Enhancing fluorophore performance for single-molecule applications and super-resolution imaging

Close, Cindy Mara (2025): Enhancing fluorophore performance for single-molecule applications and super-resolution imaging. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

To see the world, we need to see the small things. And so, science has made great efforts to observe the details of biological systems. Sophisticated microscopes have been developed to visualize structures and interactions at the dimension of proteins and other biomolecules. In fluorescence microscopy these nanometer-sized features are revealed through photon-emitting labels, attached to the molecule of interest. High performance of these dyes in terms of stability, specificity and brightness is a prerequisite for any successful experiment. At the same time, in order to observe the system in its innate state, interference of the observation method with the specimen should be minimal. Once this is ensured, it can be applied to observe biomolecules in their inherently complex environment and detect disease markers reliably. The first section of this thesis is focused on increasing fluorophore photostability. To improve the signal-to-background ratio, fluorescence microscopy is often performed at the highest possible illumination power and for as long as possible before sample and fluorescent labels degrade (photobleaching). Aside from desired cycling between singlet excited and ground state (fluorescence), fluorophores can also enter other states such as the triplet state from where photobleaching is likely to originate. Systems to prevent this pathway commonly employ oxygen removing enzymes in combination with triplet state quenchers (TSQ), which ensure a fast return to the singlet state. Sensitive biological systems, however, can be disrupted by these additives and the required concentrations. In this work a minimally-invasive strategy that adresses this issue by attaching the TSQ to single stranded DNA (ssDNA) is introduced. An extended sequence (docking site) on the molecule of interest enables the hybridization and exchange of both the label and the photostabilization strand. This approach improves the TSQ soluability and increases the local concentration near the label. By performing hour-long measurements of an otherwise photolabile dye without oxygen removal, we demonstrate enhanced fluorophore performance at 10^7 less additive concentration. This DNA-mediated stabilization is not restricted to one type of TSQ. The modularity of the technique allows for exploration of several stabilizers to match other fluorophores. Due to its adaptability and efficacy at low concentrations, our method can be applied to challenging imaging modalities such as multi-target visualization in complex biological systems. The next section explores key factors for fluorescence-based disease detection, namely specificity and brightness of fluorescent labels. Reliable diagnosis of Malaria tropica in early stages of infection is necessary to begin treatment as soon as possible. While healthy red blood cells (RBC) do not contain cellular organelles, the Plasmodium falciparum parasite introduces them upon invasion. A silicon rhodamine dye equipped with a glibenclamide moiety is first used in this study to specifically target the endoplasmic reticulum. This allows for detection and distinction of infected over uninfected RBC in two different strains. The potential for application in the field is demonstrated by experiments on a low-cost portable smartphone microscope. State-of-the-art methods for DNA detection rely on up-concentration of low-abun-dance target sequences over the background. A promising alternative is the detection of individual dye-labeled DNA molecules, which can be achieved through signal amplification using DNA origami nanoantennas. These plasmonic nanostructures bind two metallic nanoparticles (NP) and enhance fluorescence signal in the plamonic hotspot between. Fluorescently labeled disease markers can be directed towards this position by including ssDNA capture strands in the DNA origami. Two design generations for disease detection are included in this work. The chosen target sequence is responsible for an antibiotic resistant Klebsiella pneumoniae infection. The first generation introduces a cleared region in the hotspot of the DNA nanostructure to provide space for the DNA detection element and two NPs. By optimizing the target DNA hybridization with label and capture strand (sandwich assay), efficient detection and amplification is achieved and a home-built smartphone microscope can be used to detect individual disease markers. The second design (Trident) includes a more accessible hotspot to host even larger biomolecules. In comparison to the previous generation, detection speed of a 151 nucleotide ssDNA is tripled and the fraction of multiple captured molecules is doubled. Simultaneously, high fluorescence amplification is ensured. This thesis demonstrates how photostabilization improves the performance of fluorophores in state-of-the art fluorescence microscopy. Through specific labeling and fluorescence enhancement, disease detection can be performed even on low-tech microscopes.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 11. April 2025
• 1. Bericht­erstatter:in: Tinnefeld, Philip