<div><p><br></p></div><p>This study makes use of a Time-Resolved Raman Spectroscopy (TRRS) system developed in the Purdue Civil Engineering spectroscopy laboratory to advance technology critical to enable field deployment of Raman spectroscopic systems, with a primary focus on developing solutions to overcome two specific barriers to Raman analysis in the natural environment: (1) obtaining Raman spectra of chemical compounds at field-relevant concentrations, and (2) realizing economical spatial monitoring. To inform both streams of activity, this work first explores the role of component choice and apparatus design on Raman system output. A component-level Raman system transfer function is developed in terms of intensity, wavelength, and time which yields detailed insight into system performance that greatly exceeds traditional single “system factor” treatments of apparatus effects. The modelling frame provided by the transfer function is universally applicable in that it is inclusive of the majority of component choices that may be encountered in any open-path or closed-path Raman system, and is likely to be valuable in efforts to assess the performance benefits and limitations of system designs, modify or tailor apparatus layouts, facilitate experiment design, and compare results obtained on different systems. </p><p><br></p><p>The system characterization offered by the transfer function is then employed to develop a multi-photon counting algorithm realized through digital signal processing (DSP) which captures photon arrivals traditionally ignored in conventional counting methods. This approach increases acquired Raman intensity for any given analyte by using detector output voltage or a voltage-time product as an energy proxy – an approach that is likey broadly applicable to any spectroscopic techniques employing detectors that make use of the photoelectric effect. In experiments carried out on analytes (nitrate, isopropanol, and rhodamine 6G) in aqueous solutions, enhanced observations enabled by the multi-photon counting algorithm are shown to increase observed Raman intensities of low Raman-yield solutions 2.0-3.1-fold compared to single-threshold analysis, and also extend the upper observation limit of strong Raman-yield solutions that would traditionally saturate detectors using a binary photon counting scheme. Notably, the improved performance offered by the multi-photon counting algorithm is realized through comparison of multi-photon and conventional counting algorithms applied to the same data in a post-processing exercise, thus eliminating any effects of test-to-test variation on results, and highlighting the ability to employ the developed counting approach without modification of traditional systems.</p><p><br></p><p>Additional insights from the system transfer function are also used to inform exploration of a novel approach to enable spatial environmental monitoring via Raman spectroscopy by combining fiber optics, optical switch technology, and the Raman system prototype. Tests designed to evaluate the system configured as a multiplexed optically switched fiber optic network demonstrate the potential to deliver excitation and collect Raman scattering from different desired monitoring locations with a sole excitation source and a single detector over substantial distances. Using nitrate as an example compound of interest, it is demonstrated that the system has a detection limit of 5 ppm within approximately 1.5 meters, which increases to 15 ppm at 100 m, and 38 ppm at 200 m. Modelling informed using the developed system transfer function highlights that improving the prototype by eliminating fiber connectors and making use of commercially available visible-light optimized fiber can substantially extend the range of the system, offering a 15-ppm nitrate detection limit at 2100 m. As increases in laser power, testing time, and collection optic efficiency are all also straightforward and viable, the prototype demonstrates realistic potential to achieve field relevant detection sensitivity over great distance.</p><p><br></p><p>As a final demonstration of system potential, a set of experiments on aqueous nitrate solutions is performed to understand the influence of turbidity, fluorescence, optics size, and varied raw data integration lengths on Raman observations. Results demonstrate that cumulative advances in the TRRS system establish a new generation of Raman spectroscopic sensing amenable to long-term environmental monitoring over significant spatial extent in complex in-situ conditions. Specific advances made herein include enhanced power delivery and scattered light collection informed by the system transfer function, increases in sensitivity from multi-photon counting, and incorporation of optical multiplexing. Overall, the Time-Resolved Raman Spectroscopic System (TRRS) now offers a set of capabilities that bring in-field deployment within practical reach.</p>

Ein Ziel der Arbeit war die Entwicklung eines Verfahrens zur 3D-Positionierung auf Basis elektrooptischer Pseudostreckenmessung. Ein zweites Ziel war die Entwicklung eines Reflexgoniometers zur zweiachsigen Winkelmessung. Im Rahmen der Arbeit wurden die Grundlagen zur instrumentellen Umsetzung beider Verfahren erarbeitet, die Genauigkeitspotentiale ermittelt und mögliche Anwendungen für die einzelnen Schlüsseltechnologien und deren Kombination abgeleitet. In einer Prototyp-Entwicklung wurden Vorschläge für die wesentlichen Funktionselemente des räumlichen Weg- bzw. Winkelmesssystems gemacht. Hierzu gehören das kardanisch aufgehängte Etalon, die temperaturstabilisierte Laseroptik und die temperaturkalibrierte CCD der Winkelmesseinheit (Reflexgoniometer), die Systeme zur Erzeugung und Detektion ultrakurzer Laserpulse, eine elastische Optik, Möglichkeiten zur Formung des transversalen Strahlprofils, das TCSPC-System und die zur Auswertung und Kalibrierung notwendigen Algorithmen, wie z.B. die Autokollimation eines Lasers. (&amp;quot;Veröffentlicht von der Deutschen Geodätischen Kommission Reihe C (Dissertationen) unter der Nummer C 614 (München 2008; ISBN 3 7696 5053 0; 2; 144 S.&amp;quot;) / One goal of the thesis was the development of a method for three-dimensional positioning based on electro-optical measurement of pseudo ranges. Another goal was the development of a reflex goniometer for biaxial angle measurement. Within the scope of this thesis the basics for the instrumental realisation of both methods were developed, the accuracy potentials were determined and possible applications for the separate key technologies and their combination were deduced. In a prototype development proposals were made for the main functional elements of the spatial distance and angle measurement systems. These include the gimbal mounted etalon, the temperature stabilised laser optics and the temperature calibrated CCD of the angle measurement device (reflex goniometer), the systems for creation and detection of ultrashort laser pulses, an elastic optical device, possibilities of transversal beam shaping, the TCSPC system and the algorithms which are necessary for analysis and calibration, e.g. the autocollimation of a laser.

<p>We have studied fundamental aspects of time-resolved two-photon excited fluorescence depolarisation. The thesis presents experimental as well as theoretical progress. We show that a multi-photon induced instrumental response function obtained from a suspension of gold nanoparticles is appropriate for the analysis of two-photon excited fluorescence decays obtained using time-correlated single photon counting detection. Theoretical expressions have been derived for the fluorescence anisotropy decay obtained upon two-photon excitation of various molecular systems in liquid solutions: a) an anisotropic rigid rotor that undergoes rotational diffusion in the presence of ultrafast unresolved restricted reorientations, e.g. librations. b) a molecular group covalently attached to a stationary macromolecule, and undergoing local reorientation in a uniaxial ordering potential. A new approach to the analysis of two-photon excited fluorescence depolarisation experiments was developed, which combines data obtained by using linearly and circularly polarised excitation light, in a global manner. In the analysis, knowledge about unresolved reorientations was obtained from one-photon excitation studies of the corresponding systems. By means of this procedure it has been possible to obtain quantitative information about the molecular two-photon absorption tensor for perylene and two of its derivatives. Thereby the symmetry of the final excited and intermediate vibronic states could be assigned. The analysis reveals that the two-photon transition studied with the 800 nm laser exhibits mixed character. An important finding from the experiments was that the two-photon absorption tensor appears to be solvent dependent. Furthermore, the thesis presents the first theoretical treatment of two-photon excited donor-donor energy migration in the presence of molecular reorientation and which applies the extended Förster theory. Explicit expressions for molecules that belong to the point groups D_2h, D₂ and C_2v are given. Preliminary experiments are finally also reported on a two-photon excited donor-donor energy migration system consisting of a bisanthryl-bisteroid. </p>

This dissertation attempts to understand how energy transfer in a molecular wire and a spherical organic assembly are affected by molecular structure. The molecular wire is a DNA-hybrid structure composed of a strand of thymine bases appended by a cyanine dye. Hydrogen bonded to each base is a naphthalene-derivative molecule. Using time-integrated photoluminescence and time-correlated single photon counting measurements, energy transfer from the naphthalene donors to the cyanine acceptors was confirmed, and its dependence on temperature and DNA-template length investigated. Donor-thymine bonding was disrupted at temperatures above about 25 degrees Celcius resulting in poor donor template decoration and low rates of energy transfer. Increasing numbers of donors attach to the scaffold, forming an orderly array, as the template length increases due to the stabilising effects of the donor-donor pi-stacking interactions. Conversely, modelled energy transfer rates fall as the scaffold length increases because of the longer donor-acceptor distances involved. Therefore, the energy transfer rate was greatest for a template built from 30 thymines. The spherical organic assemblies (nanoparticles) are formed by fast injection of a small volume of molecularly dissolved fluorene-derivative amphiphilic molecules into a polar solvent. The amphiphilic molecules contained either a naphthalene (donor) or a benzothiadiazole (acceptor) core. The donor-acceptor mixed nanoparticles resemble an amorphous polymer film and were modelled as such using the Foerster resonance energy transfer theory. The Foerster radii extracted from the measurements depends intricately on the donor-acceptor spectral overlap and distance. The latter effect was controlled by the stacking interactions between the molecules. Altering the morphology of the structural units is the key to optimising energy transfer in molecular structures. To achieve efficient organic molecule-based devices, the importance of this property needs to be fully appreciated and effectively exploited.

Ma thèse de doctorat s’inscrit à l’interface entre la physique expérimentale et la biologie. Ce travail a été développé au sein de l’équipe imXgam du CPPM, qui a construit un prototype de micro-tomographie pour le suivi non-invasif du petit animal, équipé d’une caméra à pixels hybrides XPAD3 fonctionnant en mode comptage de rayons X. Le comptage de rayons X rendu possible par la technologie des pixels hybrides, permet de s’affranchir du bruit électronique et d’augmenter ainsi la détectabilité des tissus faiblement contrastés. Elle présente de plus la capacité d'appliquer à chaque pixel un seuil de détection en énergie permettant d’accéder à l’information spectrale des rayons X détectés et ouvre la voie au développement d’une méthode d’imagerie spectrale dite au K-edge, qui permet de différencier des agents de contraste particuliers. La caméra XPAD3 développée avec un capteur en Si présente une efficacité de détection qui limite son utilisation pour l’imagerie du vivant. Une caméra XPAD3 avec une meilleure efficacité au delà de 25 keV a été assemblée avec des capteurs en CdTe. Dans un premier temps, nous avons effectué une comparaison des caméras XPAD3/Si et XPAD3/CdTe en imagerie d’absorption standard et en imagerie au K-edge. Nous avons ensuite, en collaboration avec des biologistes de l’IBDM, assuré le suivi quantitatif et in vivo sur plusieurs mois, du développement de tumeurs hépatiques chez un modèle spécifique de souris et de l’efficacité d’un traitement ciblant les cellules tumorales. Enfin, nous avons développé un protocole d’acquisition spectrale à faible dose pour réaliser une tomographie spectrale in vivo d’un foie de souris en exploitant le K-edge du baryum. / My PhD thesis is at the interface between experimental physics and biology. This work has been developed within the imXgam team at CPPM, which has built a micro-computed tomography prototype for the non-invasive longitudinal monitoring of small animal, equipped with the XPAD3 hybrid pixel camera that operates in X-ray photon counting mode. X-ray photon counting that has been made possible by hybrid pixels, allows to free images from the electronic noise and thus to increase detectability of weakly contrasted tissues. Moreover, it provides the possibility to set an energy threshold for each pixel that allows to accessing spectral information on the detected X-rays and paving the way to the development of a spectral imaging modality also named K-edge imaging, which allows to differentiate selected contrast agents. Actually, the XPAD3 camera developed with a Si sensor presents a low detective efficiency that limits its use for biomedical imaging. A XPAD3 camera with a better efficiency above 25 keV has been assembled with high-Z CdTe sensors. Firstly, we have performed a comparison of XPAD3/Si and XPAD3/CdTe cameras for standard absorption CT and K-edge imaging. Then, in collaboration with a team of biologists from IBDM, we have carried out the quantitative and in vivo follow-up of hepatic tumour development in a specific mouse model over several months, and of the effectiveness of a treatment targeting these tumour cells. Finally, we have developed a protocol for low dose acquisition of spectral data to realize an in vivo spectral tomography of a mouse liver using the barium spectral signature.

La tomographie (CT) aux rayons X joue un rôle important dans l'imagerie non invasive depuis son introduction. Au cours des dernières années, de nombreuses avancées technologiques en tomographie par rayons X ont été observées, notamment la CT spectrale, qui utilise un détecteur à comptage de photons (PCD) pour discriminer les photons transmis correspondant à des bandes d'énergie sélectionnées afin d'obtenir une information spectrale. La CT spectrale permet de surmonter de nombreuses limitations des techniques précédentes et ouvre de nombreuses applications nouvelles, parmi lesquelles la décomposition quantitative des matériaux est le sujet le plus étudié. Un certain nombre de méthodes de décomposition des matériaux ont été rapportées et différents systèmes expérimentaux sont en cours de développement pour la CT spectrale. Selon le type de données sur lequel l'étape de décomposition fonctionne, nous avons les méthodes du domaine des projections (décomposition avant reconstruction) et les méthodes du domaine de l'image reconstruite (décomposition après reconstruction). La décomposition couramment utilisée est basée sur le critère des moindres carrés, nommée proj-LS et méthode ima-LS. Cependant, le problème inverse de la décomposition du matériau est généralement mal posé et les mesures du CT spectral aux rayons X souffrent de bruits de comptage de photons de Poisson. Le critère des moindres carrés peut conduire à un surajustement des données de mesure bruitées. Dans le présent travail, nous avons proposé un critère de moindre log-carré pour la méthode du domaine de projection afin de minimiser les erreurs sur le coefficient d'atténuation linéaire: méthode proj-LLS. De plus, pour réduire l'effet du bruit et lisser les images, nous avons proposé d'ajouter un terme de régularisation par patch pour pénaliser la somme des variations au carré dans chaque zone pour les décompositions des deux domaines, nommées proj-PR-LLS et ima -PR-LS méthode. Les performances des différentes méthodes ont été évaluées par des études de simulation avec des fantômes spécifiques pour différentes applications: (1) Application médicale: identification de l'iode et du calcium. Les résultats de la décomposition des méthodes proposées montrent que le calcium et l'iode peuvent être bien séparés et quantifiés par rapport aux tissus mous. (2) Application industrielle: tri des plastiques avec ou sans retardateur de flamme. Les résultats montrent que 3 types de matériaux ABS avec différents retardateurs de flamme peuvent être séparés lorsque l'épaisseur de l'échantillon est favorable. Enfin, nous avons simulé l'imagerie par CT spectrale avec un fantôme de PMMA rempli de solutions de Fe, Ca et K. Différents paramètres d'acquisition, c'est-à-dire le facteur d'exposition et le nombre de bandes d'énergie, ont été simulés pour étudier leur influence sur la performance de décomposition pour la détermination du fer. / X-ray computed tomography (X-ray CT) plays an important part in non-invasive imaging since its introduction. During the past few years, numerous technological advances in X-ray CT have been observed, including spectral CT, which uses photon counting detectors (PCDs) to discriminate transmitted photons corresponding to selected energy bins in order to obtain spectral information with one single acquisition. Spectral CT enables us to overcome many limitations of the conventional CT techniques and opens up many new application possibilities, among which quantitative material decomposition is the hottest topic. A number of material decomposition methods have been reported and different experimental systems are under development for spectral CT. According to the type of data on which the decomposition step operates, we have projection domain method (decomposition before reconstruction) and image domain method (decomposition after reconstruction). The commonly used decomposition is based on least square criterion, named proj-LS and ima-LS method. However, the inverse problem of material decomposition is usually ill-posed and the X-ray spectral CT measurements suffer from Poisson photon counting noise. The standard LS criterion can lead to overfitting to the noisy measurement data. In the present work, we have proposed a least log-squares criterion for projection domain method to minimize the errors on linear attenuation coefficient: proj-LLS method. Furthermore, to reduce the effect of noise and enforce smoothness, we have proposed to add a patchwise regularization term to penalize the sum of the square variations within each patch for both projection domain and image domain decomposition, named proj-PR-LLS and ima-PR-LS method. The performances of the different methods were evaluated by spectral CT simulation studies with specific phantoms for different applications: (1) Medical application: iodine and calcium identification. The decomposition results of the proposed methods show that calcium and iodine can be well separated and quantified from soft tissues. (2) Industrial application: ABS-flame retardants (FR) plastic sorting. Results show that 3 kinds of ABS materials with different flame retardants can be separated when the sample thickness is favorable. Meanwhile, we simulated spectral CT imaging with a PMMA phantom filled with Fe, Ca and K solutions. Different acquisition parameters, i.e. exposure factor and number of energy bins were simulated to investigate their influence on the performance of the proposed methods for iron determination.

<p>This project investigates a laser radar system. The system is based on the principles of time-correlated single photon counting, and by measuring the times-of-flight of reflected photons it can find range profiles and perform three-dimensional imaging of scenes. Because of the photon counting technique the resolution and precision that the system can achieve is very high compared to analog systems. These properties make the system interesting for many military applications. For example, the system can be used to interrogate non-cooperative targets at a safe distance in order to gather intelligence. However, signal processing is needed in order to extract the information from the data acquired by the system. This project focuses on the analysis of different signal processing methods.</p><p>The Wiener filter and the Richardson-Lucy algorithm are used to deconvolve the data acquired by the photon counting system. In order to find the positions of potential targets different approaches of non-linear least squares methods are tested, as well as a more unconventional method called ESPRIT. The methods are evaluated based on their ability to resolve two targets separated by some known distance and the accuracy with which they calculate the position of a single target, as well as their robustness to noise and their computational burden.</p><p>Results show that fitting a curve made of a linear combination of asymmetric super-Gaussians to the data by a method of non-linear least squares manages to accurately resolve targets separated by 1.75 cm, which is the best result of all the methods tested. The accuracy for finding the position of a single target is similar between the methods but ESPRIT has a much faster computation time.</p>

We have studied fundamental aspects of time-resolved two-photon excited fluorescence depolarisation. The thesis presents experimental as well as theoretical progress. We show that a multi-photon induced instrumental response function obtained from a suspension of gold nanoparticles is appropriate for the analysis of two-photon excited fluorescence decays obtained using time-correlated single photon counting detection. Theoretical expressions have been derived for the fluorescence anisotropy decay obtained upon two-photon excitation of various molecular systems in liquid solutions: a) an anisotropic rigid rotor that undergoes rotational diffusion in the presence of ultrafast unresolved restricted reorientations, e.g. librations. b) a molecular group covalently attached to a stationary macromolecule, and undergoing local reorientation in a uniaxial ordering potential. A new approach to the analysis of two-photon excited fluorescence depolarisation experiments was developed, which combines data obtained by using linearly and circularly polarised excitation light, in a global manner. In the analysis, knowledge about unresolved reorientations was obtained from one-photon excitation studies of the corresponding systems. By means of this procedure it has been possible to obtain quantitative information about the molecular two-photon absorption tensor for perylene and two of its derivatives. Thereby the symmetry of the final excited and intermediate vibronic states could be assigned. The analysis reveals that the two-photon transition studied with the 800 nm laser exhibits mixed character. An important finding from the experiments was that the two-photon absorption tensor appears to be solvent dependent. Furthermore, the thesis presents the first theoretical treatment of two-photon excited donor-donor energy migration in the presence of molecular reorientation and which applies the extended Förster theory. Explicit expressions for molecules that belong to the point groups D2h, D2 and C2v are given. Preliminary experiments are finally also reported on a two-photon excited donor-donor energy migration system consisting of a bisanthryl-bisteroid.

A kinetic model of electronic energy migration within pairs of photophysically non-identical fluorophores has been developed. The model applies to fluorescent groups that exhibit different photophysical and spectral properties when attached to different positions in a macromolecule. The energy migration within such asymmetric pairs is partially reversible, which leads to the case of partial donor-donor energy migration (PDDEM). The model of PDDEM is an extension of the recently developed donor-donor energy migration model (DDEM, F. Bergström et al, PNAS 96 (1999) 12477), and applies to quantitative measurements of energy migration rates and distances within macromolecules. One important distinction from the DDEM model is that the distances can be obtained from fluorescence lifetime measurements. A model of fluorescence depolarisation in the presence of PDDEM is also presented. To experimentally test the PDDEM approach, different model systems were studied. The model was applied to measure distances between rhodamine and fluorescein groups within on-purpose synthesised molecules that were solubilised in lipid bilayers. Moreover, distances were measured between BODIPY groups in mutant forms of the plasminogen activator inhibitor of type 2 (PAI-2). Measurements of both the fluorescence intensity decays and the time-resolved depolarisation were performed. The obtained distances were in good agreement with independent determinations. Finally, the PDDEM within pairs of donors is considered, for which both donors exhibit a nonexponential fluorescence decay. In this case it turns out that the fluorescence relaxation of a coupled system contains distance information even if the photophysics of the donors is identical. It is also demonstrated that the choice of relaxation model has a negligible effect on the obtained distances. The latter conclusion holds also for the case of donor-acceptor energy transfer.

This thesis concerns the development and characterisation of X-ray imaging systems based on single photon processing. “Colour” X-ray imaging opens up new perspectives within the fields of medical X-ray diagnosis and also in industrial X-ray quality control. The difference in absorption for different “colours” can be used to discern materials in the object. For instance, this information might be used to identify diseases such as brittle-bone disease. The “colour” of the X-rays can be identified if the detector system can process each X-ray photon individually. Such a detector system is called a “single photon processing” system or, less precise, a “photon counting system”. With modern technology it is possible to construct photon counting detector systems that can resolve details to a level of approximately 50 µm. However with such small pixels a problem will occur. In a semiconductor detector each absorbed X-ray photon creates a cloud of charge which contributes to the image. For high photon energies the size of the charge cloud is comparable to 50 µm and might be distributed between several pixels in the image. Charge sharing is a key problem since, not only is the resolution degenerated, but it also destroys the “colour” information in the image. This thesis presents characterisation and simulations to provide a detailed understanding of the physical processes concerning charge sharing in detectors from the MEDIPIX collaboration. Charge summing schemes utilising pixel to pixel communications are proposed. Charge sharing can also be suppressed by introducing 3D-detector structures. In the next generation of the MEDIPIX system, Medipix3, charge summing will be implemented. This system, equipped with a 3D-silicon detector, or a thin planar high-Z detector of good quality, has the potential to become a commercial product for medical imaging. This would be beneficial to the public health within the entire European Union. / Denna avhandling berör utveckling och karaktärisering av fotonräknande röntgensystem. ”Färgröntgen” öppnar nya perspektiv för medicinsk röntgendiagnostik och även för materialröntgen inom industrin. Skillnaden i absorption av olika ”färger” kan användas för att särskilja olika material i ett objekt. Färginformationen kan till exempel användas i sjukvården för att identifiera benskörhet. Färgen på röntgenfotonen kan identifieras om detektorsystemet kan detektera varje foton individuellt. Sådana detektorsystem kallas ”fotonräknande” system. Med modern teknik är det möjligt att konstruera fotonräknande detektorsystem som kan urskilja detaljer ner till en upplösning på circa 50 µm. Med så små pixlar kommer ett problem att uppstå. I en halvledardetektor ger varje absorberad foton upphov till ett laddningsmoln som bidrar till den erhållna bilden. För höga fotonenergier är storleken på laddningsmolnet jämförbar med 50 µm och molnet kan därför fördelas över flera pixlar i bilden. Laddningsdelning är ett centralt problem delvis på grund av att bildens upplösning försämras, men framför allt för att färginformationen i bilden förstörs. Denna avhandling presenterar karaktärisering och simulering för att ge en mer detaljerad förståelse för fysikaliska processer som bidrar till laddningsdelning i detektorer från MEDIPIX-projekter. Designstrategier för summering av laddning genom kommunikation från pixel till pixel föreslås. Laddningsdelning kan också begränsas genom att introducera detektorkonstruktioner i 3D-struktur. I nästa generation av MEDIPIX-systemet, Medipix3, kommer summering av laddning att vara implementerat. Detta system, utrustat med en 3D-detektor i kisel, eller en tunn plan detektor av högabsorberande material med god kvalitet, har potentialen att kunna kommersialiseras för medicinska röntgensystem. Detta skulle bidra till bättre folkhälsa inom hela Europeiska Unionen.