<p>Current gold standard practices for the diagnosis of tissue disease involve invasive tissue biopsies subjected to a time consuming histopathological examination process. An optical biopsy can offer a non-invasive diagnostic alternative by exploiting the properties of naturally occurring light-tissue interactions. A time-resolved fluorescence spectroscopy instrument (355 nm excitation) has previously been developed by our lab to capture the fluorescence response of gastrointestinal tissue (370-550 nm in 5 nm increments, 25 ns at 1000 ps/pt). Measurements were conducted ex-vivo during routine upper gastrointestinal tract biopsies on duodenum, antrum, stomach body, and esophageal tissue. The work currently presented is focused on protocol development for tissue handling, measurement collection, clinical data management, fluorescent decay modeling using Laguerre based deconvolution, instrument performance evaluation, and k-means based classification.</p> <p>Descriptive parameters derived from spectral (total signal intensity) and temporal (lifetime and Laguerre polynomial coefficients) analysis were used to evaluate the data. It was found that data were only compromised when the total signal intensity for the peak wavelength 455 nm fell blow 19.5 V·ns. The data did not exhibit any signs of photobleaching or pulse width broadening that would have otherwise distorted the lifetime from its true fluorescence response. Data for diseased tissue were limited so the clinical diagnosis was used to classify normal duodenum tissue from normal esophageal tissue. Over 400 pairs of parameters demonstrated k-means can identify duodenum tissue with 87.5 % sensitivity and 87.5 % specificity or better. With some dimensional axis transformations these results could be improved. The lifetimes are not factors here but the relative intensity and decay shape were. Protocols can be applied to diseased or other tissue types with little adaptation. Just a single set of parameters may hold the key to help surgeons choose optimum locations for traditional biopsies or perhaps one day replace them altogether.</p> / Master of Applied Science (MASc)

<p>Quantitatively measuring oxygen saturation is important to characterize the physiological or pathological state of tissue function. In this thesis, we demonstrate the possibility of using susceptibility mapping to noninvasively estimate the venous blood oxygen saturation level. Accurate susceptibility quantification is the key to oxygen saturation quantification. Two approaches are presented in this thesis to generate accurate and artifact free susceptibility maps (SM): a regularized inverse filter and a k-space iterative method. Using the regularized inverse filter, with sufficient resolution, major veins in the brain can be visualized. We found that different sized vessels show a different level of contrast depending on their partial volume effects; larger vessels show a bias toward a reduced susceptibility approaching 90% of the expected value. Also, streaking artifacts associated with high susceptibility structures such as veins are obvious in the reconstructed SM. To further improve susceptibility quantification and reduce the streaking artifacts in the SMs, we proposed a threshold-based k-space iterative approach that used geometric information from the SM itself as a constraint to overcome the ill-posed nature of the inverse filter. Both simulations and in vivo results show that most streaking artifacts inside the SM were suppressed by the iterative approach. In simulated data, the bias toward lower mean susceptibility values inside vessels has been shown to decrease from around 10% to 2% when choosing an appropriate threshold value for the proposed iterative method, which brings us one step closer to a practical means to map out oxygen saturation in the brain.</p> / Doctor of Philosophy (PhD)

<p>The annual incidence rate of tumours in the brain and central nervous system (CNS) was 19.89 per 100,000 persons between 2004 and 2008 in the United States. Surgery is a common treatment option for brain and CNS tumours. Typically, biopsy followed by histological analysis is used to confirm tumour types and margin during neurosurgery as an intraoperative diagnostic tool. However, this biopsy method is invasive, sampling number limited and not in real-time. To overcome these problems, many minimally invasive optical techniques, called optical biopsies, have been developed towards intraoperative diagnosis.</p> <p>The research work carried out in this dissertation focuses on combining the time-resolved fluorescence (TRF) and diffuse reflectance (DR) spectroscopy towards intraoperative tumour margin detection in neurosurgery. Combining these two modalities allows us to obtain additional contrast features, thus potentially improving the diagnostic accuracy. To achieve this goal, first, a clinically compatible integrated TRF-DR spectroscopy instrument was developed for <em>in vivo</em> brain tumour study. An acousto-optical-tunable-filter-based spectrometer was designed to acquire the time-resolved fluorescence signal. A dual-modality fibre optic probe was used to collect the TRF and DR signals in a small volume. The system’s capabilities of resolving fluorescence spectrum and lifetime, and optical properties were characterized and validated using tissue phantoms. Second, in order to retrieve the fluorescence impulse response function accurately from measured fluorescence signals, a robust Laguerre-based deconvolution method was optimized by using the constrained linear least squares fitting and high order Laguerre function basis. This optimized Laguerre-based deconvolution method overcomes the over-fitting problem introduced by low signal-to-noise ratio and complex fitting model. Third, an <em>ex vivo</em> clinical study of brain tumours was carried out using the TRF and DR spectroscopy. Fluorescence spectra and lifetime features were selected to classify various tumour types. The sensitivity and specificity of meningioma grade I differentiated from meningioma grade II are both 100%. Finally, in order to increase the measurement tissue volume and obtain imaging contrast features, a scanning-based hyperspectral fluorescence lifetime imaging system was developed. This setup can provide time-, space-, spectrum- resolved multi-dimensional images for tumour margin detection.</p> / Doctor of Philosophy (PhD)

Quantitative microscopy deals with the extraction of quantitative measurements from samples observed under a microscope. Recent developments in microscopy systems, sample preparation and handling techniques have enabled high throughput biological experiments resulting in large amounts of image data, at biological scales ranging from subcellular structures such as fluorescently tagged nucleic acid sequences to whole organisms such as zebrafish embryos. Consequently, methods and algorithms for automated quantitative analysis of these images have become increasingly important. These methods range from traditional image analysis techniques to use of deep learning architectures. Many biomedical microscopy assays result in fluorescent spots. Robust detection and precise localization of these spots are two important, albeit sometimes overlapping, areas for application of quantitative image analysis. We demonstrate the use of popular deep learning architectures for spot detection and compare them against more traditional parametric model-based approaches. Moreover, we quantify the effect of pre-training and change in the size of training sets on detection performance. Thereafter, we determine the potential of training deep networks on synthetic and semi-synthetic datasets and their comparison with networks trained on manually annotated real data. In addition, we present a two-alternative forced-choice based tool for assisting in manual annotation of real image data. On a spot localization track, we parallelize a popular compressed sensing based localization method and evaluate its performance in conjunction with different optimizers, noise conditions and spot densities. We investigate its sensitivity to different point spread function estimates. Zebrafish is an important model organism, attractive for whole-organism image-based assays for drug discovery campaigns. The effect of drug-induced neuronal damage may be expressed in the form of zebrafish shape deformation. First, we present an automated method for accurate quantification of tail deformations in multi-fish micro-plate wells using image analysis techniques such as illumination correction, segmentation, generation of branch-free skeletons of partial tail-segments and their fusion to generate complete tails. Later, we demonstrate the use of a deep learning-based pipeline for classifying micro-plate wells as either drug-affected or negative controls, resulting in competitive performance, and compare the performance from deep learning against that from traditional image analysis approaches.

Chapter 1 includes the Introduction and literature review which describes current developments within the field of in vitro/vivo imaging of cancers, with a particular emphasis on the techniques employing fluorescence emission-based spectroscopy and imaging modalities. Examples are cited whereby graphene and its congeners have been used in conjunction with various fluorophores and peptide sequences as a means of achieving highly specific imaging probes. This section discusses aspects of energy transfer and the possibility that molecular probes can be designed to achieve both therapeutic goals and diagnosis (Theranostics). This review concludes with a discussion of the use of organic supramolecularly assembled imaging agents as a means of achieving thermodynamically controlled nano-constructs for the functionalisation of graphenes and their potential future applications as theranostic agents. Chapters 2, 3 and 4 describe the synthesis of chiral and naphthalene diimides (NDIs) which are fluorescent. Spectroscopic investigations in the solution phase are described and the propensity for aggregation in these systems is discussed. The specific nature of the self-assembly processes involved is explored in different solvent systems and in the solid state. Fluorescence lifetime imaging microscopy (FLIM) and laser scanning confocal microscopy (LSCM) are used to investigate the cellular uptake of the NDI molecules and their capacity to image living prostate cancer cells (PC-3). The NDIs are subsequently complexed supramolecularly to poly-aromatic carbon systems such as C60 and coronene (Chapter 3), as well as thermally reduced graphene oxide (Chapter 4). Chapter 3 describes the explorations into the modelling of the donor-acceptor interactions between the NDIs and the C60/coronene in order to establish binding stoichiometry and association constants. Both Chapters 3 and 4 discuss fluorescence titration and time correlated single photon counting (TCSPC) experiments which were performed as a means of establishing the presence of excited state energy transfer mechanisms. The chapters conclude with investigations in living cells in order to establish retention of in vitro fluorescence, with particular attention being paid to confirming the graphene complex stability. Chapter 5 describes the synthesis and functionalisation of a seven amino acid sequence peptide known as the G-receptor protein (GRP) binding unit of the polypeptide bombesin. The sequence binds GRPs that are known to be up-regulated in prostate cancer carcinoma and it has been widely utilised in the literature as a means of enhancing the up-take of various cancer imaging agents that employ a variety of imaging modalities. The peptide was attached to the fluorescent NDIs via carbodiimide activation protocols with the purpose of providing added specificity to the imaging agent with respect to PC-3 cells. Prior to NDI derivatisation with bombesin, electrochemical impedance spectroscopy (EIS) has been performed to establish the extent to which the peptide sequence binds to prostate cancer cells over healthy ones. The chapter concludes with confocal microscopy of the bombesin derivative NDI complexed to thermally reduced graphene oxide as a means of validating the utility of the fluorescent targeted bioconjugate as synthetic scaffolds for future early diagnosis and sensing devices for prostate cancer. Chapter 6 constitutes the summary of this work and highlights several possible areas of future developments of relevance to the results discussed and related future experiments proposed to fully validate the device assembly for prostate cancer sensing. Chapter 7 contains the Experimental section and the relevant data gathered over the course of the investigations. Additional supporting figures or data referred to but not included in the main text of the thesis are reported in the Appendices.

Positron Emission Tomography (PET) is an imaging modality that has become increasingly beneficial in Radiotherapy by improving treatment planning (1). PET reveals tumor volumes that are not well visualized on computed tomography CT or MRI, recognizes metastatic disease, and assesses radiotherapy treatment (1). It also reveals areas of the tumor that are more radiosensitive allowing for dose painting - a non-homogenous dose treatment across the tumor (1). However, PET is not without limitations. The quantitative unit of PET images, the Standardized Uptake Value (SUV), is affected by many factors such as reconstruction algorithm, patient weight, and tracer uptake time (2). In fact, PET is so sensitive that a patient imaged twice in a single day on the same machine and same protocol will produce different SUV values. The objective of this research was to increase the capabilities of PET by exploring other quantitative PET/CT measures for Radiotherapy treatment applications. The technique of quantitative image feature analysis, nowadays known as radiomics, was applied to PET and CT images. Image features were then extracted from PET/CT images and how the features differed between conventional and respiratory-gated PET/CT images in lung cancer was analyzed. The influence of noise on image features was analyzed by applying uncorrelated, Gaussian noise to PET/CT images and measuring how significantly noise affected features. Quantitative PET/CT measures outside of image feature analysis were also investigated. The correlation of esophageal metabolic tumor volumes (tumor volume demonstrating high metabolic uptake) and endoscopically implanted fiducial markers was studied. It was found that certain image features differed greatly between conventional and respiratory-gated PET/CT. The differences were mainly due to the effect of respiratory motion including affine motion, rotational motion and tumor deformation. Also, certain feature groups were more affected by noise than others. For instance, contour-dependent shape features exhibited the least change with noise. Comparatively, GLSZM features exhibited the greatest change with added noise. Discordance was discovered between the inferior and superior tumor fiducial markers and metabolic tumor volume (MTV). This demonstrated a need for both fiducial markers and MTV to provide a comprehensive view of a tumor. These studies called attention to the differences in features caused by factors such as motion, acquisition parameters, and noise, etc. Investigators should be aware of these effects. PET/CT radiomic features are indeed highly affected by noise and motion. For accurate clinical use, these effects must be account by investigators and future clinical users. Further investigation is warranted towards the standardization of PET/CT radiomic feature acquisition and clinical application.

Les bactéries sont les organismes les plus abondants dans le monde. Des études sur les bactéries peuvent être bénéfiques pour la recherche médicale, la qualité des ressources en eau et l'industrie alimentaire. La détection et le marquage fluorescent est une des méthodes les plus utilisées pour des objectifs bioanalytiques. Dans la recherche de marqueurs luminescents et stables, des nouvelles nanoparticules fluorescentes et auto-stabilisées à base de polymères (FNPs, 60 nm) et des chaînes de polymères fluorescents (FPCs, 5nm) ont été développées. Dans un premier chapitre, une méthodologie pour insérer ces FNPs dans la bactérie E.coli a été développée. Pour contrôler si les FNPs sont en effet internalisé, nous avons développé un protocole basé sur l'extinction de luminescence des FNPs par le bleu de méthylène. Dans un second chapitre, les biotines conjuguées de FNPs peuvent être utilisées pour étudier les protéines membranaires spécifiques. En utilisant un lien streptavidine-biotine, un "sandwich" est formé pour construire un pont entre des particules, des anticorps spécifiques et des bactéries. Les images de fluorescence SPR et les images SEM ont démontré l'interaction de la biotine conjuguée de FNPs avec la bactérie E.coli. Dans un troisième chapitre, les chaînes de polymères fluorescents de couleur verte (GFPCs) peuvent facilement entrer dans des bactéries E.coli. Les GFPCs peuvent marquer le cyctoplasme mais pas l'ADN. Les chaînes de polymères fluorescents de couleur rouge (RFPCs) peuvent marquer facilement et efficacement la membrane de bactérie E.coli. Les deux FPCs sont extrêmement brillantes et non toxiques, les chaînes sont solubles dans l'eau. Ce sont de nouveaux matériaux fluorescents pour le marquage interne et externe des bactéries. Dans le dernier chapitre, il est démontré que les FANPs sont sensibles au pH et peuvent être utilisées pour mesurer la croissance de la bactérie E.coli. Les nano-objets détectent rapidement et précisément la croissance des cellules. En effet, leur fluorescence est sensible au changement de pH résultant du métabolisme cellulaire. De plus, ces particules permettent une surveillance en continu d'un grand nombre d'échantillons pour des applications de criblage à haut débit. Les nanomatériaux présentés dans ce manuscrit sont des outils prometteurs pour les applications en biocapteurs et bioimagerie en raison de leur luminosité/brillance et photostabilité élevées ainsi que les possibilités de post-fonctionnalisation. / Bacteria are the most abundant organisms in the world. Investigations and studies on bacteria can be beneficial to medical research, water resources research and food industry. Fluorescent sensing and labeling are commonly used for bioanalytical purposes. In the quest for very bright and stable labels, novel polymer-based, self-stabilized, fluorescent nanoparticles (FNPs, 60 nm) and fluorescent polymer chains (FPCs, 5 nm) have been developed. In the first part, a methodology to insert these FNPs into E.coli bacteria was developed. To control if the FNPs are indeed internalized, we developed a protocol based upon FNPs luminescence quenching by methylene blue. In the second part, a "sandwich" system is built. By using a streptavidin-biotin link, a bridge between particles (FNP), specific antibodies and bacteria is built. SPR, fluorescent images and SEM images demonstrated the interaction of biotin conjugated FNPs with E.coli bacteria. In the third part, interactions of fluorescent polymer chains with bacteria are investigated. Green fluorescent polymer chains (GFPCs) can easily enter into E.coli bacteria. GFPCs can label the cytoplasm but not the DNA. Red fluorescent polymer chains (RFPCs) can label the membrane of E.coli bacteria easily and efficiently. Both FPCs are highly water-soluble, bright and non-toxic, they are novel fluorescent labels for internal and external biological labeling of bacteria. In the last part, it is demonstrated that pH sensitive FANPs can be used to measure the growth of E.coli. They detect rapidly and accurately bacterial growth by signaling the change of pH resulting from cellular metabolism. Moreover, these particles allow for continuous monitoring a large number of samples for high-throughput screening applications. The studied fluorescent nanomaterials are promising tools for biosensing and bioimaging applications due to their brightness, high photostability and rich functionalisation ability.

Les systèmes théranostiques, consistant en un dispositif unique contenant des agents de diagnostic et des principes actifs, suscitent un interêt accru car ils peuvent améliorer le traitement de maladies telles que le cancer en réduisant les effets secondaires des médicaments et en permettant un suivi du traitement. L’objectif de ce travail était d’insérer des Quantum Dots (QDs) à base de ZnO dans des nanoparticules lipidiques pouvant délivrer un principe actif anti-cancéreux. Nous avons d’abord cherché à synthétiser des QDs présentant une structure coeur-coquille ZnO/ZnS pour améliorer leurs propriétés de luminescence. La spectroscopie d’absorption des rayons X, associée à des techniques usuelles de caractérisation, a permis de déterminer les conditions de synthèse conduisant à la formation d’une structure coeur-coquille. Néanmoins, l’émission dans le visible de ces QDs n’était pas satisfaisante. Des QDs dopés par des ions Mg ont donc été synthétisés. L’intensité de leur luminescence passe par un maximum pour une concentration molaire nominale d’ions Mg dans le milieu de réaction égale à 20%. Les QDs Zn0.8Mg0.2O présentent un rendement quantique (QY) six fois plus grand (QY ~64%) que celui des QDs de ZnO non dopés (QY ~ 10%). Les QDs dont la surface a été modifiée par de l’acide oléique (OA) forment une suspension colloidale stable dans le chloroforme et le toluène. Le rendement quantique des QDs OA-Zn0.8Mg0.2O était environ quatre fois plus élevé (Qy ~40%) que celui des QDs OA-ZnO. Les QDs Zn0.8Mg0.2O et OA-Zn0.8Mg0.2O ont été incorporés dans des nanoparticules lipidiques ayant un diamètre hydrodynamique moyen de l’ordre de 100- 220 nm. Les nanoparticules lipidiques solides (SLN) contenant des QDs sont restées stables dans différents milieux biologiques pendant trois heures à 37°C. Des mesures de fluorescence sur des suspensions de macrophages J774 ont montré une faible augmentation de l’intensité de l’émission visible pour les cellules incubées avec 2 mg/mL de SLNs luminescentes pendant 50 min, suggérant une internalisation partielle des nanoparticules par les macrophages. Malheureusement, ces résultats n’ont pas pu être confirmés par vidéo-microscopie et microscopie de fluorescence sur les cellules parce que les conditions expérimentales ( longueurs d’onde d’excitation et d’émission possibles) ne permettaient pas d’observer un signal supérieur à celui de l'auto-fluorescence des cellules. / Theranostic systems consist of a single device containing therapeutic and diagnosis agents and receive increased attention because these devices can improve the therapy of diseases such as cancer, decreasing the toxic side effects and permitting to monitor the treatment. The aim of this work was to develop theranostic systems consisting of lipid based nanocarriers containing ZnO based quantum dots (QDs) as luminescent probes, and allowing to encapsulate a model drug for cancer therapy. Firstly, the synthesis of ZnO/ZnS QDs was studied, aiming to achieve improved luminescent properties. In this step, X-Rays Absorption Spectroscopy, together with other usual characterization techniques, could identify the synthesis condition in which core-shell structures were formed. Nevertheless, the emission of ZnO/ZnS QDs in the visible range was not promising. Therefore, Mg-doped ZnO QDs were synthesized; their luminescence went through a maximum for a 20 mol% nominal concentration of Mg2+ ions in the reaction medium. Zn0.8Mg0.2O QDs presented quantum yield (QY) six times higher (QY = 64%) than undoped ones (QY = 10%). ZnO and Zn0.8Mg0.2O QDs capped by oleic acid (OA) were synthesized and formed stable colloidal dispersions in chloroform and toluene. The QY of OA-Zn0.8Mg0.2O was about 4 times (around 40%) higher than that of the OA-ZnO QDs. Zn0.8Mg0.2O QDs and OA-Zn0.8Mg0.2O QDs could be incorporated into lipid based nanocarriers of average hydrodynamic diameter around 100 – 220 nm. The luminescent solid lipid nanoparticles (SLN) were stable in different media at 37°C during 3 hours. The fluorescence study showed slightly enhanced emission of the J774 macrophage-like cells treated with 2 mg/mL of luminescent SLN during 50 min, suggesting partial internalization of the nanoparticles into the macrophages. However, the internalization studies using fluorescence video-microscopy and microscopy were not successful, because the equipment (wavelengths of excitation and emission) did not allow overcoming the cell auto-fluorescence phenomena.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22465号 / 工博第4726号 / 新制||工||1738(附属図書館) / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 関 修平, 教授 水落 憲和, 准教授 菅瀬 謙治, 教授 梶 弘典 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Exposure of hepatoma cell lines to the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) is serving as a model for a systems biological study concerning the response of cells to contaminant molecules. Several aspects of the cellular distribution of the aryl hydrocarbon receptor (AhR) and its ligand B[a]P have been addressed by different live cell imaging techniques: The intracellular distribution of the B[a]P/AhR complex is visualized by means of confocal laser scaning microscopy (cLSM) and the intracellular transport rates of the complex is investigated by fluorescence recovery after photobleaching (FRAP) technique. Furthermore, cLSM image stacks of living cells are generated for the modeling of three dimensional (3-D) cell geometries. In order to prevent photochemical damage of the living cells induced by UV excitation of B[a]P, visualization is done by B[a]P’s auto fluorescence using near infrared two-photon-excitation. Murine Hepatoma 1c1c7 cells are exposed to graded concentrations of B[a]P (50 nM to 20 μM) for different incubation time periods (15 minutes to 48 hours). The highest amounts of B[a]P were found in lipid droplets and lysosomes, where the B[a]P molecules are collected and form large aggregates. We were able to work with concentrations down to 50 nM corresponding to that used for genomic and proteomic investigations. Also, for the first time imaging of B[a]P metabolites inside lipid droplets is presented in this work. The data and the model developed in this study will provide new insights into the systematic regulation of the B[a]P, the AhR as well as the receptor-ligand-complex pathway and the study will also serve as a prototype for elucidating other stress response pathways in the future.