Direct optical nanoscopy with axially localized detection

Journal name:
Nature Photonics
Year published:
Published online
Corrected online


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.

At a glance


  1. Far- and near-field emission components.
    Figure 1: Far- and near-field emission components.

    a, The far-field emission component (UAF) has an angular distribution determined by the law of refraction and limited by the critical angle θc. This angular distribution of light can be retrieved on the BFP within a plane disk of diameter Φc, which is related to θc. b, A position of the near-field component (SAF) of a dye molecule located in the near-field region (0 to λem) is collected by the objective beyond θc. The number of SAF photons, NSAF, is potentially equal to as much as 50% of NEPI when the dye is in close proximity to the coverslip and decreases exponentially as the dye depth d increases.

  2. Direct optical nanoscopy with axially localized detection.
    Figure 2: Direct optical nanoscopy with axially localized detection.

    a, Schematic of the experimental set-up. A homemade multicolour/laser TIRF stage is connected to the input of a conventional wide-field microscope. TIRF excitation light passes through a four-colour filter set (405, 488, 561, 647 nm) and an apochromatic TIRF objective with NA = 1.49. The fluorescence emission of dye molecules is collected by the objective and reflected to the DONALD module, which splits the fluorescence into two parts. The EPI part is directly imaged on one half of an EMCCD, and the UAF part (the SAF component is blocked in the BFP) is recorded on the other half. b, DONALD data analysis. First, in the UAF and EPI portions of the frame, each PSF is super-localized in two dimensions and the number of UAF and EPI photons (NUAF and NEPI, respectively) are calculated via signal integration in a 9 × 9 area of pixels. Finally, the SAF ratio is computed and converted into the absolute dye depth d.

  3. DONALD theory.
    Figure 3: DONALD theory.

    a, Theoretical decay of the SAF ratio (black line) as a function of dye depth d for nm = 1.33 and ng = 1.515. A total of 5,000 iterations of a Monte Carlo simulation of this theory were performed for two different SNRs: 5.30 (blue error line) and 7.43 (green error line). b, Monte Carlo simulations were used to calculate the axial localization precision for both SNRs. The localization precisions of the single cylindrical lens for two extreme focal plane positions are represented using a model extracted from ref. 23. Experimental verification of the theory was performed using 20 nm red beads embedded in 3% agarose gel (nm=1.33). The DONALD module was used to measure the SAF ratio and a PSF shaping method (cylindrical lens) was applied to determine the depth. The experimental results (a, red circles) are consistent with and confirm the DONALD theory.

  4. dSTORM imaging of F-actin in CHO cells immersed in a
                    thiol + oxygen scavenger buffer using
    Figure 4: dSTORM imaging of F-actin in CHO cells immersed in a thiol + oxygen scavenger buffer using DONALD.

    a, Three-dimensional DONALD image in which the depth is colour-coded as indicated by the coloured depth scale bar. b,c, Diffraction-limited (left) and three-dimensional (right) images of sub-areas of a, indicated by the blue and yellow boxes. d, Transverse profiles of ‘Profile 1’ and ‘Profile 2’ in a and b. e, Axial profiles of various structures (zones 1–4) in a and b. Axial resolutions of 34–36, 54 and 62 nm were achieved for filaments located at depths of 104–105, 148 and 180 nm, respectively. Scale bars, 3 µm (a), 2 µm (b), 1 µm (c).

  5. dSTORM imaging of microtubules immersed in a
                    thiol + oxygen scavenger-based buffer using
    Figure 5: dSTORM imaging of microtubules immersed in a thiol + oxygen scavenger-based buffer using DONALD.

    a, Diffraction-limited image of the microtubule network of immunofluorescently labelled COS-7 cells. b, Three-dimensional super-resolved image of the same region shown in a, colour-coded as indicated by the coloured depth scale bar. c, Close-up of a microtubule indicated by the small yellow-boxed region in b. d, xz plane of tubuline Tub1 produced using ViSP software. e, Axial profiles of three different microtubules shown in b and c. Axial resolutions of 53.5, 56.2 and 80.4 nm were obtained for filaments located at depths of depths of 36.6, 63.1 and 118.1 nm, respectively. f, Close-up of a microtubule located at a depth of 35 nm and indicated by the white arrow in b. Transversal and axial profiles of this microtubule are fitted with a double Gaussian. Transversal and axial hollowness values of 33 ± 2 and 22 ± 5 nm are measured, respectively. Scale bars, 1.5 µm (a), 3 µm (b), 240 nm (c), 190 nm (f).

  6. SMLM imaging of plasma membrane immersed in a
                    thiol + oxygen scavenger-based buffer using
    Figure 6: SMLM imaging of plasma membrane immersed in a thiol + oxygen scavenger-based buffer using DONALD.

    a, Three-dimensional super-resolved image of the plasma membrane in COS-7 cells, colour-coded as indicated by the coloured depth scale bar. b, Three-dimensional visualization of the membrane nanotopography using ImageJ3D, with the same colour coding as in a. c, Axial localization precision measured from the dye molecules immobilized at the surface of the coverslip (zone σd in a: axial localization d=0.5 nm with precision σd=15.9 nm). d, Transverse profiles of ‘Profile 1’ and ‘Profile 2’ in a. Scale bar, 1.5 µm (a).

Change history

Corrected online 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.


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Author information


  1. Institut des Sciences Moléculaires d'Orsay (ISMO), Université Paris-Sud, CNRS UMR 8214, Orsay Cedex F91405, France

    • N. Bourg &
    • S. Lévêque-Fort
  2. Université Paris-Sud, Centre de Photonique BioMédicale (CPBM), Fédération LUMAT, CNRS FR 2764, Orsay Cedex F91405, France

    • N. Bourg,
    • C. Mayet,
    • G. Dupuis,
    • S. Lécart &
    • S. Lévêque-Fort
  3. Institut Langevin, ESPCI ParisTech, CNRS, PSL Research University, 1 rue Jussieu, F-75005 Paris, France

    • T. Barroca,
    • P. Bon &
    • E. Fort


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.

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The authors declare no competing financial interests.

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