Optimal de-excitation patterns for RESOLFT-Microscopy

RESOLFT microscopy has been the first method that is capable of non-invasively resolving three dimensional structures with real subdiffraction resolution using visible light. It exploits the strong nonlinear saturation of a reversible optical transition. A focal intensity pattern is essential that de-excites or de-activates dyes outside the remaining ultrasharp effective focal spot. For a given amount of available power, the steepest applied de-excitation pattern will yield the highest resolution. In this thesis, for the first time, a comprehensive search, optimization and characterization of de-excitation patterns is performed. The microscope’s pupil function is decomposed into orthonormal polynomials which allows the restriction of the space of pupil functions so that boundary conditions are fulfilled. The chosen global optimization algorithm converges reasonably well to pupil functions that can be idealized further to simple shapes. Optimal pupil functions are found according to assumptions made about the practical limitations. The optimization identified a novel, superior de-excitation pattern for circularly polarized light. Its experimental application has led to a hitherto unrivaled lateral resolution of down to 20 nm in biological systems. It is shown that an efficient resolution increase in all spatial directions is only possible by incoherent combinations of de-excitation beams. The optimal choice for current experimental conditions is identified. Finally, a new concept for fast acquisition of high resolution images is developed that is based on the simultaneous creation of compact arrays of sub-diffraction sized fluorescence spots in the sample. An optical setup that can generate the corresponding complex pupil functions is detailed.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Heidelberg

Year

2006

Author dc:creator

• Keller, Jan

Contributors dc:contributor

• Hell, Stefan W.

Repository record source_url

http://www.ub.uni-heidelberg.de/archiv/7163

OAI identifier oai:identifier

oai:archiv.ub.uni-heidelberg.de:7163