eprintid: 14477
rev_number: 13
eprint_status: archive
userid: 367
dir: disk0/00/01/44/77
datestamp: 2013-02-08 08:02:22
lastmod: 2013-02-13 15:27:12
status_changed: 2013-02-08 08:02:22
type: doctoralThesis
metadata_visibility: show
creators_name: Hartwich, Tobias Max Philipp
title: Development and Characterization of Single-Molecule Switching Nanoscopy Approaches for Deeper and Faster Imaging

subjects: 530
divisions: 120300
adv_faculty: af-13
abstract: 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
id_scheme: DOI
id_number: 10.11588/heidok.00014477
ppn_swb: 1652034757
own_urn: urn:nbn:de:bsz:16-heidok-144772
date_accepted: 2013-01-31
advisor: HASH(0x5561209c4948)
language: eng
bibsort: HARTWICHTODEVELOPMEN2013
full_text_status: public
citation:   Hartwich, Tobias Max Philipp  (2013) Development and Characterization of Single-Molecule Switching Nanoscopy Approaches for Deeper and Faster Imaging.  [Dissertation]     
document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/14477/1/Doktorarbeit_Hartwich.pdf