Biological tissues were studied with synchrotron infrared (IR) microspectroscopy, a technique that allows the spatially resolved determination and mapping of multiple components in situ at high spatial resolution. The first project involved studying brain tissue from TgCRND8 mice, a transgenic model of Alzheimer’s disease (AD). AD is the main cause of dementia in the ageing population, marked by the deposition of plaques composed of the Aβ peptide. Dense-cored and diffuse plaques were IR mapped and the results correlated with histochemistry and immunostaining. Spectral analysis confirmed that congophilic plaque cores were composed of highly aggregated protein in a β-sheet conformation. The amide I maximum of plaque cores was 1623 cm-1; there was no evidence of the high frequency (1680-1690 cm-1) peak seen in in vitro Aβ fibrils and attributed to anti-parallel β-sheet. A significant elevation in phospholipids was found around dense-cored plaques in TgCRND8 mice ranging in age from 5 to 21 months. This was due to an increase in cellular membranes from dystrophic neurites and glial cells around the core, but could also contribute to Aβ aggregation through the interaction of newly secreted Aβ with phospholipids. In contrast, diffuse plaques were not associated with infrared detectable changes in protein secondary structure or relative concentrations of other tissue components. In addition, focally elevated deposits of creatine, a molecule with a crucial role in energy metabolism, were discovered in AD brain tissue with IR microspectroscopy. The creatine deposits may be a previously undiscovered disease marker. A second project was part of a larger Natural Sciences and Engineering Research Council Collaborative Health Research Project (NSERC-CHRP) to test the hypothesis that treatment with anti-oxidants, L-2-oxo-thiazolidine-4-carboxylate (OTC) and quercetin, following spinal surgery may reduce oxidative stress, inflammation, and scarring. The effect of OTC and quercetin on scar tissue formation was evaluated in rats that had undergone laminectomy. Synchrotron IR microspectroscopy data were collected on scar tissue from OTC, quercetin and saline (control) treated animals, sacrificed at 3 and 21 days post-surgery. Spectral differences could be correlated with the stages of wound healing.

The aim of this thesis was using surface-enhanced Raman scattering (SERS) microspectroscopy for the study of biomolecules and biological systems. The main probe molecule was cationic porphyrin H2TMPyP, however, other porphyrins, tryptophan and two lipids were also used. The sensitivity and reproducibility of several solid SERS substrates: (i) Au and Ag nanoparticles (NPs) immobilized via a bifunctional linker, (ii) AgNPs immobilized by drying, (iii) highly ordered Au and Ag film-over-nanosphere (FON) and (iv) Ag-coated insect wings were compared. On most of the solid substrates, the lowest detected H2TMPyP concentration was ~10-8 M. The highest sensitivity was provided by the dried drops of AgNPs/analyte mixture, where concentrations 1×10-10 M TMPyP, 1×10-5 M tryptophan, 2×10-7 M DSPC and 3×10-7 M DMTAP were detected. Nevertheless, the spectral reproducibility was decreased due to porphyrin metallation and perturbation of the lipid spectra in comparison to their Raman spectra from solution. The highest reproducibility was achieved by AuFON and Ag-coated insect wings. Finally, the AgNPs modified by PEG polymers were tested for intracellular application using HeLa cancer cells. Metallation of H2TMPyP served to probe the accessibility of PEG- AgNPs surface. The results proved that the accessibility...

Esta dissertação de mestrado aborda dois estudos que foram desenvolvidos utilizando a técnica de microscopia FTIR (micro-FTIR): a reatividade do grafeno funcionalizado e a eletroquímica in-situ com micro-FTIR para avaliação de reações redox. A reatividade e a distribuição de cargas em materiais 2D, mais especificamente em folhas individuais de grafeno, têm sido alvo de muita investigação na última década. No entanto, ainda não é conhecido como elas se apresentam em grafeno com grandes áreas, uma vez que a maioria dos estudos utilizam áreas muito pequenas (~μm2). Neste estudo, investigou-se experimentalmente como um eletrodo formado por uma única folha de grafeno se comporta quando sua estrutura é alterada por funcionalização covalente. Utilizando microespectroscopia na região do infravermelho, avaliou-se a funcionalização de grafeno com unidades de ácido benzoico no grafeno ancorados eletroquimicamente. O mapeamento químico mostrou que a distribuição espacial dessas unidades não ocorre uniformemente, ao invés disso, existem pontos específicos de ancoramento. Por fim, observou-se que a funcionalização ocorre mais intensamente na borda da folha de grafeno, alterando as propriedades óticas e eletroquímicas deste material, reduzindo o ganho ótico proporcionado pelos plásmons e aumentando a resistência de transferência heterogênea de elétrons. Para o segundo capítulo dessa dissertação, aplicou-se a microespectroscopia FTIR multiplex ao estudo da mudança química de um eletrodo de ouro modificado com azul da prússia (AP). Para isso, observou-se que uma etapa limitante era a confecção de um porta-amostra que reduzisse a camada de eletrólito ao mínimo de forma que a água não mais absorvesse a radiação de forma majoritária. Logo, foi possível o estudo vibracional de vários pontos da superfície do eletrodo, observando-se a influência do potencial aplicado, onde tem-se uma grande dependência dos sinais referentes ao estiramento C≡N do AP com a condição de potencial imprimida no eletrodo. / This Masters dissertation approaches two studies developed using the FTIR microspectroscopy technique (micro-FTIR): the activity of graphene functionalized and the in-situ electrochemistry with micro-FTIR for the evaluation of redox reactions. Reactivity and charge distribution in 2D materials, especially in single graphene sheets, have been the focus of extensive investigation during the last decade. However, there is still no knowledge on how large-area graphene behaves, since most of the studies utilize too small areas (~μm2). In this study, we aim to investigate experimentally how an electrode composed of only one single sheet of graphene behaves when its structure is changed by covalent functionalization. Using infrared microspectroscopy, the electrochemically induced covalent functionalization of graphene with benzoic acid unities was evaluated. The chemical mapping showed that the spatial distribution of these unities does not occur uniformly, instead, there are specific anchoring points. Lastly, it was observed that the functionalization occurs more intensely on the edges of the graphene sheet and that the covalent, affecting its optical and electrochemical properties, reducing the optical gain provided by the plasmons and increasing the resistance of heterogeneous electron transfer. In the second chapter of this dissertation, multiplex FTIR microspectroscopy was applied to the study of the chemical changes of a gold electrode modified with Prussian blue (PB). It was observed that the limiting step for this type of analysis was the building of a sample holder that reduces the electrolyte layer to the minimum in a way that water did not absorb the radiation in majority. Therefore, a vibrational study of several points of the electrode surface was possible evaluating the influence of the applied potential, where there is a dependence of the signals related to the C≡N stretching mode from PB on the potential condition applied to the electrode.

Made available in DSpace on 2014-06-11T19:27:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-09Bitstream added on 2014-06-13T20:27:58Z : No. of bitstreams: 1 cardoso_m_me_sjc.pdf: 644379 bytes, checksum: 1cc6342f5a099baa07f73649f18af2d4 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A técnica da espectroscopia infravermelha por transformada de Fourier (FT-IR) vem sendo largamente empregada como uma abordagem rápida e simples para a identificação de microrganismos, incluindo a do gênero Candida. A proposta deste estudo foi avaliar o emprego da microespectroscopia FT-IR na identificação e discriminação de 5 cepas clínicas de Candida albicans e 3 de Candida glabrata, as quais foram identificadas previamente por meio de métodos convencionais, e mais duas cepas ATCC de cada espécie. As amostras foram analisadas em triplicata, a partir de culturas independentes, por meio de filmes finos obtidos da suspensão em solução salina estéril a 0,85% da biomassa da colônia que ficou incubada por 24 horas em placas com ágar Saboraud Dextrose. Dez espectros com 32 varreduras foram obtidos na forma de ponto em 10 regiões selecionadas aleatoriamente nas amostras. A média foi obtida dos dez espectros de cada amostra para a análise estatística multivariada, dada pela análise de cluster. Além disso os espectros foram transformados em primeira derivada e três janelas espectrais foram determinadas (900-1250 cm-1, 1300-1600 cm-1, 2800-3000 cm-1). A representação dos resultados foi dada pela construção de um dendograma. Nesse, foi possível separar em dois grupos distintos as duas espécies de Candida estudadas. Dessa forma, pode-se concluir que a microespectroscopia FT-IR foi capaz de identificar e discriminar cepas clínicas de Candida albicans e de Candida glabrata, sendo um método promissor para identificação de leveduras / The technique of infrared spectroscopy Fourier transform (FT-IR) has been widely used as a new approach for rapid identification and simple micro-organisms, including the genus Candida. The purpose of this study was to evaluate the use of FT-IR microspectroscopy for the identification and discrimination of 5 clinical strains of Candida albicans and Candida glabrata 3, which were previously identified by conventional methods, and two-standard strains of each species. The samples were analyzed in triplicate from independent cultures by means of thin films obtained from a suspension in sterile saline and 0.85% of the biomass of the colony that was incubated for 24 hours in Sabouraud dextrose agar plates. Ten spectra with 32 scans were obtained in 10 randomly selected regions in the samples.The average of ten spectra was obtained from each sample for the multivariate analysis, given by cluster analysis. In addition, three windows were determined spectral (900-1250 cm-1, 1300-1600 cm-1, 2800-3000 cm-1) and the spectra were transformed into first derivative. The representation of the results was given by the construction of a dendrogram. In this, we separated into two groups of two Candida species studied. Thus, one can conclude that the FT-IR microspectroscopy was able to identify and distinguish clinical isolates of Candida albicans and Candida glabrata is an important method for identification of yeasts

Orientador: Luciane Dias de Oliveira / Banca: Célia Regina Gonçalves e Silva / Banca: Antonio Olavo Cardoso Jorge / Resumo: A técnica da espectroscopia infravermelha por transformada de Fourier (FT-IR) vem sendo largamente empregada como uma abordagem rápida e simples para a identificação de microrganismos, incluindo a do gênero Candida. A proposta deste estudo foi avaliar o emprego da microespectroscopia FT-IR na identificação e discriminação de 5 cepas clínicas de Candida albicans e 3 de Candida glabrata, as quais foram identificadas previamente por meio de métodos convencionais, e mais duas cepas ATCC de cada espécie. As amostras foram analisadas em triplicata, a partir de culturas independentes, por meio de filmes finos obtidos da suspensão em solução salina estéril a 0,85% da biomassa da colônia que ficou incubada por 24 horas em placas com ágar Saboraud Dextrose. Dez espectros com 32 varreduras foram obtidos na forma de ponto em 10 regiões selecionadas aleatoriamente nas amostras. A média foi obtida dos dez espectros de cada amostra para a análise estatística multivariada, dada pela análise de cluster. Além disso os espectros foram transformados em primeira derivada e três janelas espectrais foram determinadas (900-1250 cm-1, 1300-1600 cm-1, 2800-3000 cm-1). A representação dos resultados foi dada pela construção de um dendograma. Nesse, foi possível separar em dois grupos distintos as duas espécies de Candida estudadas. Dessa forma, pode-se concluir que a microespectroscopia FT-IR foi capaz de identificar e discriminar cepas clínicas de Candida albicans e de Candida glabrata, sendo um método promissor para identificação de leveduras / Abstract: The technique of infrared spectroscopy Fourier transform (FT-IR) has been widely used as a new approach for rapid identification and simple micro-organisms, including the genus Candida. The purpose of this study was to evaluate the use of FT-IR microspectroscopy for the identification and discrimination of 5 clinical strains of Candida albicans and Candida glabrata 3, which were previously identified by conventional methods, and two-standard strains of each species. The samples were analyzed in triplicate from independent cultures by means of thin films obtained from a suspension in sterile saline and 0.85% of the biomass of the colony that was incubated for 24 hours in Sabouraud dextrose agar plates. Ten spectra with 32 scans were obtained in 10 randomly selected regions in the samples.The average of ten spectra was obtained from each sample for the multivariate analysis, given by cluster analysis. In addition, three windows were determined spectral (900-1250 cm-1, 1300-1600 cm-1, 2800-3000 cm-1) and the spectra were transformed into first derivative. The representation of the results was given by the construction of a dendrogram. In this, we separated into two groups of two Candida species studied. Thus, one can conclude that the FT-IR microspectroscopy was able to identify and distinguish clinical isolates of Candida albicans and Candida glabrata is an important method for identification of yeasts / Mestre

Master of Science / Department of Grain Science and Industry / David L. Wetzel / Fourier transform infrared microspectroscopy was used to determine protein secondary structure in hard wheat breeding lines in situ, providing a molecular means to rank endosperm hardness for the selection of wheat cultivars for a specific end-use. Mapping with a single masked spot size diameter of 4.5 [Mu]m or confocal 5 [Mu]μm on beamlines U10B and U2B, respectively, produced spectra from the subaleurone layer within each wheat kernel using the high spatial resolution available with synchrotron infrared microspectroscopy. This procedure was used for the first four crop years. A focal plane array instrument was adapted for use for the remaining two crop years with a slight reduction of spatial resolution. Deconvolution and curve fitting were applied to the amide I region of spectra selected from the interstitial protein between the starch granules, and the relative amount of [Alpha]-helix to other protein secondary structures was revealed. Over six crop years, the [Alpha]-helix to [Beta]-sheet ratio of experimental wheat varieties were compared to those of released varieties in 143 mapping experiments. The highest measurable value was 2.50 while the lowest was 1.11, a range consistent with hard wheat secondary structure determination found in previous studies (13, 16). The determination of protein secondary structure provides a means of ranking experimental breeding lines for selection in specific end-use applications. FT-IR microspectroscopic imaging was used to develop a method, using myoglobin as the model protein, to study the effects of thermal processing to 100 [degrees]C on protein secondary structure. Films cast onto highly polished stainless steel plates allowed the study of the exact same film before and after heating. Analyzing the shift in the amide I peak maxima of reflection absorption spectra for 280 pixels from myoglobin films revealed the depletion of [Alpha]-helix at the expense of other protein secondary structures. Deconvolution and curve fitting techniques were applied to the amide I region of each spectral average to model protein secondary structure components found within the region. The method developed was applied to another animal source, gelatin, and a plant source, wheat gluten.

Raman microspectroscopy enables one to acquire spectra of Raman scattering with a spatial resolution in the order of a few μm3 and thus to study the natural composition of biological objects such as tissues, single cells and cellular organelles in a non-invasive way. In this work, we used Raman microspectroscopy to investigate vacuoles of the opportunistic human yeast pathogen Candida albicans. Large sets of Raman spectra of vacuoles were collected based on different cultivation protocols. The sets of the spectra were evaluated using the multivariate statistical method of singular value decomposition. Based on the spectral analysis, we characterized the chemical composition of the vacuoles. We found out that the vacuoles of cells cultured differently or in different media vary particularly in the concentration of polyphosphate, represented in the spectra by the peak near 1155 cm-1 . Interestingly, the wavenumber position of the polyphosphate peak may also be shifted by several cm-1 . We studied these shifts in vitro with sodium hexametaphosphate as a model of vacuolar polyphosphate. Based on these experiments, we suggest that the peak position is significantly influenced by the concentration of divalent cations.

A fundamental understanding of bio-interfaces will facilitate improvement in the design and application of biomaterials that can beneficially interact with biological objects such as nucleic acids, molecules, bacteria, and mammalian cells. Currently, there exist analytical instruments to investigate material properties and report information on electrical, chemical, physical, and mechanical natures of biomaterials and biological samples. The overall goal of this research was to utilize advanced spectroscopy techniques coupled with data mining to elucidate specific characteristic properties for biological objects and how these properties imply interaction with environmental biomaterials. My studies of interfacial electron transfer (ET) of DNA-modified gold electrodes aided in understanding that DNA surface density is related to the step-wise order of which a self-assembled monolayer is created on a gold substrate. Further surface modification plays a role in surface conductivity, and I found that electro-oxidation of the DNA involved the oxidation of guanine and adenine nucleotides. Scanning tunneling microscopy (STM) was used to create topography and current images of the SAM surfaces. I also used Raman microspectroscopy (RM) to obtain spectra and spectral maps of DNA-modified gold surfaces. For studies of bacteria, atomic force microscopy (AFM) and scanning electron microscopy (SEM) images showed similar morphological features of Gram-positive and Gram-negative bacteria. Direct classical least squares (DCLS) analysis aided to distinguish co-cultured strains. Fourier transform infrared (FTIR) spectroscopy proved insightful for characteristic bands for Gram-positive bacteria and a combined AFM/RM image revealed a relationship between culture height/density and peak Raman intensity. In our mammalian cell studies we focused on human lung adenocarcinoma epithelial cells (A549), metastatic human breast carcinoma cells MDA-MB-435 (435), and non-metastatic MDA-MB-435/BRMS1 (435/BRMS1). RM revealed similarities between metastatic 435 and non-metastatic 435/BRMS1 cells compared to epithelial A549 cells. AFM showed increases in biomechanical properties for 435/BRMS1 in the areas of cell adhesion, cell spring constant, and Young’s modulus. Fluorescent staining illustrates F-actin rearrangement for 435 and 435/BRMS1. Electric cell-substrate impedance sensing (ECIS) revealed that 435 cells adhere tightly to substrata and migrate rapidly compared with 435/BRMS1. For ECIS, ≤10-fold diesel exhaust particles (DEP) concentration exposure caused clastogenic DNA degradation whereas ≥25-fold DEP exposure caused cytotoxic results. Resveratrol (RES) at 10 μM showed minimal to mild protection against DEP before and after exposure and aided in improving injury recovery.

No description available.

No description available.