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<p> Photodynamic therapy (PDn is an effective treatment option for various types of invasive tumors, the efficacy of which depends strongly on selective cell uptake and selective excitation of the tumor, which relies on proper dosage. The characterization of the fluorescence lifetimes of photosensitizers localized inside living cells may provide the basis for further investigation of in vitro PDT dosage measurements using time-domain spectroscopy and imaging. In this thesis, the fluorescence lifetimes of localized Photofrin ® and delta-aminolevulinic acid (ALA) induced protoporphyrin IX (PpiX) were investigated in living MAT-LyLu (MLL) rat prostate adenocarcinoma cells. Cells were incubated with the photosensitizers, and then treated with light under well-oxygenated conditions using a two-photon fluorescence lifetime imaging microscope (FLIM). Fluorescence lifetime images of these cells were recorded with average lifetimes of 5.5 ± 1.2 ns for Photofrin and 6.3 ± 1.2 ns for ALA-induced PpiX over 600 to 750 nm. Two channel FLIM revealed lifetimes of7.8 ± 0.5 ns for Photofrin® and 10.8 ± 1.7 ns for PpiX over 620 to 645 nm, while photoproducts observed on the second channel yielded lifetimes of 5.1 ± 0.4 ns over 650 to 670 nm for Photofrin® and 6.3 ± 1.0 ns over 670 to 690 nm for PpiX. Fluorescence lifetimes of both photosensitizers were found to be significantly shorter when localized in cells than when measured in solutions, suggesting that photosensitizers' lifetimes go through significant changes when bonded to intracellular components. These changes in lifetime also provide opportunities to quantitatively measure and monitor the binding states of the photosensitizers and their microenvironment, which may be used in real-time PDT dosimetry, as well as for diagnostic purposes. </p> / Thesis / Master of Applied Science (MASc)

The phenomenon of photobleaching of a photosensitizer during photodynamic therapy (PDT) is well known. For second generation photosensitizers it may be possible to exploit this effect to enhance the volume of damaged tissue and improve the efficacy of PDT. In addition, as a consequence of photobleaching, the fluorescence emitted by the photosensitizer will decrease during PDT. Mathematical models were developed which describe fluorescence emission, photobleaching and tissue necrosis resulting from the irradiation of tissue containing photosensitizer using an appropriate light source. Diffusion theory was used to model bleaching in a semi-infinite medium caused by broad-beam irradiation, and both pencil and broad-beam fluorescence excitation of the photosensitizer. In addition, models were developed for an isotropic point source imbedded in an infinite medium. Based on the relationship between the decreasing fluorescence signal and the increasing volume of tissue damage, these models allow the extent of necrosis achieved during treatment to be monitored. By analysing spatially resolved fluorescence measurements predictions about necrosis depth that are insensitive to treatment parameters such as photosensitizer concentration, tissue optical properties and bleach rate are possible. Tissue simulating optical phantoms that allow for relatively simple and accurate alteration to optical properties were developed. Photosensitizers which still undergo fluorescence and photobleaching in the solid medium were also added. Using these phantoms, treatment was simulated and spatially resolved fluorescence was measured as a function of time for a wide range of initial treatment parameters. Photobleaching of the photosensitizers was observed to occur through a decrease in fluorescence emission. Also, spatially resolved measurements provided information about the average photosensitizer depth, which was seen to increase with time, with little knowledge of initial treatment parameters. These experimental results were then compared with predictions from the mathematical theory, illustrating the validity of the models. The value and feasibility of this technique for photodynamic therapy dosimetry are discussed, along with planned improvements. / Thesis / Master of Science (MS)

Fullerenes and metallofullerenes have been discovered to have a lot of applications in the biomedical area, for instance, they have been shown to have antioxidant, anti-virus, anti-cancer, immunological properties, etc. However, the hydrophobicity nature of fullerenes and metallofullerenes raises the need for functionalized hydrophilic fullerenes and metallofullerenes. Also, the advancement of the purification techniques of fullerenes and metallofullerenes makes the isolation of new fullerenes and metallofullerenes possible. Therefore, discovering the biomedical applications of these newly found fullerenes and metallofullerenes is also of vital importance. In Chapter 1, we provided a comprehensive background on the history of fullerenes and metallofullerenes, synthesis and purification methods of fullerenes and metallofullerenes, and some of their biological applications, including antioxidant applications and fluorescence applications. Some important fullerene and metallofullerenes and milestones in this area were also discussed. In Chapter 2, we demonstrated the antioxidant and anti-inflammation ability of a conjugate, FIFIFK(Cy5)PEG24K(NH2)CONH2-C60, that consisted of a peptide that binds specifically to a formyl peptide receptor-1 (FPR-1), which expresses on activated macrophages, and a carboxyl-group-functionalized C60, which is the first discovered and most used fullerene is history. We showed that the fullerene-peptide conjugate had great ability as a radical scavenger and to reduce the volume of inflammatory tissue. In Chapter 3, we demonstrated the antioxidant and anti-inflammation ability of several metallofullerene derivatives, including amino-Gd3N@C80, amino-Sc3N@C80, carboxy-Gd3N@C80, and carboxyl-Sc3N@C80. Amino-group-functionalized metallofullerenes were found to have higher radical scavenging ability and anti-inflammation ability. In Chapter 4, we developed fluorescent metallofullerene derivatives, including Tb3N@C80(NH2)9(OH)4NO2 and Tb3N@C80(CH2CH2COOH)21(OH)18. The fluorescence properties of the derivatives of fullerenes or metallofullerenes were normally realized by the addition of fluorescent moieties on the carbon cage of fullerenes or metallofullerenes. However, the fluorescence of our newly developed metallofullerene derivative was realized by the tri-terbium nitride cluster inside the carbon cage. This saved the multi-step synthesis of the fluorescence probe and maintained the simplicity of the structure of the metallofullerene derivative. These derivatives of terbium-containing metallofullerene also showed radical scavenging ability towards hydroxyl radicals. In Chapter 5, we developed another fluorescent Tb3N@C80 derivative, Tb3N@C80[DiPEG2000]. The advantage of this fluorescent metallofullerene derivative was that it was easier to purify compared to small-functional-groups-functionalized Tb3N@C80 derivatives. This newly developed fluorescent Tb3N@C80 derivative also showed great fluorescent ability and radical scavenging ability. In Chapter 6, we provided a summary of the studies on the antioxidant and fluorescent properties of fullerenes and metallofullerenes' derivatives that were discussed in this dissertation. / Doctor of Philosophy / Fullerenes and metallofullerenes' have large carbonaceous outer structures that give them possibilities for a lot of chemical reactions. The conjugated carbonaceous outer structures also endow them with exceptional antioxidant ability by reacting with the oxidant species. Therefore, fullerenes and metallofullerenes can be considered as great antioxidants, which are substances that can prevent or slow damage to cells caused by oxidants, in most cases free radicals or other unstable oxidant molecules that the body produces as reactions to environmental and other pressures. Fullerenes and metallofullerenes are sometimes called "free-radical scavengers". However, fullerenes and metallofullerenes are not soluble in biological systems, therefore, it's necessary to attach functional groups onto the outer cage-like structures of fullerenes and metallofullerenes to make them water-soluble, while maintaining their great antioxidant properties. Fullerenes and metallofullerenes are not limited to antioxidant applications, and with the attachment of specialized moieties, for instance, short peptides, they can be made of additional applications. Different metals in these metallofullerenes also provide them with specialized applications. Herein, we have developed a series of water-soluble fullerenes and metallofullerenes and compared their antioxidant properties. We have also developed a water-soluble C60-peptide conjugate that can specifically bind to inflammatory cells and ease the inflammatory condition. We also synthesized a series of metallofullerene derivatives that had dual modalities fluorescent properties and antioxidant properties.

Yes / The morphology transition from micelles to vesicles of a solution-state self-assembled block copolymer, containing a fluorescent dye at the core–shell interface, has been induced by an addition–elimination reaction using a thiol, and has been shown to be coupled to a simultaneous ON-to-OFF switch in particle fluorescence. / EPSRC and the IAS at the University of Warwick

Yes / We present here a quantification of the sorption process and molecular conformation involved in the attachment of bacterial cell wall lipopolysaccharides (LPSs), extracted from Escherichia coli, to silica (SiO2) and alumina (Al2O3) particles. We propose that interfacial forces govern the physicochemical interactions of the bacterial cell wall with minerals in the natural environment, and the molecular conformation of LPS cell wall components depends on both the local charge at the point of binding and hydrogen bonding potential. This has an effect on bacterial adaptation to the host environment through adhesion, growth, function, and ability to form biofilms. Photophysical techniques were used to investigate adsorption of fluorescently labeled LPS onto mineral surfaces as model systems for bacterial attachment. Adsorption of macromolecules in dilute solutions was studied as a function of pH and ionic strength in the presence of alumina and silica via fluorescence, potentiometric, and mass spectrometry techniques. The effect of silica and alumina particles on bacterial growth as a function of pH was also investigated using spectrophotometry. The alumina and silica particles were used to mimic active sites on the surface of clay and soil particles, which serve as a point of attachment of bacteria in natural systems. It was found that LPS had a high adsorption affinity for Al2O3 while adsorbing weakly to SiO2 surfaces. Strong adsorption was observed at low pH for both minerals and varied with both pH and mineral concentration, likely in part due to conformational rearrangement of the LPS macromolecules. Bacterial growth was also enhanced in the presence of the particles at low pH values. This demonstrates that at a molecular level, bacterial cell wall components are able to adapt their conformation, depending on the solution pH, in order to maximize attachment to substrates and guarantee community survival. / The authors thank the Libyan Ministry of Education for financial support during the experimental study. We thank the EPSRC funded consortium “Hard-soft matter interfaces: from understanding to engineering” (EP/I001514/1) for financial support. Emily Caseley, who assisted in the preparation and characterization of AmNS-LPS particles as an MRC Confidence in Concept funded postdoctoral researcher at the University of Bradford, (MC_PC_16038).

Titre de l'écran-titre (visionné le 18 janvier 2024) / Les troubles neurologiques, tels que la douleur neuropathique, l'épilepsie ou la maladie d'Alzheimer, sont souvent associés à des déséquilibres de l'homéostasie des ions de chlorure. Pour étudier de façon quantitative et simultanée ce phénomène dans un grand nombre de cellules, il est crucial de développer des stratégies alliant des indicateurs génétiquement encodés bien caractérisés et des outils d'analyses quantitatifs. SuperClomeleon est un indicateur génétiquement encodé d'ions de chlorure fluorescent basé sur le transfert d'énergie par résonance de Förster (FRET) entre un fluorophore donneur (cerulean) et un fluorophore accepteur (YFP). Le FRET entre les deux fluorophores dépend de leur distance et de leur orientation dipolaire. Dans ce projet, nous avons utilisé quelques stratégies inspirées de celle présentée par Murakoshi et al. pour tenter d'améliorer l'efficacité FRET du SuperClomeleon ou modifier les propriétés photophysiques de l'accepteur. L'efficacité FRET a été mesurée à l'aide de deux méthodesdifférentes, (1) soit via un rapport entre l'intensité de la lumière émise dans la zone spectrale de la fluorescence de l'accepteur sur celle de la fluorescence du donneur, (2) soit via la microscopie d'imagerie de temps de vie de la fluorescence (FLIM) du donneur. Dans les deux cas, un microscope à balayage d'un laser multiphotonique femtoseconde a été utilisé pour imager les indicateurs fluorescents exprimés dans des neurones corticaux provenant de cultures primaires de rat. Nous avons exploré en combinant des ajustements multiexponentiels et des techniques d'analyse des plans phaseurs la gamme dynamique des différents variants des indicateurs à l'aide de protocoles précis de modifications de la concentration intracellulaire des ions de chlorure. Ces caractérisations d'indicateurs génétiques des ions de chlorure modifiés visent à fournir des outils fluorescents améliorés pour suivre les concentrations des ions de chlorure in vivo et permettre une évaluation en temps réel des équilibres ioniques dans les troubles du cerveau.

Thesis (MSc (Physics))--University of Stellenbosch, 2007. / Imaging and spectroscopy of laser induced chlorophyll fluorescence (LICF) are emerging as useful tools in plant physiology and agriculture since these methods allow an early detection of plant stress and transformation of plant tissue, before visual symptoms appear. Chlorophyll fluorescence is governed by photosynthetic efficiency and it depends on the plant species and physiological state. In addition, the laser induced fluorescence of chlorophyll molecules in the red and far red spectral range is also used to study basic processes and phenomena in photo-excited molecules. In the work reported here experimental setups used for laser induced chlorophyll fluorescence imaging and spectroscopy techniques were developed to investigate chlorophyll fluorescence under constant illumination and also to detect green-fluorescent protein (GFP) by looking at the chlorophyll fluorescence spectrum and image. He-Ne (wavelength 632 nm), tunable argon ion (wavelength 455 nm), and excimer (wavelength 308 nm) lasers were used as excitation sources. An Ocean Optics spectrometer was used to record the spectrum of the chlorophyll fluorescence and the variation of the chlorophyll fluorescence spectrum with time. The chlorophyll fluorescence spectrum of tobacco leaves expressing GFP was compared to that of control leaves. A charge-coupled device (CCD) camera was used to image the fluorescence from GFP expressing and control tobacco leaves to investigate the effect of GFP genes on chlorophyll fluorescence in relation to the state of the plant material. The spectral analysis technique and image processing procedures were elaborated in order to obtain better information on chlorophyll fluorescence. The results of this work show that the experimental setups and analytical procedures that were devised and used are suitable for laser induced chlorophyll fluorescence analysis. Fluorescence bleaching could be obtained from the time variation of the fluorescence spectrum, and plant expressing GFP can be distinguished from control plants by differences in the laser induced chlorophyll fluorescence.

Time-resolved fluorescence spectroscopy (TRFS) is a powerful analytical tool for quantifying the biochemical composition of organic and inorganic materials. The potentials of TRFS as nondestructive clinical tool for tissue diagnosis have been recently demonstrated. To facilitate the translation of TRFS technology to the clinical arena, algorithms for online TRFS data analysis are of great need. A fast model-free TRFS deconvolution algorithm based on the Laguerre expansion method has been previously introduced, demonstrating faster performance than standard multiexponential methods, and the ability to estimate complex fluorescence decay without any a-priori assumption of its functional form. One limitation of this method, however, was the need to select, a priori, the Laguerre parameter a and the expansion order, which are crucial for accurate estimation of the fluorescence decay. In this thesis, a new implementation of the Laguerre deconvolution method is introduced, in which a nonlinear least-square optimization of the Laguerre parameter is performed, and the selection of optimal expansion order is attained based on a Minimum Description Length (MDL) criterion. In addition, estimation of the zero-time delay between the recorded instrument response and fluorescence decay is also performed based on a normalized means square error criterion. The method was fully validated on fluorescence lifetime, endogenous tissue fluorophores, and human tissue. The automated Laguerre deconvolution method is expected to facilitate online applications of TRFS, such as clinical real-time tissue diagnosis.

A new technique is developed that uses planar laser-induced fluorescence (PLIF) imaging of sublimated naphthalene to image the transport of ablation products in a hypersonic boundary layer. The primary motivation for this work is to understand scalar transport in hypersonic boundary layers and to develop a database for validation of computational models. The naphthalene is molded into a rectangular insert that is mounted flush with the floor of a Mach 5 wind tunnel. The distribution of naphthalene in the boundary layer is imaged by using PLIF, where the laser excitation is at 266 nm and the fluorescence is collected in the range of 320 to 380 nm. To investigate the use of naphthalene PLIF as a quantitative diagnostic technique, a series of experiments is conducted to determine the linearity of the fluorescence signal with laser fluence, as well as the temperature and pressure dependencies of the signal. The naphthalene fluorescence at 297 K is determined to be linear for laser fluence that is less than about 200 J/m². The temperature dependence of the naphthalene fluorescence signal is found at atmospheric pressure over the temperature range of 297K to 525K. A monotonic increase in the fluorescence is observed with increasing temperature. Naphthalene fluorescence lifetime measurements were also made in pure-air and nitrogen environments at 300 K over the range 1 kPa to 40 kPa. The results in air show the expected Stern-Volmer behavior with decreasing lifetimes at increasing pressure, whereas nitrogen exhibits the opposite trend. Preliminary PLIF images of the sublimated naphthalene are acquired in a Mach 5 turbulent boundary layer. Relatively low signal-to-noise-ratio images were obtained at a stagnation temperature of 345 K, but much higher quality images were obtained at a stagnation temperature of 380 K. The initial results indicate that PLIF of sublimating naphthalene may be an effective tool for studying scalar transport in hypersonic flows. / text