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Background suppression in fluorescence nanoscopy with stimulated emission double depletion


Stimulated emission depletion (STED) fluorescence nanoscopy is a powerful super-resolution imaging technique based on the confinement of fluorescence emission to the central subregion of an observation volume through de-excitation of fluorophores in the periphery via stimulated emission. Here, we introduce stimulated emission double depletion (STEDD) as a method to selectively remove artificial background intensity. In this approach, a first, conventional STED pulse is followed by a second, delayed Gaussian STED pulse that specifically depletes the central region, thus leaving only background. Thanks to time-resolved detection we can remove this background intensity voxel by voxel by taking the weighted difference of photons collected before and after the second STED pulse. STEDD thus yields background-suppressed super-resolved images as well as STED-based fluorescence correlation spectroscopy data. Furthermore, the proposed method is also beneficial when considering lower-power, less redshifted depletion pulses.

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Figure 1: Scheme of STEDD microscopy.
Figure 2: STEDD imaging of fluorescent beads.
Figure 3: 3D STEDD imaging of a fixed HeLa cell.
Figure 4: STEDD imaging of microtubules in live COS-7 cells.
Figure 5: STED–FCS and STEDD–FCS autocorrelation functions of dye diffusion.
Figure 6: 3D STEDD–FCS on annexin A5-mGarnet diffusing within a living HeLa cell.


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The authors thank J. Kuhlmann for assistance with generating mGarnet fusion constructs. The authors acknowledge funding by the Karlsruhe Institute of Technology in the context of the Helmholtz programme Science and Technology of Nanosystems (STN). This work was also supported by the Deutsche Forschungsgemeinschaft (DFG) through grants GRK 2039 and Ni 291/12-1.

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G.U.N. supervised the project in both its conception and execution. P.G. and B.P. implemented the STEDD technique on our STED microscope, performed measurements and analysed data. L.Z. provided cell samples. G.U.N. and K.N. wrote the manuscript with input from all other authors.

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Correspondence to G. Ulrich Nienhaus.

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The authors declare no competing financial interests.

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Gao, P., Prunsche, B., Zhou, L. et al. Background suppression in fluorescence nanoscopy with stimulated emission double depletion. Nature Photon 11, 163–169 (2017).

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