Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Removing orientation-induced localization biases in single-molecule microscopy using a broadband metasurface mask

Nature Photonics volume 10, pages 459–462 (2016)Cite this article

Subjects

Abstract

Nanoscale localization of single molecules is a crucial function in several advanced microscopy techniques, including single-molecule tracking and wide-field super-resolution imaging1. Until now, a central consideration of such techniques is how to optimize the precision of molecular localization. However, as these methods continue to push towards the nanometre size scale, an increasingly important concern is the localization accuracy. In particular, single fluorescent molecules emit with an anisotropic radiation pattern of an oscillating electric dipole, which can cause significant localization biases using common estimators2,3,4,5. Here we present the theory and experimental demonstration of a solution to this problem based on azimuthal filtering in the Fourier plane of the microscope. We do so using a high-efficiency dielectric metasurface polarization/phase device composed of nanoposts with subwavelength spacing6. The method is demonstrated both on fluorophores embedded in a polymer matrix and in dL5 protein complexes that bind malachite green7,8.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Concept.
Figure 2: Details of the mask.
Figure 3: Experimental set-up.
Figure 4: Experimental results.
Figure 5: Biological demonstration.

Similar content being viewed by others

References

  1. Deschout, H. et al. Precisely and accurately localizing single emitters in fluorescence microscopy. Nature Methods 11, 253–266 (2014).

    Article  Google Scholar 

  2. Enderlein, J., Toprak, E. & Selvin, P. R. Polarization effect on position accuracy of fluorophore localization. Opt. Express 14, 8111–8120 (2006).

    Article  ADS  Google Scholar 

  3. Engelhardt, J. et al. Molecular orientation affects localization accuracy in superresolution far-field fluorescence microscopy. Nano Lett. 11, 209–213 (2011).

    Article  ADS  Google Scholar 

  4. Backlund, M. P., Lew, M. D., Backer, A. S., Sahl, S. J. & Moerner, W. E. The role of molecular dipole orientation in single-molecule fluorescence microscopy and implications for super-resolution imaging. ChemPhysChem 15, 587–599 (2014).

    Article  Google Scholar 

  5. Lew, M. D., Backlund, M. P. & Moerner, W. E. Rotational mobility of single molecules affects localization accuracy in super-resolution fluorescence microscopy. Nano Lett. 13, 3967–3972 (2013).

    Article  ADS  Google Scholar 

  6. Arbabi, A., Horie, Y., Bagheri, M. & Faraon, A. Dielectric metasurfaces for complete control of phase and polarization with subwavelength spatial resolution and high transmission. Nature Nanotech. 10, 937–943 (2015).

    Article  ADS  Google Scholar 

  7. Szent-Gyorgyi, C. et al. Malachite green mediates homodimerization of antibody V L domains to form a fluorescent ternary complex with singular symmetric interfaces. J. Mol. Biol. 425, 4595–4613 (2013).

    Article  Google Scholar 

  8. Saurabh, S., Zhang, M., Mann, V. R., Costello, A. M. & Bruchez, M. P. Kinetically tunable photostability of fluorogen activating peptide–fluorogen complexes. ChemPhysChem 16, 2974–2980 (2015).

    Article  Google Scholar 

  9. Jackson, J. D. Classical Electrodynamics (Wiley, 1962).

    MATH  Google Scholar 

  10. Dickson, R. M., Norris, D. J. & Moerner, W. E. Simultaneous imaging of individual molecules aligned both parallel and perpendicular to the optic axis. Phys. Rev. Lett. 81, 5322–5325 (1998).

    Article  ADS  Google Scholar 

  11. Backlund, M. P. et al. Simultaneous, accurate measurement of the 3D position and orientation of single molecules. Proc. Natl Acad. Sci. USA 109, 19087–19092 (2012).

    Article  ADS  Google Scholar 

  12. Stallinga, S. Effect of rotational diffusion in an orientational potential well on the point spread function of electric dipole emitters. J. Opt. Soc. Am. A 32, 213–223 (2015).

    Article  ADS  Google Scholar 

  13. Rosenberg, S. A., Quinlan, M. E., Forkey, J. N. & Goldman, Y. E. Rotational motions of macro-molecules by single-molecule fluorescence microscopy. Acc. Chem. Res. 38, 583–593 (2005).

    Article  Google Scholar 

  14. Sosa, H., Peterman, E. J. G., Moerner, W. E. & Goldstein, L. S. B. ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy. Nature Struct. Biol. 8, 540–544 (2001).

    Article  Google Scholar 

  15. Mortensen, K. I., Churchman, L. S., Spudich, J. A. & Flyvbjerg, H. Optimized localization analysis for single-molecule tracking and super-resolution microscopy. Nature Methods 7, 377–381 (2010).

    Article  Google Scholar 

  16. Mortensen, K. I., Sung, J., Flyvbjerg, H. & Spudich, J. A. Optimized measurements of separations and angles between intra-molecular fluorescent markers. Nature Commun. 6, 8621 (2015).

    Article  ADS  Google Scholar 

  17. Backer, A. S., Backlund, M. P., Diezmann, A. R., Sahl, S. J. & Moerner, W. E. A bisected pupil for studying single-molecule orientational dynamics and its application to 3D super-resolution microscopy. Appl. Phys. Lett. 104, 193701–193705 (2014).

    Article  ADS  Google Scholar 

  18. Lew, M. D. & Moerner, W. E. Azimuthal polarization filtering for accurate, precise, and robust single-molecule localization microscopy. Nano Lett. 14, 6407–6413 (2014).

    Article  ADS  Google Scholar 

  19. Zhan, Q. Cylindrical vector beams: from mathematical concepts to applications. Adv. Opt. Photon. 1, 1–57 (2009).

    Article  Google Scholar 

  20. Hashimoto, M., Yoshiki, K., Kurihara, M., Hashimoto, N. & Araki, T. Orientation detection of a single molecule using pupil filter with electrically controllable polarization pattern. Opt. Rev. 22, 1–7 (2015).

    Article  Google Scholar 

  21. Cover, T. M. & Thomas, J. A. Elements of Information Theory (Wiley, 2006).

    MATH  Google Scholar 

  22. Badieirostami, M., Lew, M. D., Thompson, M. A. & Moerner, W. E. Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane. Appl. Phys. Lett. 97, 161103 (2010).

    Article  ADS  Google Scholar 

  23. Lord, S. J. et al. Photophysical properties of acene DCDHF fluorophores: long-wavelength single-molecule emitters designed for cellular imaging. J. Phys. Chem. A 111, 8934–8941 (2007).

    Article  Google Scholar 

  24. Backer, A. S. & Moerner, W. E. Extending single-molecule microscopy using optical Fourier processing. J. Phys. Chem. B 118, 8313–8329 (2014).

    Article  Google Scholar 

  25. Novotny, L. & Hecht, B. Principles of Nano-Optics (Cambridge Univ. Press, 2007).

    Google Scholar 

  26. Liu, V. & Fan, S. S 4 A free electromagnetic solver for layered periodic structures. Comput. Phys. Commun. 183, 2233–2244 (2012).

    Article  ADS  MathSciNet  Google Scholar 

  27. Kurvits, J. A., Jiang, M. & Zia, R. Comparative analysis of imaging configurations and objectives for Fourier microscopy. J. Opt. Soc. Am. A 32, 2082–2092 (2015).

    Article  ADS  Google Scholar 

Download references

Acknowledgements

M.P.B. acknowledges support from the Robert and Marvel Kirby Stanford Graduate Fellowship. We also acknowledge support from National Institute of General Medical Sciences Grant No. 2R01GM085437 (to W.E.M.) and a National Science Foundation award (1512266) and the Defense Advanced Research Projects Agency (to A.F.). A.A. and E.A. were also supported by Samsung Electronics. We thank M. Bruchez for providing the plasmid for expressing recombinant dL5 protein and the MG ester fluorogen.

Author information

Authors and Affiliations

  1. Department of Chemistry, Stanford University, 375 North-South Mall, Stanford, 94305, California, USA

    Mikael P. Backlund, Petar N. Petrov, Saumya Saurabh & W. E. Moerner

  2. T. J. Watson Laboratory of Applied Physics, California Institute of Technology, 1200 E California Blvd, Pasadena, 91125, California, USA

    Amir Arbabi, Ehsan Arbabi & Andrei Faraon

Authors
  1. Mikael P. Backlund
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Amir Arbabi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Petar N. Petrov
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ehsan Arbabi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Saumya Saurabh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Andrei Faraon
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. W. E. Moerner
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.P.B. and W.E.M. conceived and designed the experiments. M.P.B. did the simulations and CRLB calculations. A.A., E.A. and A.F. designed, fabricated and characterized the mask. M.P.B. and P.N.P. performed experiments and analysed the data. S.S. prepared the dL5 samples. All the authors contributed to writing the paper.

Corresponding authors

Correspondence to Andrei Faraon or W. E. Moerner.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 2216 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Backlund, M., Arbabi, A., Petrov, P. et al. Removing orientation-induced localization biases in single-molecule microscopy using a broadband metasurface mask. Nature Photon 10, 459–462 (2016). https://doi.org/10.1038/nphoton.2016.93

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nphoton.2016.93

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing