Far-field optical super-resolution microscopy or nanoscopy techniques “super-resolve” features residing closer than the diffraction-limit by transiently preparing fluorophores in distinguishable (typically on- and off-) states and reading them out sequentially. In coordinate-targeted super-resolution modalities, such as stimulated emission depletion (STED) microscopy, this state difference is created by patterns of light, driving for instance all molecules to the off-state except for those residing at intensity minima. I will discuss our recent efforts to improve coordinate-targeted nanoscopy. As a specific example, I will highlight how the use of multiple off-state transitions for nanoscopy can improve repeated imaging capability and on/off state contrast, enhancing image resolution and signal-to-background ratio in an approach that we dubbed “protected STED” (1). This allowed e.g. decoding the elaborate 3D structure of dendritic spines in living brain tissue. I will also give an outlook on the activities of my recently founded group at IST Austria.

(1) Danzl, Sidenstein et al.. Coordinate-targeted fluorescence nanoscopy with multiple off-states. Nature Photonics 2016; 10: 122.