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.

Polarization-dependence provides a unique handle for extending the dimensionality of optical microscopy, with particular benefits in nonlinear optical imaging. Polarization-dependent second order nonlinear optical processes such as second harmonic generation (SHG) provide rich qualitative and quantitative information on local molecular orientation distribution. By bridging Mueller and Jones tensor, a theoretical framework was introduced to experimentally extend the application of polarization-dependent SHG microscopy measurements toward in vivo imaging, in which partial polarization or depolarization of the beam can complicate polarization analysis. In addition, polarization wavefront shaping was demonstrated to enable a new quantitative phase contrast imaging strategy for thin transparent samples. The axially-offset differential interference contrast microscopy (ADIC) was achieved as a combination of classic Zernike phase contrast and Nomarski differential interference contrast (DIC) methods. The fundamentally unique manner of this strategy also inspired rapid volumetric analysis in time dimension that is accessible for most existing microscopy systems. Finally, the dimensionality of high speed twophoton fluorescence imaging was extended to the spectral domain by spatial/spectral multiplexing, enabling beam scanning two photon fluorescence microscopy with 17 frames per second rate and over 2000 effective spectral data points.<br>

Les semi-conducteurs nitrures d'éléments III type GaN, AlN sont des matériaux extrêmement intéressants pour la photonique intégrée sur silicium. Ils sont transparents sur une gamme très étendue et possèdent des susceptibilités non linéaires non nulles, ce qui rend possible les expériences non linéaires d'ordre deux et d'ordre trois. Dans ce contexte, cette thèse a été consacrée à l'étude de circuits photoniques avec des micro-résonateurs tels que les cristaux photoniques et les microdisques en matériau GaN/AlN épitaxiés sur Si. Le dessin des microcavités et des procédés de fabrication ont été optimisés afin d’obtenir un mode résonant dans le proche infrarouge avec un facteur de qualité jusqu'à 34000 pour les cristaux photoniques et 80000 pour les microdisques. J’ai étudié sur ces circuits photoniques les propriétés de conversion harmonique telles que la génération de seconde harmonique (SHG) et la génération de troisième harmonique (THG). En utilisant les propriétés de la THG, en combinant simplement un objectif optique et une caméra CCD, j'ai effectué l'imagerie des modes de cristaux photoniques du proche infrarouge avec une résolution spatiale sub-longueur d'onde (300 nm). J'ai également effectué l'imagerie de SHG sur des microdisques avec une excitation optique en résonance avec un mode de galerie pour le laser pompe. La dernière partie porte sur l'étude de la SHG en accord de phase entre les modes TM-0-0-X et TM-0-2-2X en variant le diamètre du disque avec un pas extrêmement faible (8 nm). Cela a été effectué pour des modes résonants de facteurs de qualité autour de 10000. Ces résultats montrent le potentiel des semi-conducteurs de III-nitrures pour la réalisation de circuits optiques sur silicium à deux dimensions. / Nitride semiconductors are extremely interesting for integrated photonics on silicon. They have a large transparent window and dispose of non zero nonlinear susceptibilities which enable second and third order nonlinear experiments. In this context, this thesis has been devoted to integrated photonic circuits with microresonators such as photonic crystals and microdisks. The microcavity design and the fabrication process have been optimized in order to obtain a near infrared resonant mode with a quality factor up to 34000 for photonic crystals and 80000 for microdisks. I carried out harmonic conversion experiments such as second harmonic generation (SHG) and third harmonic generation (THG). With THG, by combining simply an optical objective and a CCD camera, I carried out near infrared photonic crystal modes imaging with a subwavelength spatial resolution (300 nm). I also did SHG imaging on microdisks with an optical excitation in resonance with the gallery mode for the pump laser. The last part of the work is dedicated to the demonstration of phase-matched SHG in microdisk photonic circuits between the TM-0-0-X and TM-0-2-2X modes by varying the microdisk diameter with a very small step (8 nm). These experiments have been done for resonant modes with quality factors around 10000.These demonstrations show the potential of III-nitride semiconductors for the realization of two dimensional optical circuits on silicon.

The unique symmetry requirements of second harmonic generation (SHG) provide exquisite selectivity to chiral crystals, enabling independent quantitative modeling of the nucleation and crystal growth of active pharmaceutical ingredients (APIs) within amorphous solid dispersions (ASDs) during accelerated in situ stability testing, and in vitro dissolution testing. ASDs, in which an API is maintained in an amorphous state within a polymer matrix, are finding increasing use to address solubility limitations of small-molecule APIs. SHG microscopy yielded limits of detection for ritonavir crystals as low as 10 ppm, which is about two orders of magnitude lower than other methods currently available for crystallinity detection in ASDs. The quantitative capabilities of SHG analysis were substantially improved further while simultaneously dramatically reducing the total sample volume and storage burden through in situ analysis. Single particle tracking of crystal growth performed in situ enabled substantial improvements in the signal to noise ratio (SNR) for recovered crystal nucleation and growth rates by nonlinear optical microscopy. Upon dissolution, the presence of solubilizing additives in biorelevant media greatly affected the generation and stabilization of supersaturated solutions. SHG microscopy was found to enable the detection of crystals even in the highly turbid Ensure Plus® system. Analysis of the SHG micrographs clearly indicated that differences in the nucleation kinetics rather than growth rates dominated the overall trends in crystallinity. For weakly basic drugs, the fate of dissolution in fasted-state simulated intestinal fluid (FaSSIF, pH 6.5) varied with the ASDs drug loading, and was highly affected by the pre-exposure to the fasted-state simulated gastric fluid (FaSSGF, pH 1.6) medium, versus the dissolution in FaSSIF medium alone. The presence of crystals during the first stage of posaconazole ASDs dissolution in FaSSGF acted as nuclei for further crystallization in the later dissolution stage in FaSSIF. The results provide insights of better formulation prediction of poorly soluble drugs, as well as understanding origins of intraluminal absorption variability for such systems

<p> Understanding the function and structure of a protein is crucial for informing on rational drug design and for developing successful drug candidates. However, this understanding is often limited by the protein pipeline, i.e. the necessary steps to go from developing protein constructs to generating high-resolution structures of macromolecules. Because each step of the protein pipeline requires successful completion of the prior step, bottlenecks are often created and therefore this process can take up to several years to complete. Addressing current limitations in the protein pipeline can help to reduce the time required to successfully solve the structure of a protein. </p><p>The field of nonlinear optical (NLO) microscopy provides a potential solution to many issues surrounding the detection and characterization of protein crystals. Techniques such as second harmonic generation (SHG) and two-photon excited UV fluorescence (TPE-UVF) have already been shown to be effective methods for the detection of proteins with high selectivity and sensitivity. Efforts to improve high throughput capabilities of SHG microscopy for crystallization trials resulted in development of a custom microretarder array (μRA) for depth of field (DoF) extension, therefore eliminating the need for z-scanning and reducing the overall data acquisition time. Further work was done with a commercially available μRA to allow for polarization dependent TPE-UVF. By placing the μRA in the rear conjugate plane of the beam path, the patterned polarization was mapped onto the field of view and polarization information was extracted from images by Fourier analysis to aid in discrimination between crystalline and aggregate protein. </p><p>Additionally, improvements to X-ray diffraction (XRD), the current gold standard for macromolecular structure elucidation, can result in improved resolution for structure determination. X-ray induced damage to protein crystals is one of the greatest sources of loss in resolution. Previous work has been done to implement a multimodal nonlinear optical (NLO) microscope into the beamline at Argonne National Lab. This instrument aids in crystal positioning for XRD experiments by eliminating the need for X-ray rastering and reduces the overall X-ray dosage to the sample. Modifications to the system to continuously improve the capabilities of the instrument were done, focusing on redesign of the beam path to allow for epi detection of TPE-UVF and building a custom objective for improved throughput of 1064 nm light. Furthermore, a computational method using non-negative matrix factorization (NMF) was employed for isolation of unperturbed diffraction peaks and provided insight into the mechanism by which X-ray damage occurs. This work has the potential to improve the resolution of diffraction data and can be applied to other techniques where X-ray damage is of concern, such as electron microscopy.</p><div><br></div>