Vulnerable plaque is a clinically silent condition of atherosclerotic plaque that leaves a large number of patients at risk of a coronary event. A method to detect vulnerable atherosclerotic plaque would greatly enhance the ability of clinicians to diagnose and treat patients at risk. Fluorescence lifetime imaging microscopy (FLIM) offers a way to extract both spatial and biochemical information from plaque by taking several wide-field images over time. The goal of this study was to determine the potential of a FLIM angioscopy system to detect and differentiate coronary atherosclerotic plaques ex-vivo into several groups including thin, fibrotic, lipid-laden, thick-cap fibroatheroma (FA), and fibrocalcified. Samples were extracted post-mortem weekly and sliced open to have their lumens imaged. For each sample, 51 time resolved wide-field images were taken over 10 nanoseconds at 390 (±40) nm, 450 (±40) nm, and 550 (±88) nm wavelengths. To analyze the samples, the intensity map and lifetime map were created at each wavelength. The intensity map was simply the wide-field images summed in time and normalized. In order to calculate lifetime at each point, a fast, model-free Laguerre deconvolution algorithm was recently developed for FLIM data analysis and was used. This allowed for fast, efficient estimations of the fluorescence decay curves at each pixel of the FLIM images and facilitated the computation of quantitative parameters describing the fluorescence emission of the tissue, specifically, the relative fluorescence intensity and lifetime at defined emission bands. Statistical analysis on these FLIM derived parameters indicated that the autofluorescence emission of the plaques allows for distinguishing relative plaque thickness: thin plaque, whose signal is dominated by elastin fluorophores, shows a marked difference between thicker plaques, such as fibrotic, fibrocalcified and thick-cap FA (who are dominated primarily by collagen). However, the ability of the current FLIM system to differentiate vulnerable plaque remains in question due to the absence of thin-cap FA samples. Further work has also been proposed; of primary concern is gathering thin-cap FA plaque samples needed to validate the system’s ability to differentiate vulnerable plaques from other common groupings.

The technique of Fluorescence Lifetime Imaging Microscopy (FLIM) has been employed to quantitatively and spatially map the fluid composition and temperature within microfluidic systems. A molecular probe with a solvent-sensitive fluorescence lifetime has been exploited to investigate and map the diffusional mixing of fluid streams under laminar flow conditions within a microfluidic device. Using FLIM, the fluid composition is mapped with high quantification and spatial resolution to assess the extent of mixing. This technique was extended to quantitatively evaluate the mixing efficiency of a range of commercial microfluidic mixers employing various mixing strategies, including the use of obstacles fabricated within the channels. A fluorescently labelled polymer has been investigated as a new probe for mapping temperature within microfluidic devices using FLIM. Time Correlated Single Photon Counting (TCSPC) measurements showed that the average fluorescence lifetime displayed by an aqueous solution of the polymer depended strongly on temperature, increasing from 3 ns to 13.5 ns between 23 and 38 oC. This effect was exploited using FLIM to provide high spatial resolution temperature mapping with sub-degree temperature resolution within microfluidic devices. A temperature-sensitive, water-soluble derivative of the rhodamine B fluorophore, effective over a wide dynamic temperature range (25 to 91 oC) has been used to map the temperature distribution during the mixing of fluid streams of different temperatures within a microchannel. In addition, this probe was employed to quantify the fluid temperature in a prototype microfluidic system for DNA amplification. FLIM has been demonstrated to provide a superior approach to the imaging within microfluidic systems over other commonly used techniques, such as fluorescence intensity and colourimetric imaging.

I have analyzed the interactions between SR proteins and splicing components that are bound at the 5’ or 3’ splice site using fluorescence resonance energy transfer (FRET) microscopy. The SR proteins interact with the U1 snRNP-associated 70 kDa protein (U170K) at the 5’splice site and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) at the 3’ splice site. These interactions have been extensively characterized biochemically in the past, and are proposed to play roles in both intron and exon definition. We employed FRET acceptor photobleaching and fluorescence lifetime imaging microscopy (FLIM) to identify and spatially localise sites of direct interactions of SF2/ASF, and other SR proteins, with U2AF35 and U1-70K in live cell nuclei. These interactions were shown to occur more strongly in interchromatin granule clusters (IGCs). They also occur in the presence of the RNA polymerase II inhibitor, DRB, demonstrating that they are not exclusively co-transcriptional. FLIM data have also revealed a novel interaction between HCC1, a factor highly related to the large subunit of the U2AF splicing factor, with both subunits of U2AF that occur in discrete domains within the nucleoplasm but not within IGCs. These data demonstrate that the interactions defining intron and exon definition do occur in living cells in a transcription-independent manner.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to non-invasively map the physical and chemical environment within microfluidic devices. In this work FLIM has been used in conjunction with a variety of other techniques to provide a greater insight into flow behaviour and fluid properties at the microscale. The pH-sensitive fluorescent dyes, fluorescein and C-SNARF 1, have been used to generate pH maps of microfluidic devices with a time-gated camera and a time-and-space-correlated single photon counting (TSCSPC) detector, respectively. Using time-gated detection and fluorescein, the fluorescence lifetime images allow for direct reading of the pH. The relative contribution to fluorescence of the acid and basic forms of C-SNARF 1 was spatially resolved on the basis of pre-exponential factors, giving quantitative mapping of the pH in the microfluidic device. Three dimensional maps of solvent composition have been generated using 2-photon excitation FLIM (2PE-FLIM) in order to observe the importance of gravitational effects in microfluidic devices. Two fluidic systems have been studied: glycerol concentration in the microfluidic device was measured using Kiton red; water concentration in a methanolic solution was measured using ANS. The density mismatch between two solutions of different composition induced a rotation of the interface between two streams travelling side by side in a microchannel. The experiment has provided evidence of non-negligible gravitational effects in microflows. 2PE-FLIM has superior capability than methods used previously to assess similar phenomena. FLIM and micro-particle imaging velocimetry (μ-PIV) have been implemented on a custom-built open frame microscope and used simultaneously for multimodal mapping of fluid properties and flow characteristics. It has been shown that viscosity mismatch between two streams induces a non-constant advective transport across the channel and results in a flow profile that deviates from the usual Poiseuille profile, characteristic of pressure driven flow in microfluidic devices.

FLIM is branch of microscopy mainly used in biology which is quickly improving thanks to a rapid enhancement of instrumentation and techniques enabled by new sensors. In FLIM, the most precise method of measuring fluorescent decays is called TCSPC. High voltage PMT detection devices together with costly and bulky optical setups which scan the sample are usually required in TCSPC instrumentation. SPADs have enabled a big improvement in TCSPC measurement setup, providing a CMOS compatible device which can be designed in wide arrays format. However, sensors providing in-pixel TCSPC do not scale in size and in large array like the time-gated SPAD pixel sensors do. Time-gated pixels offer a less precise lifetime estimation, discarding any photon information outside a given time window, but this loss in photon-efficiency is offset by gains in pixel size. This work is aimed at the development of a wide field TCSPC sensor with a pixel size and fill factor able to reduce the cost of such devices and to obtain a high resolution time-resolved fluorescence image in the shortest time possible. The study focuses on SPAD and pixel design required to maximise the fill factor in sub 10 μm pixel pitch. Multiple pixel designs are proposed in order to reduce pixel area and so enable affordable wide array TCSPC systems. The first proposed pixel performs the CMM lifetime estimation in order to reduce the frame rate needed to stream the data out of the SPAD array. This pixel is designed in a 10 μm pitch and attains with the most aggressive design a fill factor of 10:17 %. A second design proposes an analogue TCSPC which consists in a S/H TAC circuitry. This simpler pixel can achieve a higher fill factor of 19:63% as well as smaller pitch of 8 μm thanks to the adoption of SPAD n-well and electronics area sharing. This last design is implemented in a 320 x 256 SPAD array in which is included part of a novel ADC aimed at reduction of the processing time required to build a TCSPC histogram. A more conventional analogue readout is used to evaluate the pixel performance as well as a more fine TCSPC histogram. The device was used to measure the fluorescence lifetime of green micro-spheres while the 2b flash ADC is used to demonstrate rapid resolution and separation of two different fluorescence decays.

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.

No description available.

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

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.

In den vergangenen Jahren sind Lipide, Membranen und deren Organisationsformen mehr und mehr in den Fokus der biologischen Forschung gerückt. Es wurde vorgeschlagen, dass in zellulären Membranen selbstassemblierende, submikroskopische Aggregate aus Sphingolipiden, Cholesterol und bestimmten Proteinen existieren und man vermutet, dass insbesondere Viren diese “Lipid Rafts” für ihren Zusammenbau nutzen und auf diese Art ihre Proliferationseffizienz erhöhen. Gleichwohl sind die genaue biologische Funktion und auch die molekulare Basis der Assoziation bestimmter Protein mit Lipid Rafts auch weiterhin unbekannt. In der vorliegenden Arbeit wurde Fluoreszenz-Lebenszeit-Mikroskopie genutzt, um die Lipid-Raft-Anreicherung des HIV-1 Glycoproteins gp41 zu untersuchen. Förster-Resonanz-Energietransfer zwischen fluoreszenzmarkierten viralen und Raft-Marker-Proteinen wurde gemessen, um deren gemeinsame, lokale Aufkonzentrierung in Lipid Rafts nachzuweisen. Durch Verwendung verschiedener Deletions- und Mutationsvarianten des Proteins konnte nicht nur seine Lipid-Raft-Präferenz demonstriert, sondern auch das Cholesterol-Bindemotiv (CRAC) als entscheidender Faktor der lateralen Sortierung identifiziert werden. Wir haben in diesem Kontext auch eine systematische Zell-zu-Zell-Variabilität in unseren Daten bemerkt, die einen zugrundeliegenden zellbiologischen Mechanismus der Membranorganisation nahelegt. Mithilfe von Fluoreszenz-Polarisations-Mikroskopie konnte zudem eine klare CRAC-Abhängigkeit der gp41-Oligomerisierung aufgezeigt werden. Die von uns gewonnenen Daten erlauben einen tieferen Einblick in die molekulare Basis und die biologischen Folgen der cholesterol-abhängigen lateralen Proteinorganisation für Virusassemblierungsprozesse an biologischen Membranen. / In recent years, there has been a considerable interest in the molecular organization of biological membranes. It has been hypothesized that self-assembling, freely diffusing, submicroscopic domains consisting of sphingolipids, cholesterol and certain proteins exist and the prevailing view is that those lipid rafts serve as platforms for specific molecular interactions by the preferential exclusion and inclusion of proteins. It was presumed, that in particular viruses make use of plasma membrane lipid rafts to augment the infection process and spread efficiently. However, the exact biological function and physical basis of protein partitioning into microdomains remains an outstanding question in virus biology. In the present study, fluorescence lifetime imaging microscopy was used to study lipid raft partitioning of the HIV-1 glycoprotein gp41 by detecting Foerster Resonance Energy Transfer between fluorescently labeled viral and raft marker proteins in living cells. Plasma membrane microdomain association of gp41 was demonstrated and by introducing systematic mutations and truncations in different gp41 motifs, the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial determinant of the lateral sorting. Interestingly, we observed a systematic cell-to-cell variability in our raft related data that suggests underlying cell-biological mechanisms of membrane organization. Moreover, fluorescence polarization microscopy revealed a distinct CRAC requirement for gp41 oligomerization whereas other properties, such as intracellular distribution and expression efficiency were clearly demonstrated to be CRAC independent. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral protein sorting for virus assembly processes at cellular plasma membranes.