Dual-Colour STED-microscopy on the Nanoscale
Stimulated emission depletion (STED) microscopy was the first concept for breaking Abbe's diffraction barrier in optical far-field microscopy verified in biological applications. However, the theoretically infinite resolution was limited due to photobleaching of the fluorescent species. In this thesis, dark-state relaxation (D-Rex) has been traced in a comprehensive study on one- and two-photon excitation to crucially reduce photobleaching in general thus leading to a major signal increase per excitation pulse. This facilitated a successful combination of this illumination strategy with STED-microscopy making a 10-fold increase of STED-power feasible. The expansion of STED-microscopy to D-Rex conditions at 250 kHz leads to a yet unattained focal plane resolution ~20 nm, equivalent to an approximate 12-fold multilateral increase of resolution below the diffraction limit. This macromolecular resolution was exemplified in a variety of biological samples, including proteins of cell-junction and focal adhesion, or a neurofilamental protein from the human brain. Finally, the extension to a Dual-colour STED-microscope was achieved to provide nanoscale precise colocalization ability of individual protein clusters in cell biology, thereby sustainably widening the application range of STED-microscopy. The method proved to resolve hitherto uncovered nanopatterns of vesicle proteins on endosomes, as well as localized different proteins in mammalian mitochondria.
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