Graphene, a single layer of graphite, owing to its excellent mechanical, electrical, and thermal properties, has evolved as an exceptional nanomaterial in the past decade. It holds great promise in developing various novel applications from biomedical to structural composites. However, several challenges remain in realising the great potential of this material; one being the bulk scale production of graphene. This thesis has been concerned with production of pristine few-layer graphene (FLG) using liquid phase exfoliation (LPE) of graphite in various solvent media and exploring the applications of graphene-based composite coatings as optical Raman-strain sensors. LPE of natural graphite using bath sonication was used to produce highly stable pristine FLG in 1-methyl-2-pyrrolidinone (NMP) and N,N-dimethylformamide (DMF). Atomic force microscope (AFM) was used to analyse the exfoliation efficiency and lateral dimensions, while Raman spectroscopy provided an insight about the quality of the graphene flakes. Moreover, the potential for dynamic light scattering (DLS) as an efficient in situ characterisation technique for estimating the lateral dimensions of graphene flakes in dispersions was demonstrated. LPE was also employed to explore various routes to produce pristine graphene in aqueous media which can be used for toxicity studies. Aqueous dispersions were prepared by a solvent exchange method of graphene originally in organic solvents (NMP and DMF) using dialysis, achieving 0.1 v/v% organic solvent levels. Pristine aqueous graphene dispersions were also prepared by directly exfoliating graphite in biocompatible surfactant (TDOC- Sodium taurodeoxycholate) and biomolecules (Phosphatidylcholine and human serum albumin) solutions. Cell culture studies by collaborators revealed that solvent-exchanged and TDOC-exfoliated pristine FLG displayed minimal toxicity and albumin-exfoliated FLG hardly any cytotoxicity, whereas phosphatidylcholine-exfoliated FLG was cytotoxic. Raman spectroscopy is a well-established technique used to study the local deformation of carbon-based composites by following the shift rates of the Raman 2D band with strain. Raman active strain coatings were produced from epoxy composites made with the FLG produced by LPE in organic solvents and by electrochemical exfoliation method. The deformation experiments on these coatings revealed little or no strain sensitivity, due to several factors such as length of flakes, processing history, graphene loading, defects in graphene and alignment of flakes within the composites. As an alternative, composite coatings made from chemical vapour deposition (CVD) graphene were investigated. Excellent strain sensitivity was observed upon various cyclic deformational sequences and Raman mapping over 100 × 100 µm area. In comparison to the commercially available wide area strain sensors, CVD graphene composite coatings with a calculated absolute accuracy of ~ ± 0.01 % strain and absolute resolution of ~ 27 microstrains show promise for wide area Raman-based strains sensors.

This thesis explores a new technique for investigating the photochemical mechanisms of femtosecond laser inscription of permanent photonic structures in Poly(methylmethacrylate) (PMMA). The refractive index (RI) structures were fabricated with a direct writing method without ablation, and analysed using a non-invasive method - namely: Raman mapping spectrometry. The writing conditions for the photonic structures under investigation are mainly represented by 800nm and 400nm wavelength with 44fs and 100fs pulse length and a low repetition rate in the kHz domain. The mass percentage of the induced monomer and end groups modification (MMA) as a measure of the modification of the ratio of C=C and C=O Raman transition varies linearly with the total fluence (total). The mass percentage of the induced monomer and end groups change is defined by the modification of normalised ratio of the Raman intensity of C=C bond (I(C=C)) and the Raman intensity of C=O bond (I(C=O)) which is denoted by I(C=C/C=O)n. The modification of this ratio is denoted by I (C=C/C=O)n and also by MMA. MMA varies linearly with total with a positive slope for both writing conditions due to the induced main chain scission and unzipping. If total increases by 1J/cm2, it is predicted an increase in MMA, by (1.550±0.11)x10-2 (cntsxcm2)/J, for the near infrared (NIR) irradiated samples that is higher than the increase of MMA for the ultraviolet (UV) irradiated sample that show a value of (1.9200.274)x10-3 ( (cntsxcm2)/J). The same trend was found for the variation of MMA with diffraction efficiency () for NIR irradiated structures and also for UV irradiated structures. If  increases by 1cnt, it is predicted that there will be an increase in MMA, by (4.233±0.383) cnts for NIR irradiated samples that is lower than the increase of MMA for the UV irradiated sample that shows a value of (14.3922.477) cnts. The variation of MMA with  is higher for UV irradiated samples than for NIR irradiated samples, and this indicates that the nonlinear absorption of two photons produces a larger percentage of the monomer and end groups than the nonlinear absorption of three photons. Gel Permeation Chromatography (GPC), which is a destructive analytical method, was applied only for the investigation of the time dependent behaviour of the molecular weight of the photonics structures which were written with the parallel writing technique using 775nm wavelength and 160fs pulse length that shows an increase of 66 in  after seven days from the laser irradiation. Twenty-four hours after laser irradiation, the GPC results show that the weighted average molecular weight (Mw) of the exposed sample of 28,610,000 Daltons is about thirty times higher than the MW of the unexposed sample of 963,425 Daltons. This is an indication of the photo-cross-linking reaction. As a result of this reaction, the polymer chains link together through intermolecular forces to form a 3D network which produces an increase of molecular weight. It was also observed that there was a further decrease of molecular weight after three days to 437,441 Daltons due to main chain scission and unzipping. The main chain scission is actually the breaking of C-C bonds between structural units and the formation of radicals which further produce the monomer and end groups (MMA) through the unzipping reaction which leads to a decrease of the molecular weight. The main chain scission occurred with the greatest efficiency after three days following the end of irradiation, when the number of the main chain scissions (Ns) reached the maximum value of 1.193. An increase of molecular weight signifies an increase of the refractive index since the optical density has increased. The mechanical properties of PMMA optical fibres (e.g., Young's modulus) and of bulk PMMA (e.g., glass transition temperature) were investigated using Dynamical Mechanical Analysis (DMA) tests (e.g., stress-strain test and temperature ramp/frequency sweep test). These measurements were performed to study the effect of the manufacturing process that involves stretching and heating or cooling on the mechanical properties of PMMA optical fibres and unmodified PMMA material. T he ultimate aim of this section was to see the effect of the laser irradiation on the strain properties of an optical fibre sensor with gratings. The stress strain results show an increase of Young's modulus of the PMMA optical fibre of 5%, and this is an indication of decreased elasticity which is induced during the fabrication process. For a femtosecond laser irradiated region with UV wavelength, it is expected that there will be an increase of Young's modulus to 65%. This variation was obtained inthe research group from The Photon Science Institute by measuring Young's modulus for a diffraction grating which was written in PMMA with 180fs pulse length and 387nm wavelength and which was subjected to a strain. The elasticity was measured using the displacement of the first order diffracted beams as a result of a modification due to the applied strain [ ]. The temperature ramp/frequency sweep test shows an increase of glass transition temperature of the bulk PMMA of 54.12% which is also an indication of decreased elasticity induced during the fabrication process. A further increase in this temperature is expected for UV irradiated samples.

The hydrophobic effect has been a topic of research for decades, not only due to its importance as the primary building block of much of chemistry (it dictates which solvent can dissolve which solutes) and biology (guiding protein binding and gene expression) but also due to it being a fundamental physical process. The commonly held opinion is that 'like dissolve like', implying polar substances can readily mix with other polar substances, and similarly for apolar ones, but polar and apolar would separate and tend to stay isolated from one another (like oil in water). We have developed a quantitative imaging method that can be used in tandem with Raman spectroscopy in order to investigate the effect of high pressure on a model hydrophobic system - water and methane. Our study revealed an unexpectedly large increase in the amount of methane that can readily mix with water once a rather modest pressure has been applied to the system. Thus, the solubility of CH4 in H2O starts abruptly increasing at 1.3 GPa and reaches a maximum of 44(3) mole % at 2.1 GPa, showing no pressure dependence upon further compression. We have tried to reproduce the observed experimental behaviour using classical molecular dynamics simulations deploying a range of widely used water potentials (SPC/E, TIP4P, TIP3P), but unfortunately no quantitative or even qualitative agreement was reached with experiments. Finally, in order to understand the atomic level changes that enable this increased amount of methane to dissolve in water, we have performed neutron scattering measurements along with EPSR (empirical potential structure refinement) fits to the data in order to solve the structure of the fluid mixture. These revealed a tendency towards maintaining the H-bond network present in water and homogeneous mixing. Despite the network staying similar to the one found in pure fluid water at milder pressures and temperatures (close to ambient conditions), the H-bonds seem more disordered and show a greater variability in their lengths.

This project set out to examine the possibility that by acquiring Raman spectra and performing multi-photon imaging we can get better diagnosis and understanding of the biochemistry of an individual cancerous tumour and distinguish it from the healthy tissue. Within the frame of this study, colorectal primary and secondary cancer cells are examined with Raman spectroscopy in order to (i) study and distinguish them according to their chemical composition by applying multivariate methods and (ii) determine whether Raman spectroscopy can identify the cells which are the link between primary and secondary colorectal cancer cells, the so-called Cancer Stem Cells. The second part of this thesis is based on tissue studies. Human colorectal tissue sections are examined in a label-free manner with the use of multi-photon imaging modes (i) Two photon excitation fluorescence, (ii) stimulated Raman scattering and (iii) second harmonic generation, in order to determine whether these can provide fast and accurate diagnosis of colorectal cancer. These techniques were able to distinguish between healthy and cancerous tissue regions, based on the chemically-specific images of the tissue microenvironment and architecture. The hypothesis of Cancer stem cell is examined with the use of Raman spectroscopy shown that the CSCs have some small differences according to their tissue origin.

This dissertation comprises experimental studies on the synthesis and applications of metallic nanoshells. These are a class of nanoparticles composed of a dielectric core and a thin metallic shell. Metallic nanoshells play an important role in nanotechnology, particularly in nanomedicine, due to their peculiar optical properties. The overall objectives of the dissertation were to improve the fabrication of these nanoparticles, and to demonstrate new applications of these materials in cancer research and spectroscopy. The fabrication of nanoshells is a multi-step process. Previously to our work, the procedures for the synthesis of nanoshells reported in the literature lacked systematic characterization of the various steps. The procedure was extremely time-consuming and the results demonstrated a high degree of size variation. In Chapter 3, we have developed characterization tools that provide checkpoints for each step of the synthesis. We demonstrated that it is possible to control the degree of coverage on the shell for a fixed amount of reagents, and also showed important differences on the shell growth phase for gold and silver. The synthetic optimization presented in Chapter 3 led to an overall faster protocol than those previously reported. Although the improvements presented in Chapter 3 led to a higher degree of control on the synthesis of nanoshells, the variations in the resulting particle population were still too large for applications in single particle spectroscopy and imaging. In Chapter 4, the synthesis was completely reformulated, aiming to narrow the size distribution of the nanoshell colloids. Through the use of a reverse microemulsion, we were able to fabricate ultramonodisperse silica (SiO2) cores, which translate into nanoshell colloids with narrow extinction bands that are comparable to those of a single nanoshell. We then fabricate a library of colloids with different core sizes, shell thicknesses and composition (gold or silver). The localized surface plasmon resonance (LSPR) of these colloids span across the visible range. From this library, two nanoshells (18nm silver on a 50nm SiO2 core, and 18nm gold on a 72nm SiO2 core) were selected for a proof of principle cell imaging experiment. The silver nanoshells were coated with a nuclear localization signal, allowing it to target the nuclear membrane. The gold nanoshells were coated with an antibody that binds to a receptor on the plasma membrane of MCF-7 human breast cancer cells. The nanoshells were easily distinguishable by eye in a dark field microscope and successful targeting was demonstrated by hyperspectral dark field microscopy. A comparison was made between fluorescent phalloidin and nanoshells, showing the superior photostability of the nanoparticles for long-term cell imaging. The results from Chapter 4 suggest that the nanoshells obtained by our new synthetic route present acceptable particle-to-particle variations in their optical properties that enables single particle extinction spectroscopy for cell imaging. In Chapter 5 we explored the use of these nanoshells for single-particle Surface-enhanced Raman spectroscopy (SERS). Notice that particle-to-particle variations in SERS are expected to be more significant than in extinction spectroscopy. This is because particle-to-particle SERS variabilities are driven by subtle changes in geometric parameters (particle size, shape, roughness). Two types of gold nanoshells were prepared and different excitation wavelengths (λex) were evaluated, respective to the LSPR of the nanoshells. Individual scattering spectra were acquired for each particle, for a total of 163 nanoshells, at two laser excitation wavelengths (632.8 nm and 785 nm). The particle-to-particle variations in SERS intensity were evaluated and correlated to the efficiency of the scattering at the LSPR peak. Chapter 6 finally shows the application of gold nanoshells as a platform for the direct visualization of circulating tumor cells (CTCs). 4T1 breast cancer cells were transduced with a non-native target protein (Thy1.1) and an anti-Thy1.1 antibody was conjugated to gold nanoshells. The use of a transduced target creates the ideal scenario for the assessment of nonspecific binding. On the in vitro phase of the study, non-transduced cells were used as a negative control. In this phase, parameters such as incubation times and nanoshell concentration were established. A murine model was then developed with the transduced 4T1 cells for the ex vivo portion of the work. Non-transduced cells were implanted in a control group. Blood was drawn from mice in both groups over the course of 29 days. Antibody-conjugated nanoshells were incubated with the blood samples and detection of single CTCs was achieved in a dark field microscope. Low levels of nonspecific binding were observed in the control group for non-transduced cells and across different cell types normally found in peripheral blood (e.g. lymphocytes). All positive and negative subjects were successfully identified. Chapter 7 provides an outlook of the work presented here and elaborates on possible directions to further develop the use of nanoshells in bioapplications and spectroscopy. / Graduate / 2019-05-03

Carbonatos fosfatos isoestruturados do tipo sidorenkitas sÃo materiais com alto potencial de aplicaÃÃo em cÃtodos dos mais modernos tipos de baterias de sÃdio. Sua estrutura lamelar permite a inserÃÃo e extraÃÃo dos Ãons de sÃdio, que sÃo a principal fonte de forÃa eletromotriz da bateria. Utilizando um elemento muito mais abundante na crosta terrestre, esses dispositivos sÃo a grande promessa para substituiÃÃo da, jà bastante difundida, bateria de Ãons de lÃtio. PorÃm, o cÃtodo que contÃm as caracterÃsticas ideais para produzir o melhor desempenho, propiciando alta densidade de energia e estabilidade quÃmica, ainda està em desenvolvimento. A espectroscopia Raman continua sendo o melhor mÃtodo para caracterizar as propriedades destes compostos, pois contÃm a grande vantagem de ser altamente sensÃvel ao ambiente molecular das ligaÃÃes do oxigÃnio com os metais de transiÃÃo, o fÃsforo e o carbono. Nesta linha, realizamos um estudo de espectroscopia Raman em um conjunto de carbonatos fosfatos do tipo Na3MCO3PO4 (M = Mn, Fe, Co e Ni), obtidos por sÃntese hidrotÃrmica em microondas a 210 ÂC por 30 minutos. O sucesso na inserÃÃo de variados metais no interior de octaedros MO6, com raios iÃnicos distintos, produz uma alteraÃÃo no volume da cÃlula unitÃria do arranjo cristalino, modificando a distÃncia de equilÃbrio das ligaÃÃes atÃmicas, provocando uma alteraÃÃo substancial no nÃmero de onda dos seus modos normais de vibraÃÃo. Por isso, visto que informaÃÃes essenciais das propriedades quÃmicas e estruturais dos mais variados tipos de carbonatos fosfatos sÃo reveladas pela tÃcnica da espectroscopia Raman de forma confiÃvel, sua utilizaÃÃo se torna indispensÃvel para o desenvolvimento de mÃtodos e o planejamento de um material eficiente e comercialmente viÃvel para as inovadoras baterias de Ãons de sÃdio.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Uma parcela significativa da pesquisa cientÃfica hodierna baseia-se na sÃntese, caracterizaÃÃo ou aplicaÃÃo de materiais nanoestruturados. No intuito de possibilitar um maior entendimento das intrigantes propriedades destes materiais, muitas tÃcnicas com resoluÃÃo nanomÃtrica e atà subnanomÃtrica foram criadas nos Ãltimos 50 anos e tÃm passado por um processo de contÃnuo aprimoramento. Apesar do grande desenvolvimento tÃcnico e cientÃfico da instrumentaÃÃo disponÃvel, a caracterizaÃÃo Ãtica com resoluÃÃo nanomÃtrica destes materiais tem se mostrado um desafio para pesquisadores do mundo inteiro. Nesse contexto, as tÃcnicas baseadas em radiaÃÃo de campo prÃximo tÃm despontado como uma boa alternativa para quebra do limite de difraÃÃo, que constitui um limite natural para os instrumentos Ãticos convencionais. AlÃm disso, a microscopia de campo prÃximo em modo de iluminaÃÃo e contato adquire simultaneamente informaÃÃes Ãticas e topogrÃficas do material, o que permite fazer uma distinÃÃo entre as propriedades Ãticas nanomÃtricas reais e as induzidas por topografia. O fenÃmeno de domÃnios cristalogrÃficos tem sido um assunto de interesse da cristalografia desde que ela comeÃou a se desenvolver como ciÃncia. As razÃes Ãbvias para isso sÃo a beleza de formas externas de algumas estruturas de domÃnios, assim como as evidentes e desafiantes relaÃÃes de simetria entre os seus constituintes. Os domÃnios ferroelÃsticos sÃo um dos importantes tipos de domÃnios apresentados por cristais. Tais domÃnios apresentam, algumas vezes, dimensÃes muito reduzidas sem manifestar uma considerÃvel alteraÃÃo na estrutura topogrÃfica da amostra. Por outro lado, o comportamento Ãtico de domÃnios distintos pode ser bem diferente quando sujeitos a uma mesma condiÃÃo de medida. Tais caracterÃsticas tornam esta classe de materiais ideais para um estudo baseado na microscopia de campo prÃximo em modo de iluminaÃÃo e contato. Imagens de domÃnios ferroelÃsticos, utilizando microscopia de campo prÃximo em modo de iluminaÃÃo e contato, foram obtidas com resoluÃÃo nanomÃtrica. Tais imagens, em geral, apresentaram pouquÃssima relaÃÃo com a estrutura topogrÃfica do material, revelando importantes caracterÃsticas Ãticas (ocorrÃncia do efeito de guias de onda nos domÃnios) e estruturais (dimensÃes dos domÃnios ferroelÃsticos, presenÃa de defeitos de deslocamento na rede cristalina). / A significant part of the nowadays scientific research is based on the synthesis, characterization and application of the nanostructured materials. In order to make possible a further understanding of the intriguing properties of these materials, many techniques with nanometric and even subnanometric resolution have been created during the last 50 years and are passing through a process of continuous improvement. In spite of the great scientific and technical development on the available instrumentation, the optical characterization with nanometric resolution of these materials remains a challenge for researchers from all over the world. In this context, techniques based on near field radiation have emerged as a great alternative for the diffraction limit break which is a nature imposed limit for conventional optical instruments. Besides this, contact near field microscopy in illumination mode acquires optical and topographical information of the material simultaneously. This capability permits the distinction between the real and topographic induced nanometric optical features. The domain phenomenon in crystals has been a subject of interest for crystallographers since crystallography started to develop as a scientific branch. The obvious reasons are the beauty of some domain patterns as well as evident and challenging symmetry relations between their constituents. The ferroelastic domains are one of the important kinds of domains presented by crystals. They have, sometimes, very small dimensions without inducing any topographic element on the sample. On the other side the optical behavior of different domains under almost the same measurement condition may be very dissimilar. Such characteristics make then an ideal class of materials for contact near field microscopy in illumination mode based studies. Ferroelastic domains images, using near-field optical microscopy in illumination and contact mode, were obtained with a nanometric resolution. Such images, in general, presented very little relation with the topographical structure of the material, revealing optical and structural features such as occurrence of the waveguide effect in the domains, domainÂs dimensions and presence of dislocations on the crystalline structure.  

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O estudo das propriedades eletrÃnicas, mecÃnicas e vibracionais dos nanotubos de carbono com poucas camadas à importante para desenvolver e aprimorar os modelos que descrevem o comportamento dos nanotubos de parede mÃltipla, que à o tipo de nanotubo mais usado em aplicaÃÃes tecnolÃgicas. Diversos estudos teÃricos e experimentais tÃm sido realizados visando o entendimento do comportamento dos nanotubos de carbono de paredes simples e dupla (SWNT e DWNT, respectivamente) submetidos a altas pressÃes hidrostÃticas. Entretanto, o comportamento de nanotubos de carbono de parede tripla (TWNTs) sob tais condiÃÃes à ainda desconhecido. Neste trabalho, apresentamos um estudo de espectroscopia Raman ressonante em TWNTs em funÃÃo da pressÃo hidrostÃtica. Utilizamos Ãleo de parafina (Nujol) e uma mistura de metanol:etanol na proporÃÃo de 4:1 como meios transmissores de pressÃo (PTM) e uma cÃlula de pressÃo de bigorna de diamante (DAC-Diamond Anvil Cell) para a aplicaÃÃo da pressÃo. A evoluÃÃo dos modos radiais (RBM) e tangenciais (Banda G) em funÃÃo da pressÃo foi analisada de forma detalhada e comparada com os resultados existentes para os nanotubos de parede simples e dupla. Os dados de espectroscopia Raman mostram que os efeitos de blindagem internos dos TWNTs sÃo mais acentuados que nos DWNTs. As modificaÃÃes nas intensidades dos modos Raman foram interpretadas como sendo associadas as mudanÃas nas condiÃÃes de ressonÃncia dos modos RBM em funÃÃo da pressÃo. / The study of the electronic, mechanical and vibrational properties of the carbon nanotubes with few layers is important for developing and improving models that could describe the behavior of multi-walled carbon nanotubes (MWNTs), which is the kind of nanotube most used in technological applications. Several theoretical and experimental studies have been conducted for understanding the behavior of single and double walled carbon nanotubes (SWNT and DWNT, respectively) under high hydrostatic pressures. However, the behavior of triple walled carbon nanotubes (TWNTs) under such conditions is still unknown. Here, we present a study of resonance Raman spectroscopy in TWNTs as function of hydrostatic pressure. We used paraffin oil and a mixture of methanol:ethanol in 4:1 ratio as pressure transmitting media (PTM) and a diamond anvil cell (DAC) for applying pressure. The evolution of the radial breathing modes (RBM) and the tangential modes (G band) as a function of pressure was analyzed in detail and compared with existing results for SWNTs and DWNTs. The Raman spectroscopy data show that the shielding effects of inner tubes in TWNTs are more pronounced than in DWNTs. The changes in the intensities of Raman modes were interpreted as being associated with pressure induced changes in the resonance conditions.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Nesta Tese sÃo estudados por meio da tÃcnica de espectroscopia Raman dois cristais de aminoÃcidos submetidos a condiÃÃes de altas pressÃes. O primeiro deles à uma amostra de L-Valina deuterada. Inicialmente, foi feita a tentativa de identificaÃÃo de todas as bandas Raman que aparecem no espectro da L-Valina deuterada, comparando-se com os resultados da L-Valina hidrogenada existentes na literatura. A seguir foi feito o estudo da amostra comprimida com argÃnio numa cÃlula de pressÃo a extremos de diamantes no intervalo entre 0,0 GPa e 12 GPa. Deste estudo pode-se concluir que entre 0,0 GPa e 1,3 GPa a L-Valina deuterada sofre uma mudanÃa estrutural, conforme informaÃÃes fornecidas pelos espectros Raman do material na regiÃo dos modos externos. Em pressÃes mais elevadas, entre 5 GPa e 7 GPa, ocorrem modificaÃÃes nos espectros Raman em praticamente todas as regiÃes espectrais, o que aponta tambÃm para uma possÃvel mudanÃa conformacional das molÃculas na cÃlula unitÃria. Forneceu-se tambÃm os valores (∂ωj/∂P)T para os diversos modos de vibraÃÃo do cristal nas vÃrias fases apresentadas pelo mesmo. O segundo aminoÃcido investigado foi a L-Isoleucina, que foi estudada atravÃs de espectroscopia Raman sob condiÃÃes de altas pressÃes atà cerca de 7,5 GPa tendo como lÃquido compressor o Ãleo mineral. Da anÃlise na regiÃo espectral 50 1/cm - 3200 1/cm , pode-se inferir que acima de 2,5 GPa e de 5,0 GPa ocorrem grandes mudanÃas conformacionais das molÃculas de L-Isoleucina na cÃlula unitÃria. Quando se faz o grÃfico do nÃmero de onda de dois modos da rede do cristal de L-Isoleucina investigados em funÃÃo da pressÃo, observam-se descontinuidades claras em aproximadamente 2,3 GPa e 5,0 GPa. Estas mudanÃas foram associadas a modificaÃÃes nas ligaÃÃes de hidrogÃnio, uma vez que as principais variaÃÃes de frequÃncias foram observadas em bandas associadas ao rocking do NH3+ e ao rocking do CO2 . / In this thesis, are studied by Raman spectroscopy of two amino acid crystals subjected to conditions of high pressures. The first is a sample of deuterated L-Valine. Initially, an attempt was made to identify all the bands that appear in the Raman spectrum of deuterated L-Valine, comparing with the results of L-Valine hydrogenated in the litera- ture. The following study was conducted on the sample compressed in a cell with argon pressure to extremes of diamonds in the range between 0.0 GPa and 12 GPa this study can conclude that between 0.0 and 1.3 GPa, the L-Valine suffers a deuterated structural change, according to information provided by the Raman spectra of the material in the region of external modes. At higher pressures, between 5 GPa and 7 GPa, changes occur in the Raman spectra in nearly all spectral regions, which also points to a possible con- formational change of molecules in the unit cell. It also provided the values (∂ωj/∂P)T for the various modes of vibration of the crystal at various stages presented by the same. The second investigation was the amino acid L-Isoleucine, which was studied by Raman spectroscopy under high pressures up to about 7.5 GPa with the compressor as a liquid mineral oil. Analysis in the spectral region 50 1/cm - 3200 1/cm , one can infer that above 2.5 GPa and 5.0 GPa large conformational changes occur in the molecule of L-Isoleucine in the unit cell. When you make a graph of the wave number of two-mode network of the crystal of L-Isoleucine investigated as a function of pressure, there are clear discontinuities at approximately 2.3 GPa and 5.0 GPa These changes were associated with changes in the connections hydrogen, since the major changes were observed in the frequency bands associated with the NH3+ rocking and the rocking of CO2-.

PET imaging, using the bone tracer Na18F, allows the non-invasive location of atherosclerotic plaques that are at risk of rupture. However, the spatial resolution of PET is only 4-5 mm, limiting the mechanistic information this technique can provide. In this thesis, the use of fluorescence and Raman imaging to elucidate the mechanism of micro-calcification within atherosclerotic plaques has been investigated. A number of fluorescent probes to detect fluoride and calcium have been synthesised. One of the fluoride probes has been shown to be selective for fluoride however, the concentration of fluoride required to activate the probe is order of magnitudes higher than the amount of Na18F used for PET imaging making it problematic to use for future studies. On the other hand, a calcium probe has been shown to: selectively bind to hydroxyapatite (HAP); permit visualisation and quantification of HAP in both vascular and bone cell models; and effectively stain cultured aortic sections and whole mouse aorta for OPT imaging. Building on these preliminary data, fluorescence imaging and immunohistochemistry (IHC) imaging of both healthy and atherosclerotic tissue that were previously subjected to PET imaging, were successfully carried out showing the ability of the probe to detect HAP in human vascular tissue. IHC staining for Osteoprotegerin (OPG) and Osteopontin (OPN), two bone proteins recently detected in vascular tissue, showed the co-localization of OPG with the probe. Conversely, the OPN was shown to localize in areas surrounding high OPG and probe signal. To determine the exact composition of vascular calcification, Raman spectroscopy was also used. It is believed that the biosynthetic pathway to HAP passes through a series of transitional states; each of these has different structural characteristics which can be studied using Raman spectroscopy. In particular, HAP has a strong characteristic Raman peak at 960 cm-1. An increase in HAP concentration has been detected by Raman in both calcified cell models and aortic sections. When human vascular tissue was analysed, an additional peak at 973 cm-1 was present suggesting the presence of whitlockite (WTK) in this tissue as well as HAP.