The aromatic amino acids tryptophan, tyrosine, and phenylalanine have been extensively used for different label-free protein studies. These investigations extract information on protein conformations and interactions from the emitted fluorescence's intensity, wavelength, and/or polarization. Like most fluorescent organic compounds, these amino acids also undergo transitions into dark meta-stable states, including triplet and photo-radical states. These transitions are notably sensitive to the surrounding environment, offering an additional set of parameters that reflect the protein's interactions, folding states, and immediate surroundings. Transient State (TRAST) monitoring has been developed to quantify fluorophore transition dynamics by recording the average fluorescence intensity in response to a modulated excitation. In this work, we performed TRAST experiments to investigate tyrosine autofluorescence and used it to detect conformational changes in calmodulin, a calcium-binding protein containing two tyrosine residues. A photophysical model for tyrosine was established, and it was revealed how tyrosine's dark state transitions changed with excitation intensity, solvent pH, and redox conditions. The TRAST experiments demonstrated that tyrosine's dark state transitions could serve as valuable information sources for label-free analyses of protein conformations and interactions. / De aromatiska aminosyrorna tryptofan, tyrosin och fenylalanin har använts i stor utsträckning för olika inmärkningsfria proteinstudier. Dessa undersökningar extraherar information om proteinkonformationer och interaktioner från den emitterade fluorescens intensiteten, dess våglängd och/eller polarisering. Liksom de flesta fluorescerande organiska föreningar genomgår dessa aminosyror också övergångar till mörka metastabila tillstånd, inklusive triplett- och fotoradikaltillstånd. Dessa övergångar är särskilt känsliga för den omgivande miljön, och erbjuder en extra uppsättning parametrar som återspeglar proteinets interaktioner, vikningstillstånd och omedelbara omgivningar. Transient State (TRAST) monitorering har utvecklats för att kvantifiera fluoroforövergångsdynamik genom att registrera den genomsnittliga fluorescensintensiteten som svar på en modulerad excitation. I detta arbete utförde vi TRAST-experiment för att undersöka tyrosinautofluorescens och använde den för att detektera konformationsförändringar i calmodulin, ett kalciumbindande protein som innehåller två tyrosiner. En fotofysikalisk modell för tyrosin etablerades, och hur tyrosins mörka tillståndsövergångar förändrades med excitationsintensitet, lösningsmedels pH och redoxförhållanden kunde faststållas. TRAST- experimenten visade att tyrosins mörka tillståndsövergångar kan fungera som värdefulla informationskällor för inmärkningsfria analyser av proteinkonformationer och interaktioner.

Many human diseases including cancer have been associated with altered cellular metabolism and a changed oxygen consumption in cells. Fluorophores are sensitive to their local environment due to their long life times in transient dark states. A recent study successfully utilized this sensitivity to image differences in oxygen concentrations in cells using transient state (TRAST) microscopy together with fluorescent labels [1]. A natural continuation of this study is to investigate the possibilities of using this method with natural fluorophores already present in cells and thereby avoid articial labeling. Flavin adenine dinucleotide (FAD) is an auto fluorescent coenzyme that is naturally present in cells and involved in cellular metabolism. This project is an exploratory pilot study for cellular measurements with the aim to investigate if FAD can be used to probe oxygen concentrations in aqueous solution using transient state monitoring and fluorescence correlation spectroscopy (FCS). This thesis includes the results from FCS and TRAST experiments on FAD in aqueous solutions with different oxygen concentrations as well as different ascorbic acid concentrations. The performed experiments showed that FAD monitored with TRAST is sensitive to differences in oxygen concentrations for the aqueous solutions used in this study.