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Fluorescence nanoscopy by polarization modulation and polarization angle narrowing

Nature Methods volume 11, pages 579–584 (2014)Cite this article

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A Corrigendum to this article was published on 30 December 2015

This article has been updated

Abstract

When excited with rotating linear polarized light, differently oriented fluorescent dyes emit periodic signals peaking at different times. We show that measurement of the average orientation of fluorescent dyes attached to rigid sample structures mapped to regularly defined (50 nm)2 image nanoareas can, in combination with application of the SPEED (sparsity penalty-enhanced estimation by demodulation) deconvolution algorithm, provide subdiffraction resolution (super resolution by polarization demodulation, SPoD). Because the polarization angle range for effective excitation of an oriented molecule is rather broad and unspecific, we narrowed this range by simultaneous irradiation with a second, de-excitation, beam possessing a polarization perpendicular to the excitation beam (excitation polarization angle narrowing, ExPAN). This shortened the periodic emission flashes, allowing better discrimination between molecules or nanoareas. Our method requires neither the generation of nanometric interference structures nor the use of switchable or blinking fluorescent probes. We applied the method to standard wide-field microscopy with camera detection and to two-photon scanning microscopy, imaging the fine structural details of neuronal spines.

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Figure 1: Setup and principle of SPoD and ExPAN.
Figure 2: Experimental SPoD and ExPAN data from single molecules and surface-labeled nanospheres.
Figure 3: Images from immunolabeled microtubules.
Figure 4: Images from fEGFP membrane-labeled hippocampal neurons in organotypic slice culture.

Change history

  • 09 December 2015

    In the version of this article initially published, it was not clearly indicated that all the results obtained required the use of the SPEED (sparsity penalty–enhanced estimation by demodulation) deconvolution algorithm to reconstruct dye orientation data. This has been amended as follows in all versions of the article. The sentence in the abstract that was previously "We show that measurement of the average orientation of fluorescent dyes attached to rigid sample structures mapped to regularly defined (50 nm)2 image nanoareas can provide subdiffraction resolution (super resolution by polarization demodulation, SPoD)" has been amended to "We show that measurement of the average orientation of fluorescent dyes attached to rigid sample structures mapped to regularly defined (50 nm)2 image nanoareas can, in combination with application of the SPEED (sparsity penalty–enhanced estimation by demodulation) deconvolution algorithm, provide subdiffraction resolution (super resolution by polarization demodulation, SPoD)." The sentence in the introduction that was formerly "This is done by rotating the polarization of a wide-field excitation beam and detecting the periodic signals emitted with different phases from different nanoareas using wide-field camera detection (SPoD)" has been amended to "This is done by rotating the polarization of a wide-field excitation beam and detecting the periodic signals emitted with different phases from different nanoareas using wide-field camera detection (SPoD), followed by reconstruction with a deconvolution algorithm, SPEED." Finally, the following sentence has been added to the introduction: "All images generated with SPoD and ExPAN in this paper include reconstruction with the SPEED algorithm".

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Acknowledgements

This work was financially supported by the Deutsche Forschungsgemeinschaft (DFG) (INST 188/334-1 FUGG). O.M.S. thanks the International Max Planck Research School (IMPRS) “Physics of Biological and Complex Systems” for financial support. T.A., A.M., C.S. and P.J.W. acknowledge support by DFG Collaborative Research Center (CRC) 755 and 803. M.Z. and M.K. acknowledge support by DFG grant KO 1674/5-1. A.M. acknowledges support by DFG and Schweizerischer Nationalfonds (SNF) grant FOR 916.

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Author notes

  1. Nour Hafi, Matthias Grunwald, Laura S van den Heuvel and Timo Aspelmeier: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biophysical Chemistry, Institute for Physical and Theoretical Chemistry, University of Braunschweig, Braunschweig, Germany

    Nour Hafi, Laura S van den Heuvel & Peter J Walla

  2. Biomolecular Spectroscopy and Single-Molecule Detection Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Matthias Grunwald, Jian-Hua Chen & Peter J Walla

  3. Felix Bernstein Institute for Mathematical Statistics in Bioscience, Göttingen, Germany

    Timo Aspelmeier & Axel Munk

  4. Institute for Mathematical Stochastics, University of Göttingen, Göttingen, Germany

    Timo Aspelmeier & Axel Munk

  5. Statistical Inverse Problems in Biophysics Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Timo Aspelmeier & Axel Munk

  6. Division of Cellular Neurobiology, Zoological Institute, University of Braunschweig, Braunschweig, Germany

    Marta Zagrebelsky & Martin Korte

  7. Institute of Organic and Biomolecular Chemistry, University of Göttingen, Göttingen, Germany

    Ole M Schütte & Claudia Steinem

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Contributions

N.H., M.G. and L.S.v.d.H. designed and performed experiments, analyzed data and wrote the paper; T.A. developed the statistical model and analytical tools, wrote the software package SPEED, analyzed data and wrote the paper; J.-H.C. performed experiments; M.Z., O.M.S., C.S. and M.K. provided samples and edited the manuscript.; A.M. developed the statistical model and analytical tools and edited the paper; and P.J.W. designed experiments, analyzed data and wrote the paper.

Corresponding author

Correspondence to Peter J Walla.

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Competing interests

The University of Braunschweig, with which N.H., L.S.v.d.H., M.Z., M.K. and P.J.W. are affiliated, has filed a patent for parts of this work.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Notes 1 and 2 (PDF 944 kb)

Supplementary Software

Algorithm to analyse SPoD data with exemplary raw data and necessary parameters. (ZIP 7131 kb)

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Hafi, N., Grunwald, M., van den Heuvel, L. et al. Fluorescence nanoscopy by polarization modulation and polarization angle narrowing. Nat Methods 11, 579–584 (2014). https://doi.org/10.1038/nmeth.2919

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