title: Development and Characterization of Single-Molecule Switching Nanoscopy Approaches for Deeper and Faster Imaging  
creator: Hartwich, Tobias Max Philipp
subject: 530
subject: 530 Physics
description: 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.  
date: 2013
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/14477/1/Doktorarbeit_Hartwich.pdf
identifier: DOI:10.11588/heidok.00014477
identifier: urn:nbn:de:bsz:16-heidok-144772
identifier:   Hartwich, Tobias Max Philipp  (2013) Development and Characterization of Single-Molecule Switching Nanoscopy Approaches for Deeper and Faster Imaging.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/14477/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng