Tomographic STED Microscopy
by Jennifer-Rose Krüger
Date of Examination:2017-02-22
Date of issue:2017-03-09
Advisor:PD Dr. Alexander Egner
Referee:Prof. Dr. Jörg Enderlein
Referee:PD Dr. Alexander Egner
Referee:Prof. Dr. Sarah Köster
Referee:Prof. Dr. Tobias Moser
Referee:Dr. Florian Rehfeldt
Referee:Prof. Dr. Tim Salditt
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Abstract
English
Due to its non-invasive access to sub-cellular structures, far-field fluorescence microscopy constitutes one of the most prevalent methods in the life sciences. For more than one century, it had been common knowledge that the resolution is fundamentally limited by diffraction. Within the last decades, this constraint has been repealed by super-resolution concepts based on molecular transitions between a bright and a dark state of specific fluorophores. In STED (Stimulated Emission Depletion) microscopy, the excitation focus is superposed with an intensity distribution that depletes the fluorescence via stimulated emission wherever its intensity is non-zero. The depletion focus is typically doughnut-shaped, allowing for a directly accessible resolution enhancement in all lateral directions. The achievable resolution in STED microscopy is theoretically unlimited and directly linked to the STED laser power, which already raises a concern: The application of high depletion laser powers might induce additional photo-bleaching to fluorophores or photo-damage to biological samples. Consequently, there is a great demand for STED microscopes that operate at reduced depletion laser powers, while preserving resolution conditions. In the thesis presented here, a novel two-dimensional STED microscopy variant that bases on the utilization of a one-dimensional depletion focus is introduced. Contrary to the classical implementation, the STED laser power is not distributed to a doughnut-shaped depletion pattern but concentrated along a single direction. Due to higher local intensities in the depletion pattern, the same one-dimensional resolution enhancement as in the conventional STED approach can be obtained at approximately half the depletion laser power. For an isotropic resolution enhancement in all lateral directions, a sequence of images with differently oriented high-resolution axes has to be recorded. Therefore, the one-dimensional depletion pattern has to be rotated along a rotary axis that is perpendicular to the focal plane and intersects with the focal center. Via appropriate reconstruction algorithms, it is possible to transfer the high-resolution information of each individual frame of one-dimensional resolution enhancement to a final image with quasi-homogeneous resolution. Since the outlined image acquisition and processing is reminiscent of tomographic approaches, our method is referred to as ‘tomographic STED microscopy’. Besides the more efficient resolution enhancement along one direction, our new STED variant contains a further intrinsic advantage when compared to classical approaches. As the fluorescence is only constrained along one dimension, tomographic STED microscopy profits from a higher flux of fluorescence photons as compared to the conventional STED variant which is particularly advantageous when choosing the same or even a shorter total recording time. Since an image of same quality, in terms of resolution and signal, can be obtained with half the STED laser power and within equal or even lower total acquisition time when compared to the classical approach, tomographic STED microscopy has the potential to substantially reduce photo-bleaching and photo-damage. Our new STED variant thereby paves the way towards new fields of applications for STED microscopy.
Keywords: Fluorescence microscopy; Superresolution techniques; STED microscopy; PSF engineering; Biophysics; Cell imaging