Studying protein expression, localization, and behaviour within cells is invaluable to understanding biological processes. Fluorescent protein labelling can be used to easily visualize these biomolecules. However, many strategies use large fluorescent proteins or enzyme tags that disrupt the native localization and conformation of proteins of interest. FlARe is a minimalistic, chemi-genetic fluorogenic protein labelling method with two components, the FlARe probe and the dC10 tag. The probe consists of a fluorophore linked to two maleimides, which quench the fluorophore’s emission. They also react with the complementary dC10 peptide tag that is genetically fused to a specific protein and contains two cysteines positioned 10 Å apart. Reaction with dC10 anchors the FlARe probe to the tagged protein and abolishes the maleimides’ quenching mechanisms. This releases the probe’s intrinsic fluorescence, allowing the protein to be visualized by fluorescence. This thesis sets out to complete three objectives: 1) To improve the FlARe probe chemical structure; 2) To implement novel developments of the FlARe protein labelling technique into live mammalian cells; 3) To expand the scope of the FlARe method to include bioconjugation. To make the current FlARe probe design more stable in the aqueous milieu of the cell, a new FlARe probe, KT12, was synthesized. This new probe was more stable than the current YC20 FlARe probe but not more selective. Despite this, valuable insight was made into the mechanisms of maleimide hydrolysis and thiol addition. After establishing that the current methoxymaleimide FlARe probe offered the most selective design, we invested our efforts into implementing the FlARe labelling technique in live mammalian cells. To this end, a recombinant protein carrying different variants of the dC10 tag was successfully labelled with FlARe probes emitting at different wavelengths. Finally, we blended our expertise in probe design and biological implementation to develop a novel fluorogenic linker to form bioconjugates. As a result, a FlARe linker functionalized with both the dimaleimide moiety and an alkyne linker was designed. The successful synthesis and proof-of-principle demonstration of this novel FlARe linker were performed, allowing for the modular formation of fluorogenic bioconjugates. These developments to FlARe advance this minimalistic and fluorogenic protein imaging method. This improves the current capability to fluorescently label proteins in cells in a non-disruptive manner. Thus, complex questions surrounding cell biology can be answered to better understand biological processes.

In 2005, our group first reported a two-pulse multi-element analysis technique that was both sensitive and minimally destructive. The first laser pulse ablated a thin layer of the sample over a hundred m spot; the second laser pulse at 193 nm induced multi-analytes in the desorbed plume to fluoresce. Since then, this technique of laser-excited atomic fluorescence (LEAF) of ablated plumes, or PLEAF for short, had been applied to the analysis of aqueous lead colloids and metals. Sub ng/g and tens of atto-mole detection limits were demonstrated. The non-selective photoexcitation in PLEAF was believed to be due to smeared energy levels of species in dense plumes. Smearing was especially severe for highly excited states such as those reached by 193 nm excitation. As the plumes subsequently expanded, the electronic structure of the plume species evolved adiabatically from a dense gas to that of an isolated atom with the electrons still in the excited states. Signature fluorescence from multi-analytes was therefore possible. The suggested mechanism implied that ArF laser-induced PLEAF should be applicable to any sample matrix and any analyte as long as the species were imbedded in dense plumes and whose excited states could be reached by 193 nm photoexcitation. We therefore investigated the universality of PLEAF in this study by extending the analysis to ceramics, polymers, and their composites. We showed that these matrices could be successfully sampled and emissions from practically all analyte elements were observed. The detection sensitivity was orders of magnitude better than alternative laser spectrochemical probes such as laser-induced breakdown spectroscopy (LIBS). Under minimally destructive conditions, emissions from Al, Ca, Co, Cr, Cu, Fe, In, Mg, Mn, Na, Pb, Sn, and Si were observed. We also applied the technique to four practical problems: The analysis of dried paint for trace lead when g/g detection limits were achieved; the analysis of valuable potteries when two look-alike specimens were differentiated based on practically non-destructive single-shot analysis; the elemental analysis of ink when lines written with different pens could be discriminated yet without discernable sample destruction even under the microscope; and the analysis of electrode-plastic interfaces when the detection sensitivity was comparable to SIMS. In some of these applications, we found that the fluorescence intensity varied with the fluence and the timing of the ArF laser pulse in ways suggestive of particulates in the plume. Because nearly all analyte elements were excited in PLEAF, multi-analyte spectra were generated even in single-shot analysis. We showed that the rich spectral information contents could be fully exploited by chemometric techniques such as principal component analysis, SIMCA and K-means clustering. In sum, the combination of PLEAF and chemometrics paved way for ultra-sensitive and minimally destructive multi-element analysis of complex samples. The analysis was all-optical and therefore could be done in air with no restriction on sample size. No sample preparation was needed. The analysis was fast, with a turn-around time of minutes. At the end, the sample was not visibly damaged even when examined under the microscope. If the ablation could be congruent, 3-dimensional chemical profiling at tens to hundreds of m lateral resolution and tens of nm depth resolution would be possible.

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The aim of the diploma thesis was measurement and characterization of EEM (excitation emisssion spectra) of IHSS humic acids standards and soil, lignite humic and fulvic acids and humates prepared from different sources. The aim was to follow the differences in their fluorescence spectra and to determine precise location of fluorescence peaks. Target of this work was to find the optimal way for exporting of scan data to the 3D EEM spectrofluorographs. The dependence of relative intensity of fluorescence on pH value was studied further.

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Lung cancer is the leading cause of death from cancer in Canada and is typically treated with surgical resection of the tumor. To ensure good prognosis and limit metastases no cancer cells can be left behind during resection. This project uses time-resolved fluorescence and diffuse reflectance to differentiate cancerous and non-cancerous lung tissue. These differences could be used during surgical resection of tumor to ensure no positive margins are present. Using a bi-modal spectroscopy device, BEAR, optical properties were determined for 36 tumor, 36 fibrotic and 9 normal lung tissue samples. Most optical parameters showed statistically significant differences between tumor and other tissue types. Metabolic based optical parameters showed statistically significant differences between fibrotic and normal tissue while non-metabolic based parameters showed no difference. As surgical margins are likely to be between tumor and fibrotic tissue the results demonstrate success and promise for implementing this system. Future work using fresh samples would develop the system further and would be a step closer to in vivo use during surgery. / Thesis / Master of Science (MSc) / Lung cancer is the leading cause of death from cancer and is typically treated by surgically removing the tumor. To improve survival all cancer cells must be removed which can be challenging. This project uses light to extract properties that can differentiate cancerous and non-cancerous lung tissue. These differences could be used during surgery to ensure no cancer cells remain. The project tests this system on 36 tumor, 36 fibrotic and 9 normal lung tissue samples. Most parameters showed significant differences between tumor and other tissue types. Given that often times the surgical boundaries are between tumor and fibrotic tissue the results demonstrate promise in implementing this system. Future work using fresh samples would develop the system further and bring it one step closer to being used during surgery.

With their unique photochemical properties, porphyrins have remained a central research topic for decades. Porphyrins can self-assemble into tubular structures at acidic pHs. However, the possibility of the disassembly of the aggregated porphyrin has not been investigated. Furthermore, quantitative understanding of the porphyrin optical activities is complicated by the complex interplay of the photon absorption, scattering, and fluorescence emission that can concurrently occur in porphyrin samples. Using meso- Tetrakis (4-sulfonato phenyl) porphyrin (TPPS) as the model molecule, discussed herein is combined UV-vis extinction, Stokes-shifted fluorescence, and polarized resonance synchronous spectroscopy (PRS2) study of porphyrin assembly and disassembly at acidic solutions. A series of optical constants, including photon absorption, scattering, and fluorescence emission cross-sections as well as its fluorescence and light scattering depolarizations has been quantified. Compared to UV-vis and SSF methods, the PRS2 is significantly more sensitivity in the detection of both concentration- and time-dependent porphyrin aggregation.

Fluorochromes such as tetracycline have been used to label bone for histomorphometric analysis, measuring bone formation, growth, maintenance, and pathology. More recently, similar fluorescence has been observed in ancient human bone. Attributed to tetracycline (TC) exposure, this phenomenon could affect various aspects of health during life and/or preservation of remains postmortem. Standard epifluorescence microscopy is the most common tool employed in the analysis of these labels. Though valuable, this technique is limited by its inability to penetrate bone three-dimensionally and its inclusion of out-of-focus light, possibly disrupting accurate analysis. Confocal Laser Scanning Microscopy (CLSM) has been demonstrated as a valuable tool for three-dimensional histology. Its application to the study of compact bone fluorescence has been lacking, especially in archaeological and forensic sciences. In the following two papers, modern TC-controlled bone is compared to well preserved archaeological bone recovered from the Dakhleh Oasis, Egypt, using both standard wide-field and more modern confocal techniques for imaging and analysis. Spectral analysis via CLSM shows that both modern and ancient fluorescent labels in bone share the exact same fluorescence emission peak at 525 nm. Differences in the shape of the spectral curve and photobleaching characteristics are discussed. In addition, CLSM's high-resolution two- and three-dimensional imaging capabilities (in polarized light, scattered light, and fluorescence light) are found to increase the flexibility and creativity of investigations into the occurrence of tetracycline labels in archaeological bone and could have added benefits for modern medical and anatomical experimentation.

La production du sirop d'érable contribue de façon importante à l'économie du secteur agroalimentaire du Canada et tient une place de premier plan dans l'identité culturelle du pays. En 2018, le Canada a produit environ 40 millions de litres de sirop d'érable, représentant une valeur de 384 millions de dollars. L'augmentation de la production du sirop d'érable au fil des années et selon les prévisions établies conduit à développer le système de classification actuel. Cette étude porte sur la caractérisation physicochimique de 18 échantillons de sirops d'érable ayant une qualité inférieure et d'étudier en deuxième lieu l'adéquation de la spectroscopie de fluorescence frontale 3D (3D-Frontal Fluorescence Spectroscopy) sur une large gamme de longueurs d'onde d'excitation-émission comme technologie discriminante des défauts sensoriels de 90 échantillons de sirops d'érable en vrac répartis en 3 séries, chacune regroupe 6 différentes catégories de sirops (« OK », « VR4 », « VR5 », « VR12 », « VR13 », « VR42 »). Les résultats d'analyses des acides organiques montrent que l'acide malique est le plus abondant par rapport aux acides citrique et fumarique. Les échantillons de référence appartenant à la catégorie « OK » avaient toujours les concentrations moyennes les plus faibles en acide organique. La concentration en acide fumarique dans les échantillons de la catégorie « OK » est significativement plus faible que le reste des échantillons. Aucune différence n'est remarquée pour le degré de Brix et de pH entre les différentes classes analysées. Cependant, les couleurs des échantillons variaient significativement en fonction de la catégorie analysée. De plus, une corrélation négative est établie entre la concentration des échantillons en polyphénols et leur pourcentage de transmission de lumière. L'analyse en composantes principales des données physicochimiques montre qu'il existe une relation réciproque entre deux groups d'échantillons comme les sirops les plus clairs et sucrés (« VR05 » « VR42 » et « OK ») sont moins concentrés en polyphénols et en acides organiques (« VR04 », « VR12 » et « VR13 ») et vice versa. L'analyse de 18 échantillons par spectroscopie de fluorescence frontale couplée à l'analyse en composante indépendante, mène à générer deux signaux de fluorescence différents sur IC1 et IC10 permettant d'avoir une meilleure discrimination des défauts sensorielle. En plus, l'application d'une analyse factorielle discriminante sur les spectres de 90 échantillons de trois différentes séries permet de générer une fonction discriminante ayant des pourcentages de classification globale variant entre 97,78 % et 94,44 %. / Maple syrup production is an important contributor to the economy of Canada's agri-food sector and holds a prominent place in the country's cultural identity. In 2018, Canada produced approximately 10 million gallons of maple syrup, worth

Kinetic spectroscopic quantification refers to a subset of chromogenic (CG) and fluorogenic (FG) assays that deduce analyte concentration based on the UV-vis or fluorescence signal obtained during the CG/FG reaction processes. Existing kinetic spectroscopic quantification are based predominantly on reactions that can be approximated as a first-order process. Presented in this thesis is the kinetic spectroscopic quantification that uses higher order CG/FG reactions where the overall reaction can be approximated as combination of two sequential first-order processes. Included in chapter one is the theoretical model and several proof-of-concept applications. This model analyte is malondialdehyde (MDA), a lipid peroxidation biomarker of broad interest. Chapter two describes the study of the effects of the reaction solvent, temperature, acid catalyst, and calibration method on the assay performance. The most rapid MDA assays achieved so far is 3 mins, 30 times more efficient than the current equilibrium spectroscopic quantification.