Expanding the STED Principle to Multicolor Imaging and Far-Field Optical Writing

Stimulated emission depletion (STED) nanoscopy has emerged as a powerful far-field technique for subdiffraction optical imaging and is extensively used in the life sciences to investigate different protein species. In many cases, however, the relative assembly of two (or more) proteins is of interest and needs to be determined with nanometric resolution. Meanwhile, STED has also found its way into material science. Yet, the method’s potential for subdiffraction optical writing has so far remained unexplored. This work evidences the development of a dual-color STED setup with lateral resolving power of 30 nm in both color channels. The method is shown to be applicable to the study of double-stained neuronal proteins and aids in establishing a novel sample sectioning technique. As a result the first high-resolution three-dimensional reconstruction of dual-color STED images is rendered possible. Flexible operation of the individual color channels furthermore answers open questions in chemistry and biology. Another key point of this thesis is the realization of STED nanolithography. Subdiffraction-sized structures are written by bleaching a layer of fluorophores. The underlying concept of bleaching suppression through STED is experimentally introduced and theoretically described by a photophysical model. Numerical simulations corroborate the experimental findings. The presented studies take multicolor STED nanoscopy close to macromolecular resolution and thus open up new methodical perspectives in the life sciences. STED nanolithography, on the other hand, has the potential of becoming an alternative to classical photolithography, thus simplifying high-resolution optical data storage