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STED microscopy with continuous wave beams

Nature Methods volume 4, pages 915–918 (2007)Cite this article

Abstract

We report stimulated emission depletion (STED) fluorescence microscopy with continuous wave (CW) laser beams. Lateral fluorescence confinement from the scanning focal spot delivered a resolution of 29–60 nm in the focal plane, corresponding to a 5–8-fold improvement over the diffraction barrier. Axial spot confinement increased the axial resolution by 3.5-fold. We observed three-dimensional (3D) subdiffraction resolution in 3D image stacks. Viable for fluorophores with low triplet yield, the use of CW light sources greatly simplifies the implementation of this concept of far-field fluorescence nanoscopy.

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Figure 1: STED with a CW laser beam focused by a 1.4 NA lens.
Figure 2: Nanoscale imaging with CW STED.
Figure 3: Immunofluorescence CW STED microscopy shown in a side-by-side comparison with confocal microscopy.
Figure 4: 3D superresolution in axial optical sections (x-z images).
Figure 5: The 3D subdiffraction-resolution recording of the nuclear lamina of a fixed mammalian cell (immunofluorescence labeling) using the CW STED doughnut of Figure 4d.

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Acknowledgements

We thank B. Hein for sharing the setup, T. Lang (Department of Neurobiology) for providing the PC12 membrane sheets, A. Schönle, V. Westphal and J. Keller for help with the measurement and analysis software, and B. Rankin for critical reading of the manuscript.

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Author notes

  1. Katrin I Willig and Benjamin Harke: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077, Göttingen, Germany

    Katrin I Willig, Benjamin Harke, Rebecca Medda & Stefan W Hell

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  1. Katrin I Willig
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  2. Benjamin Harke
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  3. Rebecca Medda
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  4. Stefan W Hell
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Correspondence to Stefan W Hell.

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Willig, K., Harke, B., Medda, R. et al. STED microscopy with continuous wave beams. Nat Methods 4, 915–918 (2007). https://doi.org/10.1038/nmeth1108

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