Coordinate-targeted fluorescence nanoscopy with multiple off states

• Published in: Nature photonics Vol. 10; no. 2; pp. 122 - 128
• Main Authors: Danzl, Johann G., Sidenstein, Sven C., Gregor, Carola, Urban, Nicolai T., Ilgen, Peter, Jakobs, Stefan, Hell, Stefan W.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.02.2016; Nature Publishing Group
• Subjects: 631/1647/328/2238; 639/925/930/328/2238; Applied and Technical Physics; Bleaching; Chemical compounds; Fluorescence; Fluorescence microscopy; Maxima; Microscopy; Minima; Nanostructure; Photonics; Physics; Quantum Physics; Stimulated emission
• ISSN: 1749-4885; 1749-4893
• DOI: 10.1038/nphoton.2015.266

Far-field super-resolution fluorescence microscopy discerns fluorophores residing closer than the diffraction barrier by briefly transferring them in different (typically ON and OFF) states before detection. In coordinate-targeted super-resolution variants, such as stimulated emission depletion (STED) microscopy, this state difference is created by the intensity minima and maxima of an optical pattern, causing all fluorophores to assume the off state, for instance, except at the minima. Although strong spatial confinement of the on state enables high resolution, it also subjects the fluorophores to excess intensities and state cycles at the maxima. Here, we address these issues by driving the fluorophores into a second off state that is inert to the excess light. By using reversibly switchable fluorescent proteins as labels, our approach reduces bleaching and enhances resolution and contrast in live-cell STED microscopy. Using two or more transitions to off states is a useful strategy for augmenting the power of coordinate-targeted super-resolution microscopy. By exploiting a second off state of a reversibly switchable fluorophore, a general approach that can reduce photobleaching and enhance resolution of coordinate-targeted fluorescence nanoscopy has been demonstrated.