Amyloid $\beta$ (1-42) (A$\beta$42) is a seminal neuropathic agent in Alzheimer’s disease (AD), a multifaceted neurodegenerative disorder for which no preventative measures or disease modifying therapies currently exist. Aggregation of this peptide plays a key role in the synaptic dysfunction and neuronal death associated with the disease. Perturbing the aggregation process, therefore, represents a key strategy for the development of new AD therapeutics. A variety of issues with current screening methods, including lack of reproducibility, high reagent consumption and spectral interference from the test molecules, can limit efforts to identify new small molecule inhibitors. Furthermore, the lack of robust, time- and cost-efficient methods for screening compounds in cellular or in vivo models limits the throughput with which seemingly active small molecules can be validated and prioritised. Herein, this thesis describes efforts to overcome such limitations through the development of a unified in vitro to in vivo assay system, in which hits identified in the ‘nanoFLIM’ microfluidic-based assay can quickly be tested in cellular and whole organism disease models. The assay platform designed relies on the use of an amyloid aggregation fluorescence lifetime sensor. A$\beta$42 aggregation is monitored by changes in the fluorescence lifetime of an attached fluorophore, which is significantly quenched upon amyloid formation. To take advantage of the benefits associated with miniaturisation, an in vitro microfluidic platform was employed. A microfluidic chip capable of trapping 110 precisely ordered droplets was designed, allowing for increased sample size and greatly lowering reagent consumption relative to conventional assay formats. Optimisation of the lifetime sensor technique permitted real-time compound screening in SH-SY5Y neuroblastoma cells, as well as in disease model Caenorhabditis elegans (C. elegans). To demonstrate the potential of this assay, a selection of novel chemical libraries developed in the Spring research group was screened, resulting in the identification of a key library of interest. The inhibitory activity of the lead compound from this collection was validated using a variety of biophysical tests, and was also shown to suppress amyloid aggregation in the live cell fluorescence lifetime sensor assay, as well as in whole organism disease model C. elegans. Whilst assay development was underway, additional screening of structurally diverse chemical libraries was performed using a conventional Thioflavin T spectroscopic assay. Such work identified another molecular scaffold capable of exerting a strong inhibitory effect against A$\beta$42 aggregation. A selection of analogues was synthesised to improve the in vivo profile of this library, giving rise to a second lead inhibitory compound. The activity of this compound was subsequently validated in biophysical and cellular tests, and was also tested in disease model Drosophila melanogaster. The aggregation of A$\beta$42 lies at the root of Alzheimer’s disease. In light of the relatively few drug candidates in clinical trials for this disorder, the development of improved translational screening approaches and continued screening of novel chemical libraries is necessary to identify new potential therapeutics. In this study, an in vitro to in vivo fluorescence lifetime imaging assay has been established. Using this assay system and conventional screening approaches, two A$\beta$42 aggregation inhibitors have been identified and validated. These represent promising candidates for the development of new AD therapeutic agents, or for use as molecular probes to further dissect the mechanisms underlying this devastating disease.

The fluorescence spectroscopy and lifetime analysis in biological tissues has been presented as a technique of great potential for tissue characterization for diagnostic purposes. This potential is due to the main advantages of optical techniques based on fluorescence for diagnosis, which includes the possibility of evaluating the tissue metabolism in situ, without removal and processing of the biological sample, through a fast and non-invasive response. Skin lesions were the target interrogated tissue in the present study. They can be clinically classified into two major groups: pigmented and non-pigmented lesions. In each group, the clinical discrimination of benign and malignant lesions may be a complex task, especially for non-experienced clinicians. When these lesions have clinically similar features, the choice of the treatment modality becomes difficult. In this context, auxiliary diagnostic techniques are very important to improve the diagnostic resolution as well as treatment planning and success. Gold standard for skin diagnosis is obtained with the biopsy and further histological analysis. The information about these features is invasive and time consuming. When using a non-invasive procedure such as fluorescence lifetime measurements, the main interrogated fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), biomolecules involved in cellular respiration that may provide information on the metabolism of the cells. To differentiate each skin lesion, it is necessary to take into account the contribution of endogenous fluorophores emission such as collagen and elastin, and the absorption of chromophores such as melanin and hemoglobin. In addition to fluorescence decay analysis considering the contribution of fluorophores and chromophores, a stable and portable system is desired for clinical measurements and interrogation of biological tissue in vivo. In this study, we have assembled, calibrated, and characterized one of the worlds first portable time-resolved fluorescence spectroscopy system for single-point measurements. This system was designed to be robust and user-friendly for clinical applications. The system was calibrated and characterized in vitro before the clinical application. It was used for evaluation of the photoaging process in sun-exposed and non-exposed skin and for discrimination of clinically similar skin lesions. Significant statistical differences were observed for 10 parameters when comparing normal and photoaged skin (students t-test, p < 0.001), and for all combinations of non-pigmented and pigmented lesions when using tri-exponential decay parameters (Wilcoxon rank sum test, p<0.05). Both in vivo measurements showed promising results and have potential for many applications in dermatology, oncology and aesthetics. Next steps include multivariate data analysis and the determination of the diagnostic resolution of fluorescence lifetime spectroscopy. Further investigation of optical processes related to fluorescence decay changes is necessary, since fluorescence lifetime values in biological tissues reported on the literature are very scarce and heterogeneous and not completely understood. / A análise da espectroscopia e do tempo de vida da fluorescência em tecidos biológicos vem sendo apresentada como uma técnica com grande potencial para a caracterização tecidual com finalidade diagnóstica. Esse potencial é devido às principais vantagens das técnicas ópticas de diagnóstico baseadas em fluorescência, que possibilitam avaliar o metabolismo in situ, sem a necessidade de remoção e processamento da amostra biológica, com uma resposta rápida e não-invasiva. Lesões de pele foram os tecidos investigados no presente estudo. Elas podem ser clinicamente classificadas em dois grandes grupos: pigmentadas e não pigmentadas. Em cada grupo, a discriminação clínica de lesões benignas e malignas pode ser uma tarefa complexa, principalmente para médicos com pouca experiência. Quando essas lesões apresentam características clínicas semelhantes, a escolha do tipo de tratamento torna-se difícil. Nesse contexto, técnicas auxiliares de diagnóstico são de grande relevância para melhorar a resolução de diagnóstico, assim como o planejamento e o sucesso do tratamento. O padrão ouro para o diagnóstico do câncer de pele é obtido por meio da biópsia e posterior análise histopatológica. A obtenção de informações sobre essas características é invasiva e consome bastante tempo. Ao utilizar procedimentos não-invasivos como medidas de tempo de vida de fluorescência, os fluoróforos de mais investigados são o NADH (nicotinamida adenina dinucleotídeo) e o FAD (flavina adenina dinucleotídeo), biomoléculas envolvidas na respiração celular que podem fornecer informação sobre o metabolismo das células. Para diferenciar cada tipo de lesão de pele, é necessário levar em conta a contribuição da emissão de fluoróforos endógenos como o colágeno, elastina e da absorção de cromóforos como melanina e hemoglobina. Além da análise do decaimento de fluorescência considerando a contribuição de fluoróforos e cromóforos, um sistema estável e portátil é desejado para medidas clínicas e investigação de tecidos biológicos in vivo. Nesse estudo, nós montamos, calibramos e caracterizamos um dos primeiros sistemas portáteis do mundo para espectroscopia de fluorescência resolvida no tempo para medidas pontuais. Esse sistema foi projetado para ser robusto e amigável ao usuário em aplicações clínicas. O sistema foi calibrado e caracterizado in vitro antes das aplicações clínicas. Ele foi utilizado para avaliação do processo de fotoenvelhecimento em pele exposta e não-exposta ao sol e para a discriminação de lesões de pele clinicamente semelhantes. Diferenças estatísticas significativas foram observadas para 10 parâmetros na comparação entre pele normal e fotoenvelhecida (teste t-student, p<0.001) e para todas as combinações de lesões pigmentadas e não-pigmentadas ao utilizar parâmetros do decaimento triexponencial (teste Wilcoxon rank sum, p<0.05). Ambas medidas in vivo mostraram resultados promissores e um potencial para muitas aplicações em dermatologia, oncologia e estética. As próximas etapas incluem análise multivariada de dados e determinação da resolução de diagnóstico da espectroscopia de tempo de vida de fluorescência. Uma maior investigação dos processos ópticos relacionados a mudanças nos decaimentos de fluorescência é necessária, pois o número de valores de tempo de vida de fluorescência em tecidos biológicos reportados na literatura é escasso e os valores são heterogêneos e não completamente compreendidos.

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The process of spontaneous emission can be dramatically modified by optical micro- and nanostructures. We studied the modification of fluorescence dynamics using a polymer spacer layer fabricated through layer-by-layer (LbL) self-assembly. The advantages of this method are numerous: The self-assembled spacers can possess exceptional smooth surface morphology; The thickness of the spacer can be controlled with nanometer accuracy; And depending on fabrication conditions, the spacer layer is stimuli responsive and its thickness can be dynamically tuned. This thesis contains three interlinked components. First, we vary LbL spacer layer thickness and explore the change in fluorescence lifetime induced by the modified photonic density of states (PDOS), i.e., Purcell effects. Our experimental results agree well with theoretical predictions based on a classical dipole model, which also yields consistent values for the fluorophores' intrinsic fluorescence lifetime and quantum yield near a dielectric as well as a plasmonic interface. Based on this observation, we further demonstrate that self-assembled fluorophores can be used to probe the modified PDOS near optical micro- and nano-structures. These results naturally lead to the second component of our research. In particularly, based on the PDOS-induced changes in fluorescent lifetime, we develop a non-contact method that can measure morphological changes with nanoscale resolution. Our method relies on quantitatively linking fluorophore position with PDOS, and is validated through direct comparison with ellipsometry and atomic force microscopy (AFM) measurements. To demonstrate the potential application of this method, we investigated the swelling/deswelling of LbL films induced by pH changes. Our results indicate significant difference between a LbL film composed of a single polymer monolayer and a LbL film with 3 monolayers. Such stimuli-responsive polymers can be used to construct active and tunable plasmonic nano-devices. As a proof-of-principle demonstration, we experimentally confirm that it is possible to utilize the swelling/deswelling behavior of stimuli-responsive films to dynamically control the separation between Au nanoparticles and Texas Red (TR) dyes. This result is based on the strong correlation of TR fluorescence lifetime and nanoparticles-TR separation. Finally, we investigate the impact of different lithography processes on the fluorescence properties of self-assembled fluorophores. We consider three methods: direct fluorophore patterning through ultraviolet (UV) ablation, focused ion beam (FIB) milling of self-assembled fluorophores, and self-assembly of fluorescent materials over plasmonic nano-patterns. / Ph. D.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20604号 / 理博第4319号 / 新制||理||1620(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)准教授 熊﨑 茂一, 教授 林 重彦, 教授 寺嶋 正秀 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

We have synthesized dimethyl derivatives of the GFP chromophore (p-HOBDI) and several of its derivatives, and their photochemistry and photophysics were investigated using various steady-state and time-resolved techniques as follows. We first consider the effect of the £]-barrel on the optical properties of the GFP chromophore (p-HOBDI) experimentally by selective variation of the protonation state of chromophores and different solvents. Each of these forms shows a complex solvatochromic behavior and is governed by both polar and acid/base properties of the solvents. In contrast to their solution behavior, some O-alkyl GFP chromophore (alkoxy-BDI) derivatives exhibit large fluorescent enhancement in the solid state. The color of the crystalline BDI is tuned by substituent-mediated crystal packing, showing the potential applications in optoelectronic devices. Using femtosecond polarization-sensitive infrared (IR) spectrosceopy of the C=O stretching mode of the HOBDI, we have then discovered a near complete twisting around the ethylenic bridge between the phenolate and imidazolidinone groups upon electronic excitation. Cis/trans isomerization induced by the rotation around the bridge is thought to be responsible for the behavior of blinking in fluorescent protein; however, the mechanism of the thermal reverse isomerization is more problematic. Thus we synthesized BDI derivatives with decreasing para-donating ability, HO, CH3O, CH3, H, and Cl, and used a Hammett plot for the rate study. With a positive â value, we conceived, for the first time, a novel nucleophilic addition/elimination mechanism. Finally, the GFP chromophore falls into the general category of hydroxyarene photoacids, which exhibit high excited-state acidities but neutral ground states. A hydroxyl substituent at the meta position shows enhanced charge transfer and greater acidity in the excited state. As a result, we have demonstrated that the fast quenching of the excited state by internal conversion to the ground state is much slower in meta- than in para-HOBDI derivatives. This allows studies of this ultrafast intermolecular ESPT that competes with isomerization. The photoinduced dynamics of the meta isomer of GFP chromophore was further investigated using femtosecond transient absorption and fluorescence upconversion spectroscopies.

Die vorliegende Arbeit befasst sich mit zwei neuen methodischen Ansätzen auf dem Gebiet der Fluoreszenzmikroskopie. Im ersten Teil der Arbeit wir eine Methode vorgestellt, mit der die Winkelselektivität der Fluoreszenzanregung verbessert werden kann. Die ExPAN (excitation polarization angle narrowing) genannte Technik nutzt stimulierte Emission, um den Effekt der Photoselektion zu vergrößern. ExPAN lässt sich potentiell für verschiedene Methoden einsetzen, in denen fluoreszenzmarkierte Proben untersucht werden und ist insbesondere im Kontext von Fluoreszenzanisotropie-Messungen oder der Bestimmung von molekularen Orientierungen von Interesse. Solche Methoden finden in den Biowissenschaften breite Anwendung und werden z.B. zum Studium von Rezeptor-Liganden-Interaktionen oder der Proteindynamik eingesetzt. Im Rahmen der Arbeit wird ExPAN in Kombination mit einem neuen Ansatz in der Weitfeldmikroskopie untersucht, bei der die Orientierung von Farbstoffmolekülen als Kontrastmechanismus genutzt wird. Dabei wird die Polarisationsrichtung des Anregungslichts rotiert, um Informationen über die molekulare Orientierung zu gewinnen. Aufgrund der Photoselektion weist das Fluoreszenzsignal von Molekülen mit bevorzugter Ausrichtung dadurch eine periodische Modulation auf. Es wird gezeigt, dass diese Information zur Unterscheidung von Molekülen mit abweichender Orientierung genutzt werden kann, selbst wenn sich deren Signale räumlich überlagern. Für die Versuche wurde ein modifiziertes Weitfeld-Mikroskop konstruiert und die Methode zum einen experimentell an Einzelmolekülen und zum anderen mittels Simulationen erprobt. Dabei konnten Signale von Farbstoffmolekülen mit einem Abstand von bis zu 80 nm separiert werden. Darüber hinaus wurde ein moduliertes Fluoreszenzsignal bei oberflächenmarkierten Mikropartikeln in wässriger Lösung sowie bei fixierten biologischen Proben beobachtet. Eine Verbesserung der Photoselektion durch ExPAN wird experimentell nachgewiesen und gezeigt, dass mit ExPAN auch ähnlich orientierte Moleküle unterschieden werden können. Im zweiten Teil der Arbeit wird eine Methode zur Verbesserung der Auflösung von konfokalen Laser-Scanning-Mikroskopen vorgestellt, die als Multidetektor-Scanning (MDS) bezeichnet wird und auf dem Prinzip der Image-Scanning-Mikroskopie (ISM) beruht. Mit ISM lässt sich die Auflösung von Fluoreszenzmikroskopen theoretisch verdoppeln. Da ISM einen Flächendetektor voraussetzt, wurden in der Vergangenheit hauptsächlich CCD oder CMOS Kameras als Detektoren eingesetzt. In dieser Arbeit werden anstelle einer Kamera mehrere Einzelphotonendetektoren verwendet und über ein Glasfaserbündel zu einem Flächendetektor kombiniert. Dadurch ist es erstmals möglich, die Methode in Verbindung mit Fluoreszenzlebensdauer-Mikroskopie (FLIM) einzusetzen. FLIM hat sich in den Biowissenschaften als wichtige Mikroskopie-Technik etabliert und wird unter anderem bei Protein-Protein-Interaktionsstudien oder zur Untersuchung des NADH-Metabolismus eingesetzt. Die Verbesserung der räumlichen Auflösung von FLIM mit MDS ist somit für eine Reihe von biologischen Fragestellungen von potentiellem Interesse. Im Rahmen der Arbeit wurde ein Multidetektor-Scanning-Mikroskop konstruiert und durch die Vermessung von fluoreszierenden Mikropartikeln charakterisiert. Eine Verbesserung der Auflösung durch MDS wird an fixierten biologischen Proben demonstriert. Dabei wurde eine Auflösung von 168 nm mit MDS sowie 146 nm mit MDS und Dekonvolution erreicht. Schließlich wird die Kombination der Methode mit Fluoreszenzlebensdauer-Mikroskopie demonstriert.

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This thesis presents time difference (TD) circuits that are important for measuring fluorescence lifetime, building LIDAR systems, and optimizing digital systems. The contribution of this thesis is to present a systematic organization of TD circuits and to present novel designs for digital-to-time conversion (DTC) and time-to-digital conversion (TDC). Four basic time difference circuits are presented: TD adder, arbiter, time-difference MUX, and time-difference memory. Specifications, symbols, and multiple circuit implementations are presented for each block. Then the basic blocks are combined to form two compound blocks: DTC and TDC. Novel designs are presented for both blocks along with detailed simulation results. The DTC was fabricated in TSMC’s 0.35um high-voltage process. A printed circuit board was designed to interface the DTC chip to a computer and test instruments. The DTC demonstrated 80ps resolution.