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BP: Fachverband Biologische Physik
BP 2: Bioimaging and Spectroscopy I
BP 2.4: Vortrag
Montag, 20. März 2017, 10:30–10:45, HÜL 386
Coordinate-targeted fluorescence nanoscopy with multiple off-states — •Johann Georg Danzl1,2, Sven Sidenstein2, Carola Gregor2, Nicolai Urban2, Peter Ilgen2, Stefan Jakobs2, and Stefan Hell2 — 1Institute of Science and Technology Austria, 3400 Klosterneuburg, Austria — 2Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany
Far-field optical 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 superresolution 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. For high resolution, strong spatial confinement of the on-state is required. However, this also subjects fluorophores at intensity maxima to excess light intensities and state cycling. In addition, as spatial confinement of the on-state is increased, state contrast between designated on- and off-regions has to be improved, too. We show that driving fluorophores to a second off-state enables protection of fluorophores and superior state contrast. In a realization that we dubbed "protected STED", we used reversibly switchable fluorescent proteins as labels and employed both STED and reversible photoswitching as off-transitions. This directly translated into reduced bleaching and enhanced resolution in live-cell nanoscopy (J. G. Danzl, S. C. Sidenstein et al., Nature Photonics 10, 122 (2016)).