%0 Generic
%A Hartwich, Tobias Max Philipp
%D 2013
%F heidok:14477
%R 10.11588/heidok.00014477
%T Development and Characterization of Single-Molecule Switching Nanoscopy Approaches for Deeper and Faster Imaging  
%U https://archiv.ub.uni-heidelberg.de/volltextserver/14477/
%X Novel single-molecule switching super-resolution microscopy overcomes the diffraction limit of far-field fluorescence microscopy by precisely localizing individual fluorescent molecules from thousands of images of stochastic, sparse blinking-molecule distributions. However, this technique has so far mostly been limited to thin, fixed samples: usually, fluorescent molecules are activated throughout the whole depths of the sample and not just in the 1 - 2 μm thick optical section where they can be localized. In thick samples, this incurs excessive background and unwanted bleaching of probe molecules out of focus. Using two-photon absorption allows to limit activation of photo-activatable fluorescent proteins to the optical section where they can be localized. However, no spectroscopic information about the two-photon activation of the most commonly used molecules has been available so far. Live-cell imaging is additionally hampered by the typically used EM-CCD cameras which can only record up to 60 full frames/second. Novel sCMOS cameras feature much higher readout speeds and have the potential for fast live-cell imaging, but artifact-free performance at high speed has not been demonstrated yet. In this thesis, I have realized a new super-resolution microscope capable of two-photon activation of photo-activatable probes. I have characterized PAmCherry1, PA-GFP and PAmKate, three of the most popular photo-activatable fluorescent proteins, spectroscopically for two-photon activation. My results suggest a modified model of photo-activation of PAmCherry1. Super-resolution images of ring canals in thick Drosophila egg chambers have been recorded in three dimensions using this new microscope.  Furthermore, I present the first artifact-free super-resolution microscopy using a sCMOS camera. Microtubules could be imaged at 32 nm spatial resolution in only  33 ms.