Super-resolution fluorescence microscopy provides unprecedented insight into cellular and subcellular structures. However, going ‘beyond the diffraction barrier’ comes at a price, since most far-field super-resolution imaging techniques trade temporal for spatial super-resolution. We propose the combination of a novel label-free white light quantitative phase imaging with fluorescence to provide high-speed imaging and spatial super-resolution. The non-iterative phase retrieval relies on the acquisition of single images at each z-location and thus enables straightforward 3D phase imaging using a classical microscope. We realized multi-plane imaging using a customized prism for the simultaneous acquisition of eight planes. This allowed us to not only image live cells in 3D at up to 200 Hz, but also to integrate fluorescence super-resolution optical fluctuation imaging within the same optical instrument. The 4D microscope platform unifies the sensitivity and high temporal resolution of phase imaging with the specificity and high spatial resolution of fluorescence microscopy.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from $8.99

All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Change history

  • Correction 05 June 2018

    In the version of this Article originally published, there were some errors in equations in Fig. 1a; the details are shown in the correction notice. In the Acknowledgments, grant number ‘686271’ should have read ‘686271/SEFRI 16.0047’. These errors have now been corrected online.


  1. 1.

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

  2. 2.

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

  3. 3.

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

  4. 4.

    Vandenberg, W., Leutenegger, M., Lasser, T., Hofkens, J. & Dedecker, P. Diffraction-unlimited imaging: from pretty pictures to hard numbers. Cell Tissue Res. 360, 151–178 (2015).

  5. 5.

    Tinnefeld, P., Eggeling, C. & Hell, S. W. Far-Field Optical Nanoscopy (Springer, 2015).

  6. 6.

    Small, A. R. & Parthasarathy, R. Superresolution localization methods. Annu. Rev. Phys. Chem. 65, 107–125 (2014).

  7. 7.

    Liu, Z., Lavis, L. D. & Betzig, E. Imaging live-cell dynamics and structure at the single-molecule level. Mol. Cell 58, 644–659 (2015).

  8. 8.

    Dertinger, T., Colyer, R., Iyer, G., Weiss, S. & Enderlein, J. Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI). Proc. Natl Acad. Sci. USA 106, 22287–22292 (2009).

  9. 9.

    Dertinger, T., Xu, J., Naini, O. F., Vogel, R. & Weiss, S. SOFI-based 3D superresolution sectioning with a widefield microscope. Opt. Nanoscopy 1, 2–5 (2012).

  10. 10.

    Geissbuehler, S. et al. Live-cell multiplane three-dimensional super-resolution optical fluctuation imaging. Nat. Commun. 5, 5830 (2014).

  11. 11.

    Geissbuehler, S., Dellagiacoma, C. & Lasser, T. Comparison between SOFI and STORM. Biomed. Opt. Express 2, 408–420 (2011).

  12. 12.

    Girsault, A. et al. SOFI simulation tool: a software package for simulating and testing super-resolution optical fluctuation imaging. PLoS One 11, e0161602 (2016).

  13. 13.

    Geissbuehler, S. et al. Mapping molecular statistics with balanced super-resolution optical fluctuation imaging (bSOFI). Opt. Nanoscopy 1, 4 (2012).

  14. 14.

    Lukeš, T. et al. Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging. Nat. Commun. 8, 1731 (2017).

  15. 15.

    Liebling, M. Imaging the dynamics of biological processes via fast confocal microscopy and image processing. Cold Spring Harb. Protoc. 6, 783–789 (2011).

  16. 16.

    Grewe, B. F., Langer, D., Kasper, H., Kampa, B. M. & Helmchen, F. High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision. Nat. Methods 7, 399–405 (2010).

  17. 17.

    Chen, B.-C. et al. Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014).

  18. 18.

    Wolf, E. Three-dimensional structure determination of semi-transparent objects from holographic data. Opt. Commun. 1, 153–156 (1969).

  19. 19.

    Mir, M., Bhaduri, B., Wang, R., Zhu, R. & Popescu, G. Quantitative phase imaging. Prog. Opt. 57, 133–217 (2012).

  20. 20.

    Gabor, D. A New microscopic principle. Nature 161, 777–778 (1948).

  21. 21.

    Goodman, J. W. & Lawrence, R. W. Digital image formation from electronically detected holograms. Appl. Phys. Lett. 11, 77–79 (1967).

  22. 22.

    Cuche, E., Bevilacqua, F. & Depeursinge, C. Digital holography for quantitative phase-contrast imaging. Opt. Lett. 24, 291–293 (1999).

  23. 23.

    Ikeda, T., Popescu, G., Dasari, R. R. & Feld, M. S. Hilbert phase microscopy for investigating fast dynamics in transparent systems. Opt. Lett. 30, 1165–1167 (2005).

  24. 24.

    Popescu, G. et al. Fourier phase microscopy for investigation of biological structures and dynamics. Opt. Lett. 29, 2503–2505 (2004).

  25. 25.

    Wang, Z. et al. Spatial light interference microscopy (SLIM). Opt. Express 19, 1016–1026 (2011).

  26. 26.

    Reed Teague, M. Deterministic phase retrieval: a Green’s function solution. J. Opt. Soc. Am. 73, 1434–1441 (1983).

  27. 27.

    Streibl, N. Phase imaging by the transport equation of intensity. Opt. Commun. 49, 6–10 (1984).

  28. 28.

    Kou, S. S., Waller, L., Barbastathis, G. & Sheppard, C. J. R. Transport-of-intensity approach to differential interference contrast (TI-DIC) microscopy for quantitative phase imaging. Opt. Lett. 35, 447–449 (2010).

  29. 29.

    Bostan, E., Froustey, E., Nilchian, M., Sage, D. & Unser, M. Variational phase imaging using the transport-of-intensity equation. IEEE Trans. Image Process. 25, 807–817 (2015).

  30. 30.

    Kim, T. et al. White-light diffraction tomography of unlabelled live cells. Nat. Photon. 8, 256–263 (2014).

  31. 31.

    Chen, M., Tian, L. & Waller, L. 3D differential phase contrast microscopy. Biomed. Opt. Express 7, 3940–3950 (2016).

  32. 32.

    Chowdhury, S., Eldridge, W. J., Wax, A. & Izatt, J. A. Structured illumination multimodal 3D-resolved quantitative phase and fluorescence sub-diffraction microscopy. Biomed. Opt. Express 8, 2496–2518 (2017).

  33. 33.

    Choi, W. et al. Tomographic phase microscopy. Nat. Methods 4, 717–719 (2007).

  34. 34.

    Born, M. & Wolf, E. Principles of Optics, 7th (expanded) ed. (Cambridge Univ. Press, Cambridge, UK, 1999).

  35. 35.

    Mandel, L. & Wolf, E. Optical Coherence and Quantum Optics (Cambridge Univ. Press, Cambridge, UK, 1995).

  36. 36.

    McCutchen, C. W. Generalized aperture and the three-dimensional diffraction image. J. Opt. Soc. Am. A 54, 240–244 (1964).

  37. 37.

    Sheppard, C. J. R., Gu, M., Kawata, Y. & Kawata, S. Three-dimensional transfer functions for high-aperture systems. J. Opt. Soc. Am. A 11, 593–598 (1994).

  38. 38.

    Edwards, C. et al. Effects of spatial coherence in diffraction phase microscopy. Opt. Express 22, 5133–5146 (2014).

  39. 39.

    Deschout, H. et al. Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions. Nat. Commun. 7, 13693 (2016).

  40. 40.

    Borm, B., Requardt, R. P., Herzog, V. & Kirfel, G. Membrane ruffles in cell migration: Indicators of inefficient lamellipodia adhesion and compartments of actin filament reorganization. Exp. Cell Res. 302, 83–95 (2005).

  41. 41.

    Lashuel, H. A., Overk, C. R., Oueslati, A. & Masliah, E. The many faces of α-synuclein: from structure and toxicity to therapeutic target. Nat. Rev. Neurosci. 14, 38–48 (2013).

  42. 42.

    Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nat. Methods 5, 605–607 (2008).

  43. 43.

    Brakemann, T. et al. A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching. Nat. Biotechnol. 29, 942–947 (2011).

  44. 44.

    Steiner, P. et al. Modulation of receptor cycling by neuron-enriched endosomal protein of 21 kD. J. Cell Biol. 157, 1197–1209 (2002).

  45. 45.

    Mahul-Mellier, A.-L. et al. Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death. Cell Death Differ. 22, 1–16 (2015).

  46. 46.

    Volpicelli-Daley, L. A., Luk, K. C. & Lee, V. M.-Y. Addition of exogenous α-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous α-synuclein to Lewy body and Lewy neurite-like aggregates. Nat. Protoc. 9, 2135–2146 (2014).

  47. 47.

    Volpicelli-Daley, L. A. et al. Exogenous α-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron 72, 57–71 (2011).

  48. 48.

    Aitken, C. E., Marshall, R. A. & Puglisi, J. D. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys. J. 94, 1826–1835 (2008).

  49. 49.

    Abràmoff, M. D., Magalhães, P. J. & Ram, S. J. Image processing with ImageJ. Biophotonics International 11, 36–42 (2004).

Download references


We thank P. Sandoz and G. van der Goot for construction of Lifeact-Dreiklang (VDG-EPFL), the LSBG-EPFL for providing RAW 264.7 cells and M. Ricchetti from Institute Pasteur for human fibroblast cells. We are grateful to M. Sison for cell culture advice and assistance. We thank O. Peric and G. Fantner (LBNI-EPFL) for providing the technical sample and performing the AFM measurement. We acknowledge A. Radenovic and A. Nahas for support and discussion and G. M. Hagen for proofreading of the manuscript. This project has been partly funded from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No. [750528]. The research was supported by the Swiss National Science Foundation (SNSF) under grant 200020_159945/1. T.L. acknowledges the support from the Horizon 2020 Framework Programme of the European Union via grant 686271/SEFRI 16.0047.

Author information

Author notes

    • E. Bostan

    Present address: University of California, Berkeley, Computational Imaging Lab, Berkeley, CA, USA

    • M. Leutenegger

    Present address: Max-Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Göttingen, Germany

  1. These authors contributed equally: A. Descloux and K. S. Grußmayer.


  1. École Polytechnique Fédérale de Lausanne, Laboratoire d’Optique Biomédicale, Lausanne, Switzerland

    • A. Descloux
    • , K. S. Grußmayer
    • , T. Lukes
    • , A. Bouwens
    • , A. Sharipov
    • , S. Geissbuehler
    • , M. Leutenegger
    •  & T. Lasser
  2. École Polytechnique Fédérale de Lausanne, Biomedical Imaging Group, Lausanne, Switzerland

    • E. Bostan
  3. École Polytechnique Fédérale de Lausanne, Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Lausanne, Switzerland

    • A.-L. Mahul-Mellier
    •  & H. A. Lashuel


  1. Search for A. Descloux in:

  2. Search for K. S. Grußmayer in:

  3. Search for E. Bostan in:

  4. Search for T. Lukes in:

  5. Search for A. Bouwens in:

  6. Search for A. Sharipov in:

  7. Search for S. Geissbuehler in:

  8. Search for A.-L. Mahul-Mellier in:

  9. Search for H. A. Lashuel in:

  10. Search for M. Leutenegger in:

  11. Search for T. Lasser in:


A.D. and K.S.G. contributed equally to this work. T.L. and A.D. initiated the project and wrote the theory/modelling. A.D. developed the phase retrieval algorithm and simulations. K.S.G., A.D., T.L. and A.S. designed the experiments. K.S.G. prepared and performed the experiments. A.-L. M.-M. prepared the neuron samples. A.D., K.S.G. and T.Lu. analysed the data. M.L., S.G. and T.L. designed the optical system including the image splitting prism. S.G. and A.S. built the microscope set-up. E.B., A.B. and H.A.L. provided research advice. A.D., K.S.G. and T.L. wrote the manuscript with contributions from all authors.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to T. Lasser.

Supplementary information

  1. Supplementary Information

    The theory of partially coherent image formation, the retrieval of the complex three-dimensional cross-spectral density, the experimental and simulated results of the point-spread function in amplitude and phase, quantitative phase calibration using technical samples, the PRISM multi-plane platform for three-dimensional phase and SOFI imaging and supplementary figures and methods.

  2. Life Sciences Reporting Summary


  1. Supplementary Video 1

    This video shows a living human fibroblast at an imaging speed of 200 Hz as it migrates on a glass substrate.

  2. Supplementary Video 2

    This video shows long-term three-dimensional imaging of a dividing HeLa cell undergoing mitosis from the metaphase to the telophase.

  3. Supplementary Video 3

    This video shows the imaging of the vimentin network in HeLa cells and the cell dynamics by longer-term phase imaging

About this article

Publication history