Water-soluble fluorescent probes with emission in the 600-800 nm region have significant potential in biological applications such as cell imaging. Most fluorescent probes however suffer from limited fluorescence brightness in aqueous media due to aggregation and self-quenching. Their photostability in animal models for an extended period of time is also a concern. One way of improving their photophysical properties is to encapsulate them in a protective matrix to form fluorescent nanoparticles. We have synthesized a set of six through-bond energy transfer cassettes which emit in the 600-800 nm region with Fluorescein or BODIPY as donor and benzophenoxazine dye Nile Red or cyanine dye Cy5 as acceptor. Their photophysical properties in organic and aqueous media were evaluated. Some of these cassettes were encapsulated in silica or calcium phosphate nanoparticles (20 nm in diameter) to improve their solubility and photophysical properties in aqueous media. We also synthesized some water-soluble benzophenoxazine based fluorophores and the impact of different water-soluble groups on their emission characteristics in aqueous media was studied. Selected fluorophores were used for in vitro cellular imaging studies.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [147]-151).

A simple and feasible method of computer colour matching involving fluorescent dyes was developed. An ordinary abridged-spectroreflectometer with polychomatic illumination and a simulated D65 xenon light source was employed for all measurements. In addition to the normal K/S constants for non-fluorescent dyes and the non-fluorescent portion of the fluorescent dye'.. constants responsible for the fluorescent portion were necessary. Two sets of equations to relate the total radiance factors of dyeings with a fluorescent dye and its concentration were developed respectively for self and compound shades where a non-fluorescent dye is admixed. Finding constants responsible for the compound shades required a number of calibration mixture dyeings. Negative K/S constants were found useful when the total radiance factor was above that for the substrate but below one hundred. Three computer programs? s were developed to deal with calibration constants for self and compound shade and also for match prediction. Optimization was used in all cases to minimize errors in total radiance factors or colour differences. Half of the actual dyeingq formulations from the predicted were visually passed by a panel of five dyers. In this study, disperse dyes on polyester were used. Moreover, a commercial matching package was studied using non-fluorescent dyes. The dyeing system affected its accuracy. The polyester/disperse dye system was better than the cotton/reactive dye system. The sample size and luminancefactor of target colours; were also studied. The accuracy was affected slightly by the latter but not the former. / Staff-development Committee and the Institute of Textiles and Clothing of the Hong Kong Polytechnic.

The challenges in modern biological imaging applications are two-fold: (i) to develop better methods of imaging, and (ii) develop dyes that are suitable for these methods. This dissertation deals with the design and synthesis of dyes mainly by modification of known dyes to make them suitable for modern biological applications. Towards this aim, novel ways of derivatizing BODIPY dyes are explored. One method involves extending the conjugation via phenyl acetylene units, pushing fluorescence wavelengths near 600 nm. A different approach deals with C-H functionalization of BODIPY in which the fluors are functionalized with acrylate units, extending their fluorescence to the red. The BODIPY dyes developed are then incorporated in through-bond energy transfer cassettes. We examine the factors affecting energy transfer efficiencies by synthesizing analogs of the cassettes and also studying the electrochemical behavior of the donor and acceptor parts. The concept of through-bond energy transfer is incorporated into conjugated polymers by random incorporation of BODIPY dyes into polyfluorenes. The ideal ratio of fluorene to BODIPY parts was found to be 4:1. The BODIPY doping agents result in dispersed emissions when excited the polyfluorene polymers. Concurrently, the polyfluorene backbone acts as an energy harvester for the BODIPY dyes, in effect increasing their molar absorptivities. Finally the use of BODIPY dyes as photodynamic therapeutic agents was examined. We found that BODIPY dyes are efficient at producing reactive oxygen species when halogens are attached directly on the BODIPY core. Furthermore, the mechanism of cell death by using such agents was elucidated. Attachment of the most promising agent to polyglutamic acid is done to promote the EPR effect. Lastly we develop a potentially new type of PDT agent that absorbs strongly above 800 nm, permitting its use in deep tissue PDT.

A set of three benzophenoxazine dyes, two completely soluble and one partially soluble in aqueous media, has been prepared and their spectroscopic properties examined. These dyes can be used as either donor or acceptor in synthesis of through-bond energy transfer cassettes. Structural modifications prevented aggregation in water and improved their fluorescence properties in water. Their absorption and emission were studied in both organic and aqueous media. Two of the three dyes have superior quantum yields in aqueous media as compared to other reported dyes. Improved quantum yield makes these dyes attractive candidates for biological studies in aqueous media. We have also prepared alkynes and iodo derivatives of benzophenoxazines, which can be used for synthesis of water-soluble, through-bond, energy transfer cassettes. Alkynes were prepared via Sonogashira coupling.

A simple and feasible method of computer colour matching involving fluorescent dyes was developed. An ordinary abridged-spectroreflectometer with polychomatic illumination and a simulated D65 xenon light source was employed for all measurements. In addition to the normal K/S constants for non-fluorescent dyes and the non-fluorescent portion of the fluorescent dye'.. constants responsible for the fluorescent portion were necessary. Two sets of equations to relate the total radiance factors of dyeings with a fluorescent dye and its concentration were developed respectively for self and compound shades where a non-fluorescent dye is admixed. Finding constants responsible for the compound shades required a number of calibration mixture dyeings. Negative K/S constants were found useful when the total radiance factor was above that for the substrate but below one hundred. Three computer programs? s were developed to deal with calibration constants for self and compound shade and also for match prediction. Optimization was used in all cases to minimize errors in total radiance factors or colour differences. Half of the actual dyeingq formulations from the predicted were visually passed by a panel of five dyers. In this study, disperse dyes on polyester were used. Moreover, a commercial matching package was studied using non-fluorescent dyes. The dyeing system affected its accuracy. The polyester/disperse dye system was better than the cotton/reactive dye system. The sample size and luminancefactor of target colours; were also studied. The accuracy was affected slightly by the latter but not the former.

Thesis (M.Sc.). / Written for the Dept. of Microbiology and Immunology. Title from title page of PDF (viewed 2008/05/13). Includes bibliographical references.

Thesis (Ph.D.)--University of Florida, 2004. / Title from title page of source document. Document formatted into pages; contains 96 pages. Includes Vita. Includes bibliographical references.

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第21190号 / エネ博第364号 / 新制||エネ||71(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 佐川 尚, 教授 森井 孝, 教授 松田 一成 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

A espectroscopia de correlação de fluorescência (FCS) é uma das diferentes técnicas de análise por imagens de alta resolução espacial e temporal de biomoléculas em concentrações extremamente baixas. Ela se tornou uma técnica extremamente poderosa e sensível em áreas como bioquímica e biofísica. Como uma técnica bem estabelecida, ela é utilizada para medir concentrações locais de biomoléculas, através da marcação com moléculas fluorescentes. Coeficientes de difusão e constantes cinéticas também podem ser medidos através de FCS assim como detecção de molécula única. Ela também pode dar informação precisa sobre interações de antígeno-anticorpo, ácidos nucleicos e proteínas. Através de uma combinação de marcadores de alto rendimento quântico, fontes de luz estável (lasers), detecção ultrassensível e microscopia confocal, é possível realizar medidas de FCS em volumes de fentolitros (fL) e em concentrações de nanomolar (nM) em soluções aquosas ou em células vivas. Em contraste com outras técnicas de fluorescência, a sensibilidade da FCS aumenta com a diminuição da concentração do fluoróforo marcador, porque o parâmetro de interesse não é a intensidade de emissão de fluorescência, mas sim as flutuações espontâneas da fluorescência. Durante o tempo em que a partícula ou molécula atravessa o volume de medida pode ocorrer mudanças conformacionais e reações químicas e fotofísicas que alteram as características de emissão do fluoróforo e causam flutuações no sinal detectado. Estas flutuações são então monitoradas e transformadas em uma curva de autocorrelação, por intermédio de um software comercial que emprega um modelo físico apropriado para FCS. Em nosso estudo, utilizamos um marcador comercial (ALEXA 488®) para marcar proteínas. Primeiramente utilizamos a técnica de FCS para medir concentrações extremamente baixas de marcadores fluorescentes. Também realizamos um experimento testando a influência da viscosidade do meio na difusão livre do fluoróforo, assim como as melhores condições em que temos um melhor sinal de FCS. Por fim, estudamos a difusão de proteínas marcadas (PUC II e IV) em meio aquoso (PBS) e no interior de células. / Fluorescence correlation spectroscopy (FCS) is one of the many different modes of high-resolution spatial and temporal analysis of extremely low concentrated biomolecules. It has become a powerful and sensitive tool in fields like biochemistry and biophysics. As a well established technique, it is used to measure local concentrations of fluorescently labeled biomolecules, diffusion coefficients, kinetic constants and single molecule studies. Through a combination of high quantum yield fluorescent dyes, stable light sources (lasers), ultrasensitive detection and confocal microscopy is possible to perform FCS measurements in femtoliters volumes and nanomolar concentrations in aquous solution or in live cells. Unlike with other fluorescence technics, its sensibility increases with the decrease of dye concentrarion, because the main factor is not the emission intensity itself. Instead this, spontaneous statistical fluctuation of fluorescence becomes the main factor in FCS analisys. During the time that the conjugated-dye cross the volume detection can occur conformational changes, chemical reaction and photophysical processes that can change the emission properties of the dye and, then, change the detected sinal. This fluctuations are tracked and changed into a autocorrelation curve, by a specific software, appropriate to perform FCS analisys. In our study, we use comercial dye (Alexa 488) to label proteins. Firstly, we applied FCS to measure extremally diluted concentrations of dyes (~1 nM). We have performed experiments testing the influence of the viscosity medium in the free difusion of the dyes and the optical apparatus and conditions that result in the best FCS signal. We also have studied protein diffusion (PUC II e IV) in aquous medium (PBS) and toward the inner of the cells.