Direct optical nanoscopy with axially localized detection

  • An Addendum to this article was published on 29 October 2015


Evanescent light excitation is widely used in super-resolution fluorescence microscopy to confine light and reduce background noise. Here, we propose a method of exploiting evanescent light in the context of emission. When a fluorophore is located in close proximity to a medium with a higher refractive index, its near-field component is converted into light that propagates beyond the critical angle. This so-called supercritical-angle fluorescence can be captured using a high-numerical-aperture objective and used to determine the axial position of the fluorophore with nanometre precision. We introduce a new technique for three-dimensional nanoscopy that combines direct stochastic optical reconstruction microscopy (dSTORM) with dedicated detection of supercritical-angle fluorescence emission. We demonstrate that our approach of direct optical nanoscopy with axially localized detection (DONALD) typically yields an isotropic three-dimensional localization precision of 20 nm within an axial range of 150 nm above the coverslip.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Far- and near-field emission components.
Figure 2: Direct optical nanoscopy with axially localized detection.
Figure 3: DONALD theory.
Figure 4: dSTORM imaging of F-actin in CHO cells immersed in a thiol + oxygen scavenger buffer using DONALD.
Figure 5: dSTORM imaging of microtubules immersed in a thiol + oxygen scavenger-based buffer using DONALD.
Figure 6: SMLM imaging of plasma membrane immersed in a thiol + oxygen scavenger-based buffer using DONALD.

Change history

  • 15 October 2015

    The authors wish to acknowledge a highly relevant manuscript that was published during the reviewing process of this Article, which should have been cited: Deschamps, J., Mund, M., & Ries, J. 3D superresolution microscopy by supercritical angle detection. Opt. Express 22, 29081–29091 (2014). The manuscript reports interesting use of 3D DNA-PAINT origami as a ruler for super-resolution imaging.


  1. 1

    Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    ADS  Article  Google Scholar 

  2. 2

    Hess, S. T., Girirajan, T. P. K. & Mason, M. D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006).

    Article  Google Scholar 

  3. 3

    Rust, M. J., Bates, M. & Zhuang, X. Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution. Nature Methods 3, 793–795 (2006).

    Article  Google Scholar 

  4. 4

    van de Linde, S. et al. Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nature Protoc. 6, 991–1009 (2011).

    Article  Google Scholar 

  5. 5

    Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

    ADS  Article  Google Scholar 

  6. 6

    Izeddin, I. et al. PSF shaping using adaptive optics for three-dimensional single-molecule super-resolution imaging and tracking. Opt. Express 20, 4957–4967 (2012).

    ADS  Article  Google Scholar 

  7. 7

    Xu, K., Babcock, H. P. & Zhuang, X. Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton. Nature Methods 9, 185–188 (2012).

    Article  Google Scholar 

  8. 8

    Pavani, S. R. P. et al. Imaging beyond the diffraction limit by using a double-helix point spread function. Proc. Natl Acad. Sci. USA 106, 2995–2999 (2009).

    ADS  Article  Google Scholar 

  9. 9

    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).

    ADS  Article  Google Scholar 

  10. 10

    Shtengel, G. et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl Acad. Sci. USA 106, 3125–3130 (2009).

    ADS  Article  Google Scholar 

  11. 11

    Jia, S., Vaughan, J. C. & Zhuang, X. Isotropic three-dimensional super-resolution imaging with a self-bending point spread function. Nature Photon. 8, 302–306 (2014).

    ADS  Article  Google Scholar 

  12. 12

    Klein, T., Proppert, S. & Sauer, M. Eight years of single-molecule localization microscopy. Histochem. Cell Biol. 141, 561–575 (2014).

    Article  Google Scholar 

  13. 13

    Ruckstuhl, T., Enderlein, J., Jung, S. & Seeger, S. Forbidden light detection from single molecules. Anal. Chem. 72, 2117–2123 (2000).

    Article  Google Scholar 

  14. 14

    Fort, E. & Grésillon, S. Surface enhanced fluorescence. J. Phys. D 41, 013001 (2008).

    ADS  Article  Google Scholar 

  15. 15

    Ruckstuhl, T., Rankl, M. & Seeger, S. Highly sensitive biosensing using a supercritical angle fluorescence (SAF) instrument. Biosens. Bioelectron. 18, 1193–1199 (2003).

    Article  Google Scholar 

  16. 16

    Winterflood, C., Ruckstuhl, T., Verdes, D. & Seeger, S. Nanometer axial resolution by three-dimensional supercritical angle fluorescence microscopy. Phys. Rev. Lett. 105, 108103 (2010).

    ADS  Article  Google Scholar 

  17. 17

    Barroca, T., Balaa, K., Delahaye, J., Lévêque-Fort, S. & Fort, E. Full-field supercritical angle fluorescence microscopy for live cell imaging. Opt. Lett. 36, 3051–3053 (2011).

    ADS  Article  Google Scholar 

  18. 18

    Thompson, R. E., Larson, D. R. & Webb, W. W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).

    Article  Google Scholar 

  19. 19

    Barroca, T., Balaa, K., Lévêque-Fort, S. & Fort, E. Full-field near-field optical microscope for cell imaging. Phys. Rev. Lett. 108, 218101 (2012).

    ADS  Article  Google Scholar 

  20. 20

    Barroca, T., Bon, P., Lévêque-Fort, S. & Fort, E. Supercritical self-interference fluorescence microscopy for full-field membrane imaging. Proc. SPIE 8589, 858911 (2013).

    Article  Google Scholar 

  21. 21

    Izeddin, I. et al. Wavelet analysis for single molecule localization microscopy. Opt. Express 20, 2081–2095 (2012).

    ADS  Article  Google Scholar 

  22. 22

    Tang, W. T., Chung, E., Kim, Y. H., So, P. T. C. & Sheppard, C. J. R. Investigation of the point spread function of surface plasmon-coupled emission microscopy. Opt. Express 15, 4634–4646 (2007).

    ADS  Article  Google Scholar 

  23. 23

    Mlodzinaski, M. J., Juette, M. F., Beane, G. L. & Bewersdorf, J. Experimental characterization of 3D localization techniques for particle-tracking and super-resolution microscopy. Opt. Express 17, 8264–8277 (2009).

    ADS  Article  Google Scholar 

  24. 24

    Kechkar, A., Nair, D., Heilemann, M., Choquet, D. & Sibarita, J. B. Real-time analysis and visualization for single-molecule based super-resolution microscopy. PLoS ONE 8, e62918 (2013).

    ADS  Article  Google Scholar 

  25. 25

    Nanguneri, S., Flottmann, B., Herrmannsdörfer, F., Kuner, T. & Heilemann, M. Single-molecule super-resolution imaging by tryptophan-quenching-induced photoswitching of phalloidin–fluorophore conjugates. Microsc. Res. Tech. 77, 510–516 (2014).

    Article  Google Scholar 

  26. 26

    Weber, K., Rathke, P. & Osborn, M. Cytoplasmic microtubular images in glutaraldehyde-fixed tissue culture cells by electron microscopy and by immunofluorescence microscopy. Proc. Natl Acad. Sci. USA 75, 1820–1824 (1978).

    ADS  Article  Google Scholar 

  27. 27

    Olivier, N., Keller, D., Rajan, V. D., Gönczy, P. & Manley, S. Simple buffers for 3D STORM microscopy. Biomed. Opt. Express 4, 885–899 (2013).

    Article  Google Scholar 

  28. 28

    Chizhik, A. I., Rother, J., Gregor, I., Janshoff, A. & Enderlein, J. Metal-induced energy transfer for live cell nanoscopy. Nature Photon. 8, 124–127 (2014).

    ADS  Article  Google Scholar 

  29. 29

    Schachter, H., Calaminus, S., Thomas, S. & Machesky, L. Podosomes in adhesion, migration, mechanosensing and matrix remodeling. Cytoskeleton 70, 572–589 (2013).

    Article  Google Scholar 

  30. 30

    Jungmann, R. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nature Methods 11, 313–318 (2014).

    Article  Google Scholar 

  31. 31

    Tam, J., Cordier, G. A., Borbely, J. S., Alvarez, A. S. & Lakadamyali, M. Cross-talk-free multi-color STORM imaging using a single fluorophore. PLoS ONE 9, e101772 (2014).

    ADS  Article  Google Scholar 

  32. 32

    Valley, C. C., Liu, S., Lidke, D. S. & Lidke, K. A. Sequential superresolution imaging of multiple targets using a single fluorophore. PLoS ONE 10, e0123941 (2015).

    Article  Google Scholar 

  33. 33

    Wang, S., Moffitt, J. R., Dempsey, G., Xie, X. & Zhuang, X. Characterization and development of photoactivatable fluorescent proteins for single-molecule-based superresolution imaging. Proc. Natl Acad. Sci. USA 111, 8452–8457 (2014).

    ADS  Article  Google Scholar 

  34. 34

    Lukinavičius, G. et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nature Chem. 5, 132–139 (2013).

    ADS  Article  Google Scholar 

  35. 35

    Klein, T. et al. Live-cell dSTORM with SNAP-tag fusion proteins. Nature Methods 8, 7–9 (2011).

    Article  Google Scholar 

  36. 36

    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).

    ADS  Article  Google Scholar 

Download references


The authors thank J. Dompierre for help with immunofluorescence and P. Adenot for providing CellMask Deep Red stain. The authors acknowledge financial support from the AXA Research Fund, Labex WIFI, the French National Research Agency (project SMARTVIEW) and DIM Nano-K (Project NanoSAF).

Author information




N.B., G.D., E.F. and S.L.F. conceived and designed the project. N.B. performed the experiments, simulations and analysis. C.M. and N.B. developed the photoswitching buffer. C.M., N.B. and S.L. optimized the immunofluorescence protocol. T.B. and P.B. helped with the simulation and the DONALD module. All authors contributed to writing the manuscript.

Corresponding author

Correspondence to S. Lévêque-Fort.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 1655 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bourg, N., Mayet, C., Dupuis, G. et al. Direct optical nanoscopy with axially localized detection. Nature Photon 9, 587–593 (2015).

Download citation

Further reading