Automating STED microscopy for functional and structural live-cell imaging
Time: Fri 2021-12-17 13.00
Location: Sal Petrén, Wargentinhuset, Nobels väg 12B, Solna
Video link: https://kth-se.zoom.us/j/62958723396
Subject area: Biological Physics
Doctoral student: Jonatan Alvelid , Biofysik, Science for Life Laboratory, SciLifeLab, Ilaria Testa
Opponent: PhD Eggeling Christian, Friedrich-Schiller-Universität Jena
Supervisor: Universitetslektor Ilaria Testa, Biofysik, Science for Life Laboratory, SciLifeLab
Optical microscopy imaging methods are today invaluable tools for studies in life sciences as they allow visualization of biological systems, tissues, cells, and sub-cellular compartments from millimetres down to nanometres. The invention and development of nanoscopy in the past 20 years has pushed fluorescence microscopy down to the nanoscale, reaching beyond the natural diffraction limit of light that does not allow focusing of visible light below sizes of around 200 nm, and into the realm of what was previously only thought possible with electron microscopy. The superior spatial resolution does however come at a price, including complex sample preparation, prolonged recording times, increased illumination doses, and limited fields of view. Stimulated emission depletion (STED) microscopy is one of the techniques that can deliver nanoscale resolution in a range of biological systems, but with all the above-mentioned costs. However, with the right sample the technique can deliver single nanometre spatial resolution, and with the right considerations live-cell imaging is more than possible.
In this thesis I present the development of a flexible STED microscope with methodological advancements in a range of directions that aim at facilitating the use of STED microscopy in life sciences and optimising the information extraction from the image data. The developments firstly focused on automation of the data acquisition, to allow the recording of imaging data both with a higher throughput and correlated with fast dynamic processes. I also implemented improved image analysis, both in terms of high throughput and precision as well as in connection with the data acquisition. Furthermore, I worked on control software development, with novel strategies to unify the control software of microscopes and to allow development and implementation of novel acquisition schemes. I also utilized novel fluorophores, to improve live-cell and multicolour possibilities and allow a wider range of applications in STED microscopy. Lastly, I developed a novel concept that takes advantage of STED. Additionally, I present applications of the microscope and image analysis in diverse biological samples such as mammalian cells, tissue sections, and bacteria. Altogether, this work aims at presenting new tools for an imaging technique that is already well-established, to contribute to further development, facilitation of novel experiments, and expansion of the range of applications.