Adaptive Scanning for STED Microscopy
von Britta Vinçon
Datum der mündl. Prüfung:2020-01-31
Betreuer:Prof. Dr. Alexander Egner
Gutachter:Prof. Dr. Alexander Egner
Gutachter:Prof. Dr. Sarah Köster
EnglischOptical 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.
Keywords: STED microscopy; adaptive scanning; low light dose; PSF engineering; fluorescence microscopy; super-resolution techniques; biophysics; cell imaging