Single-molecule spectroscopy has provided a means to uncover pathways and heterogeneities that were previously hidden beneath the ensemble average. Such heterogeneity, however, is often obscured by the artifacts of experimental noise and the occurrence of undesired processes within the experimental medium. This has subsequently caused in the need for new analytical methodologies. It is particularly important that objectivity be maintained in the development of new analytical methodology so that bias is not introduced and the results improperly characterized. The research presented herein identifies two such sources of experimental uncertainty, and constructs objective approaches to reduce their effects in the experimental results. The first, photoblinking, arises from the occupation of dark electronic states within the probe molecule, resulting in experimental data that is distorted by its contribution. A method based in Bayesian inference is developed, and is found to nearly eliminate photoblinks from the experimental data while minimally affecting the remaining data and maintaining objectivity. The second source of uncertainty is electronic shot-noise, which arises as a result of Poissonian photon collection. A method based in wavelet decomposition is constructed and applied to simulated and experimental data. It is iii found that, while making only one assumption, that photon collection is indeed a Poisson process, up to 75% of the shot-noise contribution may be removed from the experimental signal by the wavelet-based procedure. Lastly, in an effort to connect model-based approaches such as molecular dynamics simulation to model-free approaches that rely solely on the experimental data, a coarse-grained molecular model of a molecular ionic fluorophore diffusing within an electrostatically charged polymer brush is constructed and characterized. It is found that, while the characteristics of the coarse-grained simulation compare well with atomistic simulations, the model is lacking in its representation of the electrostatically-driven behavior of the experimental system.

Fluorescence correlation spectroscopy has become a standard technique for modern biophysics and single molecule spectroscopy research. Here is presented a novel widefield extension of the established single-point technique. Flow in microfluidic devices was used as a model system for microscopic motion and through widefield fluorescence correlation spectroscopy flow profiles were mapped in three dimensions. The technique presented is shown to be more tolerant to low signal strength, allowing image data with signal-to-noise values as low as 1.4 to produce accurate flow maps as well as utilizing dye-labeled single antibodies as flow tracers. With proper instrumentation flows along the axial direction can also be measured. Widefield fluorescence correlation spectroscopy has also been utilized to produce super-resolution confocal microscopic images relying on the single-molecule microsecond blinking dynamics of fluorescent silver clusters. A method for fluorescence modulation signal extraction as well as synthesis of several novel noble metal fluorophores is also presented.

The progress of miniaturisation towards the nanoscopic scale in science and technology has also influenced the biosciences. This is particularly important, since proteins, as the smallest functional units of life, exhibit a spectacular wealth of functionalities, enabling them to fulfil complex tasks in cells and organisms. For this reason, they are often termed molecular or cellular “machines”. To be able to investigate and better understand these fascinating molecules in their native environment, new analytical methods must be developed, with appropriately high sensitivity and spatial and temporal resolution. We describe one very promising technique based on fluorescence spectroscopy, which allows a quantitative analysis of protein- protein interactions in the live cell. / Die zunehmende Miniaturisierung bis hin zum nanoskopischen Maßstab in vielen technischen Disziplinen hat auch die Lebenswissenschaften ergriffen. Dies ist insofern von großer Bedeutung, als die Proteine als kleinste funktionale Einheiten des Lebens trotz ihrer winzigen Abmessungen eine faszinierende Komplexität aufweisen, die es ihnen erlauben, hoch differenzierte und spezialisierte Aufgaben in der Zelle und im Organismus zu übernehmen. Aus diesem Grund werden sie in der modernen Biologie auch als molekulare oder zelluläre „Maschinen“ bezeichnet. Um diese kleinen Wunderwerke zu studieren und ihre Funktionsweise in ihrer natürlichen Umgebung zu analysieren, bedarf es innovativer Technologien, die es erlauben, mit maximaler räumlicher und zeitlicher Auflösung auch einzelne Moleküle in der lebenden Zelle sichtbar zu machen und zu verfolgen. Im Folgenden wird eine von uns entwickelte fluoreszenzspektroskopische Methode vorgestellt, mit deren Hilfe die komplizierten Interaktionen zwischen Proteinen in der lebenden Zelle aufgeklärt werden können.

This study combines noninvasive hybrid diffuse optical spectroscopies [near-infrared spectroscopy (NIRS) and diffuse correlation spectroscopy (DCS)] with occlusive calibration for continuous measurement of absolute blood flow (BF), tissue blood oxygenation (StO2), and oxygen consumption rate (VO2) in exercising skeletal muscle. Subjects performed rhythmic dynamic handgrip exercise, while an optical probe connected to a hybrid NIRS/DCS flow-oximeter directly monitored oxy-, deoxy-, and total hemoglobin concentrations ([HbO2], [Hb], and [tHb]), StO2, relative BF (rBF), and relative VO2 (rVO2) in the forearm flexor muscles. Absolute baseline BF and VO2 were obtained through venous and arterial occlusions, respectively, and used to calibrate continuous relative parameters. Previously known problems with muscle fiber motion artifact in optical measurements were mitigated with a novel dynamometer-based gating algorithm. Nine healthy young subjects were measured and results validated against previous literature findings. Ten older subjects with fibromyalgia and thirteen age-matched healthy controls were then successfully measured to observe differences in hemodynamic and metabolic response to exercise. This study demonstrates a novel application of NIRS/DCS technology to simultaneously evaluate quantitative hemodynamic and metabolic parameters in exercising skeletal muscle. This method has broad application to research and clinical assessment of disease (e.g. peripheral vascular disease, fibromyalgia), treatment evaluation, and sports medicine.

The diffusible soluble oligomeric amyloid β-peptide (Aβ) has been identified as a toxic agent in Alzheimer’s disease that can cause synaptic dysfunction and memory loss, indicating its role as potential therapeutic targets for AD treatment. Recently an oligomer-specific sandwich biotin-avidin interaction based assay identified the Aβ oligomer dissociation potency of a series of dihydroxybenzoic acid (DHBA) isomers. Because the sandwich assay is an ensemble method providing limited size information, fluorescence correlation spectroscopy (FCS) was employed to provide single molecule resolution of the disassembly mechanism. Using FCS coupled with atomic force microscopy, we investigated the size distribution of fluorescein labeled synthetic Aβ oligomers at physiological concentrations, and monitored in real time the change of size and mole fraction of oligomers in the presence of dissociating agents or conditions. The higher-order dissociation process caused by DHBA isomers produced no transient oligomeric intermediates, a desirable feature for an anti-oligomer therapeutic. Urea and guanidine hydrochloride, in contrast, produced a linear dissociation with a progressive decrease of size and mole fraction of oligomers. FCS allows the facile distinction of small molecule Aβ oligomer dissociators that do not produce stable potentially toxic oligomeric Aβ intermediates.

This research investigates various applications of diffuse correlation spectroscopy (DCS) on in-vivo human muscle tissue, both at rest and during dynamic exercise. Previously suspected muscle tissue relative blood flow (rBF) baseline shift during extended measurement with DCS and DCS-Near infrared spectroscopy (NIRS) hybrid optical systems are verified, quantified, and resolved by redesign of optical probe and alteration in optical probe attachment methodology during 40 minute supine bed rest baseline measurements. We then translate previously developed occlusion techniques, whereby rBF and relative oxygen consumption rV̇O2 are calibrated to initial resting absolute values by use of a venous occlusion (VO) and arterial occlusion (AO) protocol, respectively, to the lower leg (gastrocnemius) and these blood flows are cross validated at rest by strain gauge venous plethysmography (SGVP). Methods used to continuously observe 0.5Hz, 30% maximum voluntary isometric contraction (MVIC) plantar flexion exercise via dynamometer are adapted for our hybrid DCS-Imagent diffuse optical flow-oximeter in the medial gastrocnemius. We obtain healthy control muscle tissue hemodynamic profiles for key parameters BF, V̇O2, oxygen saturation (StO2), deoxyhemoglobin, oxyhemoglobin, and total hemoglobin concentrations ([Hb], [HbO2], and THC respectively), as well as systemic mean arterial pressure (MAP) and pulse rate (PR), at rest, during VO/AO, during dynamic exercise and during 15 minute recovery periods. Next, we began investigation of muscle tissue hemodynamic disease states by performing a feasibility pilot study using limited numbers of controls and peripheral arterial disease (PAD) patients using the translated methods/techniques to determine the ability of our technology to assess differences in these populations.

The fate and behaviour of hydrophobic organic compounds (e.g. pesticides) in soils are largely controlled by sorption processes. Recent findings suggest that the chemical properties of soil organic carbon (OC) significantly control the extent of sorption of such compounds in soil systems. However, currently there is no practical tool to integrate the effects of OC chemistry into sorption predictions. Therefore, the K [subscript]oc model, which relies on the soil OC content (foc), is used for predicting soil sorption coefficients (K[subscript]d) of pesticides. The K[subscript]oc model can be expressed as K[subscript]d = K[subscript]oc × foc, where K[subscript]oc is the OC-normalized sorption coefficient for the compound. Hence, there is a need for a prediction tool that can effectively capture the role of both the chemical structural variation of OC as well as foc in the prediction approach. Infrared (IR) spectroscopy offers a potential alternative to the K[subscript]oc approach because IR spectra contain information on the amount and nature of both organic and mineral soil components. The potential of mid-infrared (MIR) spectroscopy for predicting K[subscript]d values of a moderately hydrophobic pesticide, diuron, was investigated. A calibration set of 101 surface soils from South Australia was characterized for reference sorption data (K[subscript]d and K[subscript]oc) and foc as well as IR spectra. Partial least squares (PLS) regression was employed to harness the apparent complexity of IR spectra by reducing the dimensionality of the data. The MIR-PLS model was developed and validated by dividing the initial data set into corresponding calibration and validation sets. The developed model showed promising performance in predicting K[subscript]d values for diuron and proved to be a more efficacious than the K[subscript]oc model. The significant statistical superiority of the MIR-PLS model over the K[subscript]oc model was caused by some calcareous soils which were outliers for the K[subscript]oc model. Apart from these samples, the performance of the two compared models was essentially similar. The existence of carbonate peaks in the MIR-PLS loadings of the MIR based model suggested that carbonate minerals may interfere or affect the sorption. This requires further investigation. Some other concurrent studies suggested excellent quality of prediction of soil properties by NIR spectroscopy when applied to homogenous samples. Next, therefore, the performance of visible near-infrared (VNIR) and MIR spectroscopy was thoroughly compared for predicting both foc and diuron K[subscript]d values in soils. Some eleven calcareous soils were added to the initial calibration set for an attempt to further investigate the effect of carbonate minerals on sorption. MIR spectroscopy was clearly a more accurate predictor of foc and K[subscript]d in soils than VNIR spectroscopy. Close inspection of spectra showed that MIR spectra contain more relevant and straightforward information regarding the chemistry of OC and minerals than VNIR and thus useful in modelling sorption and OC content. Moreover, MIR spectroscopy provided a better (though still not great) estimation of sorption in calcareous soils than either VNIR spectroscopy or the K[subscript]oc model. Separate research is recommended to fully explore the unusual sorption behaviour of diuron in calcareous soils. In the last experiment, two dimensional (2D) nuclear magnetic resonance/infrared heterospectral correlation analyses revealed that MIR spectra contain specific and clear signals related to most of the major NMR-derived carbon types whereas NIR spectra contain only a few broad and overlapped peaks weakly associated with aliphatic carbons. 2D heterospectral correlation analysis facilitated accurate band assignments in the MIR and NIR spectra to the NMR-derived carbon types in isolated SOM. In conclusion, the greatest advantage of the MIR-PLS model is the direct estimation of Kd based on integrated properties of organic and mineral components. In addition, MIR spectroscopy is being used increasingly in predicting various soil properties including foc, and therefore, its simultaneous use for K[subscript]d estimation is a resource-effective and attractive practice. Moreover, it has the advantage of being fast and inexpensive with a high repeatability, and unlike the K[subscript]oc approach, MIR-PLS shows a better potential for extrapolating applications in data-poor regions. Where available, MIR spectroscopy is highly recommended over NIR spectroscopy. 2D correlation spectroscopy showed promising potential for providing rich insight and clarification into the thorough study of soil IR spectra. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1415416 / Thesis (Ph.D.) - University of Adelaide, School of Earth and Environmental Sciences, 2009

The fate and behaviour of hydrophobic organic compounds (e.g. pesticides) in soils are largely controlled by sorption processes. Recent findings suggest that the chemical properties of soil organic carbon (OC) significantly control the extent of sorption of such compounds in soil systems. However, currently there is no practical tool to integrate the effects of OC chemistry into sorption predictions. Therefore, the K [subscript]oc model, which relies on the soil OC content (foc), is used for predicting soil sorption coefficients (K[subscript]d) of pesticides. The K[subscript]oc model can be expressed as K[subscript]d = K[subscript]oc × foc, where K[subscript]oc is the OC-normalized sorption coefficient for the compound. Hence, there is a need for a prediction tool that can effectively capture the role of both the chemical structural variation of OC as well as foc in the prediction approach. Infrared (IR) spectroscopy offers a potential alternative to the K[subscript]oc approach because IR spectra contain information on the amount and nature of both organic and mineral soil components. The potential of mid-infrared (MIR) spectroscopy for predicting K[subscript]d values of a moderately hydrophobic pesticide, diuron, was investigated. A calibration set of 101 surface soils from South Australia was characterized for reference sorption data (K[subscript]d and K[subscript]oc) and foc as well as IR spectra. Partial least squares (PLS) regression was employed to harness the apparent complexity of IR spectra by reducing the dimensionality of the data. The MIR-PLS model was developed and validated by dividing the initial data set into corresponding calibration and validation sets. The developed model showed promising performance in predicting K[subscript]d values for diuron and proved to be a more efficacious than the K[subscript]oc model. The significant statistical superiority of the MIR-PLS model over the K[subscript]oc model was caused by some calcareous soils which were outliers for the K[subscript]oc model. Apart from these samples, the performance of the two compared models was essentially similar. The existence of carbonate peaks in the MIR-PLS loadings of the MIR based model suggested that carbonate minerals may interfere or affect the sorption. This requires further investigation. Some other concurrent studies suggested excellent quality of prediction of soil properties by NIR spectroscopy when applied to homogenous samples. Next, therefore, the performance of visible near-infrared (VNIR) and MIR spectroscopy was thoroughly compared for predicting both foc and diuron K[subscript]d values in soils. Some eleven calcareous soils were added to the initial calibration set for an attempt to further investigate the effect of carbonate minerals on sorption. MIR spectroscopy was clearly a more accurate predictor of foc and K[subscript]d in soils than VNIR spectroscopy. Close inspection of spectra showed that MIR spectra contain more relevant and straightforward information regarding the chemistry of OC and minerals than VNIR and thus useful in modelling sorption and OC content. Moreover, MIR spectroscopy provided a better (though still not great) estimation of sorption in calcareous soils than either VNIR spectroscopy or the K[subscript]oc model. Separate research is recommended to fully explore the unusual sorption behaviour of diuron in calcareous soils. In the last experiment, two dimensional (2D) nuclear magnetic resonance/infrared heterospectral correlation analyses revealed that MIR spectra contain specific and clear signals related to most of the major NMR-derived carbon types whereas NIR spectra contain only a few broad and overlapped peaks weakly associated with aliphatic carbons. 2D heterospectral correlation analysis facilitated accurate band assignments in the MIR and NIR spectra to the NMR-derived carbon types in isolated SOM. In conclusion, the greatest advantage of the MIR-PLS model is the direct estimation of Kd based on integrated properties of organic and mineral components. In addition, MIR spectroscopy is being used increasingly in predicting various soil properties including foc, and therefore, its simultaneous use for K[subscript]d estimation is a resource-effective and attractive practice. Moreover, it has the advantage of being fast and inexpensive with a high repeatability, and unlike the K[subscript]oc approach, MIR-PLS shows a better potential for extrapolating applications in data-poor regions. Where available, MIR spectroscopy is highly recommended over NIR spectroscopy. 2D correlation spectroscopy showed promising potential for providing rich insight and clarification into the thorough study of soil IR spectra. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1415416 / Thesis (Ph.D.) - University of Adelaide, School of Earth and Environmental Sciences, 2009

LIMA, Kellen Cristina Vilhena. Qualidade de gasolinas automotivas através de Espectroscopia Vibracional FT-Raman, combinada com correlação 2D generalizada. 2005. 305 f. Tese (Doutorado em Física) - Programa de Pós-Graduação em Física, Departamento de Física, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2005. / Submitted by Edvander Pires (edvanderpires@gmail.com) on 2015-05-25T22:26:07Z No. of bitstreams: 1 2005_tese_kcvlima.pdf: 10475763 bytes, checksum: e618c6deda94d946c15be6e420912ad1 (MD5) / Approved for entry into archive by Edvander Pires(edvanderpires@gmail.com) on 2015-05-27T18:52:53Z (GMT) No. of bitstreams: 1 2005_tese_kcvlima.pdf: 10475763 bytes, checksum: e618c6deda94d946c15be6e420912ad1 (MD5) / Made available in DSpace on 2015-05-27T18:52:53Z (GMT). No. of bitstreams: 1 2005_tese_kcvlima.pdf: 10475763 bytes, checksum: e618c6deda94d946c15be6e420912ad1 (MD5) Previous issue date: 2005 / It is reported the use of Generalized Two-Dimensional FT-Raman Correlation Spectroscopy (2DCOS-FT-Raman) method to monitor the quality of automotive gasoline in the gas stations. It is presented a procedure to identify the adulteration of automotive gasoline by the illicit addition of ethanol, methanol, aromatic and paraphinic compounds through the analysis of the Raman spectra obtained for different compositions of adulterants added to gasoline “A” type and to gasoline “A” type plus ethanol samples. It is discussed the data preconditioning step, the application of 2DCOS method, and the calibration curves that correlates the integrated intensities of the Raman bands of a given adulterant with its percentage in a given gasoline sample. It shown the protocol for using the 2DCOS-FT-Raman method to verify the adulteration in a given gasoline sample compared with a specific standard. This work emphasizes the flexibility and viability of using this method to have a specific standard for each gasoline distribution firm and also for each new gasoline derived from the refinery. / Relata a utilização da espectroscopia FT-Raman combinada com Correlação 2D Generalizada como método alternativo para a realização do controle de qualidade de Gasolinas automotivas diretamente nos postos de combustível. Apresenta os procedimentos para identificar a adulteração da Gasolina automotiva pela adição irregular de Etanol, Metanol e compostos aromáticos e parafínicos, através da análise dos espectros Raman obtidos para os diferentes adulterantes separadamente, misturados à Gasolina A e misturados à Gasolina A e ao Etanol. Descreve os procedimentos de pré-tratamento sobre os espectros Raman, a aplicação do método de Correlação 2D Generalizada aos sistemas estudados e a determinação das curvas de calibração que relacionam as intensidades integradas das bandas Raman com o teor de adulterante na Gasolina. Apresenta a praticidade de aplicação do método, que possibilita verificar a adulteração da Gasolina automotiva através da comparação entre o espectro Raman da amostra cuja conformidade se deseja verificar com o espectro da amostra padrão. Enfatiza a flexibilidade e a viabilidade de se ter um padrão específico para cada distribuidora, ou até mesmo para cada nova produção de Gasolina da refinaria.

Ce travail présente une nouvelle méthode de télédétection à distance de la concentration d’un gaz trace dans l’atmosphère basée sur le couplage de la méthode spectroscopique de corrélation optique (Optical Correlation Spectroscopy, OCS) et la technologie de télédétection lidar dans lequel une source laser de large étendue spectrale est considérée. Les premiers travaux furent consacrés au développement d’un nouveau formalisme pour estimer la concentration d’un gaz trace dans l’atmosphère à partir des signaux OCS-lidar. Pour évaluer les performances d’une telle méthode, un modèle numérique simulant des signaux OCS-lidar pour la mesure de la concentration de méthane et de vapeur d’eau a été réalisé. L’influence de la pression et la température sur les propriétés spectroscopiques de ces gaz sur les mesures de concentration a également été étudiée. En plus de ce travail théorique, la première démonstration expérimentale de l’OCS-lidar est présentée en utilisant un lidar basé sur une source laser femtoseconde. Les mesures OCS-lidar ont été réalisées au moyen d’un nouveau système expérimental entièrement élaboré et construit pendant cette thèse. Pour réaliser les mesures consacrées à la teneur en vapeur d’eau de l’atmosphère, la bande d’absorption de la molécule de H2O 4ν à 720 nm a été utilisée. Ainsi les résultats obtenus ont montré le potentiel de la méthode OCS5 lidar pour mesurer la concentration de vapeur d’eau. Par la suite, le développement de la méthodologie OCS-lidar dédiée à la mesure du méthane est présenté. La bande d’absorption 2ν3 à 1,66 μm est exploitée et les premières mesures des signaux lidar sont exposées. Ce travail se termine par la présentation des possibles perspectives d’évolution / In this thesis, a new active remote sensing methodology is proposed to evaluate the content of atmospheric trace gases. The new methodology is based on laser spectroscopy and consists in coupling a spectrally broadband lidar with optical correlation spectroscopy (OCS-lidar). As a first step, a new formalism has been developed to remotely evaluate the target gas concentration from the OCS-lidar signals. To evaluate the performance of this new methodology, a numerical model simulating OSC-lidar signals for methane and water vapor measurement has been developed. Moreover, the influence of the absorption spectroscopic line parameters, such as atmospheric pressure and temperature, on the retrieved gas mixing ratio has been assessed within the OCS-lidar methodology. In addition to this theoretical work, the first experimental demonstration of the OCS-lidar methodology has been performed using a femtosecond lidar system. The latter has been entirely designed, developed and implemented in the framework of this thesis. Results show the ability of the OCS-lidar methodology to monitor the water vapor using the 4ν 720 nm absorption band. Moreover, two different experimental configurations have been proposed, depending on whether the amplitude modulation is operated on the laser pulse or on the backscattered light, i.e. at the emission, with an active amplitude modulator or at the reception, with passive optical filters. The advances in developing the corresponding infrared OCS-lidar system for methane mixing ratios measurements are described and possible outlooks are given