Article | Published:

Coordinate-targeted fluorescence nanoscopy with multiple off states

Nature Photonics volume 10, pages 122128 (2016) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & Breaking the diffraction barrier: super-resolution imaging of cells. Cell 143, 1047–1058 (2010).

  2. 2.

    Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

  3. 3.

    & Breaking the diffraction resolution limit by stimulated-emission—stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).

  4. 4.

    , , , & Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Sci. USA 97, 8206–8210 (2000).

  5. 5.

    , & Concepts for nanoscale resolution in fluorescence microscopy. Curr. Opin. Neurobiol. 14, 599–609 (2004).

  6. 6.

    et al. Diffraction-unlimited all-optical imaging and writing with a photochromic GFP. Nature 478, 204–208 (2011).

  7. 7.

    et al. Resolution scaling in STED microscopy. Opt. Express 16, 4154–4162 (2008).

  8. 8.

    et al. Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores. J. Am. Chem. Soc. 32, 5021–5023 (2010).

  9. 9.

    Improvement of lateral resolution in far-field light microscopy using two-photon excitation with offset beams. Opt. Commun. 106, 19–24 (1994).

  10. 10.

    , & Nonlinear fluorescence through intermolecular energy transfer and resolution increase in fluorescence microscopy. Ann. Phys. 8, 115–133 (1999).

  11. 11.

    et al. Photoswitchable cyan fluorescent protein for protein tracking. Nature Biotechnol. 22, 1435–1439 (2004).

  12. 12.

    , & Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306, 1370–1373 (2004).

  13. 13.

    , , & On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388, 355–358 (1997).

  14. 14.

    The green fluorescent protein. Ann. Rev. Biochem. 67, 509–544 (1998).

  15. 15.

    et al. rsEGFP2 enables fast RESOLFT nanoscopy of living cells. eLife 1, e00248 (00241-00214) (2012).

  16. 16.

    et al. Nanoscopy with more than 100,000 ‘doughnuts’. Nature Methods 10, 737–740 (2013).

  17. 17.

    et al. Nanoscale resolution in GFP-based microscopy. Nature Methods 3, 721–723 (2006).

  18. 18.

    et al. Generation of monomeric reversibly switchable red fluorescent proteins for far-field fluorescence nanoscopy. Biophys. J. 95, 2989–2997 (2008).

  19. 19.

    et al. Macromolecular-scale resolution in biological fluorescence microscopy. Proc. Natl Acad. Sci. USA 103, 11440–11445 (2006).

  20. 20.

    et al. Far-field optical nanoscopy with reduced number of state transition cycles. Opt. Express 19, 5644–5657 (2011).

  21. 21.

    et al. Sharper low-power STED nanoscopy by time gating. Nature Methods 8, 571–573 (2011).

  22. 22.

    , & Efficient fluorescence inhibition patterns for RESOLFT microscopy. Opt. Express 15, 3361–3371 (2007).

  23. 23.

    , , & STED nanoscopy of actin dynamics in synapses deep inside living brain slices. Biophys. J. 101, 1277–1284 (2011).

  24. 24.

    , , & Spine neck plasticity regulates compartmentalization of synapses. Nat. Neurosci. 17, 678–685 (2014).

  25. 25.

    et al. Nanoscopy of living brain slices with low light levels. Neuron 75, 992–1000 (2012).

  26. 26.

    , & 2-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

  27. 27.

    Photochromism: memories and switches-introduction. Chem. Rev. 100, 1685–1716 (2000).

  28. 28.

    , , , & Large parallelization of STED nanoscopy using optical lattices. Opt. Express 22, 5581–5589 (2014).

Download references

Acknowledgements

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.

Author information

Author notes

    • Johann G. Danzl
    •  & Sven C. Sidenstein

    These authors contributed equally to this work

Affiliations

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

    • Johann G. Danzl
    • , Sven C. Sidenstein
    • , Carola Gregor
    • , Nicolai T. Urban
    • , Peter Ilgen
    • , Stefan Jakobs
    •  & Stefan W. Hell

Authors

  1. Search for Johann G. Danzl in:

  2. Search for Sven C. Sidenstein in:

  3. Search for Carola Gregor in:

  4. Search for Nicolai T. Urban in:

  5. Search for Peter Ilgen in:

  6. Search for Stefan Jakobs in:

  7. Search for Stefan W. Hell in:

Contributions

J.G.D. and S.C.S. built the setup, planned the experiments, and evaluated the data. S.C.S. performed the measurements shown. C.G., N.T.U., and P.I. provided samples. S.J. advised on actin and protein labelling. S.W.H. laid out the concept, and initiated and supervised the project. The paper was written by J.G.D. and S.W.H. All authors commented on the data and on the final version of the manuscript.

Competing interests

S.W.H. owns shares in the company Abberior Instruments that supplies STED and RESOLFT systems and benefits through related patents owned by the Max Planck Society.

Corresponding authors

Correspondence to Johann G. Danzl or Stefan W. Hell.

Supplementary information

PDF files

  1. 1.

    Supplementary information

    Supplementary information

Videos

  1. 1.

    Supplementary information

    Supplementary Movie 1

  2. 2.

    Supplementary information

    Supplementary Movie 2

  3. 3.

    Supplementary information

    Supplementary Movie 3

  4. 4.

    Supplementary information

    Supplementary Movie 4

  5. 5.

    Supplementary information

    Supplementary Movie 5

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nphoton.2015.266

Further reading