller için temel şablon, köşelerinde alıcı verici grupları içeren bir olarak gösterilir. Yapılan tüm çalışmalara ragmen, bugüne kadar gerçek küpü temsil edebilen bir yapı elde edilememiştir. Bu tezde, biz bu octupolar küpün electron alıcı ve verici grupları içeren ABAB tipi halkalı yapıya dayalı lantanid kompleksleri ile ortaya konabilecğini gösterdik. Bu yapılar UV-NIR ve X-Ray ile yapıları aydınlatıldı ve bugüne kadarki en yüksek 2. dereceden NLO değerleri elde edildi. Ayrıca, bu çalışma, çeşitli octupole olmayan AB3, A4, B4 ve T4 tipi lantanit çift katlı ftalosiyaninler sentezlenerek genişletildi. Ve oktupol olmayan moleküllerin 2. Dereceden NLO değerleri oldukça büyük ama beklendiği gibi oktupol Ln(ABAB)2 daha küçük olduğu gözlendi.

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.

Neste trabalho, aplicamos técnicas de óptica não-linear de segunda ordem, em particular a Espectroscopia por Geração de Soma de Freqüências (SFG) e a Geração de Segundo Harmônico (SHG), para investigar a adsorção e o ordenamento molecular de filmes automontados de polieletrólitos variando-se os parâmetros que influenciam a adsorção (pH, força iônica, densidade de carga), além de investigar a estabilidade térmica e possíveis transições de fases nesses filmes multicamadas como função do pH das soluções e o do número de camadas. A espectroscopia SFG de filmes dos polieletrólitos poly(allylamine hydrochloride) (PAH) e poly(styrene sulfonate) (PSS) permitiu acompanhar a adsorção das camadas pelo monitoramento do campo elétrico na interface (substrato+filme adsorvido)/solução. As medidas de fase do sinal SFG confirmaram a supercompensação de cargas em todos os valores de pH estudados, exceto a pH 12, onde a adsorção foi possível devido a interações eletrostáticas locais. Os filmes secos mostraram grande ordenamento e homogeneidade quando a secagem era espontânea (sem a ação de jato de N2), independentemente do pH. Nestes filmes, observou-se pela primeira vez que as camadas adsorvidas podem alterar o ordenamento e a conformação das cadeias previamente adsorvidas. A espectroscopia SFG também apontou para a redução da densidade de carga e do ordenamento molecular quando a força iônica era aumentada, devido ao efeito de blindagem eletrostática. Os filmes tornaram-se mais inomogêneos e desordenados em virtude da compensação extrínseca. O efeito da força iônica foi o de reduzir a densidade de carga e assim o ordenamento molecular, mesmo se a secagem fosse espontânea. A técnica de SHG foi aplicada a filmes automontados de PAH e PS-119 para confirmar sua estabilidade térmica em função do pH e do número de camadas, além de verificar o ordenamento molecular antes e após o tratamento térmico. Os resultados mostraram que os filmes não são termicamente estáveis: o sinal é completamente destruído à temperatura de 150oC aproximadamente, ao contrário do que relata a literatura. As medidas SHG também confirmaram a isotropia dos filmes no plano das amostras, independente do pH ou do número de camadas. Comparando-se o sinal SHG antes e depois do aquecimento, comprovou-se que após o resfriamento lento, o sinal era restituído a quase o mesmo valor que antes, mostrando que o processo de desordem térmica é reversível. Entretanto, nenhuma transição de fase foi observada, pois a redução do sinal SHG foi lenta e gradual, sem nenhuma variação brusca que caracterizasse uma transição vítrea. Enfim, nossas medidas do sinal SHG em função do número de camadas também discordaram dos resultados da literatura, pois a susceptibilidade de segunda ordem não cresceu linearmente com o número de bicamadas. Isso indica que as moléculas não adsorvem com mesmo ordenamento em cada bicamada. Desse modo, podemos concluir que as técnicas SFG e SHG fornecem informação a nível microscópico que podem levar ao aprimoramento das aplicações dos filmes automontados, e que seriam difíceis de obter com técnicas tradicionais. / In this study, we have applied second-order nonlinear optical techniques, in particular Sum-Frequency Generation (SFG) and Second-Harmonic Generation (SHG), to investigate the adsorption and the molecular ordering of self-assembled polyelectrolyte films varying the parameters which are relevant to polyelectrolyte adsorption (pH, ionic strength, charge density), besides investigating the thermal stability and possible phase transistions in these multilayer films as function of pH of the solutions and of the number of layers. SFG spectroscopy of films fabricated with the polyelectrolytes PAH and PSS allowed us to monitor the adsorption of each layer by the electric field at the (substrate+adsorbed film)/solution interface. Phase-measurements of the SFG signal confirmed that charge supercompensation occurred at all pH values investigated, except at pH 12, where the adsorption was possible by local electrostatic interactions. Dry films have shown great order and homogeneity if the drying was spontaneous (without blow-drying with N2), independently of pH. In these films, it was observed for the first time that layer adsorption can modify the order and the conformation of previously adsorbed chains. SFG spectroscopy also pointed to the reduction of the charge density and of the molecular order if the ionic strength was increased, due to the electrostatic screening effect. The films were more inhomogeneous and disordered due to extrinsic charge compensation. The effect of the ionic strength was to reduce the charge density and the molecular order, even if the drying were slow (spontaneous). The SHG technique was applied to LBL films of PAH and PS-119 to confirm their thermal stability as a function of pH of the solutions and the number of layers, besides comparing the molecular order before and after the thermal treatment. The results have shown that the films are not thermally stable, with the SHG signal nearly vanished if the temperature of 150 oC is reached, in contrast of what is reported in the literature. SHG measurements have also confirmed that the films are isotropic in the plan of the samples, independent of pH or the number of layers. Comparing the SHG signal before and after heating, it was found that the SHG signal was considerably reduced at high temperatures, but after slow cooling it was recovered to almost the same value as before heating, showing that the thermal disorder is reversible. However, no phase transistion was observed, since the SHG signal reduction was slow and gradual, without any sudden change that would characterize a glass transition. At last, our SHG measurements as a function of the number of layers also disagreed with results in the literature, therefore the second order susceptibility did not grow linearly with the number of bilayers. This indicates that the molecules do not adsorb with same order in each bilayer. Therefore, we can conclude that SFG and SHG techniques provide information on the film arrangement at the microscopic level which could be difficult to get with traditional techniques and could also lead to the improvement of applications of LBL films.

Neste trabalho, aplicamos técnicas de óptica não-linear de segunda ordem, em particular a Espectroscopia por Geração de Soma de Freqüências (SFG) e a Geração de Segundo Harmônico (SHG), para investigar a adsorção e o ordenamento molecular de filmes automontados de polieletrólitos variando-se os parâmetros que influenciam a adsorção (pH, força iônica, densidade de carga), além de investigar a estabilidade térmica e possíveis transições de fases nesses filmes multicamadas como função do pH das soluções e o do número de camadas. A espectroscopia SFG de filmes dos polieletrólitos poly(allylamine hydrochloride) (PAH) e poly(styrene sulfonate) (PSS) permitiu acompanhar a adsorção das camadas pelo monitoramento do campo elétrico na interface (substrato+filme adsorvido)/solução. As medidas de fase do sinal SFG confirmaram a supercompensação de cargas em todos os valores de pH estudados, exceto a pH 12, onde a adsorção foi possível devido a interações eletrostáticas locais. Os filmes secos mostraram grande ordenamento e homogeneidade quando a secagem era espontânea (sem a ação de jato de N2), independentemente do pH. Nestes filmes, observou-se pela primeira vez que as camadas adsorvidas podem alterar o ordenamento e a conformação das cadeias previamente adsorvidas. A espectroscopia SFG também apontou para a redução da densidade de carga e do ordenamento molecular quando a força iônica era aumentada, devido ao efeito de blindagem eletrostática. Os filmes tornaram-se mais inomogêneos e desordenados em virtude da compensação extrínseca. O efeito da força iônica foi o de reduzir a densidade de carga e assim o ordenamento molecular, mesmo se a secagem fosse espontânea. A técnica de SHG foi aplicada a filmes automontados de PAH e PS-119 para confirmar sua estabilidade térmica em função do pH e do número de camadas, além de verificar o ordenamento molecular antes e após o tratamento térmico. Os resultados mostraram que os filmes não são termicamente estáveis: o sinal é completamente destruído à temperatura de 150oC aproximadamente, ao contrário do que relata a literatura. As medidas SHG também confirmaram a isotropia dos filmes no plano das amostras, independente do pH ou do número de camadas. Comparando-se o sinal SHG antes e depois do aquecimento, comprovou-se que após o resfriamento lento, o sinal era restituído a quase o mesmo valor que antes, mostrando que o processo de desordem térmica é reversível. Entretanto, nenhuma transição de fase foi observada, pois a redução do sinal SHG foi lenta e gradual, sem nenhuma variação brusca que caracterizasse uma transição vítrea. Enfim, nossas medidas do sinal SHG em função do número de camadas também discordaram dos resultados da literatura, pois a susceptibilidade de segunda ordem não cresceu linearmente com o número de bicamadas. Isso indica que as moléculas não adsorvem com mesmo ordenamento em cada bicamada. Desse modo, podemos concluir que as técnicas SFG e SHG fornecem informação a nível microscópico que podem levar ao aprimoramento das aplicações dos filmes automontados, e que seriam difíceis de obter com técnicas tradicionais. / In this study, we have applied second-order nonlinear optical techniques, in particular Sum-Frequency Generation (SFG) and Second-Harmonic Generation (SHG), to investigate the adsorption and the molecular ordering of self-assembled polyelectrolyte films varying the parameters which are relevant to polyelectrolyte adsorption (pH, ionic strength, charge density), besides investigating the thermal stability and possible phase transistions in these multilayer films as function of pH of the solutions and of the number of layers. SFG spectroscopy of films fabricated with the polyelectrolytes PAH and PSS allowed us to monitor the adsorption of each layer by the electric field at the (substrate+adsorbed film)/solution interface. Phase-measurements of the SFG signal confirmed that charge supercompensation occurred at all pH values investigated, except at pH 12, where the adsorption was possible by local electrostatic interactions. Dry films have shown great order and homogeneity if the drying was spontaneous (without blow-drying with N2), independently of pH. In these films, it was observed for the first time that layer adsorption can modify the order and the conformation of previously adsorbed chains. SFG spectroscopy also pointed to the reduction of the charge density and of the molecular order if the ionic strength was increased, due to the electrostatic screening effect. The films were more inhomogeneous and disordered due to extrinsic charge compensation. The effect of the ionic strength was to reduce the charge density and the molecular order, even if the drying were slow (spontaneous). The SHG technique was applied to LBL films of PAH and PS-119 to confirm their thermal stability as a function of pH of the solutions and the number of layers, besides comparing the molecular order before and after the thermal treatment. The results have shown that the films are not thermally stable, with the SHG signal nearly vanished if the temperature of 150 oC is reached, in contrast of what is reported in the literature. SHG measurements have also confirmed that the films are isotropic in the plan of the samples, independent of pH or the number of layers. Comparing the SHG signal before and after heating, it was found that the SHG signal was considerably reduced at high temperatures, but after slow cooling it was recovered to almost the same value as before heating, showing that the thermal disorder is reversible. However, no phase transistion was observed, since the SHG signal reduction was slow and gradual, without any sudden change that would characterize a glass transition. At last, our SHG measurements as a function of the number of layers also disagreed with results in the literature, therefore the second order susceptibility did not grow linearly with the number of bilayers. This indicates that the molecules do not adsorb with same order in each bilayer. Therefore, we can conclude that SFG and SHG techniques provide information on the film arrangement at the microscopic level which could be difficult to get with traditional techniques and could also lead to the improvement of applications of LBL films.

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.

Polarization specific measurements are advancing the capabilities of scientific instruments looking for ever smaller effects and material parameters. For example, the magneto-optical nonlinear Faraday effect can be used to characterize various electric and magnetic polarizability parameters of an individual molecule. Another major application is detection of desired particles in a highly scattering environment, the physical effect of which has been extensively researched, and is being overcome by using time-gated and polarization techniques. The polarimeter sensitivity is limited by the extinction-ratio obtained from polarizers. Of available polarizer materials, naturally occurring Calcite crystals provide the best extinction ratios because of their good optical homogeneity and high birefringence. However, there is a need for polarization determination with higher sensitivities, and thus a necessity to find better polarizing materials and methods. I developed a next-generation polarimeter in an attempt to sensitively detect the second-order Faraday effect, along with a substance s chirality and Verdet constant. Also, I developed a device uniquely able to sensitively detect chiral signatures in the presence of massive depolarizing scattering. In addition, I begun developing a novel type of polarimeter based on the highly-polarization-sensitive nonlinear-optical process of harmonic generation, whose required crystals can be grown with extremely high quality.