Die Durchflusszytometrie und Fluoreszenzmikroskopie sind zentrale Techniken zur Analyse von Zellen, Geweben und Organen. Besonders in der Immunologie werden sie zur Identifizierung und Charakterisierung von Biomolekülen mittels fluoreszenzmarkierter Antikörper verwendet. Fluoreszenzmarker müssen je nach Anwendung hohe Helligkeit, geringe Größe und minimierte Löschung des Signals aufweisen. Stark markierte Konstrukte leiden jedoch oft unter Fluoreszenzlöschung oder großen Molekularmassen. Diese Arbeit untersucht verzweigtes Polyethylenglykol (PEG) als Träger für Fluorophore. PEG-Ketten wurden als räumliche Trennmittel identifiziert und an Aminodextran gekoppelt, wodurch hochgradig multimerisierte Fluorophor-PEG-Dextran-Zwischenprodukte entstanden. Diese Konjugate, gekoppelt mit Antikörpern, zeigen hohe Fluoreszenzintensität und wurden bei der Detektion von CAR SUP-T1-Zellen erfolgreich eingesetzt. PEG-basierte Reagenzien durchdringen jedoch oft die Zellmembran nicht, was für intrazelluläre Ziele und größere Gewebe wichtig ist. Sequentielle Multiplex-Analysen sind durch unvollständige Spaltung und Restsignale problematisch. Deshalb wurden synthetische Peptide als Rückgrat für die Fluorophor-Multimerisierung untersucht. Diese Konstrukte, verbunden mit Nanokörpern, zeigten erhöhte Helligkeit und Gewebepenetration in der Lichtblattmikroskopie von Mausorganen. Zudem wurde ein dualer Entfernungsmechanismus in die REAdyelease-Technologie integriert. Basierend auf Oligonukleotiden, Disulfiden oder Peptiden in Kombination mit Aminodextran konnte eine schnellere Signalreduktion ermöglicht werden. Dies wurde in der Konfokalmikroskopie an einer Pankreastumorzelllinie demonstriert. / Flow cytometry and fluorescence microscopy are crucial for analyzing cells and tissues, especially in immunology, where immunofluorescence is used for identifying, visualizing, and characterizing biomolecules with fluorescently labeled antibodies. These labels must meet various requirements: high brightness, small size, and the ability to be rendered non-fluorescent. However, highly labeled constructs often suffer from fluorescence self-quenching or high molecular masses, limiting their effectiveness. This work demonstrates that branched polyethylene glycol (PEG) serves as an efficient fluorophore multimerization platform for protein labeling. I explored factors critical for preventing fluorophore self-quenching in multi-fluorophore systems. Fluorescent PEGs were multimerized on an amino-dextran scaffold, generating highly multimerized fluorophore-PEG-dextran intermediates. When conjugated to antibodies, these intermediates allowed bright labeling of biomarkers on cells and tissues and were successfully used in detecting CAR SUP-T1 cells. Despite their strengths, PEG-based reagents often lack deep tissue penetration, essential for intracellular targets and 3D organ imaging. To enhance tissue penetration, I designed small peptide-based backbones for fluorophore multimerization. These constructs, coupled with nanobodies, produced homogeneous fluorescent conjugates that quickly penetrated mouse organs and enabled bright staining in light-sheet microscopy. The final part of the thesis focuses on synthesizing labels for cyclic immunofluorescence. I addressed the issue of incomplete label removal by creating erasable conjugates with two release sites. Fluorescent conjugates based on oligonucleotides, disulfides, or peptides combined with amino-dextran can be rapidly erased from labeled epitopes using a dual-release approach. This method was demonstrated in confocal microscopy and used for iterative imaging of biomarkers on a sample of a pancreatic tumor cell line.

"June, 2008". / Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2009. / Bibliography: p. 227-259. / Literature review -- Materials and methods -- Sampling sites and sampling material -- Enrichment cultures and molecular analyses -- Optical and binding characterisation of the QDs -- Applications of the QDs -- Concluding remarks. / White island, the most active volcano in New Zealand, is a poorly studied environment that represents an ideal site for the investigation of acidophilic thermophiles. The microorganisms present on here are continually exposed to extreme environmental conditions as they are surrounded by steamy sulphurous fumaroles and acidic streams. The sediment temperature ranges from 38°C to 104°C whilst maintaining pH values below 3. A survey of the volcanic hydrothermal system of White Island was undertaken in order to gain insights onto the microbial diversity using culture-dependant techniques and molecular and phylogenetic analyses. A novel liquid medium based on "soil-extract" was designed which supported growth of bacterial and archaeal mixed cultures. Molecular analyses revealed that the dominant culturable bacterial species belong to the Bacteroidetes, Firmicutes and α-Proteobacteria groups. Several previously uncultured archaeal species were also present in the mixed cultures. The knowledge gained from these studies was intended to help in the development of a novel microbial detection technique suitable for community analysis. -- Conventional molecular techniques used to study microbial biodiversity in environmental samples are both time-consuming and expensive. A novel bead-based assay employing Quantum dots (QDs) was considered to have many advantages over standard molecular techniques. These include high detection speeds, sensitivity, specificity, flexibility and the capability for multiplexed analysis. QDs are inorganic semiconductor nanoparticles made up of crystals about the size of proteins. It has been claimed that the physical and chemical properties of the QDs have significant advantages compared to organic dyes, including brighter fluorescence and resistance to photo-bleaching. Their optical properties facilitate the simultaneous imaging of multiple colours due to their flexible excitation and narrow band emission. Functionalised QDs are able to bind to different biological targets such as DNA, allowing high-throughput analysis for rapid detection and quantification of genes and cells. -- The optical and physical characteristics of the QDs as well their interaction with biomolecules are shown to be suitable for the development of a novel bead-based technique able to target the key microbial species and identify them by flow cytometric measurements (FCM). The broad absorption and narrow emission spectra of the QDs, as well as their fluorescence intensity and specify to target biomolecules, was compared to other organic fluorophores. The potential advantages and limitations of QDs as a fluorophores for biological applications are discussed. -- The data acquired during this study provides a broad overview of the microbial diversity and ecology of the volcanically-active hydrothermal systems of White Island and constitutes the baseline for the development of a novel bead-based technique based on QDs. / Mode of access: World Wide Web. / xvii, 259 p. ill. (some col.)