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Description
In this work, atomic force microscopy (AFM) and time resolved confocal fluorescence microscopy are combined to create a microscopy technique which allows for nanometer resolution topographic and fluorescence imaging. This technique can be applied to any sample which can be immobilized on a surface and which can be observed by fluorescence microscopy. Biological problems include small molecular systems, such as membrane receptor clusters, where very high optical resolutions need to be achieved. In materials science, fluorescent nanoparticles or other optically active nanostructures can be investigated using this technique. In the past decades, multiple techniques have been developed that yield high resolution optical images. Multiple far-field techniques have overcome the diffraction limit and allow fluorescence imaging with resolutions of few tens of nanometers. On the other hand, near-field microscopy, that makes use of optically active structures much smaller than the diffraction limit can give resolutions around ten nanometers with the possibility to collect topographic information from flat samples. The technique presented in this work reaches resolutions in the nanometer range along with topographic information from the sample. DNA origami with fluorophores attached to it was used to show this high resolution. The fluorophores with 21 nm distance could be resolved and their position on the origami determined within 10 nm. Not only did this work reach a new record in optical resolution in near-field microscopy (5 nm resolution in air and in water), it also gave an insight into the physics that happens between a fluorescent molecule and a dielectric nanostructure, which the AFM tip is. The experiments with silicon tips made a detailed comparison with models possible on the single molecule level, highly resolved in space and time. On the other hand, using silicon nitride and quartz as tip materials showed that effects beyond the established models play a role when the molecule is directly under the AFM tip, where quenching of up to 5 times more efficient than predicted by the model was found.
ContributorsSchulz, Olaf (Author) / Ros, Robert (Thesis advisor) / Levitus, Marcia (Committee member) / Liu, Yan (Committee member) / Lindsay, Stuart (Committee member) / Shumway, John (Committee member) / Arizona State University (Publisher)
Created2012