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Time-resolved fluorescence spectroscopy (TRFS) is a powerful analytical tool for quantifying the biochemical composition of organic and inorganic materials. The potentials of TRFS as nondestructive clinical tool for tissue diagnosis have been recently demonstrated. To facilitate the translation of TRFS technology to the clinical arena, algorithms for online TRFS data analysis are of great need. A fast model-free TRFS deconvolution algorithm based on the Laguerre expansion method has been previously introduced, demonstrating faster performance than standard multiexponential methods, and the ability to estimate complex fluorescence decay without any a-priori assumption of its functional form. One limitation of this method, however, was the need to select, a priori, the Laguerre parameter a and the expansion order, which are crucial for accurate estimation of the fluorescence decay. In this thesis, a new implementation of the Laguerre deconvolution method is introduced, in which a nonlinear least-square optimization of the Laguerre parameter is performed, and the selection of optimal expansion order is attained based on a Minimum Description Length (MDL) criterion. In addition, estimation of the zero-time delay between the recorded instrument response and fluorescence decay is also performed based on a normalized means square error criterion. The method was fully validated on fluorescence lifetime, endogenous tissue fluorophores, and human tissue. The automated Laguerre deconvolution method is expected to facilitate online applications of TRFS, such as clinical real-time tissue diagnosis.

Fluorescence based analysis is a fundamental research technique used in the life sciences. However, conventional fluorescence intensity measurements are prone to misinterpretation due to illumination and fluorophore concentration non-uniformities. Thus, there is a growing interest in time-resolved fluorescence detection, whereby the characteristic fluorescence decay time-constant (or lifetime) in response to an impulse excitation source is measured. The sensitivity of a sample’s lifetime properties to the micro-environment provides an extremely powerful analysis tool. However, current fluorescence lifetime analysis equipment tends to be bulky, delicate and expensive, thereby restricting its use to research laboratories. Progress in miniaturisation of biological and chemical analysis instrumentation is creating low-cost, robust and portable diagnostic tools capable of high-throughput, with reduced reagent quantities and analysis times. Such devices will enable point-of-care or in-the-field diagnostics. It was the ultimate aim of this project to produce an integrated fluorescence lifetime analysis system capable of sub-nano second precision with an instrument measuring less than 1cm3, something hitherto impossible with existing approaches. To accomplish this, advances in the development of AlInGaN micro-LEDs and high sensitivity CMOS detectors have been exploited. CMOS allows electronic circuitry to be integrated alongside the photodetectors and LED drivers to produce a highly integrated system capable of processing detector data directly without the need for additional external hardware. In this work, a 16x4 array of single-photon avalanche diodes (SPADs) integrated in a 0.35μm high-voltage CMOS technology has been implemented which incorporates two 9-bit, in-pixel time-gated counter circuits, with a resolution of 400ps and on-chip timing generation, in order to directly process fluorescence decay data. The SPAD detector can accurately capture fluorescence lifetime data for samples with concentrations down to 10nM, demonstrated using colloidal quantum dot and conventional fluorophores. The lifetimes captured using the on-chip time gated counters are shown to be equivalent to those processed using commercially available external time-correlated single-photon counting (TCSPC) hardware. A compact excitation source, capable of producing sub-nano second optical pulses, was designed using AlInGaN micro-LEDs bump-bonded to a CMOS driver backplane. A series of driver array designs are presented which are electrically contacted to an equivalent array of micro-LEDs emitting at a wavelength of 370nm. The final micro-LED driver design is capable of producing optical pulses of 300ps in width (full width half maximum, FWHM) and a maximum DC optical output power of 550μW, this is, to the best of our knowledge, the shortest reported optical pulse from a CMOS driven micro-LED device. By integrating an array of CMOS SPAD detectors and an array of CMOS driven AlInGaN micro-LEDs, a complete micro-system for time-resolved fluorescence analysis has been realised. Two different system configurations are evaluated and the ability of both topologies to accurately capture lifetime data is demonstrated. By making use of standard CMOS foundry technologies, this work opens up the possibility of a low-cost, portable chemical/bio-diagnostic device. These first-generation prototypes described herein demonstrate the first time-resolved fluorescence lifetime analysis using an integrated micro-system approach. A number of possible design improvements have been identified which could significantly enhance future device performance resulting in increased detector and micro-LED array density, improved time-gate resolution, shorter excitation pulse widths with increased optical output power and improved excitation light filtering. The integration of sample handling elements has also been proposed, allowing the sample of interest to be accurately manipulated within the micro-environment during investigation.

Les protéines fluorescentes sont très largement utilisées dans les études de biologie moléculaire depuis maintenant une vingtaine d’année. Pour autant, l’origine de leurs propriétés photophysiques n’est pas totalement élucidée. Dans cette thèse, nous avons essayé d’améliorer la compréhension de la photophysique de deux protéines fluorescentes particulières : Padron et EosFP.Dans la protéine Padron, nous avons étudié l’isomérisation du chromophore et cherché à déterminer si la protonation et l’isomérisation sont simultanées ou successives. Pendant l’isomérisation, le donneur de proton potentiel est le résidu Tyr159. Nous avons d’abord montré que dans le vide, le transfert de proton est peu probable quelle que soit la géométrie du chromophore. Dans la protéine (où l’effet de l’environnement n’est pas négligeable) nous avons mis en évidence par dynamique moléculaire que, durant l’isomérisation, le transfert de proton n’est presque jamais favorable et reste donc un marginal.Par ailleurs, ces mêmes dynamiques ont montré que, à la fin de l’isomérisation, il apparaît de nombreux chemins de molécules d’eau reliant le chromophore au solvant et pouvant permettre un transfert de proton. On conclut doncque l’isomérisation et la protonation ne sont pas simultanées mais successives.Dans le cas de la protéine EosFP, nous avons analysé l’effet d’une molécule d’eau présente dans une partie des structures cristallines. Les dynamiques avec le chromophore à l’état fondamental ont montré que cette molécule ne joue pas de rôle, que ce soit sur le réseau de liaison hydrogène ou sur le spectre d’absorption. Par contre, à l’état excité, les dynamiques ont montré que l’extinction de fluorescence est beaucoup plus rapide sans la molécule d’eau qu’en sa présence.Par ailleurs, ces dynamiques ont mis en évidence que la protéine bloque souvent le chromophore dans des géométries où il ne peut pas retourner à l’état fondamental ni par fluorescence, ni par conversion interne. Ces géométries « noires» jouent un rôle important dans la photophysique.Pour tenir compte de ces géométries, nous avons calculé le rendement quantique et le temps de vie de fluorescence par intégration directe le long des trajectoires et par cinétique chimique. Dans les deux cas, nous avons obtenu un accord qualitatif avec l’expérience. / Fluorescent proteins are widely used in biology studies since 20 years. Yet, the origin of their photophysical properties aren’t totally explained. Here, we try to improve the understanding of two particular fluorescent proteins: Padron and EosFP.In the protein Padron, we work on the isomerization of chromophore and try to determine whether isomerization and protonation are simultaneous or successive processes. During the isomerization, the potential donor is Tyr159.First, we show that, in vacuum, the proton transfer is quite unlikely whatever the chromophore geometry.In the protein (where the environment effect isn’t negligible) we evidence with molecular dynamics that, during isomerization, proton transfer stays marginal.In addition, these dynamics shown the appearance, at the end of isomerization, of a lot of water molecules channel between the chromophore and the solvent allowing a proton transfer. We conclude that isomerization and protonation are successive processes.In the case of the protein EosFP, we first analyze the effect of a water molecule which is found only in some of the crystallographic structures.Molecular dynamics of the protein with the chromophore in the ground state show that the water molecule doesn’t play any role neither in the hydrogen bond network nor in the absorption spectra.On the contrary, in the excited state, dynamics without this water show a significant faster decay of fluorescence that those with the molecule.In addition, those dynamics have demonstrate that during long period, the protein retains the chromophore in geometries in which it is unable to convert to the ground state, neither by fluorescence nor by internal conversion. Those “dark” geometries play a crucial role in the photophysics.To take them into account, we calculate the quantum yield and the fluorescence lifetime by direct integration along trajectories and by a kinetic scheme. We obtain a good qualitative agreement with the two methods.

La scintillation organique correspond au phénomène d’émission de lumière par un matériau moléculaire à la suite de l’excitation de celui-ci par un rayonnement externe d’énergie donnée. Lors de l’interaction, le dépôt d’énergie induit des transitions électroniques peuplant des états dont la plupart se désexcite de manière non radiative, à l’exception d’une, entre le premier état électronique singulet et l’état fondamental de la molécule. Lors de cette relaxation, un photon de fluorescence est émis. Cette émission a deux origines : i) l’excitation directe par le rayonnement primaire et les électrons secondaires ; elle donne lieu à une émission dite rapide ou prompte ; ii) l’ionisation par le rayonnement primaire et les électrons secondaires ; elle donne lieu à une émission dite lente ou différée. Ce travail de recherche fondamentale, à la fois théorique et expérimental, fait l’analyse de toutes les étapes du processus, de l’interaction primaire à l’émission de fluorescence, de manière à relier la dose déposée à la quantité de lumière émise, à des fins d’applications en dosimétrie médicale. Il repose sur la mesure des déclins de fluorescence de deux molécules modèles, l’anthracène et le paraterphényle, excitées par un flux continu de rayons X, et la séparation des contributions rapide et lente de la lumière émise, aux énergies médicales. Une modélisation analytique des processus physiques conduisant à l’émission de lumière, au regard de la dose déposée, a ensuite été effectuée, faisant apparaître de nombreux résultats originaux. Dans un premier temps, un dispositif expérimental original a été développé, basé sur la technique TCSPC (Time-Correlated Single Photon Counting), afin de pouvoir mesurer des déclins temporels de fluorescence en résolution nanoseconde et sous flux d’irradiation continu. Dans un second temps, nous avons développé une nouvelle approche mathématique permettant d’extraire finement les composantes rapides et lentes du signal. L’analyse des résultats a montré, pour la première fois, l’existence d’un rapport R constant et uniquement fonction du matériau, entre les rendements d’excitation et d’ionisation. Le caractère constant de ce rapport ne peut être attribué qu’à un mécanisme d’autoionisation moléculaire au sein d’un matériau se comportant intrinsèquement comme une chambre d’ionisation proportionnelle pour l’ionisation secondaire de basse énergie. Ceci est en accord total avec la linéarité observée entre l’intensité totale de lumière différée (ionisation) et la dose mesurée par une chambre d’ionisation proportionnelle. Une étude plus approfondie des mécanismes d’excitation, au regard du rapport R, a également permis de montrer, pour la première fois, une proportionnalité directe entre l’intensité totale de la lumière prompte et le dépôt d’une dose que nous avons baptisé dose d’excitation. Cette dose a été observée comme étant de 4 à 14 fois supérieure à celle mesurée par une chambre d’ionisation. Ce résultat original majeur devra impérativement conduire à des études futures afin de mieux comprendre les dégâts infligés à la matière organique et biologique par les excitations. / Organic scintillation is the emission of light by an organic scintillator when irradiated by an external source of radiation depositing enough energy to excite the molecule. Electronic states are populated by the electronic transitions generated by the deposited energy. The states de-excite through radiationless transitions, except for one, the transition between the first electronic state and the ground state where a photon of fluorescence is emitted. This light has two different origins: i) direct excitation caused by primary radiation or secondary electrons which leads to an emission knows as prompt; ii) ionization caused by primary radiation or secondary electrons generate what is known as the delayed component. This fundamental research was based on both theoretical and experimental work. We studied all the different processes in organic scintillation, from the interaction between the incident radiation and matter to the emission of light in order to find the relationship between fluorescence and the deposited dose, to the application to medical dosimetry. Two well known organic scintillators, anthracene and p-terphenyl, were excited using an X-ray source set at typical medical imaging parameters. The light emitted was acquired and an analytical model was used to describe the different processes that led to light emission revealing interesting new results.An experimental setup, based on the Time Correlated Single Photon Counting (TCSPC) technique, was developed to acquire fluorescence decay curves with nanosecond resolution using a continuous X-ray source. Afterwards, these curves were analyzed using an innovative mathematical approach in order to determine the prompt and delayed components.Results showed the ratio, defined as R, between the prompt and delayed components of fluorescence was constant and independent of the energy of the incident X-rays and that the response of the delayed component of fluorescence was linear to an ionization chamber. These observations were explained by considering that the only process taking place within the molecule after excitation was autoionization. Hence, the response of organic scintillator was the same as the one of an ionization chamber. Furthermore, due to the constant ration R, the response of prompt component of fluorescence was linear to the ionization chamber as well. This was the first time this behavior was observed and we referred to it as excitation dose. This dose was between 4 and 14 times bigger than the one measured with the ionization chamber. These original results suggested that energy is deposited mainly through excitation processes, suggesting the need for further studies to better understand the damage caused by excitation to the living.