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Steinhauer, Christian (2011): Imaging and Positioning of Objects at the Nanoscale. Dissertation, LMU München: Faculty of Physics
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Abstract

The process of miniaturization brings benefits in many areas of every day life. It describes the process of down-scaling mechanical, optical and electronic devices while maintaining their function. This continuous process has brought up the field of nanotechnology. It allows computers to run faster and faster while reducing their size and energy consumption. Many bio-analytical applications profit from smaller sizes to be more sensitive with lower sample volumes. Finally, each and every living cell contains the most complex nanoscopic machinery. In order to mimic nature’s highly efficient concepts, they first of all need to be understood. Their nanoscopic dimensions, however, make this task a very challenging one. Direct observation of subcellular processes by conventional light microscopy is difficult as almost all cellular components are colorless. Fluorescence microscopy introduces sufficient contrast and can be specifically applied to many different target molecules but remains limited in its spatial resolution. In this work, a new approach to increase the resolution of fluorescence microscopy is presented that is termed “blink microscopy”. It is based on the subsequent localization of single molecules as it is realized in recent techniques known as STORM or PALM, but does not require special photoswitchable fluorophores or multiple lasers. Instead of photoswitching, reversible electron transfer reactions are used to generate the required dark states. Motivated by the task to assess the resolution of blink microscopy, DNA nanotechnology is used for nano-construction of a calibration structure. The DNA origami technique, developed by Paul Rothemund in 2006, allows for arranging of individual fluorophores at distances of 1–100 nm on a DNA nanostructure. While it is impossible to resolve two spots at a distance below 200 nm with conventional fluorescence microscopy, it was possible to confidently resolve 50 nm with blink microscopy. These experiments prove both, blink microscopy to be able to reliably resolve small distances and DNA origami structures to be well suited as a rigid breadboard for fluorescence experiments. This research pioneering the combination of the two powerful tools of nano-imaging and nanoconstruction set the ground for further experiments. After the rigidity of DNA origami structures was used to characterize a fluorescence technique, single-molecule fluorescence could also help to characterize properties of the DNA origami. Namely, dynamic processes on a DNA nanostructure were exemplarily studied by reversible binding of a fluorescently labeled DNA strand while observing this process in real-time on a fluorescence microscope. With the knowledge about binding and unbinding kinetics of DNA and imaging of single molecules, another super-resolution approach was developed that is simply and flexibly implemented in DNA structures, not limited by photobleaching, easy to extend to multiple colors and that shows potential for cellular imaging.