Danzl, Johann GIST Austria ; Sidenstein, Sven; Gregor, Carola; Urban, Nicolai; Ilgen, Peter; Jakobs, Stefan; Hell, Stefan
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.
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 Körber Foundation.
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Danzl JG, Sidenstein S, Gregor C, et al. Coordinate-targeted fluorescence nanoscopy with multiple off states. Nature Photonics. 2016;10(2):122-128. doi:10.1038/nphoton.2015.266
Danzl, J. G., Sidenstein, S., Gregor, C., Urban, N., Ilgen, P., Jakobs, S., & Hell, S. (2016). Coordinate-targeted fluorescence nanoscopy with multiple off states. Nature Photonics. Nature Publishing Group. https://doi.org/10.1038/nphoton.2015.266
Danzl, Johann G, Sven Sidenstein, Carola Gregor, Nicolai Urban, Peter Ilgen, Stefan Jakobs, and Stefan Hell. “Coordinate-Targeted Fluorescence Nanoscopy with Multiple off States.” Nature Photonics. Nature Publishing Group, 2016. https://doi.org/10.1038/nphoton.2015.266.
J. G. Danzl et al., “Coordinate-targeted fluorescence nanoscopy with multiple off states,” Nature Photonics, vol. 10, no. 2. Nature Publishing Group, pp. 122–128, 2016.
Danzl JG, Sidenstein S, Gregor C, Urban N, Ilgen P, Jakobs S, Hell S. 2016. Coordinate-targeted fluorescence nanoscopy with multiple off states. Nature Photonics. 10(2), 122–128.
Danzl, Johann G., et al. “Coordinate-Targeted Fluorescence Nanoscopy with Multiple off States.” Nature Photonics, vol. 10, no. 2, Nature Publishing Group, 2016, pp. 122–28, doi:10.1038/nphoton.2015.266.