Coordinate-targeted fluorescence nanoscopy with multiple off states Journal Article

Author(s): Danzl, Johannes G; Sidenstein, Sven C; Gregor, Carola; Urban, Nicolai T; Ilgen, Peter; Jakobs, Stefan; Hell, Stefan W
Article Title: Coordinate-targeted fluorescence nanoscopy with multiple off states
Abstract: 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.
Keywords: Fluorescence; Fluorescence microscopy; Optical resolving power; Stimulated emission; Diffraction barrier; Fluorescent protein; Optical patterns; Spatial confinement; STED microscopies; Stimulated emission depletion microscopy; Super resolution; Super-resolution microscopy; Fluorophores
Journal Title: Nature Photonics
Volume: 10
Issue 2
ISSN: 1749-4893
Publisher: Nature Publishing Group  
Date Published: 2016-02-01
Start Page: 122
End Page: 128
DOI: 10.1038/nphoton.2015.266
Notes: We thank T. Gilat and E. Rothermel (both MPI) for help with preparing samples, and J. Keller for discussion. J.G.D. acknowledges support by the European Union through a Marie Curie fellowship PIEF-GA-2011-299283. S.W.H. acknowledges support by the K├Ârber Foundation.
Open access: no