| dc.description.abstracteng | Optical nanoscopy allows for highly specific imaging of biological tissue, cellular components
and even single molecules and has therefore become an integral part of modern biomedical
research. In STED microscopy, being one of these techniques, super-resolution is achieved by
employing a depletion intensity distribution to confine the fluorescence to a sub-diffraction
sized area. The high focal intensities typically employed for an efficient depletion of
fluorophores can however cause phototoxicity and photodamage to the sample. Several
techniques have been presented recently to tackle this problem. They are based on avoiding
the transition of dye molecules to any other than one of the desired molecular states or on
reducing the light dose by lowering the employed laser power or effective illumination time. For
STED microscopy, however, this reduction has not yet been translated into a faster acquisition
time, which would be of particular advantage for the investigation of fast biological processes.
Another technique that reduces both light dose and acquisition time is tomoSTED microscopy,
which drastically reduces photobleaching and sample damage. But even this form of
STED microscopy could benefit from an adaptation of the scanning process. Within this work, adaptive scanning as an approach to locally adjust the total pixel dwell time
and/or the local intensity distribution is developed and evaluated for its potential to improve
the performance of STED microscopy with respect to the beforehand outlined aspects. For this
purpose, a scan system with a fast response time is developed, implemented and characterized.
Additionally, a novel method to generate 1D depletion patterns for tomoSTED microscopy
based on conical diffraction is introduced, for which the pattern orientation can be switched on
the single pixel level by using electro-optical devices, paving the way to employing tomoSTED
microscopy with adaptive scanning.
Utilizing the new scan system, a novel technique called FastRESCue, introduced here as a
variant of RESCue, proves to allow a direct translation of a reduced light dose into
faster image acquisition, yielding imaging at only 20% of both light dose and acquisition time
at uncompromised image quality and resolution. A further reduction of the acquisition time
is realized by adapting the scanned area directly to the sample structure under investigation.
Employing the novel 1D depletion patterns for tomoSTED microscopy, this innovative adaptive
scanning technique enables a real-time detection of the structure’s position and orientation. It
is therefore successfully applied in tracing of filamentous structures in both fixed and living
cells.
In conclusion, this thesis demonstrates the successful application of adaptive scanning for STED
microscopy with the focus on low-illumination and fast-acquisition imaging schemes. The
extension of this concept to e.g. three-dimensional scanning or other sample structures will
remain the focus of future work. | de |