L’objectif de cette thèse est l’élaboration de sondes organiques pour la microscopie optique non-linéaire par fluorescence excitée à deux photons (F2P) et génération de seconde harmonique (GSH). Dans une première partie, cette thèse décrit la synthèse de sondes pour l’imagerie de potentiels de membrane par GSH, comportant un ou plusieurs motifs sucres ainsi que leurs caractérisations spectroscopiques. Les premiers essais en imagerie biologique ont permis de démontrer une bonne affinité des sondes sucres pour la membrane cellulaire et un signal de GSH sur cellule neuronale a pu être observé sur une période de temps allant jusqu'à près de trois heures. La seconde approche a consisté à synthétiser et étudier des chromophores possédant des propriétés de fluorescence à l’état solide pour des applications dans la synthèse de nanoparticules fluorescentes pour l’imagerie biologique. 18 des 21 composés synthétisés ont pu être cristallisés et leur structure résolue par diffraction des rayons X et les propriétés spectroscopique en solution et à l’état solide ont été réalisées. Cette étude a permis de montrer que l’arrangement des molécules les unes par rapport aux autres avait une grande influence sur la fluorescence à l’état solide et donc que les substituants avaient une grande importance. Enfin, cette partie se termine sur les premiers essais effectués pour synthétiser des nanoparticules fluorescentes. / The objective of this thesis is the design of new organic probes for nonlinear optical microscopy by two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). In the first part, we describe the synthesis of probes for voltage sensitive imaging by SHG, bearing one or more sugar units and their spectroscopic characterization. The first biological imaging tests have shown good affinity of the probes to the cell membrane and the SHG signal of neuronal cell was observed over a period of nearly three hours. The second part comprises the synthesis and the study of chromophores with solid state fluorescence properties for use in fluorescent nanoparticles for biological imaging. 18 of the 21 compounds synthesized have been crystallized, their crystal structures determined by X-ray diffraction and their spectroscopic properties studied in solution and in the solid state. These studies showed that the arrangement of molecules relative to each had a great influence on the solid state fluorescence and therefore that the substitution was very important. The chapter ends with the first tests of fluorescent nanoparticles synthesis.

Maleimide based highly luminescent material Cbz-MI with donor acceptor donor (D-A-D) backbone has been synthesized and characterized. An organic light emitting diode fabricated using this material as emitting layer exhibited EQE of 2.5% in the yellow region of visible spectrum. Due to the small energy gap of materials emitting in this region of spectrum, EQE of OLED is usually limited by various non-radiative decays and high EQE of OLED using this material proves that most of the nonradiative decay pathways have been avoided by the careful design of molecule and device structure. Although Cbz-MI did not show TADF properties, but if tailored with right electron donor along with maleimide as an acceptor, such derivatives may exhibit TADF properties

Resumo: O objetivo deste estudo foi verificar os procedimentos realizados pelos cirurgiões-dentistas durante a seleção de cor dental, analisar a prática de utilização do método visual para a seleção da cor, avaliar a diferença de cor entre a escala Vitapan Classical e diversas resinas compostas e a fluorescência de diferentes cores de uma resina composta e da estrutura dental. Sessenta cirurgiões-dentistas foram selecionados para responder um questionário referente à seleção de cor dental. Observou-se que todos os profissionais utilizavam o método visual, apenas 2 utilizavam o método digital e 52% desconheciam o método digital. Para a análise da prática de utilização do método visual de seleção da cor com o auxílio da escala Vitapan Classical foi verificada a concordância nas classificações de cor entre o consenso de dois observadores e a dada pelo fabricante da resina composta Helio Fill (Vigodent), obtendo um nível de concordância "sofrível" (k = 0,3774). Por meio do sistema CIE L*a*b* foram calculadas as diferenças de cor entre a escala Vitapan Classical e cada uma das diversas resinas compostas avaliadas. Observou-se que oito resinas compostas não diferem visualmente da escala Vitapan Classical e duas apresentaram esta diferença. Para a análise da fluorescência foram coletadas 16 imagens para cada uma das 11 cores da resina composta Helio Fill (Vigodent) e estrutura dental sob iluminação UV. Estas processadas matematicamente, quantificando a intensidade de fluorescência. Verificou-se que a cor A1 apresentou maior média de intensidade de fluorescência e a tonalidade C3 apresentou a menor. Porém, a estrutura dental apresentou valor médio inferior a todas as cores avaliadas. (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The purpose of this study was to evaluate the procedures carried out by dentists during dental shade selection, assess the practice of the visual method to select dental shade, to evaluate the shade difference between Vitapan Classical shade guide and several composite resins, to evaluate the fluorescence in different shades of a composite resin and dental structure. Sixty professionals were selected, who answered to questions of the form about to select dental shade. The totality of the professionals selected dental shade by visual method; only 2 used digital methods; 52% did not know about the digital method. To assess the practice of the use of visual method to select dental shade, it was observed the agreement in shade classification between two examiners and the classification established by the composite resin manufacturer Helio Fill (Vigodent) which resulted in a "suffering" level of agreement (k = 0.3774). Through CIE L*a*b* system the difference of the total shade between Vitapan Classical shade guide and the composite resin was calculated and it was observed that eight of the composite resins assessed do not differ visually from Vitapan Classical shade guide, and two showed a perceptive difference to human eye. For the fluorescence analysis sixteen images were captured by the camera for the different shades of Helio Fill (Vigodent) composite resin and dental structure under UV LED illumination, and they were processed mathematically, quantifying the fluorescence intensity. It was observed that A1 shade showed the higher mean of fluorescence intensity, whereas C3 shade showed the lower mean among the composite resin shades. However, the mean intensity of dental structure was lower than those related to all the shades evaluated. (Complete abstract, click electronic access below) / Orientador: Leonor de Castro Monteiro Loffredo / Coorientador: Alessandra Nara de Souza Rastelli / Banca: Marcelo Ferrarezi de Andrade / Banca: Mônica Campos Serra / Mestre

The discovery of the existence of the cell membrane has led to a search for its organization on a molecular scale. The advent of artificial lipid bilayers and the development of electron microscopy in the 1930's provided direct visual evidence for the existence of the cell membrane and drove forward models of membrane structure based its known composition of proteins, lipids and carbohydrates. The fluid mosaic model of membrane structure, based on thermo- dynamics and newly developed protein structural studies of the time, placed integral globular membrane proteins within a fluid phospholipid bilayer. This model allowed for the association of proteins into groups and the possible mobility of proteins within the lipid bilayer. At the the same time fluorescence microscopy demonstrated movement of proteins in the plane of the lipid bilayer. Since then experimental techniques have been developed that show protein complexes of varying sizes do exist and so this gives us the opportunity to ask how receptor proteins fit into the molecular organization of the cell membrane. This thesis presents an investigation into how the epidermal growth factor receptor (EGFR) organizes in the cell membrane of colorectal carcinoma cells. First a new cell line for studying the receptor by stably expressing the epidermal growth factor receptor conjugated to enhanced green fluorescent protein (EGFR-eGFP) in SW620 cells was developed. This is an interest- ing cell line because it originates from a colonic adenocarcinoma that during the process of metastasis has lost the ability to express the EGFR. It therefore provided an environment for the expression of the fluorescent form of the receptor more in keeping with its natural environment. The technique of total internal reflection fluorescence (TIRF) microscopy was used to visualize the fluorescently tagged receptor in the cell membrane. This technique uses the principles of total internal reflection to excite fluorescence in molecules located only 100 nm into the cell. Because sources of fluorescence from outside the illuminated area are minimized individual fluorescent molecules can be imaged. The spots in the images, produced by the fluorophores, were detected using a single molecule detection and tracking algorithm. The intensities of these detected spots were analysed and compared with that from a single molecule of enhanced green fluorescent protein (eGFP). This gave an estimate of the number of receptors contained within each receptor complex. Before ligand binding most of the receptors were found to be located in complexes containing up to eight molecules and most frequently they were found in complexes of two molecules. Larger complexes of receptors were found to have formed after activation of the receptor by its ligand.

Dysfunctional or over and under expressed enzymes play a crucial role in a variety of diseases. A tool that can identify dis-regulated enzymes in individual patients would be beneficial and would allow personalised treatment. For this purpose, a 10,000 membered ‘spit-and-mix’ PNA-encoded peptide library with a cell penetrating peptide was synthesised and interrogated with K562 cell lysate and intact K562 cells. This allowed the specific enzyme activity pattern for ABL tyrosine kinase from both inside a cell and a lysate to be obtained. Hybridisation of this library with a DNA-microarray resulted in bio-fouling by the cell lysate, thereby preventing analysis of the phosphorylation pattern. To allow extraction and purification of the peptide library from the cell lysates, a His-tag was incorporated into the library, and enabled successful library analysis. In addition to this 10,000 member library, a focused 100 PNA-encoded peptide library was synthesised. The library included peptide sequences known to be phosphorylated by specific tyrosine kinases deregulated in acute lymphoblastic leukaemia (ALL) with a PNA-tag complementary to a DNA microarray. Different ligation methods to conjugate the peptides to PNA-tags were screened – this included amide coupling, copper catalysed azide–alkyne cycloaddition, strain promoted azide–alkyne cycloaddition and Diels–Alder cycloaddition. The inverse electron demand Diels–Alder cycloaddition between a tetrazine and norbornene was chosen as the preferred ligation method, and the reaction conditions optimised. To purify the library from cell lysate, a His-tag was again coupled to each member using the strain promoted azide–alkyne cycloaddition. To test the tetrazine ligation, fluorescence in situ hybridisation (FISH) was used in cells, whereby a fluorophore was ligated onto a tetrazine–conjugated PNA probe. This was hybridised onto an mRNA in fixed cells. Results indicated that the ligation needed further optimisation.

The emergence of unique or enhanced physical, chemical and optical material properties at the nanoscale underlies the swift rise of nanomaterials science over recent decades. Within this interdisciplinary field, catalysis performed by nanomaterials (i.e. nanocatalysis) is one area where differences between nanoscale and bulk material properties offer particularly attractive opportunities for application. The consequent pursuit of viable nanomaterials with unprecedented catalytic activity has inevitably expanded across the periodic table, whereby a number of highly efficient precious metal, metal oxide and composite nanostructured catalysts have been developed for a wide range of synthetic organic and inorganic transformations. The lanthanide series has not been excluded from this search, but is still underrepresented in catalysis despite some rich chemistry and reactivity which sets these elements apart from many other metals. More recently however, the necessary paradigm shift away from commonly utilized but expensive, potentially toxic precious metal catalysts, and toward more sustainable alternatives, has seen an upsurge in the development of novel nanomaterials for heterogeneous catalysis: the general topic of this doctoral thesis. Heterogeneous nanocatalysis offers distinct advantages over homogeneous catalysis. Catalyst recyclability, ease of separation from reaction mixtures, and minimal product contamination all contribute to the higher overall effectiveness of heterogeneous catalysts relative to their homogeneous counterparts. The use of light as an abundant reagent, both in nanomaterial fabrication and for photocatalysis, is another attractive prospect. This dissertation addresses both points, describing the iterative development and application of photochemically-prepared samarium oxide based nanomaterials for heterogeneous catalysis and photocatalysis. Through a series of related peer-reviewed publications and associated commentary, the evolution of the application-driven design of a nanomaterial which is both efficient and effective for a diversity of heterogeneous catalytic and photocatalytic transformations is presented. Major findings include 1) both colloidal and supported samarium oxide nanoparticles can be prepared photochemically and comprise primarily Sm2O3 but may contain localized mixed valences or dynamic surface oxidation states; 2) colloidal samarium oxide nanoparticles possess high activity for Brønsted acid and oxidative catalysis, but recyclability and overall effectiveness is less than optimal due to a combination of polydispersity and size-dependent catalytic activity; 3) a similarly-prepared “second generation” samarium oxide/titanium dioxide nanocomposite presented several advantages over its predecessor, performing highly efficient and effective pure heterogeneous, dual photoredox-Lewis acid catalysis in two different types of synthetically relevant photocyclizations. Effects of different nanoparticle supports, rare insights into the catalytic mechanisms and behaviour of these nanomaterials‒obtained at the single molecule level by innovative application of Total Internal Reflection Fluorescence Microscopy (TIRFM) to catalysis research‒as well as advances in TIRFM data analysis protocols, are also discussed.

abstract: A method of determining nanoparticle temperature through fluorescence intensity levels is described. Intracellular processes are often tracked through the use of fluorescence tagging, and ideal temperatures for many of these processes are unknown. Through the use of fluorescence-based thermometry, cellular processes such as intracellular enzyme movement can be studied and their respective temperatures established simultaneously. Polystyrene and silica nanoparticles are synthesized with a variety of temperature-sensitive dyes such as BODIPY, rose Bengal, Rhodamine dyes 6G, 700, and 800, and Nile Blue A and Nile Red. Photographs are taken with a QImaging QM1 Questar EXi Retiga camera while particles are heated from 25 to 70 C and excited at 532 nm with a Coherent DPSS-532 laser. Photographs are converted to intensity images in MATLAB and analyzed for fluorescence intensity, and plots are generated in MATLAB to describe each dye's intensity vs temperature. Regression curves are created to describe change in fluorescence intensity over temperature. Dyes are compared as nanoparticle core material is varied. Large particles are also created to match the camera's optical resolution capabilities, and it is established that intensity values increase proportionally with nanoparticle size. Nile Red yielded the closest-fit model, with R2 values greater than 0.99 for a second-order polynomial fit. By contrast, Rhodamine 6G only yielded an R2 value of 0.88 for a third-order polynomial fit, making it the least reliable dye for temperature measurements using the polynomial model. Of particular interest in this work is Nile Blue A, whose fluorescence-temperature curve yielded a much different shape from the other dyes. It is recommended that future work describe a broader range of dyes and nanoparticle sizes, and use multiple excitation wavelengths to better quantify each dye's quantum efficiency. Further research into the effects of nanoparticle size on fluorescence intensity levels should be considered as the particles used here greatly exceed 2 ìm. In addition, Nile Blue A should be further investigated as to why its fluorescence-temperature curve did not take on a characteristic shape for a temperature-sensitive dye in these experiments. / Dissertation/Thesis / M.S. Mechanical Engineering 2011

Chlorophyll fluorescence imaging has now become a versatile and standard tool in fundamental and applied plant research. This method captures time series images of the chlorophyll fluorescence emission of whole leaves or plants upon various illuminations, typically combination of actinic light and saturating flashes. Several conventional chlorophyll fluorescence parameters have been recognized that have physiological interpretation and are useful for, e.g., assessment of plant health status and early detection of biotic and abiotic stresses. Chlorophyll florescence imaging enabled us to probe the performance of plants by visualizing physiologically relevant fluorescence parameters reporting physiology and biochemistry of the plant leaves. Sometimes there is a need to find the most contrasting fluorescence features/parameters in order to quantify stress response at very early stage of the stress treatment. The conventional fluorescence utilizes well defined single image such as F0, Fp, Fm, Fs or arithmetic combinations of basic images such as Fv/Fm, PSII, NPQ, qP. Therefore, although conventional fluorescence parameters have physiological interpretation, they may not be representing highly contrasting image sets. In order to find the effect of stress treatments at very early stage, advanced statistical techniques, based on classifiers and feature selection methods, have been developed to select highly contrasting chlorophyll fluorescence images out of hundreds of captured images. We combined sets of highly performing images resulting in images with very high contrast, the so called combinatorial imaging. The application of advanced statistical methods on chlorophyll fluorescence imaging data allows us to succeed in tasks, where conventional approaches do not work. This thesis aims to explore the application of conventional chlorophyll fluorescence parameters as well as advanced statistical techniques of classifiers and feature selection methods for high-throughput screening. We demonstrate the applicability of the technique in discriminating three species of the same family Lamiaceae at a very early stage of their growth. Further, we show that chlorophyll fluorescence imaging can be used for measuring cold and drought tolerance of Arabidopsis thaliana and tomato plants, respectively, in a simulated high ? throughput screening.

Total internal reflection fluorescence microscopy (TIRFM) has been widely applied for the study of biomolecules because of their ability to quantify biomolecules in a sample pretreatment and enrichment free manner, when compared with those costly, sample consuming and labor intensive conventional detection assay. Here, we have applied the TIRFM imaging system for the direct quantification and analysis of the biomarkers for the age-related diseases. Three research works on the quantification and study of biomarkers with the aid of TIRFM were herein described.

Title: Spectroscopic Study of Singlet Oxygen in Cells and Model Systems Author: Marek Scholz Department: Department of Chemical Physics and Optics Supervisor: doc. RNDr. Roman Dědic, Ph.D., KChFO Abstract: Singlet oxygen (1O2), the first excited state of molecular oxygen, plays many important roles in nature and technology. The work is aimed at development of novel methods for monitoring of 1O2 in cells and other biological samples. Two main ap- proaches were employed: direct detection of the very weak near-infrared phospho- rescence of 1O2, and detection of Singlet Oxygen-Feedback Delayed Fluorescence (SOFDF), which is the emission from the photosensitizer induced by energy transfer from 1O2. The first part of the thesis introduces the basic concepts of photophysics and photochemistry of 1O2: its generation, deactivation, applications, and overview of detection methods. The second part presents the experimental results. Wide-field mi- crospectroscopic detection of 1O2 phosphorescence enabled us to acquire 1O2-based images and near-infrared spectra from single cells incubated with photosensitizers. However, the direct detection suffers from the inherently very low phosphorescence quantum yield. It is shown that SOFDF may overcome this problem and become a promising alternative tool for studies of 1O2 and...