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Rozprawy doktorskie na temat „Fluorescent Bioimaging”

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Data publikacji: 7 września 2023

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1

Choi, Angela On Ki. "Fluorescent nanocrystals for bioimaging". Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114126.

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Optical imaging based on fluorescence has yet to be introduced as a clinical diagnostic tool due to the lack of reliable, photostable, and highly luminescent fluorophores. Fluorescent nanocrystals, or quantum dots (QDs), are promising alternatives to organic dyes, since QDs are small in size, resistant to photo-bleaching, and have excellent and appropriate optical properties. The main objective of this work is to use QDs for real-time imaging in live animals. Widespread use of QDs in biology is currently limited due to their questionable biocompatibility, and to the fact that some nanocrystals contain heavy metals, which are potentially hazardous, in their cores. In the present studies the mechanisms underlying the toxicity of cadmium telluride QDs was investigated in several stable cell lines. After long-term exposure to QDs, significant morphological and functional changes were observed at the cellular and subcellular levels. We showed that QD-induced toxicity includes the production of reactive oxygen species, peroxidation of membrane lipids, impairment of mitochondrial function, and changes in the genome and epigenome. Understanding how toxic QDs cause damage to the cells is a first step for i) the establishment of protocols to evaluate the safety of other nanomaterials, and ii) the development of new or improved nanocrystals that are non-toxic. We showed that modifications on QD surfaces with small drug molecules (e.g. N-acetylcysteine) or synthetic polymers can significantly decrease their toxicity, and in some cases, even render the QDs non-toxic. Utilizing a non-invasive route (i.e. intranasal) to deliver nano-probes and nano-therapeutics to the brain, we demonstrated the use of near-infrared fluorescence of non-toxic QDs to image cerebral microlesions in live animals. Repeated imaging in vivo allowed for the live monitoring of lesion size in animals; a reduction of lesion size is a measure of the effectiveness of nano-therapeutic interventions. Animals treated with micelle-incorporated nimodipine or minocycline had significantly smaller lesion volumes, and displayed better recovery of motor function. Quantitative evaluation and volume calculations were possible since the QD signal was isolated from autofluorescence and background after fluorescence lifetime gating. Taken together, the results from this work contribute to the development of QDs and fluorescence technology for biomedical imaging in two main ways: 1) by presenting in vitro measures as the first step in the evaluation of nanomaterial safety. 2) by demonstrating the advantages of using near-infrared QDs for non-invasive lifetime imaging in animals with unilateral cortical ischemic microlesions and for the determination of the spatio-temporal reduction of lesions upon nano-therapeutic interventions. These findings support the use of carefully designed and rigorously tested fluorescent QDs for lifetime optical imaging of the brain in experimental animals, and eventually extending to clinical studies.
L'imagerie par fluorescence reste à introduire dans les cabinets médicaux en raison du manque de fluorophores photo-stables, à haute intensité lumineuse, disponibles sur le marché. Les nanocristaux fluorescents ou boîtes quantiques (BQ), représentent une alternative intéressante par rapport aux teintures organiques car les BQ sont très petits, résistants au photoblanchiment et ont d'excellentes propriétés optiques. L'objectif principal de cette étude est d'utiliser les BQ pour une imagerie en temps réel sur les animaux vivants. L'usage étendu des BQ en biologie est limité en raison de leur biocompatibilité discutable et également en raison du fait que quelques nanocristaux sont composés en partie de métaux lourds. Dans cette étude, les mécanismes cellulaires impliquant la toxicité des BQ de cadmium telluride sont examinés. Après une exposition prolongée aux BQ, des modifications morphologiques et fonctionnelles significatives ont été observées à l'échelle cellulaire et infracellulaire. Nous démontrons que la toxicité induite par les BQ peut entrainer la production d'espèces réactives de l'oxygène, la peroxydation des lipides de la membrane biologique, l'altération du fonctionnement mitochondrial mais aussi des changements du génome et de l'épigénome. Comprendre comment les BQ toxiques endommagent les cellules est un premier pas dans l'établissement de protocoles d'évaluation de la sécurité des nanomatériaux et dans le développement de nouveau nanocristaux non-toxiques. Nous démontrons que la modification de la surface des BQ grâce à des médicaments (ex : N-acetylcysteine) ou des polymères synthétiques peut grandement diminuer leur toxicité, et dans quelques cas, peut aussi rendre les BQ non-toxiques. En utilisant de tel BQ non-toxiques, nous effectuons une démonstration de l'utilisation de la fluorescence infrarouge proche pour effectuer des clichés en temps réel de microlésions cérébrales sur des animaux vivants, à l'aide de méthodes non effractives (ex : voie intra-nasale) pour insérer des nano-sondes ou administrer des nano-thérapies au niveau du cerveau. Des imageries répétées permettent de surveiller la taille des lésions sur les animaux, et prouvent l'efficacité des nano-thérapies dans la prévention de l'expansion de la lésion. Les animaux traités par micelles chargées de nimodipine ou de minocycline ont des lésions moins volumineuses et une meilleure récupération de la fonction motrice. Une évaluation quantitative et un calcul de volume ont été possibles car le signal BQ était séparé de l'autofluorescence tissulaire grâce à de la synchronisation d'image fondé sur la durée de vie fluorescence. L'ensemble des résultats de ces études contribue au développement des BQ et des technologies par fluorescence en imagerie biomédicale, et ceci de deux façons : 1) en présentant des résultats in vitro qui constituent une première étape dans l'évaluation de la sécurité des nanomatériaux. 2) en démontrant des avantages de l'utilisation les BQ infrarouges proches pour l'imagerie non effractives sur les animaux vivants avec des lésions cérébrales et pour la détermination de la réduction des lésions après des nano-thérapies. Ces constatations appuient l'utilisation des BQ fluorescentes créés avec soin et ayant subi des essais précliniques rigoureux pour l'imagerie encéphalique in vivo et s'étendant finalement aux études cliniques.
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2

Si, Yang. "Fluorescent Nanomaterials for Bioimaging and Biosensing : Application on E.coli Bacteria". Thesis, Cachan, Ecole normale supérieure, 2015. http://www.theses.fr/2015DENS0038/document.

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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
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3

Kong, Yifei. "Multifunctional fluorescent nanoparticle-bioconjugates : preparation, characterisation and bioimaging applications". Thesis, University of Leeds, 2015. http://etheses.whiterose.ac.uk/12252/.

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4

Trofymchuk, Kateryna. "Controlled switching of fluorescent organic nanoparticles through energy transfer for bioimaging applications". Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAJ121/document.

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Les performances des techniques de bioimagerie et de biodétection peuvent être améliorées grâce aux nanoparticules fluorescentes (NPs) permettant un transfert d’énergie résonante de type Förster (FRET) efficace. Le but de mon projet de thèse est le développement de NPs polymériques brillantes et ultrastables encapsulant des fluorophores, capables de produire un FRET au-delà du rayon de Förster. Il a été montré que les groupements encombrés sont essentiels pour minimiser l’auto-extinction et le blanchiment des fluorophores encapsulés. Par ailleurs, la matrice polymérique joue un rôle crucial dans le contrôle de l’effet collaboratif entre fluorophores du au transfert d’énergie d’excitation. Puis, en utilisant cet effet collaboratif entre fluorophores, nous avons conçu des NPs présentant une photocommutation efficace, ainsi qu'un phénomène de "light harvesting" très important. Enfin, de très petites NPs avec un FRET efficace à leur surface ont été élaborées et appliquées pour la détection ultra-sensible de protéines. Les résultats obtenus fournissent de nouvelles perspectives dans le développement des nanoparticules brillantes avec un transfert d'énergie efficace, ainsi que des nano-sondes pour la détection de molécules uniques
Performance of biosensing and bioimaging techniques can be improved by fluorescent nanoparticles (NPs) capable of efficient Förster resonance energy transfer (FRET). The aim of my PhD project is to develop bright and photostable dye-loaded polymer NPs capable to undergo efficient FRET beyond the Förster radius. We showed that bulky groups are essential for minimizing self-quenching and bleaching of encapsulated dyes. Moreover, polymer matrix plays a crucial role in controlling the inter-fluorophore communication by excitation energy transfer. Then, by exploiting communication of dyes, we designed NPs exhibiting efficient photoswitching as well as giant light-harvesting. Finally, very small NPs with efficient FRET to their surface were developed and applied for ultra-sensitive molecule detection of proteins. The obtained results provide new insights in the development of bright nanoparticles with efficient energy transfer as well as nano-probes for single-molecule detection
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Mastrodonato, Cristiano Matteo. "Elaboration of fluorescent molecular probes and molecular-based nanoparticles for bioimaging purposes". Thesis, Bordeaux, 2017. http://www.theses.fr/2017BORD0652/document.

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Les techniques de fluorescence sont des outils de choix pour l’étude et la compréhension fine des processus biologiques. Ceci requiert toutefois l’utilisation de sondes fluorescentes parfaitement adaptées au but visé et répondant aux différentes exigences requises pour l’application visée. Dans ce cadre, nous nous sommes plus particulièrement intéressés à l’élaboration de sondes biphotoniques de pH adaptées à une mesure très sensible de faibles variations de pH autour du pH neutre. Les variations et gradients de pH sont en effet impliqués dans un certain nombre de processus biologiques importants et peuvent être associées à des dysfonctionnements liés à certaines maladies. Dans ce cadre, nous avons développé de nouvelles sondes fluorescentes de pH fluorescentes présentant à la fois un comportement ratiométrique, une forte sensibilité autour du pH neutre et facilement excitables dans le proche IR par absorption à deux photons. Ces sondes de structure quadrupolaire et bolamamphiphile permettent ainsi la détection ratiométrique du pH dans des environnements biologiques au moyen d'une excitation biphotonique dans le proche IR. En parallèle, nous nous sommes intéressés à l’élaboration de nanoparticules hyperbrillantes dédiées à l’imagerie biologique par microscopie de fluorescence induite par excitation à deux photons. Nous nous sommes plus particulièrement attachées au design de nanoparticules organiques fluorescentes constituées de molécules organiques de bas poids moléculaire (FONs). Cette approche offre en effet une grande flexibilité et la possibilité d’accéder à des nanosondes ayant des brillances comparables aux très populaires quantum dots mais moins toxiques et plus facilement dégradables. L’ingénierie moléculaire des fluorophores utilisés pour la préparation des FON est cruciale puisqu’elle influence fortement à la fois les propriétés photophysiques (brillance, couleur…) et leur propriétés physico-chimiques (stabilité chimique et structurale, stabilité colloïdale). Dans ce contexte, une librairie de nouveaux chromophores dipolaires a été synthétisée et utilisées pour la préparation de FON par la méthode de nano-précipitation. Leurs propriétés ont été étudiées afin de déterminer la relation entre la structure du chromophore et les propriétés globales des nanoparticules constituées de ces colorants. Ce travail a permis d’identifier les paramètres structuraux permettant d’accéder à des nanoparticules présentant à la fois une brillance exceptionnelle, une émission modulable du vert au rouge et proche IR et une remarquable stabilité colloïdale. Ces nanoparticules présentent des potentialités majeures pour l’imagerie in vivo par excitation et détection dans le proche IR
Fluorescence-based techniques are popular tools for the study and understanding of biological processes. This has prompted continuous research aimed at the development of a wide range of fluorescent probes specifically designed for specific applications. Among them, fluorescent pH probes are of much interest as pH variations or gradients are involved in many biological events and anomalous alterations are often related to the onset of dysfunctions and diseases. In this framework we have developed a series of promising two-photon pH fluorescent molecular probes. These quadrupolar bolaamphiphilic probes are of great interest, as they combine a steep pH dependence of their optical properties close to neutral pH, ratiometric behavior and large response to two-photon (2P) excitation in the NIR region. As such they offer much promise for ratiometric detection of the pH in biological environments and in situ monitoring of acidification. In parallel, we have been interest in the design of ultrabright nanoparticles for bioimaging purpose (in particular highly sensitive optical imaging). We chose to focus on Fluorescent Organic Nanoparticles made of organic molecules with low molecular weight (FONs) as they offer a flexible route and promising alternatives to toxic quantum dots. In this case the design of the dye used as building blocks of the FONs is of crucial importance and strongly influence the chemical and physical properties of the nanoparticles generated, such as their one and two-photon brightness and both their structural and colloidal stability. In that context a library of novel dipolar chromophores have been synthesized and used to prepare FONs using the nanoprecipitation method. Their properties were thoroughly investigated in order to determine the relationship between the molecular design of the isolated dye and the overall properties of the nanoparticles made of these dyes. As a result, Hyperbright FONs emitting in the green to NIR region and combining giant brightness and remarkable stability have been achieved. They offer major promise for bioimaging based on both excitation and detection in the NIR region
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Ardizzone, Antonio. "New fluorescent nanovesicles, by self-assembly of organic fluorophores, sterols and surfactants, as probes for bioimaging". Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/403924.

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El estudio de nuevas nanopartículas orgánicas fluorescentes (FONs) que puedan superar los límites de las comunes sondas fluorescentes como fluoróforos moleculares, proteínas fluorescentes y nanopartículas inorgánicas es un tema de gran interés para los científicos de materiales que desarrollan nuevas sondas para microscopía de fluorescencia y teranóstica. En los últimos años, se han desarrollado nuevas nanovesículas no liposómicas, basadas en el autoensamblaje de tensioactivos y esteroles, denominados Quatsomes (QSs), que constituyen unos prometedores vehículos de fármacos multifuncionales. Dentro de este escenario, el objetivo principal de esta Tesis (realizada en el marco del proyecto Marie Skłodowska-Curie ITN “Nano2Fun”) es explorar la posibilidad de utilizar los Quatsomes como vehículos para la nano-estructuración en medios acuosos de varias moléculas fluorescentes, independientemente de sus propiedades físico-químicas y ópticas, con el fin de obtener nuevas nanopartículas orgánicas fluorescentes (FONs) con elevada estabilidad coloidal y características fluorescentes optimales, especialmente en relación al brillo. El auto-ensamblaje de fluoróforos orgánicos moleculares, esteroles y tensioactivos de amonio cuaternario en Quatsomes fluorescentes se logró mediante el método DELOS-SUSP, un proceso a base de CO2 comprimido, que garantiza una composición de membrana y una disposición supramolecular altamente homogéneas y, como consecuencia, nanovesículas con elevadas propiedades ópticas. Se han explorado diferentes estrategias para la nano-estructuración en medios acuosos, por medio de QSs, de moléculas fluorescentes con diferentes propiedades fisicoquímicas, incluyendo aquellas solubles y no solubles en agua, analizando el impacto de la nano-estructuración sobre las propiedades ópticas de las FONs obtenidas. De esta manera, los fluoroforos aniónicos solubles en agua, como la fluoresceína, fueron nano-estructurados encima de los QSs. Por otra parte, los fluoróforos lipófilos y no solubles en agua, modificados con largas cadenas alquílicas pueden incorporarse de forma estable en la membrana de los QSs, como se muestra en el caso de varias familias de colorantes, por ejemplo, las cianinas, los diketopirrolopirroles (DPP) y los derivados del fluorene. Los QSs fluorescentes mostraron una estabilidad óptica coloidal excepcional (hasta varios meses), un alto grado de homogeneidad estructural y altas eficiencias de fluorescencia, mostrando mejores prestaciones en comparación con otras nanoestructuras de los mismos fluoroforos. Además, con el objetivo de obtener nanopartículas multicolores, los Quatsomes permitieron cargar simultáneamente diferentes fluoroforos dentro de sus membranas, mostrando un mecanismo de transferencia de energía de resonancia de fluorescencia (FRET) altamente eficiente, una interesante herramienta para monitorear la integridad del carrier durante la administración del fármaco y para la adquisición de imágenes multicolores. En conclusión, los Quatsomes fluorescentes se probaron como nano-sondas para la obtención de imágenes de células in vitro. Se ha demostrado que los Quatsomes que incorporan derivados del fluorene (denominados LysoQS) constituyen una sonda lisosómica altamente específica, ideal para la adquisición de imágenes en tiempos largos. Además, los Quatsomes cargados de cianinas se utilizaron como sondas para técnicas de microscopía superresolución (STORM), que permitió la visualización y resolución de Quatsomes individuales tras la internalización en las células. Los resultados de esta tesis muestran que los Quatsomes fluorescentes, gracias a las ventajas ofrecidas en comparación con otros marcadores fluorescentes comúnmente empleados, son unas nanosondas altamente prometedoras, con posibles aplicaciones futuras en bioimagen, teragnostica y, en general, nanomedicina.
Finding new fluorescent organic nanoparticles (FONs) with the potential to overcome the limits of common fluorescent probes as molecular fluorophores, fluorescent proteins and inorganic nanoparticles is a subject of strong interest for materials scientists developing new probes for fluorescence microscopy and theranostics. In the recent years, innovative non-liposomal nanovesicles, based on the self-assembly of quaternary ammonium surfactants and sterols, named Quatsomes (QSs), have been developed as promising candidates for applications as multifunctional drug carriers. Within this scenario, the main objective of this Thesis (conducted in the framework of the Marie Skłodowska-Curie ITN “Nano2Fun”) is to explore the possibility of using Quatsomes as a vehicle for nanostructuring in aqueous media several dye molecules, irrespective of their physicochemical and optical properties, in order to obtain new fluorescent organic nanoparticles (FONs) with superior colloidal stability and enhanced fluorescent features, especially with high brightness, in relation to single molecule flurofores and other type of FONS. The self-assembly of molecular organic fluorophores, sterols and quaternary ammonium surfactants into fluorescent Quatsomes was achieved by the DELOS-SUSP method, a compressed CO2 –based process which guarantees a highly homogeneous membrane composition and supramolecular arrangement, which have impact on the optical properties of the obtained FONs. Different strategies have been explored to nanostructurate in aqueous media, by mean of QSs, molecular dyes with different physicochemical properties, including those water- and non-water soluble, analyzing the impact of their nanostructuration on the optical properties of the obtained FONs. Thus, anionic water-soluble dyes, such as fluorescein, were nanostructured over QSs surface, taking advantage of anionic/cationic interaction among dye and vesicles surface. On the other hand, lipophilic and non-water soluble dyes modified with long alkyl chains can be stably incorporated into QSs membrane, as shown in the case of several dyes families, including cyanine, diketopyroolopyrrole (DPPs) and fluorene derivatives. The fluorescent QSs showed superior colloidal and optical stability (up to several months), a high degree of structural homogeneity and high fluorescence performances, overcoming those of other nanostructures of the same dyes. Furthermore, aiming to obtain multicolor nanoparticles, Quatsomes allowed the simultaneous loading within their membrane of different dyes, which showed a highly efficient fluorescence resonance energy transfer (FRET) mechanism, an interesting tool for monitoring the carrier integrity during the drug delivery and for multiplexed imaging applications. Finally, fluorescent Quatsomes were tested as nanoprobes for in vitro cells imaging. It has been demonstrated that fluorene-based Quatsomes (named LysoQS) constitute a strongly specific lysosomal probe ideal for long-term imaging. Furthermore, cyanines-loaded Quatsomes were used as probes for super-resolution microscopy technique (STORM) which allowed visualizing and resolving single Quatsomes structures upon internalization in cells. The results of this Thesis showed that fluorescent Quatsomes, thanks to the advantages offered in comparison with other commonly employed fluorescent labels, constitute a promising fluorescent nanoprobes with possible future applications in bioimaging, theranostics and, generally, nanomedicine.
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Nilsson, Ruben. "Optical properties of fluorescent quantum dots for super-resolution bioimaging". Thesis, KTH, Tillämpad fysik, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-169624.

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Lesani, Pooria. "Novel Carbon Dot-Based Fluorescent Nanomaterials for Biosensing and Bioimaging". Thesis, The University of Sydney, 2022. https://hdl.handle.net/2123/27346.

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Carbon dots (CDs)-based nanoparticles have been extensively explored for biological applications in sensing and bioimaging. However, the major translational barriers to CDs for imaging and sensing applications include optimal synthetic strategies to obtain monodisperse CDs with tunable structural, electronic, and optical properties in order to achieve high-resolution deep-tissue imaging, intracellular detection, and sensing of metal ions with high sensitivity down to nanomolar levels. In this thesis we have presented the synthesis and development of a series of novel carbon dot based nanoprobes with unique photophysical and biological properties for bioimaging and biosensing applications. These properties include water dispersibility, superior photostability and thermal stability, high quantum yield, excellent two-photon excitability, ease in surface functionalization, rapid cellular uptake, good biocompatibility, rapid detection of targeted molecules with a low detection limit, and high-resolution bioimaging capability. The CD-based probes developed in this study were used for two-photon intensity-based and ratiometric exogenous and endogenous ferric ions sensing in living cells, single- and two-photon deep tissue imaging in synthetic scaffold and complex biological tissue, and two-photon ratiometric real-time intracellular pH monitoring in 3D environment. Furthermore, the influence of CDs synthetic and post-synthetic parameters on parameters on photophysical properties and biological behavior of CDs were comprehensively investigated.
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Kilic, Nüzhet Inci. "Graphene Quantum Dots as Fluorescent and Passivation Agents for Multimodal Bioimaging". Thesis, KTH, Tillämpad fysik, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-298302.

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Zero-dimensional graphene (carbon) quantum dots have been drawing attention in bio-related applications since their discovery, especially for their optical properties, chemical stability, and easily modifiable surface.  This thesis focuses on the green synthesis of nitrogen-doped graphene quantum dots (GQDs) for dual-mode bioimaging with X-ray fluorescence (XRF) and optical fluorescence. Both conventional and microwave- (MW-)assisted solvothermal methods were followed to investigate the precursors’ effect on the synthesized GQDs. The MW-assisted method permitted the synthesis of uniform GQDs with an excitation-independent behavior, due to highly controllable reaction conditions. It was demonstrated that the molecular structure of the precursors influenced the optical fluorescence properties of the GQDs. Thus, both blue- (BQDs) and red-emitting (RQDs) GQDs were obtained by selecting specific precursors, leading to emission maxima at 438 and 605 nm under the excitation wavelengths of 390 and 585 nm, respectively.  Amine-functionalized Rh nanoparticles (NPs) were chosen as the X-ray fluorescence (XRF) active core, synthesized via MW-assisted hydrothermal method with a custom designed sugar ligand as the reducing agent. These NPs were conjugated with BQDs using EDC-NHS treatment. The hybrid Rh-GQDs NPs exhibited green emission (520 nm) under 490 nm excitation and led to a reduced cytotoxicity with respect to bare Rh NPs, highlighting the passivation role of the GQDs via the real-time cell analysis (RTCA) assay. The hybrid complex constituted a multimodal bioimaging contrastagent, tested with confocal microscopy (in vitro) and XRF phantom experiments.
Sedan deras upptäckt har nolldimensionella kvantprickar av grafen (kol) uppmärksammats inom biorelaterade applikationer, särskilt för deras optiska egenskaper, kemiska stabilitet och enkelt modifierbara yta. Denna avhandling fokuserar på en grön syntesmetod av kvävedopade grafen-kvantprickar för bimodal bioavbildning med röntgenfluorescens och optisk fluorescens. Både konventionella och mikrovågs-assisterade solvotermiska syntesmetoder användes för att undersöka metodernas effekt på de syntetiserade kvantprickarna. Den mikrovågs-assisterade metoden möjliggjorde syntes av uniforma kvantprickar med exciteringsoberoende egenskaper på grund av mycket kontrollerbara reaktionsförhållanden. Det demonstrerades att den molekylära strukturen hos prekursorerna påverkade de optiska fluorescensegenskaperna hos grafen-kvantprickarna. Genom att välja specifika prekursorer erhölls kvantprickar som emitterar i både blått och rött ljus, motsvarande emissionsmaxima vid 438 respektive 605 nm under excitering vid 390 respektive 585 nm. Amin-funktionaliserade Rh-nanopartiklar valdes som en aktiv kärna för röntgenfluorescens, syntetiserad genom en mikrovågs-assisterad hydrotermisk metod med en specialdesignad sockerligand som reduktionsmedel. Dessa nanopartiklar konjugerades med blåemitterande kvantprickar genom EDC-NHS-behandling. De hybrida nanopartiklarna uppvisade grön emission (520 nm) under 490 nm excitation och ledde till en minskad cytotoxicitet uppmätt genom cellanalys i realtid (RTCA) jämfört med endast Rh-nanopartiklar, vilket framhävde passiveringsrollen som kvantprickarna spelar. Hybridkomplexet utgjorde ett multimodalt kontrastmedel för bioavbildning, vilket demonstrerades med konfokalmikroskopi (in vitro) och fantomexperiment med röntgenfluorescens.
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10

Banerjee, Anusuya. "Novel, Targettable Bioimaging Probes Using Conjugates of Quantum Dots and DNA". Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066376/document.

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Les boîtes quantiques (ou Quantum Dots en anglais - QD) sont une nouvelle génération de sondes polyvalentes pour la biologie, en particulier pour l’imagerie. Pour des applications de marquage des voies intra-cellulaires, les QDs peuvent être conjugués à des biomolécules telles que des acides nucléiques ou des protéines. En partant des travaux du LPEM portant sur le développement de ligands permettant la dispersion des QDs dans l’eau et leur fonctionnalisation, une nouvelle méthode de conjugaison de l'ADN sur les QDs a été développée dans cette thèse. Cette méthode utilise les motifs présents sur les polymères des QDs pour le greffage d'ADN. Les paramètres affectant cette réaction ont été étudiés et cette stratégie de couplage a été étendue à d'autres nanoparticules et biomolécules. En partant de ces QDs-ADN, des protéines modifiées ADN ont pu être attachées aux QDs en utilisant le principe d’hybridation de l’ADN. Les propriétés des conjugués ainsi générés ont été mises en évidence en utilisant la Transferrine (QD-ADN-Tf) et ces complexes ont été étudiés in vitro et in cellulo. Ces conjugués ont ensuite été utilisés pour le suivi de la dynamique des endosomes, exploitant ainsi pleinement le potentiel des QDs pour l’imagerie directe. Dans la dernière partie, des études supplémentaires sur les facteurs influençant la «performance biologique» des QDs ont été réalisées. Pour cela, une large gamme de ligands polymères développée par le groupe a été utilisée pour sonder l'interaction de la surface des QDs avec l'interface biologique. Des expériences biochimiques et cellulaires ont permis de démontrer que les QDs revêtus de divers polymères ont des comportements différents
Quantum dots (QD) are new generation of versatile probes for biology, particularly for bioimaging. For specific applications, QDs are conjugated to biomolecules such as nucleic acid or proteins and subsequently targeted to unique intra-cellular pathways. Building upon the state-of-the-art ligands for water-dispersible QDs developed by the lab, a novel and highly generalizable method to conjugate DNA to QD is developed in this thesis. This method employs thiols present on polymers on QDs for conjugation to maleimide-functionalized DNA. Extensive characterization of parameters affecting this reaction is carried out and the strategy is extended to other nanoparticles and biomolecules. Following this, a novel method to conjugate proteins to QD via DNA hybridization is discussed. Using a model protein Transferrin (Tf), the unique properties of thus generated QD-DNA-Tf conjugates are studied in-vitro and in-cellulo. These conjugates are subsequently used for tracking endosomal dynamics for up-to 20 minutes, exploiting the fullest potential of QDs for live imaging. In the last part, additional studies on factors affecting the ‘biological performance’ of QDs are carried out. Using a range of highly adaptable polymeric ligands developed by the group, interactions of surface-modified QDs with the biological interface are probed. Systematic biochemical and cellular experiments demonstrate that QDs coated with zwitterionic polymers have superior antifouling properties compared to poly(ethylene glycol)-based polymers and stability in diverse biological contexts
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11

Kanyuk, M. I. "New Fluorescent Nanomaterial Based on Silver Atoms and Organic Dye for Biosensing and Bioimaging Applications". Thesis, Sumy State University, 2012. http://essuir.sumdu.edu.ua/handle/123456789/34966.

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We report on synthesis and studies of strongly fluorescent nanostructures formed of several Ag atoms with the aid of organic dye Thioflavin T that serves as sensitizer and molecular support. The 1:1 stoichiometry between silver atoms and dye molecules is observed in this complex formation. These novel structures are formed in a simple one-step way by photoreduction on illumination by UV light and are characterized by excitation and emission maxima at 340 nm and 450 nm correspondingly. These materials offer good prospects for different applications in biosensing and bioimaging technologies. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/34966
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12

Andrade, Carolina D. "Design, synthesis and characterization of new two-photon absorbing (2PA) fluorescent dyes and bioconjugates, and their applications in bioimaging". Doctoral diss., University of Central Florida, 2010. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4553.

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The development of new multiphoton absorbing materials has attracted the attention of researchers for the last two decades. The advantages that multiphoton absorbing materials offer, versus their one-photon absorbing counterparts, rely on the nature of the nonlinearity of the absorption process, where two photons are absorbed simultaneously offering increased 3D resolution, deeper penetration, and less photobleaching and photodamage as a result of a more confined excitation. The applications of efficient two-photon absorbing materials have been extensively expanding into the fields of photodynamic therapy, microscopy, and optical data storage. One of the fields where an increased interest in multiphoton absorbing materials has been most evident is in bioimaging, in particular, when different cellular processes and organelles need to be studied by fluorescence microscopy. The goal of this research was to develop efficient two-photon absorption (2PA) compounds to be used in fluorescence bioimaging, meaning that such compounds need to posses good optical properties, such as high fluorescence quantum yield, 2PA cross section, and photostability. In the first chapter of this dissertation, we describe the synthesis and structural characterization of a new series of fluorescent donor-acceptor and acceptor-acceptor molecules based on the fluorenyl ring system that incorporated functionalities such as alkynes and thiophene rings, through efficient Pd-catalyzed Sonogashira and Stille coupling reactions, in order to increase the length of the conjugation in our systems. These new molecules proved to have high two-photon absorption (2PA), and the effect of these functionalities on their 2PA cross section values was evaluated. Finally, their use in two-photon fluorescence microscopy (2PFM) imaging was demonstrated. One of the limitations of the compounds described in Chapter 1 was their poor water solubility; this issue was addressed in Chapter 2.; The use of micelles in drug delivery has been shown to be an area of increasing interest over the last decade. In the bioimaging field, it is key to have dye molecules with a high degree of water solubility to enable cells to uptake the dye. By enclosing a hydrophobic dye in Pluronic F-127 micelles, we developed a system that facilitates the use of 2PA molecules (typically hydrophobic) in biological systems for nonlinear biophotonic applications, specifically to image the lysosomes. Furthermore, we report in this chapter the efficient microwave-assisted synthesis of the dye used in this study. In addition, linear photophysical and photochemical parameters, two-photon absorption (2PA), and superfluorescence properties of the dye studied in Chapter 2, were investigated in Chapter 3. The steady-state absorption, fluorescence, and excitation anisotropy spectra of this dye were measured in several organic solvents and aqueous media. In Chapter 4, we describe the preparation and the use of an efficient and novel two-photon absorbing fluorescent probe conjugated to an antibody that confers selectivity towards the vascular endothelial growth factor receptor 2 (VEGFR-2) in porcine aortic endothelial cells that express this receptor (PAE-KDR). It is known that this receptor is overexpressed in certain cancer processes. Thus, targeting of this receptor will be useful to image the tumor vasculature. It was observed that when the dye was incubated with cells that do not express the receptor, no effective binding between the bioconjugate and the cells took place, resulting in very poor, nonspecific fluorescence images by both one and two-photon excitation. On the other hand, when the dye was incubated with cells that expressed VEGFR-2, efficient imaging of the cells was obtained, even at very low concentrations (0.4micrometer]). Moreover, incubation of the bioconjugate with tissue facilitated successful imaging of vasculature in mouse embryonic tissue.
ID: 030422918; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Thesis (Ph.D.)--University of Central Florida, 2010.; Includes bibliographical references (p. 145-158).
Ph.D.
Doctorate
Department of Chemistry
Sciences
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13

Huth, Katharina [Verfasser]. "Synthesis and characterization of water-soluble dendronized dye systems as fluorescent labels for bioimaging applications / Katharina Huth". Berlin : Freie Universität Berlin, 2018. http://d-nb.info/1171431244/34.

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14

Stadler, Charlotte. "Towards subcellular localization of the human proteome using bioimaging". Doctoral thesis, KTH, Proteomik, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-103616.

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Since the publication of the complete sequence of the human genome in 2003 there has been great interest in exploring the functions of the proteins encoded by the genes. To reveal the function of each and every protein, investigation of protein localization at the subcellular level has become a central focus in this research area, since the localization and function of a protein is closely related. The objective of the studies presented in this doctoral thesis was to systematically explore the human proteome at the subcellular level using bioimaging and to develop techniques for validation of the results obtained. A common imaging technique for protein detection is immunofluorescence (IF), where antibodies are used to target proteins in fixated cells. A fixation protocol suitable for large-scale IF studies was developed and optimized to work for a broad set of proteins. As the technique relies on antibodies, validation of their specificity to the target protein is crucial. A platform based on siRNA gene silencing in combination with IF was set-up to evaluate antibody specificity by quantitative image analysis before and after suppression of its target protein. As a proof of concept, the platform was then used for validation of 75 antibodies, proving it to be applicable for validation of antibodies in a systematic manner. Because of the fixation, there is a common concern about how well IF data reflects the in vivo subcellular distribution of proteins. To address this, 500 proteins were tagged with green fluorescent protein (GFP) and used to compare protein localization results between IF to those achieved using GFP tagged proteins in live cells. It was concluded that protein localization data from fixated cells satisfactory represented the situation in vivo and together exhibit a powerful approach for confirming localizations of yet uncharacterized proteins. Finally, a global analysis based on IF data of approximately 20 % of the human proteome was performed, providing a first overview of the subcellular landscape in three different cell lines. It was found that the intracellular distribution of proteins is complex, with many proteins occurring in several organelles. The results also confirmed the close relationship between protein function and localization, which in a way further strengthens the accuracy of the IF approach for detection of proteins at the subcellular level.

QC 20121017


The Human Protein Atlas
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15

Cepraga, Cristina. "Two-photon chromophore-polymer conjugates grafted onto gold nanoparticles as fluorescent probes for bioimaging and photodynamic therapy applications". Phd thesis, INSA de Lyon, 2012. http://tel.archives-ouvertes.fr/tel-00863765.

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Photodynamic therapy (PDT) is an alternative treatment of cancer requiring the use of chromophore molecules (photosensitizers), which can induce cell death after light excitation. Gold nanoparticles (AuNP), exhibiting localized Surface Plasmon Resonance, can enhance the photophysical response of chromophores located in their vicinity, and thus improve their therapeutic action. Moreover, the use of highly localized two-photon chromophores (photosensitizers and fluorophores), capable to undergo a localized excitation by light in the Near InfraRed region, should increase the penetration depth into tissues, thus improve the treatment efficiency (by PDT) and the imaging (by fluorescence microscopy) of cancer tissues.In this work, we describe the elaboration of water-soluble hybrid nano-objects for PDT and fluorescence bioimaging applications, composed of two-photon chromophore-polymer conjugates grafted onto gold nanoparticles. In order to obtain these nano-objects we follow a multistep strategy: i) the synthesis of a well-defined water-soluble chromophore-polymer conjugates; ii) the end-group oriented grafting of chromophore-polymer conjugates onto 20 nm AuNP. The coupling of hydrophobic two-photon chromophores on linear water-soluble copolymer chains (poly(N-acryloylmorpholine-co-N-acryloxysuccinimide)), obtained by controlled/living RAFT polymerization, resulted in well-defined water-soluble chromophore-polymer conjugates, with different polymer lengths (2 000 g.mol-1 < Mn < 37 000 g.mol-1) and architectures (random or block), and a controlled number of chromophores per chain (varying between 1 and 21). Their grafting onto 20 nm AuNP gave water-soluble hybrid nano-objects with high grafting densities (~0.5 chains/nm²). The role of the polymer chain being to tune the distance between chromophores and AuNP surface, we have evidenced the increase in the polymer corona thickness of grafted AuNP (estimated by TEM) with the increasing polymer Mn, corroborating with the corresponding distance-dependent fluorescence properties of those. Finally, the in cellulo biological properties of two-photon chromophore-polymer conjugates, before and after grafting onto AuNP, have been investigated, highlighting their potential for two-photon bioimaging and PDT applications.
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16

Tian, Yayang. "Elaboration of New Layer by Layer (LbL) Fluorescent thin films and their functionalization for the sensitive detection of bacteria". Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLN029/document.

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Les antibiotiques ont été utilisés pour le traitement des infections bactériennes depuis plus de 70 ans, sauvant des millions de vies. Cependant, leur mauvaise et sur-utilisation ont conduit à l’émergence de la résistance bactérienne. Outre le développement de nouvelles familles d'antibiotiques, la détection rapide et sensible de bactéries est très importante pour le diagnostic médical. Les polymères fluorescents représentent un grand potentiel, car ils sont faciles à fonctionnaliser, synthétiser et greffer. Les films sont plus pratiques, faciles à manipuler et peuvent être réutilisés, ce qui n'est pas le cas des méthodes de détection en solution. L’objectif de ce travail est de développer un film de polymère nanostructuré fluorescent et sensible sur des surfaces de verre pour la détection bactérienne. Sur la base de la méthode de polymérisation radicalaire par transfert de chaîne réversible par addition-fragmentation (RAFT), trois types de polyélectrolytes fluorescents à base de BODIPY (FPC) ont été synthétisés : des chaînes relativement courtes à caractère polyélectrolyte faible (SW FPC), des chaînes courtes à caractère polyélectrolyte fort (SS FPC) et enfin des chaînes longues à caractère polyélectrolyte faible (FPC LW). Les films FPC LbL ont été élaborés sur des lames en verre par interaction électrostatique. Les propriétés photophysiques et de surface des FPC LbL ont été contrôlées en ajustant les conditions de dépôt. Les films FPC LbL à base de BODIPY ont été utilisés comme dispositif de première génération pour la détection de E. coli. Dans l'étape suivante, la sensibilité des films a été augmentée en utilisant le principe de fluorescence exaltée par plasmon (Metal Enhanced Fluorescence MEF). Un film LbL -MEF a été préparé et testé pour la détection de bactéries. Des nanoparticules d'or sphériques (Au NPs) ont été synthétisées et recouvertes de poly(chlorhydrate d'allylamine) (PAH). Le FPC LW a été sélectionné comme couche fluorescente. Différents films contenant des Au NPs et LW FPC- ont été fabriqués. La distance entre les NPs Au et LW FPC- a été ajustée par l'ajout de deux polymères de charge opposée (PC + et PC-). Les deux surfaces de AuNP / 4 couches PC / LWFPC- et Au NPs / 8 couches PC / LWFPC- ont montré que E. coli peut être ciblée par LW FPC-.La sélectivité des films LbL a été ajoutée en introduisant un anticorps comme site de reconnaissance spécifique. Le polyanion et le polycation avec le groupe fonctionnel 4-dibenzocyclooctynol (DIBO) ont été assemblés sur des lames de verre activées. L'anticorps anti-E. coli a ensuite été introduit sur la surface en une seule étape via la réaction de cycloaddition azide-alcyne (SPAAC). Le nombre d'E. coli capturées dépend de la concentration d’anticorps sur la surface. La surface a montré une sélectivité significative pour E. coli, comparée à B. subtilis.La croissance bactérienne peut être détectée sur un film mince LbL en introduisant un fluorophore sensible au pH (fluorescéine). En effet, la croissance des bactéries est souvent associée à une diminution du pH du milieu due à une libération de métabolites acides. Nous avons préparé avec succès différents types de films LbL sensibles au pH. Dans un premier temps, la synthèse de différents polyanions fonctionnalisés (chaîne courte et longue de DIBO-PC et polymère fluorescent rouge) a été achevée. Ensuite, trois types de surfaces sensibles au pH contenant de la fluorescéine (DIBO-SWPC- / fluorescéine, DIBO-LW PC- / fluorescéine et ratiométrique RFPC- / fluorescéine) ont été préparés sur la base d'assemblage LbL et de chimie click. Enfin, trois surfaces sensibles au pH ont été étudiées pour la détection de la croissance des bactéries. Toutes les surfaces étaient biocompatibles, le nombre de E. coli augmentait même après plusieurs heures d'incubation sur chaque surface. La détection par le changement de fluorescence est en cours de développement
Antibiotics have been used for the treatment of bacterial infections for over 70 years, saving millions of lives. The current antibiotic resistance crisis has been attributed to the overuse and misuse of these medications. Therefore, the prevention of infection transmission by the rapid and sensitive detection of antibiotic resistant strains is needed in managing this crisis. Fluorescent polymers show great potential for bacteria detection, because they are easy to functionalize, reproduce and graft. Compared with the methods used for bacterial detection in liquid, bacterial detection on a film surface is more convenient, easier to handle and is applied in devices that can be easily reused. The goal of my PhD work is to develop fluorescent and sensitive nanostructured polymer films on surfaces for bacterial detection. Three types of BODIPY-based fluorescent polyelectrolytes (FPC) with different features were synthetized based on reversible addition-fragmentation transfer (RAFT) polymerization: relatively Short chains and Weak polyelectrolytes (SW FPC), Short chains and Strong polyelectrolytes (SS FPCs) and Long chains and Weak polyelectrolytes (LW FPCs). FPC LbL films were fabricated on activated glass slides by means of electrostatic attraction. The photophysical and surface properties of FPC LbL fims were easily controlled by adjusting the deposition conditions.The following step aimed at increasing the films’ sensitivity by using the metal-enhanced fluorescence (MEF) principle. A MEF based LbL film was prepared and tested for bacteria detection. Spherical gold nanoparticles (Au NPs) were synthesized and coated with poly(allylamine hydrochloride) (PAH). The LW FPC- was selected as the fluorescent layer. Different films containing Au NPs and LW FPC- were fabricated and the distance between the Au NPs and LW FPC- was adjusted by changing the numbers of layers with two oppositely charged polymers (PC+ and PC-). Both Au NPs/4 layers PCs/LWFPC- and Au NPs/8 layers PCs/LWFPC- surfaces indicated that E. coli can be detected by LW FPC-.The selectivity of LbL films was added by introducing an antibody on the surface of the film to provide specific recognition of a chosen bacterial strain. This LbL surface achieved a rapid, effective and specific detection of E. coli bacteria. The polyanion and polycation with a 4-dibenzocyclooctynol (DIBO) functional group were assembled on the activated glass slides and an anti-E. coli antibody containing an azide group was efficiently introduced on the surface in a single step based on the azide-alkyne cycloadditions (SPAAC) reaction. The number of E. coli captured on the surface was shown to be dependent on the amount of antibody on the surface. The anti-E. coli antibody surface showed significant selectivity for E. coli, compared with B. subtilis. An alternative approach is to detect bacterial growth on thin LbL film by introducing pH sensitive fluorophore (fluorescein). The growth of bacteria is often associated with a decrease in pH of the growth medium due to a release of acidic metabolites. Different types of pH sensitive LbL film were prepared and tested for the detection of bacterial growth. Firstly, the synthesis of different functionalized polyanions (short and long chain of DIBO-PC- and red fluorescent polymer) was carried out. Three types of pH sensitive surfaces containing fluorescein (DIBO-SWPC-/fluorescein, DIBO-LW PC-/fluorescein and ratiometric RFPC-/fluorescein surfaces) were prepared based on the combination of LbL assembly and copper-free click chemistry. Finally, three pH sensitive surfaces were studied for bacteria growth detection. All the surfaces were shown to be biocompatible, the number of E. coli increased after several hours of incubation on each surface, as detected by brightfield microscopy imaging. The application for the fluorophore-dependent detection of bacterial growth remains to be developed
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17

Shulov, Ievgen. "Synthesis of fluorescent organic nanoparticles for biological applications". Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAJ001/document.

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Boîtes quantiques (QDs) et nanoparticules fluorescentes de silice (NPs) ont influencé le domaine de la bioimagerie de par leur forte luminosité et photostabilité. Par rapport aux QDs, les NPs organiques peuvent s’avérer être encore plus brillantes et entièrement biodégradables, avec une bonne biocompatibilité et sans contenir aucun élément toxique. Nous avons développé quatre types de ces NPs : en premier, des nano-gouttelettes lipidiques chargées de colorants lipophiles (flavone et Nil Rouge) pour l'imagerie in vivo chez le poisson zèbre ; en second, l’association ionique entre rhodamine B alkylée et tétraphénylborate fluoré (TPB) donne des NPs de 11-20 nm avec un rendement quantique de ~60% ; une troisième type de NPs consiste en des micelles de 7 nm obtenus par co-assemblage de cyanine amphiphiles et contre-ions TPB ; enfin, la polymérisation de micelles de calix[4]arène par agents de réticulation bi-fonctionnels à base de cyanine donne des NPs de 7 nm présentant un comportement fluorogène et une bonne stabilité en milieu intracellulaire. Ces NPs plus brillantes et de taille inférieure aux QDs apparaissent comme des outils prometteurs en bioimagerie
Quantum dots (QDs) and fluorescent silica nanoparticles (NPs) have impacted the domain of bioimaging by their high brightness and robust photostability. In comparison to QDs, organic NPs can be even brighter and fully biodegradable, as well biocompatible and not containing toxic elements inside. Herein, we developed four types of these NPs. At first, lipid nano-droplets loaded with lipophilic flavone and Nile Red dyes for in vivo imaging in zebrafish; second, ion-association of alkyl rhodamine B with fluorinated tetraphenylborate (TPB) counterions result in 11-20 nm NPs with fluorescence quantum yield up to 60%; third, 7 nm micellar NPs obtained by co-assembly of cyanine amphiphiles with TPB counterions; finally, polymerization of calix[4]arene micelles using bi-functional cyanine crosslinkers giving 7 nm NPs, that show fluorogenic behavior and high intracellular stability. These NPs, being of smaller size and brighter than QDs, have emerged as promising tools for bioimaging
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18

John, Sween. "A Study of the Synthesis and Surface Modification of UV Emitting Zinc Oxide for Bio-Medical Applications". Thesis, University of North Texas, 2009. https://digital.library.unt.edu/ark:/67531/metadc10990/.

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This thesis presents a novel ZnO-hydrogel based fluorescent colloidal semiconductor nanomaterial system for potential bio-medical applications such as bio-imaging, cancer detection and therapy. The preparation of ZnO nanoparticles and their surface modification to make a biocompatible material with enhanced optical properties is discussed. High quality ZnO nanoparticles with UV band edge emission are prepared using gas evaporation method. Semiconductor materials including ZnO are insoluble in water. Since biological applications require water soluble nanomaterials, ZnO nanoparticles are first dispersed in water by ball milling method, and their aqueous stability and fluorescence properties are enhanced by incorporating them in bio-compatible poly N-isopropylacrylamide (PNIPAM) based hydrogel polymer matrix. The optical properties of ZnO-hydrogel colloidal dispersion versus ZnO-Water dispersion were analyzed. The optical characterization using photoluminescence spectroscopy indicates approximately 10 times enhancement of fluorescence in ZnO-hydrogel colloidal system compared to ZnO-water system. Ultrafast time resolved measurement demonstrates dominant exciton recombination process in ZnO-hydrogel system compared to ZnO-water system, confirming the surface modification of ZnO nanoparticles by hydrogel polymer matrix. The surface modification of ZnO nanoparticles by hydrogel induce more scattering centers per unit area of cross-section, and hence increase the luminescence from the ZnO-gel samples due to multiple path excitations. Furthermore, surface modification of ZnO by hydrogel increases the radiative efficiency of this hybrid colloidal material system thereby contributing to enhanced emission.
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19

Chen, Esther. "Investigation of Experimental Variation of Bovine Sphingomyelin as a Novel Ingredient for Ultraviolet Protection". DigitalCommons@CalPoly, 2020. https://digitalcommons.calpoly.edu/theses/2167.

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Skin cancer is a prevalent disease that globally affects 2-3 million people per year [1]. This number is expected to grow tenfold as depletion of the ozone layer contributes to harsher rays reaching Earth’s surface [2]. A common way to protect against those ultraviolet waves is to apply sunscreen, however, recent reports call into question the safety of some active ingredients as they can enter through the skin into the bloodstream [3]. This thesis aims to investigate an alternative solution that uses bovine sphingomyelin (BSM) as photoprotective solution against UV irradiation. In order to evaluate the effectiveness of BSM against UV radiation, p21 intensity was measured on a monolayer of keratinocytes, as the intensity directly correlates to cell damage. Additionally, fluorescent sphingomyelin (FSM) was added as a treatment because it was created to be an analog to BSM and allowed for visualization of sphingomyelin within the cell. Differences in p21 intensities were observed with BSM and FSM showing a reduced p21 intensity compared to the no sphingomyelin case. FSM helped locate sphingomyelin within the cell and a mechanism was proposed for how it reduces cell damage. Lastly, high variation was seen between experimental designs. Further measures were needed to reduce this intra-subject standard deviation, so additional experimental parameters were tested such as min/max intensity values, cell count, and nucleus circularity to explain this variation.
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20

Colon, Gomez Maria. "Squaraine dyes for two-photon fluorescence bioimaging applications". Honors in the Major Thesis, University of Central Florida, 2013. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/834.

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21

Ronzitti, E. "NANOBIOPHOTONICS APPROACHES TOWARDS ADVANCED BIOIMAGING". Doctoral thesis, Università degli Studi di Milano, 2011. http://hdl.handle.net/2434/155507.

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Due to its nature, imaging is dependent on data visualization. More specifically, when dealing with optical microscopy imaging, diffraction limited spatial resolution and the presence of out-of-focus contributions are two of the key elements for the generation of 4D (x,y,z,t) data sets oriented to study molecular processes at the nanoscale. Within the medical nanotechnology scenario, optical fluorescence techniques have a pivotal role thanks to their non invasive capability to address biological questions in three dimensions with a remarkable capability to distinguish fine details, tremendously improved up to the nanoscale level in the last decade. Within this scenario, this Thesis concerns the study and the development of advanced fluorescence optical methods for the improvement of the imaging capability and a better exploitation of its potentialities. Two approaches have been followed in fluorescence microscopy to gather information, namely: a direct diffraction-limited observation of the sample allowed by the optical architecture and the analysis of those fluorescence signals particularly sensitive to the environmental condition. Consequently, two strategies have been pursued to improve such capabilities. They are related to the design of novel optical arrangements utilizing light interferences pathways to improve the diffraction resolution limit and to the study of novel fluorescent molecules and/or fluorescent techniques which allows the investigations of subresolved molecular interactions. In particular, following a fluorescent probe approach, a nanostructured polyelectrolyte system has been designed to study the fluorescence quenching effect induced by specific quencher molecules on the fluorescence emission process. The realized system allows entrapping the fluorescent molecules and monitoring fluorescence signal variations to probe quencher metal ions at microrange concentrations, significantly higher with respect to the current fluorescent quenching based technologies. As well, following an optical approach, the interferences effect induced by structuring the illumination light with different masks have been studied, in order to improve some features of the imaging capability of the fluorescence microscope. In the Two-Photon Excitation (2PE) and in the Confocal Single Photon Laser Scanning Microscope, the insertion of a ring shaped mask in the illumination pathway is proposed to enhance the signal to noise ratio of the optical system at the high spatial frequencies. Since in these optical systems the transmission of the high spatial frequencies is particularly weak, such features allow to improve the overall practical capability of the confocal and 2PE system to distinguish fine details of the image. In the widefield microscope, the insertion of a periodic grid to structure the light has been investigated in order to confer a 3D optical sectioning capability comparable to that of the confocal microscope, with major advantages in terms of the efficient use of the light, simplicity of construction, speed of imaging acquisition, versatility, and low cost. The proposed scheme allows to quickly collect a pure sectioned image without any computational demodulation thanks to a novel optical architecture where both the illumination and the detection light is structured by a spinning grid.
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22

Nilsson, Ruben. "Optical properties of fluorescence quantum dots for super-resolution bioimaging". Thesis, KTH, Tillämpad fysik, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-172345.

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23

Xu, Hao. "Fluorescence Properties of Quantum Dots and Their Utilization in Bioimaging". Doctoral thesis, KTH, Tillämpad fysik, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-191985.

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Quantum dots (QDs), especially colloidal semiconductor QDs, possess properties including high quantum yields, narrow fluorescence spectra, broad absorption and excellent photostability, making them extremely powerful in bioimaging. In this thesis, we studied the fluorescence properties of QDs and attempted multiple ways to boost applications of QDs in bioimaging field. By time-correlated single photon counting (TCSPC) measurement, we quantitatively interpreted the fluorescence mechanism of colloidal semiconductor QDs. To enhance QD fluorescence, we used a porous alumina membrane as a photonic crystal structure to modulate QD fluorescence. We studied the acid dissociation of 3-mercaptopropionic acid (MPA) coated QDs mainly through electrophoretic mobility of 3-MPA coated CdSe QDs and successfully demonstrated the impact of pH change and Ca2+ ions. Blinking phenomena of both CdSe-CdS/ZnS core-shell QDs and 3C-SiC nanocrystals (NCs) were studied. A general model on blinking characteristics relates the on-state distribution to CdSe QD surface conditions. The energy relaxation pathway of fluorescence of 3C-SiC NCs was found independent of surface states. To examine QD effect on ciliated cells, we conducted a 70-day long experiment on the bioelectric and morphological response of human airway epithelial Calu-3 cells with periodic deposition of 3-MPA coated QDs and found the cytotoxicity of QDs was found very low. In a brief summary, our study of QD could benefit in bioimaging and biosensing. Especially, super-resolution fluorescent bioimaging, such as, stochastic optical reconstruction microscopy (STORM) and photo-activated localization microscopy (PALM), may benefit from the modulation of the QD blinking in this study. And fluorescence lifetime imaging (FLIM) microscopy could take advantage of lifetime modulation based on our QD lifetime study.

QC 20160905

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24

Gortari, Antu Nehuen. "Metasurfaces for bioimaging". Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS416/document.

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Au cours des dernières années, des efforts importants ont été déployés pour développer des métasurfaces (MSs) électromagnétiques avec la possibilité de changer de manière abrupte les propriétés de la lumière. Ces avancées ont ouvert une nouvelle gamme de possibilités pour contrôler la lumière en utilisant des dispositifs optiques ultra-minces. Dans ce contexte, et plus spécifiquement dans le spectre visible, les applications en bio-imagerie s’avèrent particulièrement intéressantes. Une technique qui est particulièrement bien adaptée à l'étude de molécules proches d'une membrane cellulaire est la microscopie à fluorescence par réflexion interne (TIRFM), qui repose sur un champ évanescent d'excitation. Dans ce cas la lumière incidente est totalement réfléchie sur une interphase (typiquement verre/eau) en raison de son angle d'incidence élevé. À ce jour, la TIRFM est généralement mise en œuvre à l'aide d'objectifs volumineux de grande ouverture numérique et de petit champ de vision.Dans ce travail de thèse, nous réalisons de substrats pour la microscopie TIRF à base de métasurfaces constituées de réseaux périodiques de structures asymétriques fabriquées en dioxyde de titane (TiO2) sur du verre borosilicaté. Ces structures, aussi petites que 48 nm, ont été optimisées à l’aide de simulations numériques "Rigorous coupled-wave analysis” (RCWA) dans le but de coupler de 50 à 90% de la lumière incidente dans le premier ordre de diffraction avec des angles élevés (θ > 63deg). Le fait de pouvoir utiliser des objectifs de faible grossissement et d'avoir une grande zone de champ évanescent fournit des conditions TIRF uniques qui ne sont pas accessibles par les méthodes traditionnelles. De plus, ces structures sont compatibles avec la lithographie par nanoimpression UV, ce qui permet d’envisager une fabrication à bas coût et à grande échelle. Outre la conception, et la fabrication, dans cette thèse nous aboutissons à une preuve de principe de la microscopie TIRF basée sur des métasurfaces en milieu biologique en imageant notamment des membranes fluorescentes de cellules souches. Ces métasurfaces permettent ainsi l’implémentation TIRFM à contraste élevé et à faible photo-blanchissement compatible avec des microscopes à champ large peu coûteux
In recent years there has been a significant effort to push electromagnetic metasurfaces with the ability to abruptly change light properties into visible wavelengths. These advancements have opened a new range of possibilities to reshape light using ultra-thin optical devices and there is one field that is starting to gather attention: bioimaging. One technique particularly well suited for the study of molecules near a cell membrane is Total Internal Reflection Fluorescence (TIRF) microscopy, which relies on an evanescence field created by light being totally internally reflected within a glass substrate due to its high incidence angle. As of today, TIRF is generally implemented using bulky high-NA, small field of view oil objectives.In this project we present the realization of metasurface-based TIRF microscopy substrates consisting of periodic 2D arrays of asymmetric structures fabricated in titanium dioxide on borosilicate glass. These patterns, as small as 48nm, were optimized through rigorous coupled-wave analysis to couple 50-90% of the incoming normally incident light into the first diffraction order, which outputs at an angle that suffices total internal reflection in water and eliminates the requirement for high NA objectives or prisms to achieve TIRF. Being able to utilize lower-magnification air objectives and having a large evanescence field area provide unique TIRF conditions not accessible by traditional methods. Additionally, these structures are compatible with soft UV nanoimprint lithography, for cost-effective scale production, to give TIRF’s high contrast, low photodamage and low photobleaching capabilities to inexpensive wide-field microscopes
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25

Asem, Heba. "Synthesis of Polymeric Nanocomposites for Drug Delivery and Bioimaging". Licentiate thesis, KTH, Funktionella material, FNM, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-186300.

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Nanomaterials have gained great attention for biomedical applications due to their extraordinary physico-chemical and biological properties. The current dissertation presents the design and development of multifunctional nanoparticles for molecular imaging and controlled drug delivery applications which include biodegradable polymeric nanoparticles, superparamagnetic iron oxide nanoparticles (SPION)/polymeric nanocomposite for magnetic resonance imaging (MRI) and drug delivery, manganese-doped zinc sulfide (Mn:ZnS) quantum dots (QDs)/ SPION/ polymeric nanocomposites for fluorescence imaging, MRI and drug delivery.Bioimaging is an important function of multifunctional nanoparticles in this thesis. Imaging probes were made of SPION and Mn:ZnS QDs for in vitro and in vivo imaging. The SPION have been prepared through a high temperature decomposition method to be used as MRI contrast agent. SPION and Mn:ZnS were encapsulated into poly (lactic-co-glycolic) acid (PLGA) nanoparticles during the particles formation. The hydrophobic model drug, busulphan, was loaded in the PLGA vesicles in the composite particles. T2*-weighted MRI of SPION-Mn:ZnS-PLGA phantoms exhibited enhanced negative contrast with r2* relaxivity of 523 mM-1 s-1. SPION-Mn:ZnS-PLGA-NPs have been successfully applied to enhance the contrast of liver in rat model.The biodegradable and biocompatible poly (ethylene glycol)-co-poly (caprolactone) (PEG-PCL) was used as matrix materials for polymeric nanoparticles -based drug delivery system. The PEG-PCL nanoparticles have been constructed to encapsulate SPION and therapeutic agent. The encapsulation efficiency of busulphan was found to be ~ 83 %. PEG-PCL nanoparticles showed a sustained release of the loaded busulphan over a period of 10 h. The SPION-PEG-PCL phantoms showed contrast enhancement in T2*-weighted MRI. Fluorescein-labeled PEG-PCL nanoparticles have been observed in the cytoplasm of the murine macrophage cells (J774A) by fluorescence microscopy. Around 100 % cell viability were noticed for PEG-PCL nanoparticles when incubated with HL60 cell line. The in vivo biodistribution of fluorescent tagged PEG-PCL nanoparticles demonstrated accumulation of PEG-PCL nanoparticles in different tissues including lungs, spleen, liver and kidneys after intravenous administration.

QC 20160516

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26

Needham, Lisa-Maria. "Next-generation fluorophores for single-molecule and super-resolution fluorescence microscopy". Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/283232.

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The development of single-molecule and super-resolution fluorescence techniques has revolutionised biological imaging. Nano-scale cellular structures and heterogeneous dynamic processes are now able to be visualised with unprecedented resolution in both time and space. The achievable localisation precision and therefore the resolution is fundamentally limited by the number of photons a single-fluorophore can emit. The ideal super-resolution dye would emit a large number of photons over a short period of time. On the contrary, an optimal single-molecule tracking probe would be highly photostable and undergo no transient dark-state transitions. Single-molecule instrument development is beginning to reach technological saturation and as the frontiers of bioimaging expand, exorbitant demands are placed on the gamut of available probes that often cannot be met. Thus, the next key challenge in the field is the development of the better fluorophores that underlie these techniques; this includes both the synthesis of new chemical derivatives and alternative novel strategies to augment existing technologies. The results of this thesis are divided into two distinct parts; Project One details the development of new synthetic fluorescent probes for the study of amyloid protein aggregates implicated in neurodegenerative diseases. This includes a study of the photophysical and binding properties of a novel fluorophore library based on the amyloid dye Thioflavin-T. Following on from this, is the presentation of novel bifunctional dyes capable of simultaneously identifying hydrogen peroxide and amyloid aggregates by combining existing tools for the independent detection of these species. The sensing capabilities of these dyes are explored at the bulk and single-molecule levels. Project Two describes a new photo-modulatable fluorescent-protein fusion construct that can undergo Förster resonance energy transfer (FRET) to an organic dye molecule. This FRET cassette is comprised of a photoconvertible fluorescent protein donor, mEos3.2 and acceptor fluorophore, JF646. This strategy imparts a strong photostabilising effect on the fluorescent protein and a resistance to photobleaching. The functionality of this approach is demonstrated with in vitro single-molecule fluorescence studies and its biological applicability shown by tracking single proteins in the nuclei of live embryonic stem cells. Furthermore, initial characterisations of the excited state dynamics in effect are presented through the systematic modification of parameters.
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27

Muñoz, Resta Ignacio. "Pyrylium salts for bioimaging, sensing and solid-state emission". Doctoral thesis, Universitat Jaume I, 2021. http://dx.doi.org/10.6035/14104.2021.782064.

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The thesis entitled "Pyrylium salts for bioimaging, sensing and solid-state emission" is structured in 7 chapters that cover different aspects related to several applications of pyrylium salts. Two chapters correspond to biological applications, in which the synthesis and characterization of molecules that are used as mitochondrial probes and nitric oxide sensors is carried out. A third one introduces an analytical application, where pyrylium compounds are used for water sensing in organic solvents. And in the last one, an optic approach is proposed and the development of a family of compounds as solid-state emitters is presented. Due to its synthetic ease and good photophysical properties, it's concluded that the use of pyrilium salts could be expanded even further in the future into fields in which they have not been fully exploited, such as bioanalysis.
La tesis titulada "Pyrylium salts for bioimaging, sensing and solid-state emission" se encuentra estructurada en 7 capítulos en los que se desarrollan nuevas aplicaciones para compuestos de pirilio. Dos capítulos se corresponden con aplicaciones biológicas, en los que se lleva a cabo la síntesis y caracterización de moléculas que son utilizadas como sondas mitocondriales y sensores de óxido nítrico. Un tercero introduce una aplicación analítica, donde los pirilios se utilizan para el sensado de agua en solventes orgánicos. Y en un cuarto se plantea un enfoque desde la óptica y se presenta el desarrollo de una familia de compuestos como emisores en estado sólido. Se concluye que, debido a su facilidad sintética y sus buenas propiedades fotofísicas, el uso de sales de pirilio puede continuar expandiéndose hacia campos en los que no han sido completamente explotados, como el bioanálisis.
Programa de Doctorat en Ciències
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28

Mu, Huiying. "Studies on Activatable Chemical Probes Based on Sulfur Nucleophilicity for Fluorescence and/or Photoacoustic Bioimaging". Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263676.

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29

Bu, Lulu. "Synthesis of NIR Dyes and Nanoparticles for in vivo Bioimaging and Dicyanovinyl Dyes for Primary Amine Detection". Thesis, Lyon, 2020. http://www.theses.fr/2020LYSEN086.

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L’imagerie de fluorescence et l'imagerie photo-acoustique (PA) sont deux outils puissants pour la visualisation des tissus et organes biologiques de manière non invasive. Toutefois, ces technologies sont actuellement limitées par le manque d'agents de contraste efficaces. Utiliser des longueurs d’onde de domaine du proche infrarouge (NIR, 650-900 nm), dont l'absorption et la diffusion dans les organismes est relativement faible, permet une imagerie in vivo plus profonde, induit moins d’auto-fluorescence et apporte un bon rapport signal/bruit. Par conséquent, la conception et la synthèse de nouveaux colorants organiques NIR efficaces revêt une importance fondamentale pour l’imagerie de fluorescence ou photo-acoustique. L'encapsulation de colorants organiques dans des nanoparticules dispersibles dans l'eau présente un grand potentiel en imagerie bio-optique, offrant les avantages d'une haute luminosité, d'une bonne photo-stabilité, d'une excellente biocompatibilité et d'une capacité potentielle de ciblage, etc. Notre objectif principal dans cette thèse est de synthétiser de nouvelles molécules organiques pouvant servir d’agents de contraste pour l'imagerie in vivo par fluorescence ou PA
Fluorescence and photoacoustic (PA) imaging are both powerful tools for visualization of biological tissues and organs in non-invasive ways. However, these technologies are limited by the lack of efficient contrast agents. NIR light (650-900 nm) with relatively low absorption and scattering in organisms allows for deeper in vivo imaging, lower auto-fluorescence as well as a good signal to noise ratio. Hence, design and synthesis of efficient NIR organic dyes are of great significance for fluorescence or PA bio-imaging. Meanwhile, encapsulation of organic dyes in nanoparticles dispersible in water present great potential in bio-optical imaging, offering the advantages of high brightness, good photo-stability, excellent biocompatibility and potential targeting ability, etc. Our main goal in this thesis is to synthesize novel organic contrast agents for in vivo fluorescence or PA imaging
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30

Hales, Katherine J. "Synthesis, Characterizations, and Evaluation of New Reactive Two-Photon Absorbing Dyes for Two-Photon Excited Fluorescence Imaging Applications". Doctoral diss., University of Central Florida, 2005. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/3575.

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Recent, cooperative advances in chemistry, biology, computing, photophysics, optics, and microelectronics have resulted in extraordinary developments in the biological sciences, resulting in the emergence of a novel area termed 'biophotonics'. The integrative and interdisciplinary nature of biophotonics cuts across virtually all disciplines, extending the frontiers of basic cellular, molecular, and biology research through the clinical and pharmaceutical industries. This holds true for the development and application of the novel imaging modality utilizing multiphoton absorption and its extraordinary contribution to recent advances in bioimaging. Intimately involved in the revolution of nonlinear bioimaging has been the development of optical probes for probing biological function and activity. The focus of this dissertation is in the area of probe development, particularly conjugated organic probes, optimized for efficient two-photon absorption followed by upconverted fluorescence for nonlinear, multiphoton bioimaging applications. Specifically, [pi]-conjugated fluorene molecules, with enhanced two-photon absorbing (2PA) properties and high photostability, were prepared and characterized. Contemporary synthetic methods were utilized to prepare target fluorene derivatives expected to be highly fluorescent for fluorescence imaging, and, in particular, exhibit high two-photon absorptivity suitable for two-photon excitation (2PE) fluorescence microscopy. The flexibility afforded through synthetic manipulation to integrate hydrophilic moieties into the fluorophore architecture to enhance compatibility with aqueous systems, more native to biological samples, was attempted. Incorporation of functional groups for direct covalent attachment onto target biomolecules was also pursued to prepare fluorene derivatives as efficient 2PA reactive probes. Linear and two-photon spectroscopic characterizations on these novel compounds reveal they exhibit high 2PA cross-sections on the order of ~100 GM units, nearly an order of magnitude greater than typical, commonly used fluorophores utilized in nonlinear, multiphoton microscopy imaging of biological samples. Photostability studies of representative fluorene derivatives investigated and quantified indicate these derivatives are photostable under one- and two-photon excitation conditions, with photodecomposition quantum yields on the order of 10[super-5]. Preliminary cytotoxicity studies indicate these fluorene derivatives exhibit minimal cytotoxic effects on proliferating cells. Finally, their ultimate utility as high-performance, 2PA fluorescent probes in 2PE fluorescence microscopy imaging of biological samples was demonstrated in both fixed and live cells. Due to the low cytotoxicity, high photostability, efficient 2PA, and high fluorescence quantum yield, the probes were found suitable for relatively long-term, two-photon fluorescence imaging of live cells, representing a significant advance in biophotonics.
Ph.D.
Department of Chemistry
Arts and Sciences
Biomolecular Sciences: Ph.D.
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31

Bsaibess, Talia. "Nanoparticules organiques ultra-brillantes pour l'imagerie biologique". Thesis, Bordeaux, 2015. http://www.theses.fr/2015BORD0055/document.

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Les nanoparticules inorganiques luminescentes ont suscité un intérêt croissant au cours des dernières décennies, notamment pour leur application en imagerie biologique. Un certain nombre d’entre elles présentent toutefois des limitations telles que toxicité, absence de biodégradabilité, faible brillance, clignotements…. Dans cette optique, les nanoparticules fluorescentes à base de petites molécules organiques (FONs) offrent une solution alternative prometteuse aux nanoparticules inorganiques pour l'imagerie biologique. Le principal défi réside dans l'élaboration des nanoparticules organiques possédant une brillance élevée, une bonne stabilité dans l'eau (y compris en milieu biologique), une bonne biocompatibilité ainsi qu'une émission accordable dans le visible et au-delà dans le proche infrarouge (pour une détection plus aisée en milieu diffusant). Dans cette optique, nous avons utilisé une stratégie basée sur l’utilisation de chromophores dipolaires de type "push pull" « adaptés ». Au cours du travail, la synthèse de séries de chromophores homologues bâtis sur le même système conjugué et ayant en commun un groupe donneur de type triphénylamine (destiné à préserver les propriétés de luminescence) présentant ou non des motifs encombrants positionnés a été réalisée. Les nanoparticules correspondantes ont été préparées selon un protocole classique, simple et rapide à mettre en oeuvre (précipitation). L’étude des propriétés photophysiques des nanoparticules organiques fluorescentes ainsi obtenues a été réalisée et mise en perspective avec celles des chromophores en solution dans des solvants organiques de polarité variable. Une étude systématique de l’évolution dans le temps des propriétés optiques des nanoparticules organiques a été réalisée permettant de mettre en lumière des relations entre la structure des sous-unités chromophoriques et la stabilité colloïdale et « optique » des nanoparticules. Ces études ont permis d’identifier des nanoparticules émettant dans le proche infrarouge extrêmement brillantes et présentant une stabilité colloïdale remarquable dans l’eau, une photostabilité accrue et une très bonne biocompatibilité. De ce fait, ces nanoparticules ont pu être utilisées avec succès dans l'imagerie biologique des cellules et le suivi (tracking) à l'échelle de la particule unique, démontrant l'intérêt de la démarche d'ingénierie mise en oeuvre
During the last decades, luminescent inorganic nanoparticles have attracted a large interest in different fields including biological imaging. However, a number of them have drawbacks such as toxicity and absence of biodegradability. Recently, molecular-based fluorescent organic nanoparticles (FONs) have emerged as a promising alternative to inorganic nanoparticles for bioimaging. The main challenge lies in the elaboration of organic nanoparticles that combine large brightness, good colloidal stability in biological environments) and biocompatibility as well as NIR emission (to allow improved detection in thick tissues). To achieve this objective, we have implemented a molecular engineering strategy based on dedicated polar and polarizable "push pull" chromophore built from a triphenylamine donor moiety and a specific pi-conjugated system. The corresponding nanoparticles were readily prepared by the reprecipitation method. In the present manuscript, the synthesis of the chromophores and the preparation and characterization of the organic fluorescent nanoparticles is described. A comprehensive investigation of their photophysical properties and study of their colloidal stability is presented allowing to derive structure-property relationships. The implemented study led to innovative NIR-emitting nanoparticles combining large brightness (superior to those of QDs and NIR-emitting organic dyes), remarkable colloid stability and suitable photostability. These nanoparticles have been successfully used for single particle tracking and imaging in cells, while no toxic effect was observed
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32

Nguyen, Dao. "Design, Synthesis, and Characterization of Novel Hydrophilic Fluorene-Based Derivatives for Bioimaging Applications". Doctoral diss., University of Central Florida, 2009. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2842.

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In this work, hydrophilic fluorene-based derivatives that contain ethylene oxide substituents, have been synthesized and characterized for potential use as new fluorophores for bioimaging applications and for fluorescence sensing of heavy metals. Symmetrical and unsymmetrical fluorene derivatives based on structural types of acceptor-pi-acceptor, acceptor-pi-donor, and donor-pi-donor were characterized by TGA, UV-vis absorption, fluorescence emission, lifetime, anisotropy, and two-photon absorption (2PA) cross section. They were found to possess high thermal stability, high photostability, high fluorescence quantum yields, and generally large two-photon absorption cross sections, making them quite suitable for new probes in single-photon absorption and two-photon absorption fluorescence microscopy imaging. Novel hydrophilic fluorene derivatives were synthesized from fluorene in multiple steps employing the metal-catalyzed Heck coupling reaction, the Stille reaction, the Sonogashira reaction, the Ullmann condensation reaction, and "click" chemistry. To increase the hydrophilicity of the new compounds, ethylene oxide substituents were utilized for to impart water solubility. An alternative alkylation methodology using ethyleneoxy tosylates was introduced for the synthesis of ethylene oxide-containing fluorene derivatives. Several of these hydrophilic derivatives were incubated into various cell lines as new probes for both conventional and two-photon absorption fluorescence bioimaging. These compounds were biocompatible, exhibiting low cytotoxicity as determined by cell viability studies, and displayed colocalization for selected cellular organelles. In addition, hydrophilic bis(1,2,3-triazolyl)fluorene derivatives were found to exhibit sensitive fluorescence responses in the presence of certain heavy metal, and were selective for sensing zinc and mercury over other a number of other metal ions relevant to living cells or other biological environments. The UV-vis absorption and fluorescence emission spectra of the complexes exhibited a blue-shifted absorption and emission for selective metal chelation upon binding to zinc and mercury(II) ions, resulting in an approximately two-fold enhanced fluorescence response. Fluorescence titration studies revealed that the complexes of 1:2 and 1:3 ligand to metal formed with binding constant values of 108 and 1014 for zinc and mercury ions, respectively. Finally, preliminary experiments were performed to explore the possibility of employing select hydrophilic fluorene-based derivatives in the synthesis of hydrophilic fluorescent gold nanoparticles. Although results are very preliminary, the aim is to use such materials for other biomedical applications, such as surface enhanced scattering resonance and noninvasive photothermal therapy to diagnose and to treat cancers. Thus, this research had led to the discovery of alternative methodologies for synthesis of hydrophilic fluorene derivatives by alkylation with alkyl tosylates and synthesis of hydrophilic fluorescent molecule capped gold nanoparticles. Furthermore, several novel hydrophilic fluorene-based derivatives were synthesized and characterized for their linear and nonlinear photophysical properties, and are now available for further examination of their bioimaging and sensing applications.
Ph.D.
Department of Chemistry
Sciences
Chemistry PhD
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33

Tyson, James Abner. "Investigations into the supramolecular chemistry of graphene biocomposites : towards prostate cancer theranostics design, imaging and biosensing". Thesis, University of Bath, 2016. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.715277.

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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.
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34

Pagano, Paolo. "Design and synthesis of ultra-bright organic nanoparticles (ONPs) for bioimaging". Thesis, Bordeaux, 2017. http://www.theses.fr/2017BORD0634/document.

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L’utilisation de nano-objets luminescents en milieu biologique est devenue très répandue, notamment en vue d’applications biomédical est elles que l’imagerie, la thérapie et le diagnostic. Jusqu’à récemment, les principaux travaux réalisés dans ce domaine concernaient les nanoparticules de silice dopées ou fonctionnalisées avec des molécules organiques, les nanoparticules d’or et les nanoparticules semi-conductrices (quantum dots, i.e., QDs). Toutefois, un certain nombre de limitations demeurent pour les applications dans le domaine du vivant, en lien notamment avec des problèmes de stabilité, de biocompatibilité et de toxicité ou encore de biodégradabilité. En parallèle,un certain nombre de molécules organiques fluorescentes non-toxiques ont été utilisées comme sondes fluorescentes en milieu biologique, mais leur brillance demeure limitée. L’idée directrice de la thèse est de concevoir et synthétiser de nouveaux chromophores organiques présentant une émission modulable (du visible au proche infrarouge) et adaptés à la préparation de nanoparticules organiques fluorescentes (FONs) combinant à la fois une brillance extrêmement élevée, une excellente stabilité colloïdale et une photostabilité adaptée à leur utilisation en imagerie in vitro et in vivo. De tels nanoobjets ultra-brillants pourraient alors représenter une alternative très intéressante aux nanoparticules actuellement les plus utilisées en imagerie de fluorescence du vivant (QDs). Le manuscrit décrit la synthèse et les propriétés de plusieurs classes de molécules fluorescentes spécifiquement conçues pour former des telles FONS par auto-assemblage dans l’eau. La préparation de ces FONs est présentée et leurs propriétés étudiées et discutées. Enfin des applications concrètes en bio-imagerie sont présentées
Nowadays the use of bright luminescent nano-objects in biological environment is a topic that is gaining more and more importance, especially for biomedical applications such as imaging, the rapyand diagnostic. So far, numerous studies have been conducted with gold nanoparticles, silica nanoparticles (doped or functionalized with organic molecules), as well as semiconductor nanoparticles (quantum dots, i.e., QDs). However, most of these nanoparticles suffer from drawbacks (in terms of stability, biocompatibility, eco-toxicity or degradability). On the other hand, several nontoxic fluorescent molecular probes have been widely used, but most of the time their brightness remain modest in biological environments compared to QDs. Our idea is to engineer new organicchromophores with tunable emission wavelength (from visible to near infrared) for further preparation of organic fluorescent nanoparticles (so called FONs) that display giant one-photon and two-photonbrightness, as well as good colloidal and chemical stability, and suitable photostability for in vitro andin vivo imaging. As such, these FONs would represent interesting alternatives to QDs for use in bioimaging. This manuscript describes the synthesis and characterization of new classes of fluorescent molecules specifically engineered as building blocks for the fast preparation of such nanoparticles byself-aggregation in water. The FONs were fully characterized from both morphological and photophysical points of view and further used in bioimaging
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35

Dušan, Milojkov. "Dobijanje nanofosfora na bazi fluorapatita dopirani Pr3+ jonima za bio-medicinske primene". Phd thesis, Univerzitet u Novom Sadu, Tehnološki fakultet Novi Sad, 2020. https://www.cris.uns.ac.rs/record.jsf?recordId=114851&source=NDLTD&language=en.

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    Luminescentni nanokristali (nanofosfori) na bazi fluorapatita (FAP-a) dopirani elementima retkih zemalja idealni su kontrastni agenti za bio-medicinske primene, kao što su detekcije, snimanja, praćenja i terapije ćelija kancera. Kancer je jedna od najčešćih bolesti modernog doba čiji uspeh lečenja zavisi od rane dijagnostike i neinvazivnog tretmana. Luminescentne nanočestice mogu uneti inovativnu paradigmu u lečenje kancera kombinovanjem biosnimanja, dijagnostike i tretmana. Za studije fluorescentnih biosnimanja nanokristali fluorapatita dopirani retkim zemljama kao kontrastni agenti pružaju značajne prednosti u vidu velikih kontrasta i dugotrajnosti luminescencije, i što je još važnije visoke biokompatibilnosti, netoksičnosti i bioaktivnosti. Glavni ciljevi ove doktorske disertacije su sinteza novih luminescentnih multifotonskih bionanomaterijala na bazi fluorapatita dopiranih jonima prazeodimijuma (Pr3+), njihova karakterizacija i evaluacija  primene za fluorescentna biosnimanja kancera. Sintezom nanoprahova u umerenim uslovima metodom ko-precipitacije, a potom sušenjem na 110 oC i kalcinacijom na temperaturama od 700 i 1000 oC očekuje se pronalaženje najboljih uslova za dobijanje novih nanofosfora koji bi našli i različite bio-medicinske primene u oblasti fluorescentnih biosnimanja. Proučavane su tri vrste PrFAP nanokristala, sa 0,1%, 0,5% i 1% atomskih procenta Pr3+, zajedno sa nedopiranim FAP kontrolnim uzorkom. Nivoi energije aktivator jona Pr3+ sadrže metastabilna multipletna stanja koja nude mogućnosti efikasnih emisionih linija u više boja u FAP nanokristalima, kao i u infracrvenoj i ultravioletnoj oblasti spektra. Metodom ko-precipitacije na sobnoj temperaturi (25 oC), a potom sušenjem na 110 oC, sintetisani su monofazni heksagonalni nanokristali PrFAPs nepravilnog sfernog oblika. Termičkom analizom sintetisanih uzoraka, na osnovu detektovanih temperaturnih opsega procesa dekarbonacije i dehidroksilacije, utvrđene su temperature kalcinacije od 700 i 1000 oC. Termička analiza i karakterizacija uzoraka su pokazale da Pr3+ joni dovode do stabilizacije FAP strukture na višim temperaturama, što je pripisano unosu lantanoidnih jona sa specifičnim magnetnim osobinama u sistem i stvaranju jačih privlačnih sila sa O2- anjonima. Nanokristali sušeni na 100 oC i kalcinisani na 1000 oC, zbog prisustva defekata kristalne rešetke koji zadržavaju emisiju Pr3+ jona, nisu pokazali luminescentne karakteristike od značaja za primene u medicinskim fluorescentnim biosnimanjima. Kalcinacijom uzoraka na 700 oC izrađen je novi tip aktiviranih fluorapatitnih nanokristala dopiranih prazeodimijumom (PrFAPa) sa ekscitaciono-emisionim profilima u vidljivom delu spektra. Fizičko-hemijska karakterizacija potvrdila je sferne kristale heksagonalne strukture do nanometrske veličine od oko 20 nm. Kvantno-hemijske kalkulacije predvidele su da se joni Pr3+ ugrađuju u kristalnu rešetku FAP nanokristala na položaju Ca2 (6h), što je praćeno deformacijama pozicije F- jona. Pretpostavljeni mehanizam supstitucije je jedan jon Pr3+ za jedan Ca2+, s delimičnom supstitucijom anjona F– sa O2– i OH– i stvaranjem vakansi usled postizanja neutralnosti sistema. Rezultati in vitro biokompatibilnosti i hemokompatibilnosti pokazali su da nanokristali PrFAPa nisu toksični za žive ćelije. Pored toga, internalizacija PrFAPa nanokristala od strane ćelija kancera kože (A431) i pluća (A549) je proučavana korišćenjem konfokalne mikroskopije i mikroskopije širokog polja zasnovane na fluorescenciji. Nanokristali pokazuju karakterističnu zelenu emisiju na 545 nm (3P0→3H5 tranzicija Pr3+ jona) i narandžastu emisiju na 600 nm (1D2→3H4), koje su korišćene za razlikovanje od pozadinske autofluorescencije ćelije. Studije dobijenih slika konfokalnom mikroskopijom u plavom, zelenom i crvenom kanalu su otkrile da nanokristali mogu da prepoznaju ćelijsku površinu i da se lepe za nju, ali nisu potvrdile ulazak nanokristala u ćelije. Mikroskopija širokog polja je detektovala emisione prelaze u zelenoj i narandžastoj boji i potvrdila da luminescentni signal dolazi iz unutrašnjosti ćelija. Korišćenjem rezonantne ekscitacije od 488 nm i emisije od 600 nm PrFAPa nanokristala, konfokalnom mikroskopijom ekstrahovan je signal fluorescencije iz unutrašnjosti ćelija kancera. Ortogonalne projekcije u 3D konfokalnim ravnima pokazuju da su nanokristali u stanju da uđu u ćelije kancera i da se raspoređuju po citoplazmi. Sveukupno, ovako dobijeni nanokristali PrFAPa su biokompatibilni i od testiranih uzoraka, aktivirani nanokristali dopirani sa 0,5% Pr3+ pokazuju najviše potencijala za primenu u medicinskim fluorescentnim biosnimanjima kao kontrastni agenti.  
Luminescent nanocrystals (nanophosphorus) based on fluorapatite (FAP) doped with rare earth elements are ideal contrast agents for biomedical applications such as cancer cell detection, imaging, tracking and therapy. Cancer is one of the most common diseases of the modern times whose success of the cure depends on early diagnosis and non-invasive treatment. Luminescent nanoparticles can bring an innovative paradigm into the treatment of cancer by combining bioimaging, diagnostics and treatment. Rare earth doped fluorapatite nanocrystals as contrast agents for studies of fluorescence bioimaging, offer significant advantages in terms of high contrasts and long-term luminescence, and more importantly high biocompatibility, non-toxicity and bioactivity. The main objectives of this doctoral dissertation are the synthesis of novel luminescent multiphoton bionanomaterials based on fluorapatites doped with praseodymium ions (Pr3+), their characterization and evaluation of their application for cancer fluorescence bioimaging. Synthesis of nanopowders under moderate conditions by the co-precipitation method, followed by dried at 110 °C and calcination at 700 and 1000 °C, is expected to find the best conditions for obtaining new nanophosphors that would find different bio-medical applications in the field of fluorescence bioimaging. Three types of PrFAP nanocrystals were studied, with 0,1%, 0,5%, and 1% atomic percentages of Pr3+, together with an undoped FAP control sample. Energy levels of the Pr3+ ion activator contain metastable multiplet states that offer the possibility of efficient multi-color emission lines in FAP nanocrystals as well as in the infrared and ultraviolet regions of the spectrum. Single-phase hexagonal nanocrystals PrFAPs of irregular spherical shape were synthesized by the method of co-precipitation at room temperature (25 oC) and then drying at 110 oC. Thermal analysis of the synthesized samples, based on the detected temperature ranges of the decarbonation and dehydroxylation processes, determined calcination temperatures of 700 and 1000 oC. Thermal analysis with characterization showed that Pr3+ ions lead to stabilization of the FAP structure at higher temperatures, which was attributed to the entry of lanthanoid ions with specific magnetic properties into the system and the creation of stronger attractive forces with O2- anions. Nanocrystals dried at 100 oC and calcined at 1000 oC, due to the presence of crystal lattice defects that quench the emission of Pr3+ ions, did not show luminescent characteristics of significance for applications in medical fluorescence imaging. Calcination of the samples at 700 oC produced a new type of activated praseodymium doped fluorapatite nanocrystals (PrFAPa) with excitation-emission profiles in the visible part of the spectrum. Physicochemical characterization confirmed spherical crystals of hexagonal structure up to a nanometer size of about 20 nm. Quantum-chemical calculations predicted that Pr3+ ions would be embedded in the crystal lattice of FAP nanocrystals at the Ca2 position (6h), which was followed by deformations of the F- ion position. The assumed substitution mechanism is one Pr3+ ion for one Ca2+, with partial substitution of F– anions with O2– and OH– and creation of vacancies due to achieving system neutrality. The results of in vitro biocompatibility and hemocompatibility showed that PrFAP nanocrystals were not toxic to living cells. In addition, the internalization of PrFAPa nanocrystals by skin (A431) and lung (A549) cancer cells was studied using fluorescence-based confocal microscopy and wide-field microscopy. The nanocrystals show characteristic green emission at 545 nm (3P0→3H5 transition of Pr3+ ion) and orange emission at 600 nm (1D2→3H4), which we use to discriminate from cell autofluorescence. Studies of the images obtained by confocal microscopy in the blue, green, and red channels revealed that nanocrystals could recognize the cell surface and adhere to it, but they did not confirm the entry of nanocrystals into the cells. The wide-field microscopy detected emission transitions in green and orange color, and confirmed that the luminescent signal was coming from inside the cells. Using resonant excitation of PrFAP nanocrystals at 488 nm and emission of 600 nm, confocal microscopy extracted the fluorescence signal from inside the cancer cells. Orthogonal projections across 3D confocal stacks show that the nanocrystals are able to enter the cells positioning themselves within the cytoplasm. Overall, the obtained PrFAPa nanocrystals are biocompatible and of the tested types, the 0,5% Pr3+ doped nanocrystals show the highest promise as a tracking nanoparticle probe for bioimaging applications.
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36

Grimes, Logan. "DEVELOPENT OF A PHOSPHOLIPID ENCAPSULATION PROCESS FOR QUANTUM DOTS TO BE USED IN BIOLOGIC APPLICATIONS". DigitalCommons@CalPoly, 2014. https://digitalcommons.calpoly.edu/theses/1237.

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The American Cancer Society predicts that 1,665,540 people will be diagnosed with cancer, and 585,720 people will die from cancer in 2014. One of the most common types of cancer in the United States is skin cancer. Melanoma alone is predicted to account for 10,000 of the cancer related deaths in 2014. As a highly mobile and aggressive form of cancer, melanoma is difficult to fight once it has metastasized through the body. Early detection in such varieties of cancer is critical in improving survival rates in afflicted patients. Present methods of detection rely on visual examination of suspicious regions of tissue via various forms of biopsies. Accurate assessment of cancerous cells via this method are subjective, and often unreliable in the early stages of cancer formation when only few cancer cells are forming. With fewer cancer cells, it is less likely that a cancer cell will appear in a biopsied tissue. This leads to a lower detection rate, even when cancer is present. This lack of detection when cancer is in fact present is referred to as a false negative. False negatives can have a highly detrimental effect on treating the cancer as soon as possible. More accurate methods of detecting cancer in early stages, in a nonsubjective form would alleviate these problems. A proposed alternative to visual examination of biopsied legions is to utilize fluorescent nanocrystalline biomarker constructs to directly attach to the abnormal markers found on cancerous tissues. Quantum dots (QDs) are hydrophobic nanoscale crystals composed of semiconducting materials which fluoresce when exposed to specific wavelengths of radiation, most commonly in the form of an ultraviolet light source. The QD constructs generated were composed of cadmium-selenium (CdSe) cores encapsulated with zinc-sulfide (ZnS) shells. These QDs were then encapsulated with phospholipids in an effort to create a hydrophilic particle which could interact with polar fluids as found within the human body. The goal of this thesis is to develop a method for the solubilization, encapsulation, and initial functionalization of CdSe/ZnS QDs. The first stage of this thesis focused on the generation of CdSe/ZnS QDs and the fluorescence differences between unshelled and shelled QDs. The second stage focused on utilizing the shelled QDs to generate hydrophilic constructs by utilizing phospholipids to bind with the QDs. Analysis via spectroscopy was performed in an effort to characterize the difference in QDs both prior to and after the encapsulation process. The method generated provides insight on fluorescence trends and the encapsulation of QDs in polar substances. Future research focusing on the repeatability of the process, introducing the QD constructs to a biological material, and eventual interaction with cancer cells are the next steps in generating a new technique to target and reveal skin cancer cells in the earliest possible stages without using a biopsy.
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37

Irwin, Daniel. "MULTIMODAL NONCONTACT DIFFUSE OPTICAL REFLECTANCE IMAGING OF BLOOD FLOW AND FLUORESCENCE CONTRASTS". UKnowledge, 2018. https://uknowledge.uky.edu/cbme_etds/50.

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In this study we design a succession of three increasingly adept diffuse optical devices towards the simultaneous 3D imaging of blood flow and fluorescence contrasts in relatively deep tissues. These metrics together can provide future insights into the relationship between blood flow distributions and fluorescent or fluorescently tagged agents. A noncontact diffuse correlation tomography (ncDCT) device was firstly developed to recover flow by mechanically scanning a lens-based apparatus across the sample. The novel flow reconstruction technique and measuring boundary curvature were advanced in tandem. The establishment of CCD camera detection with a high sampling density and flow recovery by speckle contrast followed with the next instrument, termed speckle contrast diffuse correlation tomography (scDCT). In scDCT, an optical switch sequenced coherent near-infrared light into contact-based source fibers around the sample surface. A fully noncontact reflectance mode device finalized improvements by combining noncontact scDCT (nc_scDCT) and diffuse fluorescence tomography (DFT) techniques. In the combined device, a galvo-mirror directed polarized light to the sample surface. Filters and a cross polarizer in stackable tubes promoted extracting flow indices, absorption coefficients, and fluorescence concentrations (indocyanine green, ICG). The scDCT instrumentation was validated through detection of a cubical solid tissue-like phantom heterogeneity beneath a liquid phantom (background) surface where recovery of its center and dimensions agreed with the known values. The combined nc_scDCT/DFT identified both a cubical solid phantom and a tube of stepwise varying ICG concentration (absorption and fluorescence contrast). The tube imaged by nc_scDCT/DFT exhibited expected trends in absorption and fluorescence. The tube shape, orientation, and localization were recovered in general agreement with actuality. The flow heterogeneity localization was successfully extracted and its average relative flow values in agreement with previous studies. Increasing ICG concentrations induced notable disturbances in the tube region (≥ 0.25 μM/1 μM for 785 nm/830 nm) suggesting the graduating absorption (320% increase at 785 nm) introduced errors. We observe that 830 nm is lower in the ICG absorption spectrum and the correspondingly measured flow encountered less influence than 785 nm. From these results we anticipate the best practice in future studies to be utilization of a laser source with wavelength in a low region of the ICG absorption spectrum (e.g., 830 nm) or to only monitor flow prior to ICG injection or post-clearance. In addition, ncDCT was initially tested in a mouse tumor model to examine tumor size and averaged flow changes over a four-day interval. The next steps in forwarding the combined device development include the straightforward automation of data acquisition and filter rotation and applying it to in vivo tumor studies. These animal/clinical models may seek information such as simultaneous detection of tumor flow, fluorescence, and absorption contrasts or analyzing the relationship between variably sized fluorescently tagged nanoparticles and their tumor deposition relationship to flow distributions.
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38

Silva, Uéslen Rocha. "Nanopartículas multifuncionais de fluoreto de lantânio dopadas com Nd3+ como agentes de contrastes e terapêuticos". Universidade Federal de Alagoas, 2014. http://www.repositorio.ufal.br/handle/riufal/1702.

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In this work, we investigated the possible applications of Nd3+ ions doped lanthanium trifluoride (LaF3) nanocrystals as infrared constrast agents in the first and second biological windows of the electromagnetic spectrum, which extend from 700 to 1400 nm. For this, we use the three emissions of Nd3+ ions centered around 900, 1060, and 1330 nm, corresponding to transitions generated from the metastable state 4F3/2. In comparison with other fluorescent nanoparticles (NPs) used as biolables agents, such as semiconductor quantum dots and multiphotonic luminescent NPs, the Nd3+ doped LaF3 NPs present several advantages such as high fluorescence quantum efficiency and high chemical and spectral stabilities. We have demonstrated that, with the emission around 1060 nm is possible to obtain high brightness images of cancer cells and high penetration images of animal models (mices). Additionally, we have demonstrated that the emission around 900 nm has an appreciable thermal sensitivity that allows the use of such NPs as optical nanothermometers. As the Nd3+ concentration is increased to values around 25 mol%, this thermal sensitivity comes with a high conversion efficiency of light-to-heat, so that the NPs work as multifunctional agents capable of generating heat and measuring, simultaneously, induced local temperature. This has allowed the development of real time controlled thermal therapies of cancerous tumors in animal models (mices).
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Nesta tese avaliamos as possíveis aplicações de nanocristais de trifluoreto de lantânio (LaF3) dopados com íons de Nd3+ como agentes de contrastes infravermelho na primeira e segunda janelas biológicas do espectro eletromagnético, as quais se estendem de 700 a 1400 nm. Para isso usamos as três emissões de íons de Nd3+ centradas em torno de 900, 1060 e 1330 nm, correspondentes a transições geradas a partir do estado metaestável 4F3/2. Na comparação com outras nanopartículas (NPs) fluorescentes usadas como agentes de bio-contrastes, tais como, pontos quânticos de semicondutores e NPs multifotônicas luminescentes, as NPs de LaF3 dopadas com íons de Nd3+ apresentam diversas vantagens, tais como, alta eficiência quântica de fluorescência e altas estabilidades química e espectral. Nós demonstramos com a emissão em torno de 1060 nm que é possível obter imagens de alto brilho de células cancerígenas e imagens de alta penetração de modelos animais (ratos). Adicionalmente, demonstramos que a emissão em torno de 900 nm apresenta uma apreciável sensibilidade térmica que permite utilizar tais NPs como nanotermômetros ópticos. Quando a concentração de íons de Nd3+ é elevada para valores em torno de 25 mol%, esta sensibilidade térmica vem acompanhada de uma alta eficiência de conversão luz-calor, fazendo as NPs se comportarem como agentes multifuncionais capazes de gerar calor e medir, de forma simultânea, a temperatura local induzida. Isto tem permitido o desenvolvimento de terapias térmicas, controladas em tempo real, de tumores cancerígenos em modelos animais (ratos).
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39

Linarès-Loyez, Jeanne. "Développement de la microscopie par auto-interférences pour l'imagerie super-résolue tridimensionnelle au sein de tissus biologiques épais". Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0167/document.

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Le travail de cette thèse a été consacré au développement d’un nouvelle technique SELFI (pour self-interferences, auto-interférences en anglais). Cette méthode permet d’obtenir une localisation tridimensionnelle d’émetteurs fluorescents individuels. Nous avons démontré que cela permet l'imagerie super-résolue en 3D et le suivie 3D de molécules uniques en profondeur dans des échantillons biologiques denses et complexes. La technique SELFI se base sur l'utilisation des interférences auto-référencées (également appelées « auto-interférences ») pour remonter à la localisation 3D d’un émetteur en une seule mesure. Ces interférences sont générées via l’utilisation d'un réseau de diffraction placé en sortie du microscope de fluorescence : le signal de fluorescence diffracte sur le réseau et les ordres interfèrent, après une courte propagation, sur le détecteur. Les interférences ainsi formées sont décodées numériquement pour remonter à la localisation 3D d'une molécule fluorescente au sein de l'échantillon. Une molécule unique peut ainsi être localisée avec une précision d'une dizaine de nanomètre, et cela jusqu'à une profondeur d'au moins 50µm au sein d'un échantillon biologique vivant épais (par exemple un tissu biologique).En combinant la méthode SELFI à différentes techniques de super-résolution (PALM, dSTORM et uPAINT), nous montrons que cette méthode de localisation tridimensionnelle permet de retrouver la hiérarchie et l'organisation de protéines dans des objets biologiques. En effectuant du SELFI-PALM, nous avons pu observer différentes protéines des points focaux d’adhésion (talin-C terminale et paxiline) et retrouver les différences de hauteur attendues, et ceux sur des échantillons de cellules vivantes. Ces résultats confirment la résolution accessible avec la technique SELFI (environ 25nm) même pour un faible nombre de photons collectés (environ 500 photons par molécule).Nous mettons en évidence la robustesse de la technique SELFI en reconstruisant des images de super-résolution 3D de structures denses en profondeur dans des échantillons tissulaires complexes. En effectuant du SELFI-dSTORM, nous avons observé le réseau d’actine sur des cellules cultivées en surface de la lamelle dans un premier temps, et à différentes profondeurs (25 et 50 microns) au sein de tissus artificiels dans un second temps.Du suivi 3D de particule unique a aussi été effectué sein de tissus biologiques vivants. Nous avons observé la diffusion libre de quantum dots à différentes profondeurs (jusqu’à 50 microns, limité par l’objectif utilisé) dans des tranches vivantes de cerveau.Nous avons appliqué la technique SELFI à la détection de récepteurs postsynaptiques NMDA. Cela nous a permis d'observer, sur des échantillons de neurones en culture primaire mais aussi au sein de tranches de cerveaux de rats, une différence d'organisation entre les deux sous-unités GluN2A et GluN2B de ce récepteur au glutamate.Enfin, nous avons démontré l'importance de suivre l'évolution de l'environnement des échantillons biologiques vivants lors des acquisitions permettant la détection de molécules individuelles. Grâce à l'utilisation additionnelle et simultanée de l'imagerie de phase quantitative, nous avons pu étudier la dynamique de la membrane cellulaire durant l’activation par un facteur de croissance. L'analyse corrélative entre les images de phase quantitative en lumière blanche et les détections de molécules fluorescentes uniques permet d'obtenir de nouvelles informations pertinentes sur l'échantillon étudié
The work of this thesis was devoted to the development of a new technique SELFI (for self-interferences). This method unlocks the three-dimensional localization of individual fluorescent emitters. We have demonstrated that this allows 3D super-resolved imaging and 3D tracking of single molecules deep into dense and complex biological samples. The SELFI technique is based on the use of self-referenced interference to go back to the 3D location of a emitter in a single measurement. These interferences are generated using a diffraction grating placed at the exit of the fluorescence microscope: the fluorescence signal diffracts on the grating and, after a short propagation, the orders interfere on the detector. The formed interferences are digitally decoded to extract the 3D location of a fluorescent molecule within the sample. A single molecule can thus be localized with a precision of approximatively ten nanometers up to a depth of at least 50 µm in a thick living biological sample (for example a biological tissue).By combining the SELFI method with different super-resolution techniques (PALM, dSTORM and uPAINT), we show that this three-dimensional localization method grants the access to the hierarchy and organization of proteins in biological objects. By performing SELFI-PALM, we observed different proteins of the adhesion focal points (talin C-terminal and paxilin) and found the expected elevation differences, and those within living cell samples. These results confirm the resolution capability of the SELFI technique (about 25 nm) even for a small number of photons collected (about 500photons per molecule).We highlight the robustness of the SELFI technique by reconstructing 3D super-resolution images of dense structures at depth in complex tissue samples. By performing SELFI-dSTORM, we observed the actin network in cells grown on the surface of the coverslip at first, and at different depths (25 and 50 microns) within artificial tissues in a second time.3D single particle tracking has also been performed in living biological tissues. We observed the free diffusion of quantum dots at different depths (up to 50 microns) in living brain slices.We applied the SELFI technique to the detection of NMDA postsynaptic receptors. We observed, in primary culture of neurons but also within slices of rat brains, a difference in organization between the two subunits GluN2A and GluN2B of this glutamate receptor.Finally, we show the importance of following the evolution of the living biological sample environment during the acquisition of images leading to detections of single molecules. Thanks to the additional and simultaneous use of quantitative phase imaging, we were able to study cell membrane dynamics during the activation by a growth factor. The correlative analysis between white light quantitative phase images and single fluorescent molecule detections provides new relevant information on the sample under study
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40

Zhang, Yuanwei. "Squaraine dyes, design and synthesis for various functional materials applications". Doctoral diss., University of Central Florida, 2013. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/6392.

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This dissertation contains the synthesis and characterization of squaraine based new functional materials. In the first part of this thesis work, a water soluble benzothiazolium squaraine dye was synthesized with pyridium pendents, and controlled aggregation properties were achieved. After formation of partially reversible J-aggregation on a polyelectrolyte (poly(acryl acid) sodium salt) template, the nonlinear, two-photon absorption cross section per repeat unit was found to be above 30-fold enhanced compared with nonaggregate and/or low aggregates. Using a similar strategy, sulfonate anions were introduced into the squaraine structure, and the resulting compounds exhibited good water solubilities. A 'turn on' fluorescence was discovered when these squaraine dyes interacted with bovine serum albumin (BSA), titration studies by BSA site selective reagents show these squaraine dyes can bind to both site I and II of BSA, with a preference of site II. Introduction of these squaraine dyes to BSA nanoparticles generated near-IR protein nano fabricates, and cell images were collected. Metal sensing properties were also studied using the sulfonates containing a benzoindolium squaraine dye, and the linear response of the absorption of the squaraine dye to the concentration of Hg2+ makes it a good heavy metal-selective sensing material that can be carried out in aqueous solution. Later, a squaraine scaffold was attached to deoxyribonucleosides by Sonogashira coupling reactions, in which the reaction conditions were modified. Iodo-deoxyuridine and bromo-deoxyadenosine were used as the deoxyribonucleosides building blocks, and the resulting squaraine dye-modified deoxyribonucleosides exhibited near-IR absorption and emission properties due to the squaraine chromophore. Interestingly, these non-natural deoxyribonucleosdies showed viscosity dependent photophysical properties, which make them nice candidates for fluorescence viscosity sensors at the cellular level. After incubation with cells, these viscosity sensors were readily uptaken by cell, and images were obtained showing regions of high viscosity in cells.
Ph.D.
Doctorate
Chemistry
Sciences
Chemistry
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41

Bourdolle, Adrien. "Conception de sondes et nano-sondes à base de lanthanides émettant dans le proche infrarouge pour la microscopie biphotonique". Thesis, Lyon, École normale supérieure, 2011. http://www.theses.fr/2011ENSL0657.

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L’objectif de cette thèse est l’élaboration de sondes à base de lanthanide pour la microscopie optique biphotonique. Cette technique d’imagerie complémentaire à l’IRM et au scanner permet une analyse rapide et facile de tissus épais. Afin de permettre l’observation en profondeur, l’absorption et l’émission de la sonde doit se situer dans la zone de transparence biologique [700 – 1200 nm]. L’absorption à deux photons (ADP) est un phénomène d’optique linéaire de troisième ordre par lequel l’état excité est atteint par absorption simultanée de deux photons. De fait, l’excitation à énergie moitié se situe dans la zone de transparence biologique. Les sondes envisagées combineraient les propriétés optiques uniques des lanthanides, telles que des bandes d’émission très étroites allant du visible à l’infrarouge et des durées de vie de luminescence longues, et les avantages de l’ADP, permettant une excitation dans l’IR et une résolution tridimensionnelle. Dans ce contexte, cette thèse décrit l’élaboration de complexes d’europium et d’ytterbium à ligands macrocycliques stables en milieu aqueux et dont la luminescence peut être sensibilisée à deux photons. Ces complexes ont permis l’imagerie de la vascularisation de cerveaux de souris par microscopie biphotonique dans le proche infrarouge. La seconde approche consiste à encapsuler un complexe luminescent dans des nanoparticules desilice formées par la technique sol-gel (collaboration A. Ibanez, institut Néel, Grenoble) afin de protéger le complexe du milieu biologique. Enfin la dernière approche consiste à greffer des complexes de lanthanides à la surface d’une particule de silice par chimie organométallique de surface. Ces travaux ont conduit à la formation de nano-objet très brillants dans le rouge et le proche infrarouge, détectables à l’échelle de l’objet unique par microscopie à deux photons
Two Photon Scanning Laser Microscopy (TPSLM) has evolved as an emerging bio-imaging technique widely used in academic research and in medical diagnosis. This technique requires the design of bioprobes specially optimized for such purpose. A particular attention is actually devoted bio-probes featuring both two-photon absorption (TPA) and emission in the near infra-red (NIR) spectral range [700 – 1200 nm], also called biological window that is particularly promising for thick tissues imaging. In this context, europium complexes emitting in the red (615 nm) has been recently sensitized by two photon antenna effect and used for TPSLM in cells combining the advantages of lanthanide emission (sharp line and long lived) and those of TPA. Based on this preliminary results, this thesis describe the design of europium and ytterbium complexes which have an improved stability in water and good emission properties sensitized by TPA. Theses complexes allow the imaging of mice’s brain vascularisation in the NIR. An another approach to stabilize lanthanide complexes was also used by encapsulating theses fluorophores in silica nanoparticle (collaboration with A. Ibanez, Institut Néel, Grenoble). Then the last approach consists on the grafting of the chromophores on silica sphere using surface organometallic chemistry methods. The nanoparticles obtained by both way are really luminescent in the red or infrared and can be imaged as single nanoparticle by TPSLM
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42

Fiorini, Federica. "Soft hybrid materials for cell growth and proliferation". Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAF027/document.

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Le travail de recherche consiste à développer des hydrogels pour la prolifération et la migration cellulaires in vitro et in vivo en trois dimensions (3D). Des hydrogels à base de polyamidoamines avec d'intéressantes propriétés physicochimiques et une remarquable biocompatibilité ont été développés pour différentes applications biomédicales. Un hydrogel avec des sondes luminescentes d’iridium(III) incorporés de manière covalente, a été conçue comme plate-forme 3D de culture cellulaire, pour la visualisation directe des cellules vivantes en temps réel, et a démontré être un puissant outil de bioimagerie in vitro. En outre, un hydrogel nanocomposite, capable d'induire la chimiotaxie des cellules souches, a été développé et testé in vivo, en confirmant son potentiel en tant qu’implant pour l’ingénierie tissulaire. Finalement, un hydrogel injectable et biodégradable a été réalisé comme un nouvel agent pour la dissection sous-muqueuse endoscopique des lésions néoplasiques digestives
The research work focuses on the development of hydrogels to investigate three-dimensional (3D) cell proliferation and migration in vitro and in vivo. Polyamidoamines-based hydrogels with interesting physicochemical properties and high biocompatibility have been developed for different biomedical applications. An hydrogel with covalently incorporated iridium(III) fluorescent probes, has been conceived as a 3D cell culture platform for the direct visualization of living cells in real-time, demonstrating to be a powerful tool for in vitro bio-imaging. Moreover, a nanocomposite hydrogel, able to induce chemotaxis of stem cells, was developed andtested in vivo, confirming its potential as a tissue engineering implant. Finally, an injectable biodegradable nanocomposite hydrogel was realized as a novel agent for endoscopic submucosal dissection of large neoplastic lesions of the gastro-intestinal tract
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43

Huisman, Maximiliaan. "Vision Beyond Optics: Standardization, Evaluation and Innovation for Fluorescence Microscopy in Life Sciences". eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1017.

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Fluorescence microscopy is an essential tool in biomedical sciences that allows specific molecules to be visualized in the complex and crowded environment of cells. The continuous introduction of new imaging techniques makes microscopes more powerful and versatile, but there is more than meets the eye. In addition to develop- ing new methods, we can work towards getting the most out of existing data and technologies. By harnessing unused potential, this work aims to increase the richness, reliability, and power of fluorescence microscopy data in three key ways: through standardization, evaluation and innovation. A universal standard makes it easier to assess, compare and analyze imaging data – from the level of a single laboratory to the broader life sciences community. We propose a data-standard for fluorescence microscopy that can increase the confidence in experimental results, facilitate the exchange of data, and maximize compatibility with current and future data analysis techniques. Cutting-edge imaging technologies often rely on sophisticated hardware and multi-layered algorithms for reconstruction and analysis. Consequently, the trustworthiness of new methods can be difficult to assess. To evaluate the reliability and limitations of complex methods, quantitative analyses – such as the one present here for the 3D SPEED method – are paramount. The limited resolution of optical microscopes prevents direct observation of macro- molecules like DNA and RNA. We present a multi-color, achromatic, cryogenic fluorescence microscope that has the potential to produce multi-color images with sub-nanometer precision. This innovation would move fluorescence imaging beyond the limitations of optics and into the world of molecular resolution.
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44

Panagiotopoulou, Maria. "Organic-inorganic composite materials for specific recognition and optical detection of environmental, food and biomedical analytes". Thesis, Compiègne, 2016. http://www.theses.fr/2016COMP2315/document.

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Cette thèse décrit l'état de l'art des sondes et nanoparticules fluorescents traditionnels utilisés en imagerie de fluorescence ainsi que le développement de nouveaux nanomatériaux à base de polymère à empreinte moléculaire, aussi dénommé ‘anticorps plastique’, pour le ciblage et la bioimagerie. En biologie et en médecine, il y a un besoin constant de diagnostiquer diverses maladies pour leur éventuel traitement et prévention. Une distribution anormale et un taux élévé de glycosylation (e.g. acides hyaluronique et sialique) à la surface ou dans les cellules sont indicateurs d’une infection ou d’un cancer. Généralement, l’imagerie par fluorescence permet de visualiser, localiser et quantifier les biomarqueurs de pathologie mais à l’heure actuelle, il n’existe pas d’outil analytique fiable pour cibler spécifiquement les molécules de glycosylation car les anticorps et les lectines vendus dans le commerce ont une faible affinité et sélectivité vis-à-vis de ces cibles. Dans ce contexte, les polymères à empreintes moléculaires (MIPs) pourraient apporter une solution. Les MIPs sont des récepteurs synthétiques possédant des affinités et sélectivités comparables à ceux des anticorps, mais exhibant une stabilité physique, thermique et chimique bien plus accrue. De plus, leur fabrication est peu coûteuse et ne nécessite pas de tuer des animaux comme pour l’obtention des anticorps biologiques. Dans cette thèse, nous avons optimisé et synthétisé des MIPs biocompatibles pour leur utilisation en bioimagerie afin de détecter et quantifier l’acide hyaluronique et l’acide sialique sur les cellules et les tissus de peau humaine. L’acide glucuronique, une composante de l’acide hyaluronique et l’acide N-acétylneuraminique, l’acide sialique le plus commun, ont été utilisés comme molécules ‘patron’, générant des MIPs très sélectifs envers leur cible en milieu aqueux. Deux types de nanoparticules de MIPs fluorescents ont été synthétisés: (1) en incorporant un colorant rhodamine polymérisable dans la solution de pré-polymérisation et (2) en encapsulant des boîtes quantiques InP/ZnS générant ainsi des MIPs de type cœur-coquille. Pour cela, nous avons adopté une stratégie innovante qui consiste à synthétiser les coquilles de MIPs directement autour des boîtes quantiques en utilisant l’énergie de l’onde fluorescente émise par l’excitation des points quantiques, pour initier la polymérisation. Un protocole d'immunocoloration standard a ensuite été optimisé afin d’imager des kératinocytes humains fixés et vivants ainsi que des tissus de peau, par microscopie à épifluorescence et confocale. Les résultats étaient similaires à ceux obtenus par la méthode de référence utilisant une protéine biotinylée reconnaissant l'acide hyaluronique. L'imagerie multiplex en combinant deux MIPs couplés à deux couleurs de boîtes quantiques et l’imagerie des cellules cancéreuses ont également été démontrées. Bien que les MIPs n’étaient pas cytotoxiques aux concentrations utilisées pour la bioimagerie, la toxicité des différentes composantes du MIP pourrait être un frein à leur utilisation dans le domaine biomédical. Afin de rendre ces MIPs plus ‘inoffensifs’, nous avons supprimé l’amorceur de polymérisation, une molécule considérée comme toxique. Les MIPs ont été synthétisés en employant des monomères qui s’auto-initient sous l’effet de l’UV ou de la chaleur. La spécificité et la sélectivité des MIPs obtenus étaient similaires à ceux préparés avec des amorceurs. En conclusion, cette thèse décrit la première utilisation des MIPs comme anticorps synthétique pour la bioimagerie de fluorescence. Ce travail ouvre la voie à de nouvelles applications en détection, diagnostique et thérapie par des MIPs
This thesis describes the state of the art in nanomaterials-based targeted bioimaging and introduces molecularly imprinted polymers, also termed ‘plastic antibodies’ as novel biorecognition agents for labeling and imaging of cells and tissues. In fundamental biology and medical diagnostics, there is a constant need to localize and quantify specific molecular targets. Abnormal glycosylation levels or distributions of hyaluronan or sialic acids on cells are indicators of infection or malignancy. In general, bioimaging with fluorescent probes enables the localization and qualitative or quantitative determination of these pathological biomarkers. However, no reliable tools for the recognition of glycosylation sites on proteins exist, because the commercially available antibodies or lectins have poor affinity and selectivity for these targets. In this context, tailor-made molecularly imprinted polymers (MIPs) are promising synthetic receptor materials since they present a series of advantages over their natural counterparts such as the ease and low cost of preparation and their physical and chemical stability. Thus, MIPs could provide a robust and specific imaging tool for revealing the location/distribution, time of appearance and structure of glycosylation sites on/in cells, which would lead to a better insight of the tremendously diverse biological processes in which these molecules are involved. Herein, we describe the synthesis of water-compatible MIPs for the molecular imaging of hyaluronan and sialylation sites on cells and tissues. Since molecular imprinting of entire biomacromolecules like oligosaccharides is challenging, we opted for what is commonly called the ‘epitope approach’, which was inspired by nature. The monosaccharides, glucuronic acid and N-acetylneuraminic acid were imprinted, and the resulting MIPs were able to bind these molecules when present and accessible on the terminal unit of hyaluronan and sialylation sites. Fluorescent MIPs were synthesized as rhodamine-labeled nanoparticles and as MIP-coated InP/ZnS core-shell quantum dot (QD) particles. For the coating of the QDs, a novel versatile solubilization and functionalization strategy was proposed, which consists of creating polymer shells directly on QDs by photopolymerization using the particles as individual internal light sources. A standard immunostaining protocol was then successfully adapted for the application of the fluorescently labeled MIPs to image fixed and living human keratinocytes and skin tissues, by epifluorescence and confocal fluorescence microscopy. The results were comparable to those obtained with a reference method where staining was done with a biotinylated hyaluronic acid binding protein. Multiplexed and cancer cell imaging were also performed, demonstrating the potential of molecularly imprinted polymers as a versatile biolabeling and bioimaging tool. Although the MIPs were not cytotoxic at the concentrations used for bioimaging, in order to render them generally applicable in biomedicine, where toxicity of the polymerization precursors is a matter of concern, we suppressed the initiator, a toxic chemical. Initiator-free MIPs were thus synthesized by using monomers that can self-initiate under UV irradiation or heat. The specificity and selectivity of the obtained MIPs were as good as the ones prepared with initiators. In conclusion, we have demonstrated for the first time the great potential of MIPs as synthetic antibody mimics for bioimaging. The possibility to associate other functionalities such as QDs and additionally attach drugs to the same material appears rather straightforward due to the synthetic polymeric nature of MIPs, which paves the way to new potential applications in theranostics
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45

Tzeng, Yan-Kai, i 曾彥凱. "Manipulation and Bioimaging Applicationsof Fluorescent Nanodiamonds". Thesis, 2014. http://ndltd.ncl.edu.tw/handle/15044126303809867087.

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博士
國立臺灣大學
化學研究所
102
Diamond is an allotrope of carbon. A unique property that distinguishes it from other carbon materials is that diamond is optically transparent and often contains point defects as color centers. Negatively charged nitrogen-vacancy (NV&;#8722;) defects are the most noteworthy color centers in diamond because it emits far-red fluorescence with high photostability. This unique optical property combined with good biocompatibility makes nanoscale diamonds a promising fluorescent probe for bioimaging, particularly cell tracking studies. Firstly, we measured the efficiency of Forster resonance energy transfer (FRET) with sub-20-nm fluorescent nanodiamonds (FNDs) as the FRET donors and near-infrared dyes as the acceptors. A FRET efficiency of ~7% was found. Next, we built a super-resolution stimulated emission depletion (STED) microscopy system for FNDs and demonstrated the nanoscale precision for fluorescence imaging. FND is an ideal candidate for STED, since it does not photobleach. In contrast, organic dyes or fluorescent proteins are easily photodamaged by the high-power STED laser beam. Another distinct feature of FND is that its fluorescence lifetime is more than 13 ns, significantly longer than that of common organic dyes or green fluorescent proteins (GFPs) as well as cell auto-fluorescence. Using a time-gating technique, which successfully reduces cell and tissue auto-fluorescence background signals, we applied FNDs as long-term cell trackers and demonstrated the homing and engraftment capacity of lung stem cells transplanted in mice. Finally, the NV&;#8722; center in diamond is a very unique quantum system, and can be manipulated by optical detected magnetic resonance (ODMR), a technique applicable to measure environmental changes such as temperature shifts. With the ODMR technique, we achieved high-sensitivity detection of the surrounding temperature of 100-nm FND particles at the nanoscale. All the experimental results demonstrate that FNDs are ideal candidates for potential applications in modern biomedical science and biotechnologies as nanotechnology-enabled imaging agents and sensors.
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46

Lin, Ching-Shiuan, i 林青璇. "The Study of Green Fluorescent ProteinApplications for E.coli Bioimaging System". Thesis, 2006. http://ndltd.ncl.edu.tw/handle/92178940810334994512.

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碩士
國立成功大學
化學工程學系碩博士班
94
To　develop　a　rapid　reporter　system　for　estimating　the　promoter　efficiency　in　Escherichia　coli　XL1B,　the　expression　pattern　of　the　green　fluorescent　protein(GFP)　in　the　giant　protoplast　was　investigated.　In　addition, the　methods　for quantifying　the　fluorescent　intensity　of　GFP　by　fluorescent　microscopy as　well　as　digital　camera　were　illustrated.　The　results　showed　that　the　protoplasts　of　E. coli　was　able　to　express　GFP,　indicating　that　giant　protoplasts　possess　cell　functionality.　The　expression　level　of　GFP　in　E. coli　protoplasts　was　affected　by　the　air　level,　i.e.　the　fluorescence　intensity　increases　as　the　air　level　does.　The　digital　imaging　method　with　the　advantages　of　rapidity,　directness,　and　easiness　established　in　the　work　could　digitalize　the　intensity　of　fluorescence　emitted　by　GFP.　The　results　suggested　that　the　tested　plasmids　were　stable　in　recombinant　cells,　no　matter　cells　in　normal　situation　or　becoming　the　giant　protoplasts.　In　conclusion,　the　bioimaging　system　of　the　giant　protoplast　of　E. coli　could　be　a　suitable　method　to　estimate　the　promoter　efficiency　and　promise　to　be　a　tool　for　studying　the　protein　expression　and　cellular　activity.
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47

Kao, Chia-Yun, i 高嘉鄖. "Optical Properties of Fluorescent Proteins and Their Applications in Bioimaging". Thesis, 2008. http://ndltd.ncl.edu.tw/handle/16348386449264065643.

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碩士
中原大學
應用物理研究所
95
Recently, the exogenous fluorescent agent has been applied to bio-imaging techniques widely. In different fluorescent agents, the green fluorescent agent has high-stability and high-luminescence efficiency superior in itself, thus, it usually used to the lable of noninvasive molecular and cell biology. It also use to monitor the gene phenomenon, rotein distribution, and protein interaction at the same time. In the thesis, we offer a spectrum analyzing system to use to analysis the fluorescent property changing of the sample in different temperature, and then, we also offer a algorithm to analysis the phenomenon of the fluorescent spectrum distributions. Furthermore, we will treat the thermal effect of the samples. The more research of optical coherence tomography (OCT) is mainly the systems improvement. One is the resolution of the systems, and the other is imaging speed improvement. In the thesis, we focus on improve the imaging speed and provide a real time all reflective optical delay line system to alternate the traditional stepper motor. The best distinguishing of this system is closed, stable, easily fabricated, and in scanning process, it can lower the light loss. We applied this system to the OCT, when the scanning mirror of the system tuned ± 9.6°, the scanning depth of the sample will arrived 2.9 mm by calculated. Then we will identify the system capability by the experiment. In the future, we hope to combine with these two system, and use OCT to analyze the changing with thermal effect inside structure, and treat to inside optical properties.
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48

Chen, Yen-Hao, i 陳彥豪. "Design and Synthesis of Nitric Oxide and Cysteine/Homocysteine Fluorescent Probes for Intracellular Bioimaging". Thesis, 2014. http://ndltd.ncl.edu.tw/handle/c4a56v.

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博士
國立交通大學
生物科技系所
102
Molecular imaging provides the ability to study cellular and molecular processes that have the potential to impact many facets of biomedical research. Fluorescence imaging, one technique of molecular imaging, is generally superior in terms of sensitivity, low-price, and ease of use. To promote the specificity of the target molecule, the developments of fluorescent probes is essential. In this thesis, the basic concepts including components, signal transduction principles, and design strategies of fluorescent probes were illustrated. This thesis is composed of two parts, nitric oxide fluorescent probes and cysteine/homocysteine fluorescent probe. In the first part of this thesis, two fluorescent probes, RH and FA-OMe, were developed for the visualization of nitric oxide (NO). The design strategy of RH is based on the NO-induced spirolactam ring opening. The ring opening reaction gives rise to strong fluorescence emission. RH possesses a ~1000-fold fluorescence turn-on from a dark background, which is higher than that of the commercialized probe, DAF-2. In the other hand, FA-OMe is designed by the concept of NO-induced reductive deamination of aromatic primary monoamine. After reacting with NO, the photoinduced electron transfer (PeT) effect is suppressed due to the disappearance of the electron donating amino group, and the fluorescence is restored. The PeT effect was demonstrated by density functional theory (DFT) calculations of the components forming FA-OMe and dA-FA-OMe. FA-OMe successfully improves the shortcoming of the condensation of o-phenylenediamine moiety by dehydroascorbic acid and ascorbic acid. Compared to the DAF-2, FA-OMe is more specific to NO. The concentration-dependent studies of these two probes showed an excellent linearity between fluorescence intensities and NO concentrations. The detection limit of RH and FA-OMe for NO is 20 and 44 nM, respectively. Furthermore, both probes are highly stable at physiological pH, and can be applied to monitor the endogenous nitric oxide. In the second part of this thesis, a push-pull fluorogenic reagent, NBD-SCN, was developed and applied for specific detection of cysteine (Cys) and homocysteine (Hcy). Replacing thiocyanato group with Cys/Hcy increased the intramolecular charge transfer (ICT) characteristic of the probe and resulted in emission of fluorescence. The fluorescent response of the probe toward Cys/Hcy was significantly higher than toward glutathione and other amino acids. The probe showed 470- and 745-fold fluorescence enhancement at 550 nm and detection limit of 2.99 and 1.43 nM for Cys and Hcy, respectively. Time-dependent fluorescence assays showed that the fluorescence intensity reached a plateau within 20 seconds after addition of Cys and within 10 minutes after addition of Hcy. Furthermore, the fluorescence images of biothiol in Raw 264.7 cells were obtained successfully by the employment of NBD-SCN.
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49

Liu, Chih-Wei, i 劉智偉. "The Study of Vibrio vulnificus Blue Fluorescent Protein Applications for Gram Negative Bacteria Bioimaging System". Thesis, 2005. http://ndltd.ncl.edu.tw/handle/27077609491115446733.

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碩士
國立成功大學
化學工程學系碩博士班
93
The expression of the wild type (bfp) and mutation gene (bfp-D7) of blue fluorescent protein (BFP) isolated from Vibrio vulnificus CKM-1 in the giant protoplasts of Escherichia coli XL1B and V. vulnificus CKM-1 were investigated, respectively. In addition, the method for quantifying of the fluorescent intensity of BFP by fluorescent microscopy as well as digital camera was illustrated. The results first showed that both the protoplasts of E. coli and V. vulnificus were able to express bfp as well as bfp-D7, indicating that giant protoplasts possess cell functionality. The fluorescent intensity of the fluorescent protein expressed by bfp-D7 gene was stronger than that expressed by bfp gene. The growth and death rates of V. vulnificus protoplasts were higher than that of E. coli ones. As compared with E. coli, the expression level of BFP in V. vulnificus protoplasts was higher. The death rate of V. vulnificus protoplasts could be slow down at the expense of cellular activity when decreasing the incubation temperature from 30 to 25°C. The digital imaging method with the advantages of rapidity, directness, and easiness established in the work could digitalize the intensity of fluoresce emitted by BFP. The fluorescence produced by giant protoplasts is not only stronger but also longer than that emitted by normal cells. The result suggested that the plasmids were stable when the recombinant microbial cells becoming the giant protoplasts. Additionally, the yield of fluorescent protein produced by giant protoplasts was higher than normal cells. In conclusion, the bioimaging system of the giant protoplast of Gram-negative strains is promising to be a tool for studying the protein expression and cellular activity.
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50

Chou, Chih-Hung, i 周志鴻. "Sulfonyl Triazole-Directed Synthesis of Heterocyclic molecules and Its Application in Fluorescent Turn-On Bioimaging". Thesis, 2018. http://ndltd.ncl.edu.tw/handle/ug4nsa.

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博士
國立中山大學
化學系研究所
106
Environmental sensitive fluorophores are widely used to detect protein microenvironment, cell staining, and protein labeling in living organisms. This type of fluorescent molecule has a large Stokes shift, which will facilitate the detection of the emission spectrum without interference from the absorption wavelength. In this research, we used 1,4-tosyltriazole under heating or Cu (I) catalyst to generate high electrophilic center, keteneimine, induced denitrogenative annulation for the synthesis of dihydroquinoline-4-imine (DQI). After DFT simulation, we can modify electron donating or withdrawing group on benzene ring to modulate HOMO and LUMO’s energy gap to change the emission wavelength. DQI in different polarity solvent system showed dramatic change, which shifts of the fluorescence emission from 573 nm in methanol to 478 nm in n-hexane, as well as diminishes quantum yield from 62% in dichloromethane to 6% in methanol. It matches environmental sensitive properties. In order to apply DQI fluorophore for biological system, SA-DQI was synthesized successfully and used to detect human carbonic anhydrase II (hCAII) with a fifteen-fold enhancement of fluorescence intensity. We also utilized SA-DQI to stain transfected HEK 293T cell. It shows fluorescence signal on cell membrane. Finally, more water soluble aryl-suldonamide conjugated DQI (SA-TEG-DQI) molecule was successfully track CA in vivo in zebrafish.
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