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Nanometric axial localization of single fluorescent molecules with modulated excitation

A Publisher Correction to this article was published on 23 February 2021

An Author Correction to this article was published on 01 February 2021

This article has been updated


Distance measurements are commonly performed by phase detection based on a lock-in strategy. Super-resolution fluorescence microscopy is still striving to perform axial localization but through entirely different strategies. Here we show that an illumination modulation approach can achieve nanometric axial localization precision without compromising the acquisition time, emitter density or lateral localization precision. The excitation pattern is obtained by shifting tilted interference fringes. The molecular localizations are performed by measuring the relative phase between each fluorophore response and the reference modulated excitation pattern. We designed a fast demodulation scheme compatible with the short emission duration of single emitters. This modulated localization microscopy offers a typical axial localization precision of 6.8 nm over the entire field of view and the axial capture range. Furthermore, the interfering pattern being robust to optical aberrations, a nearly uniform axial localization precision enables imaging of biological samples by up to several micrometres in depth.

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Fig. 1: ModLoc principle and experimental implementation.
Fig. 2: The performances of ModLoc.
Fig. 3: Three-dimensional imaging of the microtubules network in the first 600 nm in COS-7 cell in dSTORM.
Fig. 4: Three-dimensional ModLoc imaging in COS-7 cells labelled with AF647 in dSTORM.
Fig. 5: Three-dimensional imaging of COS-7 cell microtubule grown in collagen matrix in dSTORM.

Data availability

The data that supports the images and plots, within this paper and other findings are available from the corresponding author upon reasonable request.

Code avaibility

Processing code are based on already published solutions as described in the Supplementary Information.

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

    Article  ADS  Google Scholar 

  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  ADS  Google Scholar 

  3. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–796 (2006).

    Article  Google Scholar 

  4. Heilemann, M. et al. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew. Chem. Int. Ed. 47, 6172–6176 (2008).

    Article  Google Scholar 

  5. von Diezmann, A., Shechtman, Y. & Moerner, W. E. Three-dimensional localization of single molecules for super-resolution imaging and single-particle tracking. Chem. Rev. 117, 7244–7275 (2017).

    Article  Google Scholar 

  6. Ram, S., Prabhat, P., Chao, J., Ward, E. S. & Ober, R. J. High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cells. Biophys. J. 95, 6025–6043 (2008).

    Article  ADS  Google Scholar 

  7. Juette, M. F. et al. Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat. Methods 5, 527–529 (2008).

    Article  Google Scholar 

  8. Hajj, B., El Beheiry, M., Izeddin, I., Darzacq, X. & Dahan, M. Accessing the third dimension in localization-based super-resolution microscopy. Phys. Chem. Chem. Phys. 16, 16340–16348 (2014).

    Article  Google Scholar 

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

    Article  ADS  Google Scholar 

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

    Article  ADS  Google Scholar 

  11. Shechtman, Y., Weiss, L. E., Backer, A. S., Sahl, S. J. & Moerner, W. E. Precise three-dimensional scan-free multiple-particle tracking over large axial ranges with tetrapod point spread functions. Nano Lett. 15, 4194–4199 (2015).

    Article  ADS  Google Scholar 

  12. 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 

  13. Bourg, N. et al. Direct optical nanoscopy with axially localized detection. Nat. Photon. 9, 587–593 (2015).

    Article  ADS  Google Scholar 

  14. Deschamps, J., Mund, M. & Ries, J. 3D superresolution microscopy by supercritical angle detection. Opt. Express 22, 29081 (2014).

    Article  ADS  Google Scholar 

  15. Cabriel, C. et al. Combining 3D single molecule localization strategies for reproducible bioimaging. Nat. Commun. 10, 1–10 (2019).

    Article  Google Scholar 

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

    Article  ADS  Google Scholar 

  17. Wang, G., Hauver, J., Thomas, Z., Darst, S. A. & Pertsinidis, A. Single-molecule real-time 3D imaging of the transcription cycle by modulation interferometry. Cell 167, 1839–1852 (2016).

    Article  Google Scholar 

  18. Aquino, D. et al. Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores. Nat. Methods 8, 353–359 (2011).

    Article  Google Scholar 

  19. Huang, F. et al. Ultra-high resolution 3D imaging of whole cells. Cell 166, 1028–1040 (2016).

    Article  Google Scholar 

  20. Bon, P. et al. Self-interference 3D super-resolution microscopy for deep tissue investigations. Nat. Methods 15, 449–454 (2018).

    Article  Google Scholar 

  21. Burke, D., Patton, B., Huang, F., Bewersdorf, J. & Booth, M. J. Adaptive optics correction of specimen-induced aberrations in single-molecule switching microscopy. Optica 2, 177–185 (2015).

    Article  Google Scholar 

  22. Abbott, B. P. et al. (LIGO Scientific Collaboration and Virgo Collaboration).Observation of gravitational waves from a binary black hole merger. Phys. Rev. Lett. 116, 061102 (2016).

    Article  ADS  MathSciNet  Google Scholar 

  23. Zernike, F. How I discovered phase contrast. Science 121, 345–349 (1955).

    Article  ADS  Google Scholar 

  24. Beaurepaire, E., Boccara, A. C., Lebec, M., Blanchot, L. & Saint-Jalmes, H. Full-field optical coherence microscopy. Opt. Lett. 23, 244–246 (1998).

    Article  ADS  Google Scholar 

  25. Taylor, R. W. et al. Interferometric scattering microscopy reveals microsecond nanoscopic protein motion on a live cell membrane. Nat. Photon. 13, 480–487 (2019).

    Article  ADS  Google Scholar 

  26. TOF Range-Imaging Cameras (Springer, 2013).

  27. Cappello, G. et al. Myosin V stepping mechanism. Proc Natl Acad. Sci. USA 104, 15328–15333 (2007).

    Article  ADS  Google Scholar 

  28. Busoni, L., Dornier, A., Viovy, J.-L., Prost, J. & Cappello, G. Fast subnanometer particle localization by traveling-wave tracking. J. Appl. Phys. 98, 064302 (2005).

    Article  ADS  Google Scholar 

  29. Reymond, L. et al. SIMPLE: structured illumination based point localization estimator with enhanced precision. Opt. Express 27, 24578–24590 (2019).

    Article  ADS  Google Scholar 

  30. Cnossen, J. et al. Localization microscopy at doubled precision with patterned illumination. Nat. Methods 17, 59–63 (2020).

    Article  Google Scholar 

  31. Gu, L. et al. Molecular resolution imaging by repetitive optical selective exposure. Nat. Methods 16, 1114–1118 (2019).

    Article  Google Scholar 

  32. Wang, Y. et al. Localization events-based sample drift correction for localization microscopy with redundant cross-correlation algorithm. Opt. Express 22, 15982 (2014).

    Article  ADS  Google Scholar 

  33. Weber, K., Rathke, P. C. & 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).

    Article  ADS  Google Scholar 

  34. Zwettler, F. U. et al. Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM). Nat. Commun. 11, 3388 (2020).

  35. Gold, V. A. M. et al. Visualizing active membrane protein complexes by electron cryotomography. Nat. Commun. 5, 4129 (2014).

    Article  ADS  Google Scholar 

  36. Xu, F. et al. Three-dimensional nanoscopy of whole cells and tissues with in situ point spread function retrieval. Nat. Methods 17, 531–540 (2020).

    Article  Google Scholar 

  37. Bratton, B. P. & Shaevitz, J. W. Simple experimental methods for determining the apparent focal shift in a microscope system. PLoS One 10, e0134616 (2015).

    Article  Google Scholar 

  38. Balzarotti, F. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606–612 (2017).

    Article  ADS  Google Scholar 

  39. Gwosch, K. C. et al. MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells. Nat. Methods 17, 217–224 (2020).

    Article  Google Scholar 

  40. Arigovindan, M., Sedat, J. W. & Agard, D. A. Effect of depth dependent spherical aberrations in 3D structured illumination microscopy. Opt. Express 20, 6527–6541 (2012).

    Article  ADS  Google Scholar 

  41. Booth, M., Andrade, D., Burke, D., Patton, B. & Zurauskas, M. Aberrations and adaptive optics in super-resolution microscopy. Microscopy 64, 251–261 (2015).

    Article  Google Scholar 

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

    Article  Google Scholar 

  43. Klevanski, M. et al. Automated highly multiplexed super-resolution imaging of protein nano-architecture in cells and tissues. Nat. Commun. 11, 1–11 (2020).

    Article  Google Scholar 

  44. Lampe, A., Haucke, V., Sigrist, S. J., Heilemann, M. & Schmoranzer, J. Multi-colour direct STORM with red emitting carbocyanines. Biol. Cell 104, 229–237 (2012).

    Article  Google Scholar 

  45. Zhang, Y. et al. Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging. Nat. Methods 17, 225–231 (2020).

    Article  Google Scholar 

  46. Gómez-García, P. A., Garbacik, E. T., Otterstrom, J. J., Garcia-Parajo, M. F. & Lakadamyali, M. Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT. Proc. Natl Acad. Sci. USA 115, 12991–12996 (2018).

    Article  Google Scholar 

  47. Bowman, A. J., Klopfer, B. B., Juffmann, T. & Kasevich, M. A. Electro-optic imaging enables efficient wide-field fluorescence lifetime microscopy. Nat. Commun. 10, 4561 (2019).

    Article  ADS  Google Scholar 

  48. Cabriel, C., Bourg, N., Dupuis, G. & Lévêque-Fort, S. Aberration-accounting calibration for 3D single-molecule localization microscopy. Opt. Lett. OL 43, 174–177 (2018).

    Article  ADS  Google Scholar 

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

    Article  ADS  Google Scholar 

  50. Przybylski, A., Thiel, B., Keller-Findeisen, J., Stock, B. & Bates, M. Gpufit: an open-source toolkit for GPU-accelerated curve fitting. Sci. Rep. 7, 1–9 (2017).

    Article  Google Scholar 

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P.J. acknowledges Master’s funding from GDR ImaBio and PhD funding from IDEX Paris Saclay (grant no. ANR-11-IDEX-0003-02). M.B. was funded by the Labex PALM (ANR-10-LABX-0039-PALM). We acknowledge the advices of the Centre de Photonique pour la Biologie et les Matériaux to cell culture and labelling. We also thank G. Dupuis for discussion and S. Sreenivas for a careful reading of the manuscript. We thank Abbelight for the free use of NEO software and dSTORM buffers. This work was supported by the AXA research fund, the ANR (grant nos. LABEX WIFI, ANR-10-LABX-24), ANR MSM-modulated super-resolution microscopy (grant no. ANR-17-CE09-0040), the valorization programme of the IDEX Paris Saclay and of Labex PALM.

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Authors and Affiliations



P.J, C.C., N.B., C.P., E.F. and S.L.F. conceived the project. P.J. designed the optical set-up, performed the acquisitions, CRLB calculations. P.J. and E.F performed the data analysis and carried out simulations. N.B. developed the dSTORM buffer. N.B., C.C. and P.J. optimized the immunofluorescence protocol. P.J., C.C. and S.L.F prepared the COS-7 and U2OS cells samples. M.B designed the 3D sample protocol. All authors have contributed to the manuscript. E.F and S.L.F. equally contribute to this work.

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Correspondence to Sandrine Lévêque-Fort.

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Competing interests

The CNRS has deposited a patent FR3054321-A1 on the 25 July 2016 to protect this work, currently under international extension. S.L.F, E.F. and N.B. are co-inventors.

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Jouchet, P., Cabriel, C., Bourg, N. et al. Nanometric axial localization of single fluorescent molecules with modulated excitation. Nat. Photonics 15, 297–304 (2021).

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