TY  - THES
AB  - The diffraction barrier of focused light has been overcome by modern superresolution fluorescence microscopy techniques. These methods harness transitions between signaling and non-signaling states of the marker molecules to make them discernible at length scales of few tens of nanometers. Although superresolution methods like STED (STimulated Emission Depletion) and RESOLFT (REversible Saturable Optical Linear Fluorescence Transitions) microscopy proved to be valuable tools for cell biology, neurobiology and other fields, further improvements are needed. For example, lower fluorophore bleaching rates and enhanced state contrasts of the molecule ensemble are desired. Moreover, biological studies ask for better multicolour schemes allowing simultaneous imaging of multiple cellular structures with nanoscopic resolution. In this thesis, novel methods for coordinate-targeted superresolution microscopy are presented, which take up these challenges. Superresolution imaging with Multiple Off-State Transitions (MOST) is introduced and realized by reversible photoswitching and STED of fluorescent proteins leading to a new method called ´protected STED´. On the basis of measurements in different biological samples, it can be shown that in protected STED both fluorophore bleaching is reduced and state contrast is enhanced compared to conventional coordinate-targeted superresolution methods. Also, in this theses, a novel multicolour scheme is presented, which is based on a STED wavelength of 620 nm and which is valuable for imaging of both fixed and living samples. Furthermore, with multilevel STED, a new method was developed, which in multicolour imaging with a single STED beam improves both the image quality and avoids unnecessary STED light doses. Three-colour superresolution imaging harnessing multilevel STED was utilized to study nanoscale protein distributions in mature neurons. Periodic actin/βII spectrin lattices were found to be present along dendrites and thick spine necks, but they are absent from pre- and post-synaptic sites. These findings add new pieces to the picture of the nanoscale cytoskeletal arrangement in neurites.
AU  - Sidenstein, Sven-Christian
DA  - 2016
KW  - STED-Mikroskopie
KW  - Hochauflösende Mikroskopie
KW  - Fluoreszenzmikroskopie
KW  - STED
KW  - Neuron
KW  - Superresolution Microscopy
KW  - Nanoscopy
KW  - Fluorescence Microscopy
LA  - eng
PY  - 2016
TI  - Protected STED and multicolour multilevel STED nanoscopy
UR  - https://nbn-resolving.org/urn:nbn:de:hbz:467-10354
Y2  - 2024-11-22T05:16:54
ER  -