STED-4Pi Microscopy

Saturated stimulated emission depletion (STED) has been shown to break Abbe's resolution limit in fluorescence microscopy. This technique suppresses fluorescence at the focal rim by use of a depleting laser pulse. In this thesis, theoretical and experimental examinations show that the combination with another technique, namely 4Pi-confocal microscopy, leads to a yet unreached axial resolution in light microscopy. The central part of this work is the performance of a STED-4Pi experiment. Simulations predict a superresolution of about 30 nm that is confirmed in experiments with ultrathin fluorescent layers. These results are so far considered to have the highest axial resolution for a light microscope. First biological images are recorded with membrane labeled bacteria at 40-50 nm resolution. A major issue is the photostability of the fluorescent dyes. With the bacterial samples, it can be exemplarily demonstrated that longer depletion pulses increase photostability. An amino-reactive dye, that is stable under STED conditions, enabled the first superresolved mammalian immuno-fluorescence images. They reveal a resolution of 50 nm along the optic axis. Finally, a further implementation of an additional STED beam for lateral resolution enhancement is demonstrated. In future, this will enable simultaneous superresolution of at least 100 nm in the lateral and 50 nm in the axial direction.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Heidelberg

Year

2004

Author dc:creator

• Dyba, Marcus

Contributors dc:contributor

• Hell, Stefan W.

Repository record source_url

http://www.ub.uni-heidelberg.de/archiv/4438

OAI identifier oai:identifier

oai:archiv.ub.uni-heidelberg.de:4438