Visualisation of nanoscropic interactions in living systems using subdiffraction fluorescence microscopy

The proposed research aims to create a framework for the visualization of nanoscopic structures and interactions in complex systems, such as living cells and tissues, using diffraction-unlimited fluorescence microscopy. The project builds on a technique developed by the applicant, photochromic stochastic optical fluctuation imaging (pcSOFI), which uses genetically encoded labels and is considerably more robust and more widely applicable than other diffraction-unlimited techniques. In the proposed research the applicant will aim to expand pcSOFI to a wide range of genetically-encoded labels by making use of strongly conserved photophysical processes. This capability will then be applied to extend subdiffraction imaging to the study of molecular interactions by combining pcSOFI microscopy with Förster resonant energy transfer (FRET). This superresolution visualization of interactions, which is very difficult or impossible to achieve using established techniques, will be applied to address relevant questions in the biophysics of signaling using pcSOFIFRET.