Over the past two decades a body of evidence concerning residual biological contamination on cleaned surgical instruments has accumulated. This is substantiated by the number of yearly surgery cancellations due to visible residue on instruments in surgical packs and incidences of iatrogenic Creutzfeldt-Jakob disease (iCJD). It is therefore imperative to develop a method of protein detection for use in clinical sterile services departments (SSDs) for monitoring of decontamination quality. This Thesis describes the development and use of an epifluorescence surface scanner (EFScan) technology in the assessment of proteinaceous residue on surgical instruments, by detecting protein pre-labelled with fluorescein isothiocyanate (FITC), and exploratory studies on the feasibility of label-free detection, using intrinsic protein fluorescence. Measurements using FITC labelling showed that residual protein on the order of micrograms can be found on new, single-use instruments (i.e. prior to use). This is comparable to the amount of residual protein found on retired, reusable instruments. To confirm the suitability of fluorescence techniques in the detection and quantification of proteinaceous residue, a blind, pilot study was carried out in conjunction with groups from Queen Mary University and the University of Southampton. Each University used a different labelling and detection method, and results showed good agreement between these methods. This showed that fluorescence is a suitable technique for the detection and quantification of proteinaceous contamination on surgical instruments. The next step in the project focussed on detection of contamination via intrinsic protein fluorescence from tryptophan residues, with a view to elimination of the labelling step. Intrinsic fluorescence of proteins in solution is widely characterised; however, fluorescence characteristics of solid or surface-bound protein have been little studied. Therefore, the characterisation of solid protein fluorescence and the emission characteristics of protein adsorbed onto stainless steel was undertaken. Analysis of the commonly used protein standard bovine serum albumin (BSA) showed that the two tryptophan residues it contains are highly susceptible to photo-oxidation in the solid state, resulting in conversion to the fluorescent photoproducts n-formylkynurenine (NFK) and kynurenine. Therefore, BSA is not suitable for use as a standard in the development of intrinsic fluorescence detection of surface-bound protein. The 72-tryptophan protein fibrinogen, as well as a series of other multi-tryptophan proteins, were assessed and it was found that photo-oxidation of the tryptophan residues did not occur on the irradiation timescale of 1 hour utilized. Therefore, it was concluded that lysozyme or gamma-globulins, a prominent group of serum proteins, would be more suitable candidates as a standard in subsequent research into the intrinsic detection and quantification of proteinaceous contamination. A third study explored the potential use of fluorescence in the early diagnosis of cataract. This involved the fluorescence characterisation of healthy porcine lenses and the use of UV irradiation of the lens to attempt to create cataract in vitro. There was found to be a large variation in fluorescence characteristics from lens to lens, suggesting that fluorophore concentrations can vary significantly. This implies that identification of a suitable standard for the early detection of cataract may be problematic. Attempts to create cataract resulted in the photo-oxidation pathway which had been observed in BSA, and although NFK and kynurenine play a role in cataractogenesis, the accumulation of these photoproducts is not analogous to a natural cataract. It was found that these products could be destroyed by irradiation of the lens at appropriate photo-bleaching wavelengths. However, this also destroyed intrinsic, protective fluorophores within the lens, suggesting that a light-based method of cataract treatment may not be achievable.

This thesis is focused on possibility of studying stability and fusion of catanionic vesicles with Förster resonance energy transfer. The mainly used technique was Time-Correlated single photon counting. Firstly, excitation and emission spectra of chosen probes were measured and donor-acceptor pairs were suggested: 5-hexadecanoylaminofluorescein with Octadecyl Rhodamine B, Bodipy 493/503 with rhodamine or DiI, perylene with fluorescein, DiO with DiI. Then, time-resolved measurements of suggested pairs from environment of catanionic vesicles with different content of cholesterol were made in order to track the FRET associated with fusion of vesicles. It was found out, that it is not possible to use DiO as a donor because of it’s inefficient solubilisation into vesicles. It is also not possible for Bodipy to be used as a donor, because of it‘s excimer formation. In case of using fluorescein as a donor, it was found, that there is ongoing homo-fret between fluorescein molecules. Thanks to this, fusion was tracked by addition of unstained vesicles. It was also possible to track fusion in longer period of time. Also perylene-fluorescein pair was found to be capable of tracking the fusion, but with the exception of vesicles with content of cholesterol of 43 mol. %, tracking of fusion was possible only in short period of time.

No description available.

L'étude de l'émission intégrée et résolue en temps de quatre configurations d'hétérostructures quantiques de type points-dans-un-fil d'InGaN/GaN nous a permis de déterminer la nature de la localisation et du mécanisme de recombinaison des porteurs de charge dans ces nanofils. Des mesures de comptage de photon unique correlés en temps (TCSPC) étendues sur une plage temporelle allant de 210 à 26000ns ont permis d'observer un comportement fortement non exponentiel de l'émission que nous avons déterminé être une loi de puissance. Nous avons trouvé que le temps de vie de l'émission diminue rapidement avec l'énergie d'émission. Par contre, l'observation d'un effet de la puissance d'excitation sur le temps de vie semble indiquer qu'à une énergie d'émission ne soit pas associée une seule dynamique d'émission à long temps. En utilisant une densité d'excitation laser de seulement quelques dizaines de watt par cm au carré, nous avons pu démontrer, en régime non perturbatif, que le profil des spectres d'émission intégrés en temps ainsi que la dynamique de l'évolution temporelle de l'émission étaient tout à fait compatibles avec une recombinaison radiative centrée sur une distribution de nano-agrégats riches en indium naturellement formés lors de la croissance des nanofils par MBE assistée par plasma. Cette conclusion est supportée par notre incapacité à observer l'effet Stark à confinement quantique, le succès d'un modèle de séparation de charges parfaitement compatible avec l'image des nano-agrégats d'indium et, finalement, par l'observation d'une émission principalement isotrope en polarisation. / We have performed time-integrated and time-resolved photoluminescence measurements on four different configurations of InGaN/GaN dot-in-a-wire heterostructures in order to further our understanding of the localization and radiative recombination mechanism involved in the process of emission. Time correlated single photon counting (TCSPC) measurements from 100 ns to 26000 ns have allowed us to observe a strong non-exponential decay which follows a power law on long time scale. The characteristic exponent of this power law is strongly correlated with the emission energy, causing the life-time of the emission to fall rapidly with increasing of its energies. The observation that the excitation power has an effect on the life-time shows that other factors such as the growth conditions must be involved in the coupling between life-time and energy. Using a low power density of a few tens of watts per cm squared, we have shown, in a non perturbative regime, that the shape of the time-integrated spectra and the dynamics of the time-resolved decay curves were consistent with a radiative recombination process centered on In-rich nanocluster. These nanoclusters naturally occur in the embedded InGaN inclusions during the growth by plasma-assisted MBE. This conclusion is supported by the absence of Quantum confined Stark effect. The success of a charge separation model is perfectly consistent with the emission centered on In-rich nanocluster and the observation of a quasiperfect isotropic emission. / Mesures effectuées dans le laboratoire de caractérisation optique des semi-conducteurs du Prof. Richard Leonelli du département de physique de l'université de Montréal. Les nanofils d'InGaN/GaN ont été fournis par le groupe du Prof. Zetian Mi du département de génie électrique et informatique de l'université McGill.

This thesis presents an investigation of fluorescence resonance energy transfer (FRET) as a reporting signal for protein-protein interactions. Quantitative optical assays to measure protein binding, conjugation and deconjugation are developed and results validated by conventional biochemical techniques. The optical techniques developed provide fast, cheap, quantitative and accurate alternatives to conventional methods. Fluorescent protein fluorophores ECFP and Venus-EYFP were chosen as they are a non-interfering FRET pair and provide an inexpensive and convenient cloning-based labelling method. The small ubiquitin-like modifier SUMO and the SUMOylation pathway leading to its conjugation to target proteins is investigated as a model system. These assays are hence particularly relevant to research on post-translational modification and ubiquitin systems. In protein-protein binding assays we utilise both steady-state and time-resolved FRET detection to measure the equilibrium binding constant of the well-characterised pair SUMO1 and Ubc9. An assay in multi-well plate format is also presented, which uniquely enables repeat measurements under varying conditions and under the addition of further substances. The multi-protein binding interactions of the SUMOylation pathway including RanBP2 are analysed in binding inhibition assays. Our results clarify the role of RanBP2: a covalent SUMO1-Ubc9 link is required for the formation of a trimeric complex, although mutual binding sites are present on all three proteins. Furthermore, the binding of SUMO1 and Ubc9 is disrupted by RanBP2, which may be an essential step in transferring SUMO1 to its target protein. A FRET-based kinetic study of this conjugation process to RanGAP1 is presented. An assay to monitor the deconjugation of SUMO1 by specific proteases is established using a doubly-tagged SUMO construct. This enables a quantitative analysis of protease and substrate specificity based on real-time kinetic data, a characterisation of crude cell extracts and a high-throughput screen for protease inhibitors using FRET. A screen of the National Cancer Institute (NIC) diversity set for SenP1 inhibition reveals nine suitable compounds, which are potential anti-cancer drugs. The results of two further projects, the study of protein-protein binding by measuring small refractive index changes and the autofluorescence of normal and neoplastic cervical tissue models are also presented. In the latter, principal component analysis was used to systematically identify emission regions of significant variation between samples, enabling discrimination between healthy and pre-cancerous tissue models.

Mesures effectuées dans le laboratoire de caractérisation optique des semi-conducteurs du Prof. Richard Leonelli du département de physique de l'université de Montréal. Les nanofils d'InGaN/GaN ont été fournis par le groupe du Prof. Zetian Mi du département de génie électrique et informatique de l'université McGill. / L'étude de l'émission intégrée et résolue en temps de quatre configurations d'hétérostructures quantiques de type points-dans-un-fil d'InGaN/GaN nous a permis de déterminer la nature de la localisation et du mécanisme de recombinaison des porteurs de charge dans ces nanofils. Des mesures de comptage de photon unique correlés en temps (TCSPC) étendues sur une plage temporelle allant de 210 à 26000ns ont permis d'observer un comportement fortement non exponentiel de l'émission que nous avons déterminé être une loi de puissance. Nous avons trouvé que le temps de vie de l'émission diminue rapidement avec l'énergie d'émission. Par contre, l'observation d'un effet de la puissance d'excitation sur le temps de vie semble indiquer qu'à une énergie d'émission ne soit pas associée une seule dynamique d'émission à long temps. En utilisant une densité d'excitation laser de seulement quelques dizaines de watt par cm au carré, nous avons pu démontrer, en régime non perturbatif, que le profil des spectres d'émission intégrés en temps ainsi que la dynamique de l'évolution temporelle de l'émission étaient tout à fait compatibles avec une recombinaison radiative centrée sur une distribution de nano-agrégats riches en indium naturellement formés lors de la croissance des nanofils par MBE assistée par plasma. Cette conclusion est supportée par notre incapacité à observer l'effet Stark à confinement quantique, le succès d'un modèle de séparation de charges parfaitement compatible avec l'image des nano-agrégats d'indium et, finalement, par l'observation d'une émission principalement isotrope en polarisation. / We have performed time-integrated and time-resolved photoluminescence measurements on four different configurations of InGaN/GaN dot-in-a-wire heterostructures in order to further our understanding of the localization and radiative recombination mechanism involved in the process of emission. Time correlated single photon counting (TCSPC) measurements from 100 ns to 26000 ns have allowed us to observe a strong non-exponential decay which follows a power law on long time scale. The characteristic exponent of this power law is strongly correlated with the emission energy, causing the life-time of the emission to fall rapidly with increasing of its energies. The observation that the excitation power has an effect on the life-time shows that other factors such as the growth conditions must be involved in the coupling between life-time and energy. Using a low power density of a few tens of watts per cm squared, we have shown, in a non perturbative regime, that the shape of the time-integrated spectra and the dynamics of the time-resolved decay curves were consistent with a radiative recombination process centered on In-rich nanocluster. These nanoclusters naturally occur in the embedded InGaN inclusions during the growth by plasma-assisted MBE. This conclusion is supported by the absence of Quantum confined Stark effect. The success of a charge separation model is perfectly consistent with the emission centered on In-rich nanocluster and the observation of a quasiperfect isotropic emission.

Nucleic acids, such as DNA, play an essential role in all known forms of life; however, despite their fundamental importance, there is still a significant lack of understanding surrounding their functional behaviour. This thesis explores the structure and dynamics of DNA by employing methods based on fluorescence and through the use of computational calculations. Time-resolved fluorescence experiments have been performed on dinucleotides containing 2-aminopurine (2AP) in various alcohol-water mixtures. 2AP, a fluorescent analogue of the nucleobase adenine, has been used extensively to investigate nucleic acids because of its ability to be incorporated into their structures with minimal perturbation and its high sensitivity to its local environment. Direct solvent effects on 2AP were established through measurements on the free fluorophore. Analysis of the complex fluorescence decays associated with the dinucleotides was challenging but has provided insight into their conformational dynamics. Solvent polarity was found to play a significant role in determining both photophysical and conformational properties in these systems. The complicated fluorescence decay of 2AP in nucleic acids highlights the need for accurate and unbiased analysis methods. Various time-resolved fluorescence analysis methods, including iterative reconvolution and the exponential series method, have been investigated with real and simulated data to obtain an overview of their benefits and limitations. The main outcome of the evaluation is that no single method is preferred in all situations and there is likely to be value in using a combination when there is ambiguity in the interpretation of the results. Regardless of the analysis technique used, the parameterised description of the observed fluorescence decay is meaningless if the underlying physical model is unrealistic. The advance of computational methods has provided a new means to rigorously test the viability of proposed models. Calculations have been performed at the M06-2X/6-31+G(d) level of theory to investigate the stability of 2AP-containing dinucleotides in conformations similar to those observed in the double-helical structure of DNA. The results help to explain the similarity of the time-resolved fluorescence behaviour of 2AP in dinucleotide and DNA systems but also bring to light subtle differences that could perhaps account for experimental discrepancies. The recent emergence of advanced optical microscopy techniques has offered the prospect of being able to directly visualise nucleic acid structure at the nanoscale but, unfortunately, limitations of existing labelling methods have hindered delivery of this potential. To address this issue, a novel strategy has been used to introduce reversible fluorescence photoswitching into DNA at high label density. Photophysical studies have implicated aggregation and energy-transfer as possible quenching mechanisms in this system, which could be detrimental to its future application. The reliability of fluorescence photoswitching was investigated at ensemble and single-molecule level and by performing optical lock-in detection imaging. These developments lay the foundations for improved and sequence-specific super-resolution microscopy of DNA, which could offer new insights into the 3D nanoscale structure of this remarkable biopolymer. In summary, the work presented in this thesis outlines important observations and developments that have been made in the study of the structure and dynamics of nucleic acids.

Gegenstand dieser Diplomarbeit ist die Untersuchung der Dynamik in dünnen Polymerfilmen anhand von einzelnen Molekülen. Zu diesem Zweck wurden dünne Filme (kleiner 100nm) hergestellt und mittels Einzelmoleküldetektion und zeitaufgelöster Einzelphotonenzählung analysiert, was eine orts- und zeitaufgelöste Untersuchung einzelner Farbstoffmoleküle ermöglicht. Ziel war es, festzustellen ob, und auf welchem Weg, die Dynamik der Umgebung in Fluktuationen der Fluoreszenzlebensdauer einzelner Moleküle sichtbar wird. Neben der Evaluierung der Untersuchungsmethoden wurden in dieser Arbeit zwei Arten von Sensormolekülen - DiD und Malachit Grün - näher untersucht. / The subject of this diploma thesis is the analysis of dynamics in thin polymer films using single molecules. Thin polymer films (less than 100nm) were produced and analysed by Single Molecule Detection (SMD) and Time Correlated Single Photon Counting (TCSPC). This allows a spatial and time resolved investigation of the single dye molecule. The aim was to ascertain if, and in which way, the dynamics of the environment are reflected by fluctuations of the fluorescence lifetime of the single molecule. In addition to evaluating the investigation methods two kinds of molecules - DiD and Malachite Green - were also analysed.

This thesis focuses on the study of UV light-generated zinc complexes with cadmium and organic molecules SAM, SAH, CYS, HCYS and GSSG, specifically at 375 nm. Furthemore, the aim of the work is to characterize the precursors spectrally and temporally before and after irradiation in the transilluminator at 250 nm. Study of genesis these complexes was performed by FLIM. Thanks to this method, it was found that the formation of complexes occurs only with Zn:SAH, Zn:GSSG and Zn:Cd. The formation of complexes is influenced by the method of preparation. The spectral characteristic was performed on a fluorimeter where the increase in fluorescence intensity of the irradiated solution with the precursors was expected. These were turbid solutions where sedimentation of the particles was observed and the intensity of fluorescence was changed. In the Zn:SAM and Zn:CYS sample, the sedimentation increased in intensity, while in Zn:SAH and Zn:HCYS decreased. The Zn:Cd precursor solution was clear and there was no change in intensity. Zn:Cd showed the best spectral properties, while the Zn:SAM sample, whose excitation and emission maxima are very close to each other, appeared to be the worst. A sample with Zn:CYS and Zn:HCYS showed almost the same spectra and respective peak results. Based on lifetime characteristics by TCSPC, the sample with Zn:CYS, Zn:HCYS and Zn:GSSG, which showed 3 lifetimes, was best treated. Lifetime could not be unambiguously determined for SAM and SAH samples. Zn:Cd had 4 lifetimes

Fluorescence lifetime imaging microscopy (FLIM) is an imaging modality that is able to provide key insights into subcellular processes. When used to measure Förster resonance energy transfer (FRET), for instance, it can discern protein-protein interactions and conformational changes. This kind of information is highly useful in the drug screening process in order to determine the effectiveness of drug leads and their mechanisms of action. FLIM has yet to be successfully translated to high-content screening (HCS) platforms due to the high throughput and fine temporal and spatial resolution requirements of HCS. Our prototype HCS FLIM system uses a time-resolving instrument called a streak camera to multiplex the FLIM scanning process, allowing for 100 confocal spots to be simultaneously scanned across a sample. There have been a few major advancements to the prototype. First the fiber array used to connect the fluorescence channels to the streak camera was characterized. Its alternating fiber delay scheme was successful in greatly reducing optical crosstalk between adjacent channels. Next, an optical beam scanner for parallel excitation beams was designed and implemented, greatly improving the possible scan speeds of the system. The streak camera was upgraded to a higher repetition rate sweep, and modifications to system components and reconstruction procedures were made to accommodate the new sweep unit. A single-photon avalanche diode array was also tested as a possible replacement for the streak camera, and was found to offer photon detection efficiency advantages. Finally, improvements were made to the excitation power and optical throughput of the system in order to reduce the required exposure time. These advances to the prototype system bring it closer to realizing the requirements of HCS FLIM, and provide a clear picture for future improvements and research directions. / Thesis / Doctor of Philosophy (PhD) / Fluorescent proteins are commonly used to tag subcellular targets so that they can easily be distinguished with a fluorescence microscope. While this can help visualize where different organelles and proteins are located in the cell, a great deal more information can be gained by measuring the fluorescence lifetime at each point in the sample, which is highly sensitive to the microenvironment. Fluorescence lifetime imaging microscopy (FLIM) has the potential to be a powerful technique for testing drug leads in the drug discovery process, although current FLIM systems are not able to provide the high throughput speeds and high temporal resolution required for drug screening. This thesis project has succeeded in improving a highly parallel FLIM microscope by reducing inter-channel crosstalk, implementing an optical scanner, improving power and optical throughput, and investigating future time-resolving instruments. This progress has brought the prototype setup closer to being used in a drug screening environment.