Fluorescence imaging has been increasingly relied upon as the method of choice for many biological and medical applications. As demands for more sensitive and higher resolution imaging are ever-increasing, it is critical that photostable, and robust fluorophores capable of delivering high emission rates are available. Fluorescent silver nanoclusters offer an attractive compromise between the photostability and brightness of quantum dots and the compact versatility of organic chromophores. They have been shown to be superior in many roles, including as single molecule fluorophores and bulk multiphoton biological staining agents. The two-photon absorption cross sections are several orders of magnitude larger than commercially-available dyes, and they have demonstrated superior photostability under high intensity irradiation. In addition to the endogenous effects of the cluster, its small size of only a few atoms renders it highly susceptible to surface and environmental effects, which manifests, for example, in the observed photoinduced charge transfer between the silver cluster and oligonucleotide. This state has been shown to be highly advantageous in imaging applications, as control of this state enables better control over the time-averaged emission rate of the molecule. The mechanism of charge transfer, and the possible means by which this state can be controlled will be also be investigated in this work.

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Se describe la aplicación de pulsos láser ultracortos (≤10fs) en espectroscopía y microscopía de dos fotones llevada a cabo en nuestro grupo de investigación, subrayando las ventajas y requerimientos de este enfoque. Además se presenta una breve descripción de la manipulación de pulsos, de las distorsiones de fase experimentadas por los pulsos láser de femtosegundo y de cómo corregir tales distorsiones utilizando manipuladores de pulsos. / Femtosecond laser pulses in two-photon spectroscopy and microscopy: The application of shaped ultrashort laser pulses (≤10fs) in two-photon spectroscopy and microscopy carried out in our group is  described, highlighting the advantages and requirements of this approach. In addition, a brief description of pulse shaping, phase distortions experienced by femtosecond laser  pulses and how to correct these distortions using a pulse shaper is also presented.

A larga banda espectral, característica de pulsos ultracurtos de luz laser, tem possibilitado o controle coerente da interação da luz com a matéria através da manipulação das componentes espectrais da fase do pulso. Esta nova área de pesquisa tem sido responsável por avanços no entendimento e controle de fenômenos foto-induzidos, especialmente no que diz respeito a processos ópticos não lineares. Nesta tese de doutorado, estudamos o controle coerente da absorção de dois fótons (A2F) em compostos orgânicos usando pulsos de femtossegundos. O processo de A2F em derivados de perilenos foi investigado utilizando pulsos com chirp linear (máscara de fase quadrática), a partir do monitoramento da fluorescência excitada por dois fótons. A otimização da A2F desses compostos, através da formatação da fase do pulso via algorítmo genético, revelou que pulsos limitados por transformada de Fourier induzem maior A2F. Cálculos de Química Quântica, empregando o formalismo da teoria do funcional densidade, foram utilizados para caracterizar a estrutura eletrônica e determinar as transições permitidas por dois fótons nos derivados de perilenos, fundamentando nossos resultados experimentais. Além disso, estudamos também o controle coerente da A2F de compostos orgânicos aplicando uma máscara de fase senoidal ao pulso. Neste caso, demonstramos que a eficiência do controle depende da posição relativa entre o comprimento de onda central do pulso e da banda de A2F do material. Finalmente, o controle coerente da A2F foi investigado com o uso de uma máscara de fase do tipo degrau. Nossos resultados evidenciam a importância da relação entre a banda espectral do pulso e a largura de linha da A2F do material para atingir o controle da A2F. Em conclusão, os resultados obtidos neste trabalho ajudam a esclarecer aspectos do controle coerente, obtido com pulsos ultracurtos formatados, em sistemas moleculares. / The broad spectral band of ultrashort laser pulses has been used to coherently control the lightmatter interaction, by acting on the spectral phase of pulses using the so called pulse shaping methods. This new research area has been held responsible for advances in the understanding and controlling of photo-induced phenomena, especially in nonlinear optics. In this work, we studied the coherent control of two-photon absorption (2PA) processes in organic compounds, employing femtosecond pulses. We investigated the 2PA of perylene derivatives using chirped pulses (quadratic phase mask), by monitoring the two-photon excited fluorescence. Optimization of 2PA in perylene derivatives was achieved by shaping the pulse using a genetic algorithm, which revealed that Fourier transform limited pulses lead to higher 2PA. Quantum chemical calculations, using Density Functional Theory, were carried out to characterize the electronic structure and determine the allowed two-photon transitions of perylene derivatives, backing up our experimental results. Furthermore, we also studied the coherent control of 2PA in organic molecules applying a cosine-like phase mask. In such case, we demonstrated that the control efficiency depends on the detuning between the pulse central wavelength and materials 2PA band. Finally, coherent control of 2PA was explored using a step-like phase mask. Our results indicate that, in this situation, control of 2PA is only attained if a specific ratio between the pulse bandwidth and the 2PA transition bandwidth is used. In conclusion, the results obtained in this work help the understanding of coherent control in molecular systems.

No presente trabalho produzimos microestruturas, através da técnica de fotopolimerização via absorção de dois fótons, dopadas com nanofios de ZnO, um material que vem sendo amplamente explorado devido as suas interessantes propriedades ópticas e elétricas. Para a fabricação das microestruturas, utilizamos um oscilador laser de Ti:safira que produz pulsos de aproximadamente 100 fs em 800 nm. A intensidade dos pulsos de femtossegundos é alta o suficiente para induzir a absorção¬ de dois fótons em torno do volume focal, localizando a polimerização a esta região. Portanto, através da varredura do feixe na resina polimérica fabrica-se a estrutura desejada. Neste trabalho, desenvolvemos uma metodologia para introduzir nanofios de ZnO às microestruturas fabricadas, a partir da mistura do pó de nanofios de ZnO à resina acrílica. A resina utilizada é uma combinação de duas resinas, o etoxilated(6)trimethylolpropane triacrylate (SR-499) e tris(2-hydroxy ethyl)isocyanurate triacrylate (SR-368). Como fotoiniciador utilizamos o Lucirin TPO-L (2,4,6-trimetilbenzoiletoxifenil phosphine oxide). As microestruturas produzidas foram caracterizadas pelas técnicas de microscopia óptica, microscopia eletrônica de varredura, espectroscopia de energia dispersiva, difração de Raios X e espectroscopia de espalhamento micro-Raman. Através destas técnicas, foi possível observar a presença dos nanofios nas microestruturas, bem como caracterizar suas propriedades morfológicas que se mostram adequadas para o desenvolvimento de microdispositivos. Observamos também a emissão de fluorescência das microestruturas excitadas por um e dois fótons. Sendo assim, a metodologia de fabricação descrita aqui pode ser usada como mais uma opção na concepção de novos dispositivos tecnológicos. / In this study we fabricated microstructures, using the two-photon polymerization technique, containing ZnO nanowires, a material that has been widely exploited due to their interesting optical and electrical properties. For the microstructures fabrication, we used Ti:Sapphire laser oscillator operating at 800 nm with 100 fs pulses. The intensity of the fs-pulses is high enough to induce two-photon absorption, confining the excitation and thus the polymerization to the focal volume. By scanning the beam across the resin the desired microstructure is fabricated. In this work, we developed a method to introduce ZnO nanowires in the fabricated microstructure by mixing the ZnO nanowires powder to the acrylic resin. The used resin is a combination of two compounds, etoxilated(6)trimethylolpropane triacrylate (SR-499) and tris(2-hydroxy ethyl)isocyanurate triacrylate (SR-368). As a photoinitiator we have used Lucirin TPO-L (2,4,6-trimetilbenzoiletoxifenil phosphine oxide).The produced samples were characterized by optical microscopy, scanning electron microscopy, energy dispersive spectroscopy, X-ray diffraction spectroscopy and micro-Raman scattering. From these techniques it was possible to observe the presence of nanowires in the microstructures, as well as to characterize the morphological properties, which has been shown to be interesting for developing microdevices. We have also observed fluorescent emission of the microstructures excites by one and two-photons absorption. Therefore, the methodology described here can be used as an alternative in the design of new optical devices.

Submitted by Isaac Francisco de Souza Dias (isaac.souzadias@ufpe.br) on 2016-03-02T19:08:33Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese José Ferraz.pdf: 2743054 bytes, checksum: 9154337dac4a587934aab71697b77a90 (MD5) / Made available in DSpace on 2016-03-02T19:08:33Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese José Ferraz.pdf: 2743054 bytes, checksum: 9154337dac4a587934aab71697b77a90 (MD5) Previous issue date: 2008-05-29 / CNPQ / Neste trabalho, utilizamos as técnicas de controle temporal coerente e de manipulação da fase óptica do campo óptico para investigar e controlar os diferentes caminhos quânticos induzidos em transições de dipolo elétrico envolvendo a absorção de dois fótons no átomo de rubídio. As transições foram excitadas por pares de pulsos de separação temporal variável e analisamos a resposta do meio atômico em função dessa separação. Três situações experimentais são analisadas envolvendo transições com diferentes características. No primeiro experimento, utilizamos luz incoerente de um laser de corante com duração temporal de nanosegundos, mas com tempo de coerência de picosegundos, para excitar a transição de dois fótons, envolvendo níveis altamente excitados, níveis de Rydberg. A resposta do sistema é analisada através de um processo de mistura de quatro ondas, resolvida no tempo, e a seleção dos diferentes caminhos quânticos envolvidos no processo é feita a partir do controle da polarização dos campos do laser incidente. Interferências na freqüência central do laser, interferências "ópticas", e no dobro dessa freqüência, interferências quânticas, são observadas. Nos outros dois estudos, a transição de dois fótons é excitada por pulsos com duração temporal da ordemde 100 fentosegundos. No primeiro caso, investigamos uma transição de dois fótons pura, entre os níveis 5S e 7S do rubídio, onde uma fase externa, dependente da freqüência, é adicionada em um dos pulsos, enquanto o outro tem seu atraso temporal controlado. A fluorescência detectada é uma medida direta da população do estado excitado. Novamente, um sinal interferométrico é observado, cujo controle coerente é efetuado por uma combinação da fase externa e do atraso temporal. O último experimento envolve uma transição seqüencial, cuja ressonância de um fóton leva a efeitos de propagação observados no sinal de interesse. Outro aspecto importante é que a taxa de repetição do laser era maior que as taxas de relaxação dos níveis envolvidos, de forma que efeitos de acumulação na população e na coerência também estão presentes. A resposta do sistema é analisada através do processo de mistura paramétrica de quatro ondas, resolvido no tempo. O sinal interferométrico, com controle de polarização e da freqüência de detecção, permite uma demonstração clara da origem quântica nas interferências "ópticas". / In this work, we use the techniques of temporal coherent control and phase manipulation of the optical field to investigate and control different quantum pathways in the two-photon absorption in rubidium atoms. Three experimental situations are studied involving transitions with different characteristics. In all of them, the medium response is analyzed as a function of the temporal delay between the pulse pairs responsible for the two-photon transition. In the first experiment, we use incoherent light from a dye laser with pulse duration in the nanosecond scale, but with coherence time in the picosecond scale, to excite the two-photon transition, which involves highly excited levels, Rydberg states. The system response is analyzed through a time-resolved four-wave mixing process, and the quantum pathway selection is realized by polarization control of the incident laser fileds. Interferences in the central laser frequency - "optical" interferences - and at twice this frequency - quantum interferences - are observed. In the subsequent two studies, the two-photon transition is excited with 100 fs pulses. In the first case, we investigate a pure two-photon transition, the 5S - 7S rubidium transition, where an external frequency depedent phase is added in one pulse, while the temporal delay of a second pulse is controlled. We observe an interferometric signal, in which coherent control is achieved with external phase and temporal delay combination. The last experiment involves a sequential transition, which has a one-photon resonance that leads to propagation effects observed in the signal. The laser repetition rate is greater than the atomic system relaxation rates, leading to accumulation effects in the population and coherence, which leads to important effects. A time-resolved parametric four-wave mixing is used to investigate the system response. The interferometric signal, with polarization and detection frequency control, allows us to clearly demonstrate the quantum origin of the "optical" interferences.

Orientador: Carlos Lenz Cesar / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin / Made available in DSpace on 2018-08-17T16:12:03Z (GMT). No. of bitstreams: 1 Pelegati_VitorBianchin_M.pdf: 3778666 bytes, checksum: d19d947cc4b4206345d5c2da244362d6 (MD5) Previous issue date: 2010 / Resumo: Biologia celular é um novo mundo promissor com enorme impacto social, econômico e na saúde. Organismos vivos são capazes de, produzir a própria energia a partir da luz do sol, se reproduzir, de se auto-reparar, sinalizar e navegar por sinais bioquímicos, biomecânicos, luminosos, térmicos, e outros, e produzir materiais à temperatura ambiente. As possibilidades abertas por essa área incluem, desde bactérias e protozoários usados para destruir células de câncer, regeneração de órgãos inteiros, produção de etanol a partir de algas, e outros. Entretanto, para o entendimento da biologia em seu nível mais profundo, ferramentas de observação não destrutivas fazem-se necessária para seguir os processos celulares durante seu tempo de vida. A óptica tem a única onda não destrutiva capaz de fornecer informações em tempo real com suficiente resolução espacial de eventos acontecendo internamente à célula. Ademais, porque feixes de luz não colidem, a óptica permite a integração de diferentes técnicas capazes de reunir informações simultâneas de processos celulares. Óptica não linear é especialmente adequada para tal, pois não requer marcação ou processamentos especiais de amostras que poderiam destruir, ou modificar, os processos celulares. Além disso, técnicas elásticas, como a geração de segundo e terceiro harmônicos não liberam energia no material que é, portanto, preservado após a observação. O objetivo dessa tese é desenvolver uma plataforma multimodal para observação de processos biológicos pelo uso de microscopias de fluorescência excitada por absorção de dois fótons, geração de segundo harmônico e geração de terceiro harmônico no mesmo instrumento. Nosso grupo foi pioneiro em demonstrar a aquisição de imagens de geração de segundo harmônico no Brasil e, essa tese é a primeira a realizar a aquisição de imagens por geração de terceiro harmônico. Estas três técnicas juntas fornecem informações complementares a respeito da organização de células e tecidos. Enquanto a fluorescência pode ser específica para algumas proteínas alvo, o segundo harmônico pode observar a rede de colágeno da matriz extra celular e, o terceiro harmônico pode observar os núcleos e gotículas de lipídios internas às células. Esta tese descreve o sistema experimental para realizar essas aquisições multimodais de imagens, a física por trás dos sinais não lineares, importantes para entender seu significado biológico, e mostra aplicações das técnicas para diferentes amostras biológicas e inorgânicas / Abstract: Cell biology is promising a brave new world with enormous social economic and health impacts. Living organisms are capable of producing their own energy from sun light, reproduce, self-repair, signalize and travel in response to biochemical, biomechanical, light and thermal signals among others, and to produce materials at room temperature. The possibilities opened by this area range from bacteria and protozoa used to destroy cancer cells, whole organs regeneration, ethanol produced from algae, and others. However, to actually understand biology at its deepest level no destructive observation tools are necessary to follow cell processes during their time course. Optics is about the only wave capable to provide non destructive real time information with enough spatial resolution of the events happening inside the cells. Moreover, because light beams do not collide, optics allows the integration of different techniques capable to gather simultaneous information during a cell process. Non linear optics is specially suited for that in the sense that it does not require staining or special sample processing that would destroy, or change, the process. Besides, elastic techniques such as second and third harmonic generation do not release energy at the material which is therefore preserved after the observation. The objective of this thesis is to develop a multimodality platform for biology process observation by using Two Photon Excited Fluorescence, Second Harmonic Generation and Third Harmonic Generation Microscopy with the same instrument. Our group was the first one to demonstrate the acquisition of Second Harmonic Generation images in Brazil and this thesis is the first one to perform the acquisition of third harmonic generation images. These three techniques together provide complementary information respect to cell and tissue organization. While fluorescence can be specific target to some proteins, second harmonic can observe the collagen network of extra cellular matrix and the third harmonic can observe the nucleus and lipid droplets inside the cells. This thesis describe the experimental setup to perform these multimodal image acquisition, the physics behind the non linear signals, important to understand their biological mean, and shows applications of these techniques for different biological and inorganic samples / Mestrado / Física / Mestre em Física

La nanomédecine activée à deux-photon est devenue l'un des principaux candidats à l'accomplissement de la sélectivité spatiotemporelle nécessaire pour la nanomédecine. En effet, la raison d'être de l'application médicale de nanotechnologie dans le domaine du traitement du cancer est de diminuer et supprimer les effets secondaires causés par les techniques actuelles telles que la chimiothérapie et la radiothérapie, à cause de leur manque de sélectivité. Parmi diverses nanoparticules (NPs), les nanoparticules de silice mésoporeuse (MSN) ont attiré une attention croissante dans la dernière décennie pour leur faible cytotoxicité, leur internalisation cellulaire et excrétion, et leur capacité de combiner de nombreuses fonctions à la fois pour le diagnostique et la thérapie de cancers via un seul nanovéhicule : l'ainsi appelée nanomédecine théranostique.Dans cette thèse, des MSN pour l'activation à un et deux-photon d'imagerie par fluorescence, de délivrance de principe actifs et d'acides nucléiques, et de photothérapie dynamique (PTD), seront présentées. Premièrement, le relargage contrôlé de molécules encapsulées dans des MSN fonctionnalisées avec des nanovalves est considéré par effet plasmonique. La photodégradation contrôlée de la silice soumise à l'effet photothermique de NPs d'or est ensuite étudiée. Deuxièmement, l'activation biphotonique est considérée pour la délivrance contrôlée de molécules anticancéreuses in-vitro par avec des nano-rotor et des nano-valves, ainsi que la fonctionnalisation de surface des NPs par des dérivés d'ammonium- azobenzene pour la délivrance d'acides nucléiques. Troisièmement, des MSN multifonctionnelles incorporant des photosensibilisateurs à deux-photon sont systématiquement étudiées en termes de leurs propriétés optiques et photophysiques; la sélection du meilleur matériau est suivie d'applications biomédicales in-vitro.De plus, deux types de nanomatériaux émergeant sont également élaborés pour la nanomédecine activée à deux-photon, des NPs de polysilsesquioxane pontés (BS) et d'organosilice mésoporeuse périodiques (PMO). Ces matériaux furent élaborés sans précurseur de silice (tétraéthoxysilane par exemple), et seulement à partir de bis- ou multi-organoalkoxysilane, afin d'obtenir le plus haut pourcentage de matière organique pour l'application ciblée. En conséquence, des NPs de BS et de PMO hybrides à base de disulfures se révélèrent être des outils biodégradables, et les NPs à base de photosensibilisateurs furent appliquées pour la PTD à deux-photon. Des NPs de BS et de cœur-coquille d'or-BS sont synthétisées pour d'efficaces imagerie et PTD à deux-photon, tandis que des NPs de PMO servirent de nano-plateformes théranostiques. En outre, diverse NPs de PMO multipodes à surface spécifique très élevées sont présentées pour la construction de structuration complexe à l'échelle nanométrique.Enfin, des nano-conteneurs d'MSN composées de cœur d'oxyde de fer (Fe3O4@MSN) sont décrites pour de multiples applications. D'une part, l'élaboration de NPs MSN (et PMO) magnétiques sensibles à deux-photon est étudiée en tant que perspective pour la délivrance de gène combinant l'imagerie par résonance magnétique. D'autre part, les conteneurs de Fe3O4@MSN sont misent en œuvre et appliqués pour la dépollution de métaux lourds via la fonctionnalisation d'un ligand de type acide diéthylène triamine penta acétique. L'augmentation de l'efficacité de la dépollution est étudiée par la fonctionnalisation de la surface extérieure et/ou des pores des Fe3O4@MSN. / Two-photon actuated nanomedicine has become one of the main proponents for the achievement of the spatiotemporal selectivity needed for nanomedicine. Indeed, the raison d'être of the medical application of nanotechnology in the field of cancer treatment is to lower and suppress the side effects caused by current techniques such as chemotherapy and radiotherapy, due to their lack of selectivity. Among various nanoparticles (NPs), mesoporous silica nanoparticles (MSN) have attracted increasing attention over the past decade for their low cytotoxicity, cellular internalization and excretion, and the ability to carry multiple features for both the diagnosis and therapy of cancers in a single nanovehicle: the so-called theranostic nanomedicine.In this dissertation, I will describe MSN for one and/or two-photon-actuated fluorescence imaging, drug-delivery, gene delivery and photodynamic therapy (PDT). First, plasmonically-triggered cargo delivery via MSN nanovalves and designed mesoporous silica photodegradation is presented. Then, in-vitro two-photon-triggered drug delivery with azobenzene-functionalized MSN such as nanoimpellers and fluorescent nanovalves, along with preliminary studies of gene delivery via ammonium-functionalized nanoimpellers are discussed. Multifunctional MSN incorporating a two-photon photosensitizer are systematically studied in terms of the resulting optical and photophysical properties of the NPs, and then used for in-vitro biomedical applications.Furthermore, two kinds of emerging nanomaterials are also designed for two-photon actuated nanomedicine, bridged silsesquioxane (BS) and periodic mesoporous organosilica (PMO) NPs. These nanomaterials are elaborated without silica precursor (e.g. tetraethoxysilane) and solely with bis- or tetra-organoalkoxysilanes, thus providing materials with the highest organic content for the targeted applications. Consequently, disulfide-based hybrid BS and PMO NPs were elaborated as biodegradable nanomedical tools, and photosensitizer-based BS and PMO NPs were used for efficient in-vitro PDT. BS and gold-BS core-shells NPs are constructed for ultrabright two-photon imaging and efficient PDT, while two-photon functionalized PMO NPs serve as theranostic nanocarriers. Besides, versatile multipodal ethylene-benzene PMO NPs with very high surface areas are presented as a promising strategy for the design of structural complexities at the nanoscale.Finally, iron oxide core MSN shell (Fe3O4@MSN) nanocontainers are described for versatile applications. The design of two-photon-sensitive magnetic MSN and PMO core-shell nanovehicles is presented as a perspective for gene delivery and magnetic resonance imaging. Furthermore, Fe3O4@MSN containers are constructed for heavy metal removal of twelve of the most toxic metal ions through the diethylene triamine pentaacetic acid (DTPA) ligand. The enhancement of the pollutant removal efficiency is studied by selective surface and/or porous DTPA functionalizations.

The first aim of this thesis is to perform relativistic calculation of atomic and ionic polarizabilities and two-photon decay rates. Hydrogenic systems are treated by the Lagrange-mesh method. The extension to alkali-like systems is realized by means of a semiempirical-core-potential approach combined with the Lagrange-mesh method. The studied systems are partitioned into frozen-core electrons and an active valence electron. The core orbitals are defined by a Dirac-Hartree-Fock (DHF) calculation using the GRASP2K package. The valence electron is described by a Dirac-like Hamiltonian involving a core-polarization potential to simulate the core-valence electron correlation. Polarizabilities appear in a large number of fields and applications, namely in cold atoms physics, metrology and chemical physics. Two-photon transitions are part of a priori highly unlikely processes and are therefore called forbidden radiative processes. Experimental situations report decays from metastable excited states through these channels. Long lifetimes were measured for highly charged Be-like ions in recent storage-ring experiments, but their interpretation is problematic. The study of the competition between forbidden (one-photon beyond the dipole approximation, or multi-photon) and unexpected (hyperfine-induced or induced by external magnetic fields) radiative processes is all obviously relevant. The second aim of this thesis is to perform relativistic ab initio calculations of electronic isotope-shift (IS) factors by using the multiconfiguration DHF (MCDHF) method implemented in the RIS3/GRASP2K and RATIP program packages. Using the MCDHF method, two different approaches are adopted for the computation of electronic IS factors for a set of transitions between low-lying levels of neutral systems. The first one is based on the estimate of the expectation values of the one- and two-body nuclear recoil Hamiltonian for a given isotope, including relativistic corrections derived by Shabaev, combined with the calculation of the total electron densities at the origin. In the second approach, the relevant electronic factors are extracted from the calculated transition shifts for given triads of isotopes. These electronic quantities together with observed ISs between different pairs of isotopes provide the changes in mean-square charge radii of the atomic nuclei. Within this computational approach for the estimation of the mass- and field-shift factors, different models for electron correlation are explored in a systematic way to determine a reliable computational strategy, and to estimate theoretical error bars of the IS factors. / Doctorat en Sciences de l'ingénieur et technologie / info:eu-repo/semantics/nonPublished