New Concepts for STED Microscopy
von Julia Kratz
Datum der mündl. Prüfung:2021-10-25
Erschienen:2022-01-04
Betreuer:Prof. Dr. Alexander Egner
Gutachter:Prof. Dr. Alexander Egner
Gutachter:Prof. Dr. Jörg Enderlein
Dateien
Name:Dissertation_Kratz_Julia.pdf
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Format:PDF
Zusammenfassung
Englisch
Fluorescence nanoscopy allows to non-invasively resolve three-dimensional cellular structures beyond the diffraction limit. One of these high resolution imaging techniques is stimulated emission depletion (STED) microscopy. However, the practically achievable resolution of a STED microscope is often limited by photobleaching. One method to overcome this limitation is tomographic STED (tomoSTED) microscopy. In tomoSTED microscopy, excited fluorophores in the sample plane are depleted by 1D STED patterns, which lead to an effective narrowing of the fluorescence-allowed area in a single direction. As the effective 1D STED-PSFs exhibit both a higher resolution in the respective direction as well as a higher signal as compared to conventional STED, the STED laser power as well as the exposure time can be reduced, leading to a lower light dose. A highly resolved image in two dimensions is reconstructed from multiple images, each exhibiting a different orientation of the 1D STED-PSF. The number of required pattern orientations depends in this context on the ratio of the resolutions in the depleted and non-depleted direction. Since the resolution per pattern orientation is only increased along a single direction, imaging along the other direction is still diffraction-limited. Therefore, the resolution along this direction can be increased by utilizing the concept of image scanning microscopy (ISM) and the number of required pattern orientations can be accordingly reduced. This leads to a lower overall acquisition time and translates directly into a lower light dose. Within this thesis, the combination of tomoSTED and ISM was investigated for the first time, both in theory and experiment. Furthermore, it was investigated whether the tomoSTED principle can be extended from 2D to 3D.
Keywords: STED microscopy; image scanning microscopy; superresolution imaging; fluorescence microscopy