%0 Generic
%A Winter, Franziska Ruth
%D 2016
%F heidok:20199
%K multicolour, hyperspectral, unmixing
%R 10.11588/heidok.00020199
%T Multicolour STED nanoscopy with hyperspectral detection
%U https://archiv.ub.uni-heidelberg.de/volltextserver/20199/
%X Within the scope of this thesis, a new multicolour STED microscope with a hyperspectral detection was designed and built. The challenges regarding the limitation of usable fluorophores as well as the stability of the optical set-up were overcome. Different existing spectral unmixing methods were compared to a self-implemented algorithm, both in simulations and using experimental data. A detailed study of the best separation conditions for several dye combinations was carried out for each of the methods. During the experiments, a spectral blue shift of several dyes was observed and its mechanisms were investigated. The optical resolution of the set-up was dye dependent and better than 80nm for all dyes. Moreover, an excellent separation for three fluorescent bead species and good a separation for a four-colour fixed-cell sample was achieved. This was done for the first time by purely distinguishing the different emission spectra using only one wavelength for excitation and a single STED beam for depletion.