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<p> The potential of a mode-locked semiconductor laser oscillator as a short pulse source for two-photon fluorescence microscopy is explored. Amplification of the 1075 nrn laser is performed with a single pass semiconductor optical amplifier or a ytterbium-doped fibre amplifier. The mode-locked diode oscillator amplified by the Yb-doped fibre amplifier has been shown to produce uncompressed pulses of 4-10 ps with an average power of up to ~0.8 W. Compression with a single pass modified grating pair compressor reduces the pulse duration to as short as 860 fs. The output power level can be easily scaled to higher values. </p> <p> The ability to tightly focus the Yb-doped fibre amplifier beam and semiconductor optical amplifier beam for the purpose of microscopy is studied. Results indicate that the fibre performs close to an ideal Gaussian laser beam source. The semiconductor optical amplifier beam does not focus as well. Measurements suggest that regions of the beam, when focused, do not significantly contribute to the generation of two-photon fluorescence. </p> <p> The efficiency of two-photon fluorescence generation of the two amplifiers is compared to that of the conventional two-photon excitation source: the mode-locked titanium sapphire laser. Results illustrate the need to improve certain operating parameters of the laser oscillator and two amplifiers to be considered practical for two-photon fluorescence microscopy. The mode-locked semiconductor laser oscillator amplified by the Yb-doped fibre amplifier is deemed to be close to being ready for two-photon imaging applications. </p> / Thesis / Master of Applied Science (MASc)

Gold nanoparticles (GNP) have been widely used as optical imaging and photothermal therapy agents due to their biocompatibility, simplicity of conjugation chemistry, optical tunability and efficient light conversion to heat. A number of in vitro and in vivo studies have demonstrated that they can be used as effective thermal therapy and imaging contrast agents to treat and diagnose cancer. As clinical applications of GNPs for cancer imaging and therapy have gained interest, efforts for understanding their accumulation kinetics has become more important. Given the recent demonstration of intrinsic two-photon induced photoluminescence (TPIP) of gold nanoshells (GNSs) and gold nanorods (GNRs), TPIP imaging is an efficient tool for investigating the microscopic distribution of the GNPs at intra-organ level. The following work explores these GNPs’ physical and optical properties for effective use of GNPs in TPIP imaging and examines the feasibility of using intrinsic TPIP imaging to investigate GNP’s biodistribution in bulk tumors and thin tissue slices processed for standard histology. Our results showed that GNPs yield a strong TPIP signal, and we found that the direct luminescence-based contrast imaging of GNPs can image both GNPs and nuclei, cytoplasm or vasculature simultaneously. Also, we present the effect of GNP morphology on their distribution within organs. Collected images showed that GNPs had a heterogeneous distribution with higher accumulation at the tumor periphery. However, GNRs had deeper penetration into tumor than GNRs due to their shape and size. In addition, GNPs were observed in unique patterns close to vasculature. Finally, we introduce single- and multiple-dose administrations of GNPs as a way of increasing GNP accumulation in tumor. Our results show that multiple dosing can increase GNP accumulation in tumor 1.6 to 2 times more than single dosing. Histological analysis also demonstrated that there were no signs of acute toxicity in tumor, liver and spleen excised from the mice receiving 1 injection, 5 injections of GNPs and trehalose injection. / text

Fluorescent nucleic acid base analogues (FBAs) are an important class of molecule used to study the structure and dynamics of DNA and RNA. These base analogues are molecules with structures that resemble one of the natural bases but which, unlike the natural bases, have high fluorescence quantum yields. 2-Aminopurine (2AP) has long been the most widely used fluorescent base analogue and is one of the few base analogues commercially available. One problem with 2AP is that it undergoes significant quenching when incorporated into DNA: the quantum yield decreases 100 fold from that of the free base, thus becoming too low for use in, for example, single molecule studies. A secondary problem is that the 305 nm absorption peak requires excitation in the UV. A variety of new fluorescent base analogues are being produced, with a view to remedying the deficiencies of 2AP and expanding the current range of use. The first part of this thesis explores the one-photon photophysical properties of several of these novel FBAs. The first of these novel FBAs is the 6-aza-uridine family. These compounds, analogues of uridine, have large Stokes shifts and their absorption and emission spectra are red-shifted in comparison to 2AP; their quantum yields as free bases have been shown to exceed that of 2AP and their environmental sensitivity has been demonstrated. Time-resolved measurements reported in this thesis indicate the presence of multiple emitting species. A density functional theory (DFT) study has been carried out to rationalise these emitting species as rotational isomers. Similar fluorescence lifetime measurements were made on a second class of FBAs, the quadracyclic adenine analogues, qANs; these results also indicated the presence of multiple emitting species. Experimental results show that these FBAs undergo excited-state proton transfer. The final FBA studied in this thesis is pentacyclic adenine, pA. This FBA showed some of the most promising characteristics of all the analogues investigated, such as a high quantum yield in both polar and non-polar solvents. A time-resolved investigation into pA-containing oligonucleotides indicated that in an oligonucleotide pA adopts multiple stacked conformations and its behaviour is highly sequence dependent. Several of these aforementioned fluorescent base analogues have absorption spectra in a region that makes them accessible to two-photon (2P) excitation with a Ti:Sapphire laser. In biological systems, multiphoton excitation has several advantages over one-photon excitation. By avoiding the use of ultraviolet light there is reduced phototoxicity. Out of focus photobleaching and autofluorescence are also minimised which leads to a higher signal-to-background ratio and allows deeper tissue penetration to be achieved. Fluorescent base analogues tend to have small Stokes shifts; this is another problem that can be overcome by using two-photon excitation. To be of potential use in multiphoton microscopy, a FBA must have a high two-photon absorption cross-section and a high two-photon brightness. Previously, the highest two- photon brightness measured for a fluorescent base analogue was less than 2 GM. Amongst the base analogues investigated here, are several that have higher two-photon brightness than ever reported for FBAs; these include pA which is shown to have the highest 2P brightness of a FBA in an oligonucleotide, 1.3 GM, and a member of the 6-azauridine family which as a free base has a 2P brightness of 18 GM. Detection of individual molecules represents the ultimate level of sensitivity and enables details about a molecular system that would otherwise be concealed using conventional ensemble techniques to be revealed. With the improved 2P brightness of the molecules measured in this thesis, it has become feasible to detect single FBA molecules using 2P excitation. To maximise the chance of detection, ultrafast, shaped laser pulses have been used as the excitation source. For the first time, the signal has been high enough and the molecule of interest sufficiently photostable such that 2P fluorescence correlation spectroscopy of a fluorescent base analogue in an oligonucleotide could be measured. In summary, this thesis reports the fluorescence lifetimes and two-photon cross-sections of a series of novel fluorescent base analogues, as well as fluorescence correlation spectroscopy measurements of the most promising candidates.

In this study, we have developed a high performance multi-photon microscopic system to perform second- harmonic (SH) imaging on a tooth and a KTP crystal . The high sensitivity of the system allows acquisition rate of 300 seconds/frame with resolution at 512¡Ñ512 pixels. The surface SH signal generated from the tooth and the KTP crystal is also carefully verified through micro-spectroscopy, polarization rotation and wavelength tuning. In this way, we can ensure the authenticity of the signal. KTP crystal and the enamel that encapsulates the dentine is known to possess highly ordered structures. The anisotropy of the structure is revealed in the microscopic SH images of the tooth and the KTP crystal samples.

In the atomic, molecular, optical physics field, processes involving two photons are very well understood and used in applications ranging from spectroscopy to laser cooling technics. In this thesis is presented the study and experimental realization of two two-photon processes. Using sodium atoms trapped in a magnetic optical trap we could demonstrate two-photon cooperative absorption, and that the creation of a molecular bound state using only light fields, or photonic bound state, is possible. Two-photon cooperative absorption, very common in solid-state physics, is a process where a pair of atoms initially in the ground state is excited to the double excited state, via absorption of two photons with frequecy that is not ressonant with any excited state. Its experimental realization with cold atoms may open new and exciting possibilities to better understand nonlinear effects, and it is a new way to create correlated atoms and photons in cold atomic physics. This absorption was observed by ionization of the pair after the excitation. A simple model that considers only dipole-dipole interactions between the atoms allows us to understand the basic features observed in the experimental data. A photonic bound state uses two photons to create the two basic features of a molecular bound state: a repulsive part and an attractive part. A blue photon, blue detuned from the atomic transition, connects the ground state of the pair to the repulsive part of the first excited molecular state 1, and a red photon, red detuned from the atomic transition, connects the connects the ground state of the pair to the attractive part of the first excited molecular state. In the dressed state picture, when the light fields are strong, this three-states-two-photon system creates adiabatic bound potentials that are strongly dependent of the photon properties. Using a theoretical model we could study how this bound energies changes when we change the photon properties, and the experimental data shows that this photos are indeed dressing the potentials with a efficiency that would enable the creation of photonic molecules. / No campo da física atômica, molecular e ótica processos envolvendo dois fótons são bem compreendidos e usados em diversas aplicações. Nesta tese apresentamos o estudo e a realização experimental de dois processos de dois-fótons. Usando átomos de sódio aprisionados em uma armadilha magneto ótica, demonstramos a absorção cooperativa de dois fótons e que a criação de um estado ligado molecular usando somente campos de luz, ou estado ligado fotônico, é possível. Absorção cooperativa de dois fótons, um processo bem comum em física de estado sólido, acontece quando um par de átomos inicialmente no estado fundamental é excitado para o estado duplamente excitado, via absorção de dois fótons de frequência não ressonante com a dos estados excitado. A realização experimental deste processo em um sistema de átomos frios pode abrir novas, e excitantes possibilidades para entender melhor processos não lineares, e é um novo método de criar átomos e fótons correlacionados. Essa absorção foi observada através da ionização do par depois da absorção, e um modelo simples que considera somente interação dipolo-dipolo entre os dois átomos nós ajuda entender as características básicas dos dados obtidos. Um estado ligado fotônico usa dois fótons para criar as duas características básicas dos estados ligados moleculares: a parte repulsiva e a parte atrativa. Um fóton azul, deslocado para o azul da transição atômica, conecta o estado fundamental do par a parte repulsiva do primeiro estado excitado molecular 1, e um fóton vermelho, deslocado para o vermelho da transição atômica, conecta o estado fundamental a parte atrativa do deslocado para o azul da transição atômica. No contexto de estados vestidos, quando os campos de luz são intensos, esse sistema de três estados e dois fótons cria potenciais ligantes adiabáticos que são fortemente dependentes das propriedades desses fótons. Usando um modelo teórico para esses potenciais pudemos estudar como é essa dependência, com as características do fótons, e os dados experimentais mostram que esses fótons estão de fato vestindo os estados com uma eficiência que viabiliza a criação de moléculas fotônicas.

This thesis provides an extensive study of the first molecular hyperpolarizability b of charge-transfer chromophores using hyper-Rayleigh scattering (HRS). The charge-transfer chromophores used in present work involve the tricyanohydrofuran¡]TCF¡^group as an electron acceptor, and/or thiophene in the pi-electron bridge. TCF is a very strong electron acceptor and thiophene greatly lowers the resonance energy. Their presence significantly increases the beta value of the chromophore, therefore enhancing potentials in applications. In hyper-Rayleigh scattering experiments, the laser radiation with tunable wavelengths is used as an excitation source for measuring the frequency dependence of beta. The experiment shows beta exhibiting a significant dispersion in the two-photon resonance region. Using the linear absorption spectrum in coordination with theory, we show that it is possible to use Kramers-Kronig (K-K) transform to reproduce the experimental beta value in the two-photon resonance region. The K-K approach provides an extension to the conventional Oudar-Chemla equation, which is invalid in the spectral region in which two-photon resonance occurs. Using the new approach, it is shown that reliable values of intrinsic hyperpolarizabilities beta_zero of charge-transfer chromophores can be extracted. The coordination of beta_zero with molecular structure provides one with an insight for the origin of the enhancement of the first molecular hyperpolarizability of charge-transfer chromophores. This thesis examines the variation of beta_zero with molecular structure. The same technique is also applied to a dendrimer that has charge-transfer nonlinear optical chromophores incorporated in the dendritic structure. The measured frequency dependent hyperpolarizability of the dendrimer is compared with that calculated from the linear absorption spectrum by the KK transform technique. The intrinsic hyperpolarizability beta_zero of the dendrimer obtained is compared with that of the single chromophore having a structure similar to that incorporated in the dendrimer. The comparison shows that the 3D dendritic structure is effective in reducing the interaction between chromophores by providing sufficient space between them, hence avoiding the possibility of aggregation formation due to attractive interactions between chromophores. The topic of two-photon fluorescence (TPF), which is related to HRS, is also investigated. The intensity of TPF is generally proportional to the square of the incident excitation intensity. Careful measurements of the TPF intensity of a nonlinear optical chromophore in conjunction with required auxiliary parameters have been used as a technique for determining the two-photon absorption cross-section. The TPF intensity measurement carried out in this thesis uses a variety of intensities. At low intensity excitation, the TPF intensity follows the usual quadratic intensity law (QIL), whereas deviations from the QIL are observed at higher incident intensities. The observation of similar lineshape associated with one- and two-photon fluorescence spectra suggests a 3-level model for the description of TPF excited by the incident intensity at various strengths. It is shown that by fitting the observed TPF intensity to an equation developed from the three-level model, it is possible to deduce the two-photon absorption cross section of the nonlinear optical chromophore in solution. The new technique developed using the three-level model is tested on a Rhodamine B/Chloroform solution. The two-photon absorption cross-section obtained by using the new technique is found in agreement with that reported in the literature. Having demonstrated the suitability of the new technique, it is used to determine the two-photon absorption cross-section of a novel nonlinear optical chromophore. The two-photon absorption cross-section using the new technique is then compared with that obtained by the nonlinear transmittance method. The two results are in good agreement, indicating the applicability of the new technique. The new technique is more convenient than the conventional low excitation TPF method as it does not require various auxiliary parameters, some of them are difficult to obtain. The second harmonic generation (SHG) of a chromophore/polymer film which is optically poled by using a coherent superposition of a fundamental and its second harmonic beams. The growth rate of the SHG intensity is found to be proportional to the fourth power of the incident intensity of the fundamental beam, and the plateau intensity SHG is proportional to the square of the incident intensity. These observations are not in agreement with the published theory. While the reason for disagreement is yet to be clarified, the information obtained is useful for the development of nonlinear optical devices.

This dissertation describes the investigation of the synthesis and characterization of new squaraine-based photonic and electronic materials. In the first part of this thesis, squaraine dyes with large conjugation systems, including extended squaraines consisting of bis(donor)substituted vinylene-heterocycles and bis(indolinylenemethyl)squaraine-based oligomers linking through different π-bridges were designed, synthesized and characterized to exhibit strong two-photon absorption (2PA) for femotosecond and nanosecond optical-power limiting applications in the near-infrared (NIR). One of the dendronized squaraine forms smooth and high optical quality films with large NIR transparency window. In the second part, a series of squaraine- and phthalocyanine-based metal complexes were studied. Those dyes did not show large triplet quantum yield but significantly improved photovoltaic performance compared to the metal-free compounds. In the last part, an effective approach on optimizing bis(indolinylenemethyl)-based squaraine sensitizers with various surface anchor groups and π-linkers, achieved high power conversion efficiencies (PCEs) of 6.7% in liquid dye-sensitized solar cells (DSSCs) and 2.7% in solid-state DSSCs, which stand out all the previous reported squaraine-based sensitizers.

Photodynamic therapy (PDT) is an alternative treatment of cancer requiring the use of chromophore molecules (photosensitizers), which can induce cell death after light excitation. Gold nanoparticles (AuNP), exhibiting localized Surface Plasmon Resonance, can enhance the photophysical response of chromophores located in their vicinity, and thus improve their therapeutic action. Moreover, the use of highly localized two-photon chromophores (photosensitizers and fluorophores), capable to undergo a localized excitation by light in the Near InfraRed region, should increase the penetration depth into tissues, thus improve the treatment efficiency (by PDT) and the imaging (by fluorescence microscopy) of cancer tissues.In this work, we describe the elaboration of water-soluble hybrid nano-objects for PDT and fluorescence bioimaging applications, composed of two-photon chromophore-polymer conjugates grafted onto gold nanoparticles. In order to obtain these nano-objects we follow a multistep strategy: i) the synthesis of a well-defined water-soluble chromophore-polymer conjugates; ii) the end-group oriented grafting of chromophore-polymer conjugates onto 20 nm AuNP. The coupling of hydrophobic two-photon chromophores on linear water-soluble copolymer chains (poly(N-acryloylmorpholine-co-N-acryloxysuccinimide)), obtained by controlled/living RAFT polymerization, resulted in well-defined water-soluble chromophore-polymer conjugates, with different polymer lengths (2 000 g.mol-1 < Mn < 37 000 g.mol-1) and architectures (random or block), and a controlled number of chromophores per chain (varying between 1 and 21). Their grafting onto 20 nm AuNP gave water-soluble hybrid nano-objects with high grafting densities (~0.5 chains/nm²). The role of the polymer chain being to tune the distance between chromophores and AuNP surface, we have evidenced the increase in the polymer corona thickness of grafted AuNP (estimated by TEM) with the increasing polymer Mn, corroborating with the corresponding distance-dependent fluorescence properties of those. Finally, the in cellulo biological properties of two-photon chromophore-polymer conjugates, before and after grafting onto AuNP, have been investigated, highlighting their potential for two-photon bioimaging and PDT applications.