Beschreibung:
The diffraction limit in far-field fluorescence microscopy can be overcome by photoswitching the marker molecules between a fluorescent and a non-fluorescent state. Photoswitching enables the successive generation and readout of nano-sized fluorescent areas in the sample. Subsequently, these areas can be assembled to form a super-resolution image of the specimen. For the first time in this work, one and the same molecular switching mechanism of a reversibly switchable fluorescent protein (RSFP) was used to realize two alternative variations of this general nanoscopy principle: one based on the targeted switching of ensembles of molecules and the other based on the stochastic switching of single emitters. RSFPs are of particular interest for these techniques because they allow the non-invasive study of processes in living cells and can be efficiently switched using low light intensities, thus minimizing photo stress and possible photo-induced damage of the cell. Furthermore, the protein"s photoswitching properties can be improved via targeted mutagenesis. In the case of both nanoscopy techniques presented here, image acquisition could be significantly improved and accelerated as compared to related approaches. For the first time, two-color far-field fluorescence nanoscopy based on the photoswitching of single emitters was successfully realized in biological samples.