Neste trabalho, demonstramos a fabricação de microambientes tridimensionais para investigar o crescimento celular. Inicialmente, desenvolvemos um sistema de microfabricação que utiliza fotopolimerização via absorção de dois fótons, com o qual se pode fabricar um conjunto de microestruturas com formas e espaçamentos pré-determinados. Este sistema de fabricação utiliza pulsos de femtossegundos, provenientes de um laser de Ti:safira operando em 790 nm. A intensidade destes pulsos é alta o bastante para induzir a absorção de dois fótons no fotoiniciador, o qual é responsável por promover a polimerização em uma resina acrílica. A natureza não linear da absorção de dois fótons confina a excitação ao volume focal, permitindo a fabricação de estruturas tridimensionais com alta resolução espacial. Para a obtenção dos microambientes, foi necessário o desenvolvimento de um sistema opto-mecânico de movimentação, tanto do feixe quando do substrato da amostra. Com esta técnica, fabricamos microambientes compostos de estruturas com diferentes formas (paralelepípedos, cilindros e cones) e espaçamentos, os quais foram caracterizados através de microscopia óptica e eletrônica. Para demonstrar a viabilidade destes microambientes para a investigação do crescimento celular, estes foram utilizados para monitorar o desenvolvimento da célula Michigan Cancer Foundation-7 (MCF-7), uma linhagem celular de adenocarcinoma de mama que apresenta fenótipo tumoral amplamente utilizada como modelo de estudo para câncer de mama. Observamos, via microscopia óptica de transmissão e fluorescência, o desenvolvimento das células MCF-7 nos distintos microambientes. Nossos resultados indicam uma melhor aderência e, portanto, desenvolvimento celular nas microestruturas cilíndricas. Observamos ainda uma maior densidade de células nos microambientes com estruturas separadas de 12 µm, a qual diminui com o aumento do espaçamento, de tal forma que para o microambiente com 30 µm, por exemplo, poucas células são observadas. Portanto, nossos resultados demonstram que os microambientes desenvolvidos são viáveis para estudos mais aprofundados em biologia celular, com potenciais aplicações em engenharia de tecido. / In this work we have demonstrated the fabrication of tridimensional microenvironments for the investigation of cell growth. Initially we have developed a two-photon absorption photopolymerization microfabrication system, which allows producing a set of microstructures with predetermined forms and spacing. This fabrication system uses femtosecond pulses from a Ti: sapphire laser operating at 790 nm. These pulses are intense enough to induce two-photon absorption by the photoinitiator, that is responsible for promoting the polymerization in an acrylic resin. The nonlinear nature of the two-photon absorption confines the excitation to the focal volume, allowing the fabrication of tridimensional structures with high spatial resolution. In order to obtain the microenvironments, it was necessary to develop a movement system for both the laser beam and the sample substrate. With this technique we have fabricated microenvironments composed by structures with different geometries (parallelepipeds, cylinders and cones) and spacing, which were characterized by optical and scanning electron microscopes. To demonstrate the feasibility of the microenvironments for the investigation of cell growth, the samples were used to monitor de development of the Michigan Cancer Foundation-7 cell (MCF-7), a lineage of breast adenocarcinoma that has a tumoral phenotype and is highly used as a model in breast cancer studies. We have observed, through conventional optical and fluorescence microscopy, the growth of the MCF-7 cells in the various microenvironments. Our results indicate a better adhesion and, therefore, better development of cells on the cylindrical microstructures. We also observe a higher cell density in the microenvironments with microstructures having a spacing of 12 µm, which reduces as the distance between microstructures increases, in such a way that for the microenvironment with 30 µm spacing, for example, just a few cells are observed. Thus, our results demonstrate that the produced microenvironments are applicable in deeper studies in microbiology, with potential application in tissue engineering.

Highly correlated quantum chemical methods have been appliedto study the photophysical properties of substituted benzenes.With the inclusion of spin-orbit coupling, the phosphorescencesof these molecules have been calculated usingMulti-CongurationalSelf- Consistent Field (MCSCF) quadraticresponse theory. The Herzberg-Teller approximation has beenadopted to evaluate the vibronic contributions tophosphorescence. The performance of hybrid density functional theory (DFT) atthe B3LYP level is examined in comparison to the MP2, CCSD andCCSD(T) methods for the geometry and permanent dipole moment ofp-aminobenzoic acid. The time-dependent DFT/B3LYP method isapplied to calculate the two-photon absorption of a series ofZinc-porphyrin derivatives in combination with a two-statemodel. The transitions between excited singlet and tripletstates of Zinc and Platinum based organometallic compounds havebeen computed using DFT quadratic response theory. The resultsare used to simulate the non-linear propagation of laser pulsesthrough these materials utilizing a dynamical wave propagationmethod.

This thesis presents a novel method of modulating silicon photonic circuits using 3D printed microfluidic devices. The fluids that pass through the microfluidic device interact directly with the silicon waveguides. This method changes the refractive index of the waveguide cladding, thus changing the effective index of the system. Through using this technique we demonstrate the shift in resonant wavelength by a full free spectral range (FSR) by increasing the concentration of the salt water in the microfluidic device from 0% to 10%. On a 60 μm microring resonator, this equals a resonant wavelength shift of 1.514 nm when the index of the cladding changes by 0.017 refractive index units (RIU), or at a rate of 89.05 nm/RIU. These results are confirmed by simulations that use both analytical and numerical methods. This thesis also outlines the development of a process that uses two-photon absorption(TPA) in silicon to produce a photoelectrochemical (PEC) etching effect. TPA induces free carriers in silicon that then interact with the Hydroflouric Acid (HF) solution that the wafer is submerged in. This interaction removes silicon away from the wafer, which is the etching observed in our experiments. Non-line-of-sight PEC etching is demonstrated. The optical assemblies used in these experiments are presented, as are several of the results of the etching experiments.

This dissertation reports on the investigation of two-photon absorption (2PA) in a series of fluorenyl molecules. Several current and emerging technologies exploit this optical nonlinearity including two-photon fluorescence imaging, three-dimensional microfabrication, site-specific photodynamic cancer therapy and biological caging studies. The two key features of this nonlinearity which make it an ideal candidate for the above applications are its quadratic dependence on the incident irradiance and the improved penetration into absorbing media that it affords. As a consequence of the burgeoning field which exploits 2PA, it is a goal to find materials that exhibit strong two-photon absorbing capabilities. Organic materials are promising candidates for 2PA applications because their material properties can be tailored through molecular engineering thereby facilitating optimization of their nonlinear optical properties. Fluorene derivatives are particularly interesting since they possess high photochemical stability for organic molecules and are generally strongly fluorescent. By systematically altering the structural properties in a series of fluorenyl molecules, we have determined how these changes affect their two-photon absorbing capabilities. This was accomplished through characterization of both the strength and location of their 2PA spectra. In order to ensure the validity of these results, three separate nonlinear characterization techniques were employed: two-photon fluorescence spectroscopy, white-light continuum pump-probe spectroscopy, and the Z-scan technique. In addition, full linear spectroscopic characterization was performed on these molecules along with supplementary quantum chemical calculations to obtain certain molecular properties that might impact the nonlinearity. Different designs in chemical architecture allowed investigation of the effects of symmetry, solvism, donor-acceptor strengths, conjugation length, and multi-branched geometries on the two-photon absorbing properties of these molecules. In addition, the means to enhance 2PA via intermediate state resonances was investigated. To provide plausible explanations for the experimentally observed trends, a conceptually simple three level model was employed. The subsequent correlations found between chemical structure and the linear and nonlinear optical properties of these molecules provided definitive conclusions on how to properly optimize their two-photon absorbing capabilities. The resulting large nonlinearities found in these molecules have already shown promise in a variety of the aforementioned applications.

Back in 1894 Lord Kelvin coined the term "chiral" in order to refer to molecules whose mirror images were not superimposable with themselves. Over the years, research has demonstrated the important role that chiral molecules play in life, chemistry, and biology as well as their importance in the development of new drugs and technologies. The efforts to understand chiral systems have been mainly driven by spectroscopic methods that leverage on the opposite responses that enantiomers have to linear or circularly polarized light of both handedness. More specifically, Electronic Circular Dichroism (ECD) which measures the differences in linear absorption of left and right circularly polarized light has been the method par excellence for the spectroscopic characterization of chiral compounds. Unfortunately, the fact that ECD is based on linear absorption severely limits the use of this method in the near to far UV region. This is mainly due to the interferences generated by the strong linear absorption of common organic solvents and buffers in this portion of the light spectrum. Nevertheless, the fact remains that many chiral biomolecules of interest related to deceases like Alzheimer and Parkinson, exhibit most of their linear absorption in the near to far UV region where ECD cannot be employed for their study. Therefore, it has become an urgent necessity to develop spectroscopic methods to study chiral molecules that can circumvent the limitations of ECD at shorter wavelengths. In order to overcome the existent limitations in linear chiral spectroscopy, the nonlinear equivalent of ECD arises as a promising alternative, i.e. Two-Photon Circular Dichroism (TPCD). Although, this phenomenon was theoretically predicted in 1975, it was not until 2008, with the introduction of the double-L scan, that a reliable and versatile method for the measurement of TPCD was introduced. The high sensitivity of this method is based on the use of "twin" pulses that allow accounting for fluctuations in the excitation source that prevented the experimental realization of the measurement. The first measurement of a full TPCD spectrum was performed on BINOL enantiomers and the results were supported and discussed with the help of theoretical calculations. After that seminal work, we embarked in expanding the understanding of the structure-property relationship of TPCD by performing, systematically, a series of theoretical-experimental studies in chiral biaryl derivatives and compounds with helical chirality. In Chapter 2 we present the theoretical-experimental study of the effect of the π-electron delocalization curvature on the TPCD of molecules with axial chirality. The targeted molecules for this part of our investigation were S-BINOL, S-VANOL, and S-VAPOL. Our findings revealed that an increase in the TPCD signal, within this series of compounds, was related to the curvature of the π–electron delocalization. The contributions of the different transition moments to the two-photon rotatory strength support our outcomes. Then, in Chapter 3 we introduce the development of the Fragment-Recombination Approach (FRA) for the calculation of the TPCD spectra of large molecules. This simple but powerful method is based on the additivity of the TPCD signal, and is subject to a strict conditional fragmentation approach. FRA-TPCD is demonstrated, theoretically, in two hypothetical molecular systems from the biaryl derivatives family. Afterward, in Chapter 4 we show the first experimental demonstration of FRA-TPCD through the conformational analysis of an axially-chiral Salen ligand in solution (AXF-155). The FRA-TPCD spectra calculated for the different isomers of AXF-155 allowed narrowing the number of possible isomers of this complex molecule in THF solution to only two. This represents a significant improvement from previously reported results using ECD. Subsequently, in Chapter 5 we present the study of the effect of intramolecular charge transfer (ICT) in S-BINAP, an axially dissymmetric diphosphine ligand with strong ICT. The evaluation of the performance of two different exchange-correlation functional (XCF) confirmed that in order to properly predict the theoretical TPCD spectrum of a molecule exhibiting strong ICT, it is required to use an XCF such as CAM-B3LYP. In addition, our findings revealed the importance of considering an adequate number of excited states in order to be able to fully reproduce the experimental TPCD spectrum, thus avoiding wrong assignments of theoretical transitions to experimental spectral features. Finally, and expanding on our previous study, in Chapter 6 we investigated the effect of the nature of ICT on two hexahelicene derivatives. Our investigation demonstrated that the TPCD signal of chiral molecules with strong ICT does not only depend on the strength of this effect but on its nature, i.e. extension of the π–electronic delocalization increasing beyond (EXO-ICT) or within (ENDO-ICT) the helicene core. In summary, with the results presented in this thesis we closed a first loop in the understanding of the structure-property relationship of TPCD. In the future, we expect to deepen in our knowledge of the structure-property relationship of this phenomenon by studying further helicene derivatives with donor-acceptor motif, and through the application of FRA-TPCD to the conformational analysis of amino acids in peptides. We foresee numerous applications of TPCD for the study of optically active molecules with implications in biology, medicine, and the drug and food industry, and applications in nanotechnology, asymmetric catalysis and photonics.

Multi-photon absorption processes have received significant attention from the scientific community during the last decade, mainly because of their potential applications in optical limiting, data storage and biomedical fields. Perhaps, one of the most investigated processes studied so far has been two-photon absorption (2PA). These investigations have resulted in successful applications in all the fields mentioned above. However, 2PA present some limitations in the biomedical field when pumping at typical 2PA wavelengths. In order to overcome these limitations, three-photon absorption (3PA) process has been proposed. However, 3PA in organic molecules has a disadvantage, typical values of σ3' are small (10-81 cm6s2/photon2), therefore, 3PA excitation requires high irradiances to induce the promotion of electrons from the ground state to the final excited state. To overcome this obstacle, specific molecules that exhibit large 3PA cross-section must be designed. Thus far, there is a lack of systematic studies that correlate 3PA processes with the molecular structure of organic compounds. In order to fill the existent gap in 3PA molecular engineering, in this dissertation we have investigated the structure/property relationship for a new family of fluorene derivatives with very high three-photon absorption cross-sections. We demonstrated that the symmetric intramolecular charge transfer as well as the -electron conjugation length enhances the 3PA cross-section of fluorene derivatives. In addition, we showed that the withdrawing electron character of the attractor groups in a pull-pull geometry proved greater 3PA cross-section. After looking for alternative ways to enhance the effective σ3' of organic molecules, we investigated the enhancement of two- and three-photon absorption processes by means of Surface Plasmon. We demonstrated an enhancement of the effective two- and three-photon absorption cross-section of an organic compound of 480 and 30 folds, respectively. We proved that the enhancement is a direct consequence of the electric field enhancement at a metal/buffer interface. Next, motivated by the demands for new materials with enhanced nonlinear optical properties, we studied the 3PA of Hematoporphyrin IX and J-aggregate supramolecular systems. As a result, we were able to propose the use of 3PA in photodynamic therapy using Photofrin, the only drug approved by the FDA for PDT.

This dissertation reports on the investigation of the relationships between molecular structure and two-photon absorption (2PA) properties for a series of polymethine and squaraine molecules. Current and emerging applications exploiting the quadratic dependence upon laser intensity, such as two-photon fluorescence imaging, three-dimensional microfabrication, optical data storage and optical limiting, have motivated researchers to find novel materials exhibiting strong 2PA. Organic materials are promising candidates because their linear and nonlinear optical properties can be optimized for applications by changing their structures through molecular engineering. Polymethine and squaraine dyes are particularly interesting because they are fluorescent and showing large 2PA. We used three independent nonlinear spectroscopic techniques (Z-scan, two-photon fluorescence and white-light continuum pump-probe spectroscopy) to obtain the 2PA spectra revealing 2PA bands, and we confirm the experimental data by comparing the results from the different methods mentioned. By systematically altering the structure of polyemthines and squaraines, we studied the effects of molecular symmetry, strength of donor terminal groups, conjugation length of the chromophore chain, polarity of solvents, and the effects of placing bridge molecules inside the chromophore chain on the 2PA properties. We also compared polymethine, squaraine, croconium and tetraon dyes with the same terminal groups to study the effects of the different additions inserted within the chromophore chain on their optical properties. Near IR absorbing squaraine dyes were experimentally observed to show extremely large 2PA cross sections ([approximately equal to] 30000GM). A simplified three-level model was used to fit the measured 2PA spectra and detailed quantum chemical calculations revealed the reasons for the squaraine to exhibit strong 2PA. In addition, two-photon excitation fluorescence anisotropy spectra were measured through multiple 2PA transitions. A theoretical model based on four-levels with two intermediate states was derived and used for analysis of the experimental data.

The purpose of this dissertation is to provide a study and possible applications of two-photon absorption (2PA), in direct-gap semiconductors and quantum-well (QW) semiconductor structures. One application uses extremely nondegenerate (END) 2PA, for mid-infrared (mid-IR) detection in uncooled semiconductors. The use of END, where the two photons have very different energies gives strong enhancement comapared to degenerate 2PA. This END-2PA enhanced detection is also applied to mid-IR imaging and light detection and ranging (LIDAR) in uncooled direct-gap photodiodes. A theoretical study of degenerate 2PA (D-2PA) in quantum wells, QWs, is presented, along with a new theory of ND 2PA in QWs is developed. Pulsed mid-IR detection of femtosecond pulses is investigated in two different semiconductor p-i-n photodiodes (GaAs and GaN). With the smaller gap materials having larger ND-2PA, it is observed that they have better sensitivity to mid-IR detection, but unwanted background from D-2PA outweighs this advantage. A comparison of responsivity and signal-to-background ratio for GaAs and GaN in END-2PA based detection is presented. END-2PA enhancement is utilized for CW IR detection in uncooled GaAs and GaN p-i-n photodiodes. The pulsed mid-IR detection experiments are further extended to perform mid-IR imaging in uncooled GaN p-i-n photodetectors. A 3-D automated scanning gated imaging system is developed to obtain 3-D mid-IR images of various objects. The gated imaging system allows simultaneous 3-D and 2-D imaging of objects. The 3-D gated imaging system described in the dissertation could be used for examination of buried structures (microchannels, defects etc.) or laser written volumetric structures and could also be suitable for in-vivo imaging applications in biology in the mid-IR spectral region. As an example, 3-D imaging of buried semiconductor structures is presented. A theoretical study of D-2PA of QWs for transverse electric (TE) and transverse magnetic (TM) fields is carried out and an analytical expression for the D-2PA coefficient in QWs using second-order perturbation theory is derived. A theory for ND-2PA in QW semiconductor using second-order perturbation theory is developed for the first time and an analytical expression for the ND-2PA coefficient for TE, TM, and the mixed case of TE and TM is derived. The shape of the 2PA curve for the D-2PA and ND-2PA for QWs in the TE case is similar to that of bulk semiconductors. As governed by the selection rules both the D-2PA and ND-2PA curves for the TE case does not show a step-like signature for the density of states of the QWs whereas 2PA curve for the TM case shows such step like sharp features. The ND-2PA coefficient for TE, TM, and the mixed case is compared with that obtained for bulk semiconductors. Large enhancement in ND-2PA of QW semiconductors for the TM case over bulk semiconductors is predicted.

Despite not being a tangible substance, light is becoming an increasingly valuable tool in numerous areas of science and technology: the use of laser excitation of a fluorescent probe can generate incredibly detailed images of cellular structures without the need for large amounts of dissection; new types of solar cells are being produced using organic dyes to harvest light; computer data can be stored by inducing a chemical change in a compound through irradiation with light. However, before any of these materials can be applied in such a way, their properties must first be analyzed for them to be deemed viable. The focus of this dissertation is the photophysical characterization, linear and nonlinear, of a several novel organic compounds, and a europium complex, as well as using quantum chemical calculation techniques to understand some of the phenomena that are witnessed and begin to develop predictive capability. The nonlinear characterization of compounds utilizes wavelengths outside of their linear absorption range, where a focused beam can achieve the same excitation as one at half the wavelength, though this effect has a quadratic dependence on power. The potential for nonlinear excitation, or two-photon absorption (2PA), is becoming of increasing interest and importance for organic chromophores. Exciting only a small volume of material at a focal point makes it possible to nondestructively image samples in 3-dimensions, record data in multiple layers, and fabricate intricate structures through photopolymerization reactions. Lanthanides such as europium are known to exhibit sharp emission bands when excited, typically through an antenna effect due to the low probability of achieving direct excitation. This emission is long-lived, and through gating systems can readily be separated from background noise and autofluorescence (often observed in biological samples) that have much shorter lifetimes. Thus, one of the foci of this dissertation is the photophysical investigation of a series of novel lanthanide complexes, with particular attention to a europium complex.

The two-photon absorption (2PA) phenomenon has attracted attention from various fields ranging from chemistry and biology to optics and engineering. Two of the common NLO applications in which organic materials have been used are three-dimensional (3D) fluorescence imaging and optical power limiting. Two-photon absorbing materials are, therefore, in great demand to meet the needs of emerging technologies. Organic molecules show great promise to meet this need as they can be customized through molecular engineering, and as the development of two-photon materials that suit practical application intensifies, so does research to meet this need. However, there remains some uncertainty in the particulars of design criteria for molecules with large 2PA cross sections at desired wavelengths, as such research to understand structure-property relationships is matter of significant importance. As a result, the full potential of 2PA materials has not been fully exploited. Several strategies to enhance the magnitude and tune the wavelength of 2PA have been reported for ?-conjugated organic molecules. On this account, we have designed novel fluorophores using the fluorene moiety and modified it to tune the properties of the compounds. Chapter 2 of this dissertation reports the successful application of fluorene-based compounds in photocatalysis; a process that involves the decomposition of organic compounds into environmentally friendly carbon dioxide and water attesting to the photostability of the fluorene moiety. A facile organic nanoparticle preparation method is reported in chapter 3 using the reprecipitation method, whose surface was then modified using a naturally occurring surfactant, Lecithin, and were then successfully used in fluorescence cell imaging. Chapter 4 reports the design and synthesis of a fluorene-based compound using an acceptor, s-indacene-1, 3, 5, 7(2H, 6H)-tetra one, or Janus Dione, a moiety that is relatively new and that has not been fully exploited despite its very attractive features. Owing to the hydrophobicity of this compound, notwithstanding its unprecedented 2PA cross section, it was not applicable in fluorescence cell imaging but provided the tenets for the design of related derivative. This limitation was circumvented in the concluding chapter by tuning the compound's hydrophilicity. The hydrophilic Janus dione probe was then used as envisioned for cell imaging as the dual prerequisites for fluorescence imaging probes; large 2PA cross sections and high fluorescence quantum yields were met.