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Osseforth, Christian (2014): Simultaneous dual-color 3D stimulated emission depletion microscopy: from design to applications. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



Super-resolution imaging, the ability to resolve structures well below the diffraction limit, has changed fluorescence microscopy as we know it. Diffraction-unlimited microscopy, termed nanoscopy, is able to image deep inside the cell with a resolution approaching that of electron microscopes. A variety of different methods currently exist, the first proposed and demonstrated technique being stimulated emission depletion (STED) microscopy. In STED microscopy, the resolution limit of a laser scanning microscope is overcome by transiently silencing fluorescence in parts of the focus. Practical implementations of this method are limited by optical aberrations and the characteristics of the fluorophores used. In this thesis, I present a fully working, custom built, 3D dual-color STED microscope. A super-continuum source is used to provide all spectral bands necessary for excitation and efficient depletion to achieve a lateral and axial resolution of ~ 35 nm and ~ 90 nm, respectively. I characterize the system’s performance by imaging colloidal particles and single fluorescent molecules. Its biological applicability is demonstrated by imaging of nuclear pore complexes in U2OS and yeast cells, replication complexes in C2C12 cells, C2 toxin component C2I in HeLa cells and scaffold proteins in chemical synapses of hippocampal neurons. Advice on how to build such a microscope is given in the appendix. In addition, a theory is presented which demonstrates that the resolution of STED microscopes can be further enhanced by using the arrival time of spontaneous emission by time-gating the detection.