Marker-free phase nanoscopy

  • A Corrigendum to this article was published on 29 April 2013

Abstract

We introduce a microscopic method that determines quantitative optical properties beyond the optical diffraction limit and allows direct imaging of unstained living biological specimens. In established holographic microscopy, complex fields are measured using interferometric detection, allowing diffraction-limited phase measurements. Here, we show that non-invasive optical nanoscopy can achieve a lateral resolution of 90 nm by using a quasi-2π-holographic detection scheme and complex deconvolution. We record holograms from different illumination directions on the sample plane and observe subwavelength tomographic variations of the specimen. Nanoscale apertures serve to calibrate the tomographic reconstruction and to characterize the imaging system by means of the coherent transfer function. This gives rise to realistic inverse filtering and guarantees true complex field reconstruction. The observations are shown for nanoscopic porous cell frustule (diatoms), for the direct study of bacteria (Escherichia coli), and for a time-lapse approach to explore the dynamics of living dendritic spines (neurones).

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Figure 1: Marker-free nanoscopy by 2π-DHM in real and spatial frequency domains.
Figure 2: Side-by-side comparisons of resolution in traditional DHM and 2π-DHM.
Figure 3: Super-resolved phase images of nanoscopic porous cell frustule (diatoms).
Figure 4: Refraction tomogram of bacteria (E. coli).
Figure 5: Three-dimensional remodelling of a synaptic network from observations over a long time.

Change history

  • 16 April 2013

    In the version of this Letter originally published online, no competing financial interests were declared. However, the authors wish to acknowledge a relevant patent. The competing financial interests statement has been modified in the HTML and PDF versions of the Letter.

References

  1. 1

    Hell, S. W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

    ADS  Article  Google Scholar 

  2. 2

    Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).

    Article  Google Scholar 

  3. 3

    Rappaz, B. et al. Measurement of the integral refractive index and dynamic cell morphometry of living cells with digital holographic microscopy. Opt. Express 13, 9361–9373 (2005).

    ADS  Article  Google Scholar 

  4. 4

    Charrière, F. et al. Living specimen tomography by digital holographic microscopy: morphometry of testate amoeba. Opt. Express 14, 7005–7013 (2006).

    ADS  Article  Google Scholar 

  5. 5

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

    Article  Google Scholar 

  6. 6

    Kemper, B. et al. Investigation of living pancreas tumor cells by digital holographic microscopy. J. Biomed. Opt. 11, 034005 (2006).

    ADS  Article  Google Scholar 

  7. 7

    Pavillon, N. et al. Early cell death detection with digital holographic microscopy. PLoS ONE 7, e30912 (2012).

    ADS  Article  Google Scholar 

  8. 8

    Mir, M. et al. Visualizing Escherichia coli sub-cellular structure using sparse deconvolution spatial light interference tomography. PLoS ONE 7, e39816 (2012).

    ADS  Article  Google Scholar 

  9. 9

    Rappaz, B., Depeursinge, C. & Marquet, P. in Biomedical Optical Phase Microscopy and Nanoscopy (eds Shaked, N. T., Zalevskey, Z. & Satterwhite, L.) Ch. 5.1, 71–95 (Elsevier, 2012).

  10. 10

    Cuche, E., Marquet, P. & Depeursinge, C. Simultaneous amplitude-contrast and quantitative phase-contrast microscopy by numerical reconstruction of Fresnel off-axis holograms. Appl. Opt. 38, 6994–7001 (1999).

    ADS  Article  Google Scholar 

  11. 11

    Gureyev, T. E., Roberts, A. & Nugent, K. A. Phase retrieval with the transport-of-intensity equation: matrix solution with use of Zernike polynomials. J. Opt. Soc. Am. A 12, 1932–1941 (1995).

    ADS  MathSciNet  Google Scholar 

  12. 12

    Popescu, G. in Methods in Nano Cell Biology vol. 90 (eds Jena, B. P.) 87–115 (Academic, 2008).

    Google Scholar 

  13. 13

    Bon, P., Maucort, G., Wattellier, B. & Monneret, S. Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells. Opt. Express 17, 13080–13094 (2009).

    ADS  Article  Google Scholar 

  14. 14

    Lauer, V. New approach to optical diffraction tomography yielding a vector equation of diffraction tomography and a novel tomographic microscope. J. Microsc. 205, 165–176 (2002).

    MathSciNet  Article  Google Scholar 

  15. 15

    Paturzo, M. et al. Super-resolution in digital holography by a two-dimensional dynamic phase grating. Opt. Express 16, 17107–17118 (2008).

    ADS  Article  Google Scholar 

  16. 16

    Mico, V., Zalevsky, Z., Ferreira, C. & García, J. Superresolution digital holographic microscopy for three-dimensional samples. Opt. Express 16, 19260–19270 (2008).

    ADS  Article  Google Scholar 

  17. 17

    Debailleul, M., Georges, V., Simon, B., Morin, R. & Haeberle, O. High-resolution three-dimensional tomographic diffractive microscopy of transparent inorganic and biological samples. Opt. Lett. 34, 79–81 (2009).

    ADS  Article  Google Scholar 

  18. 18

    Kuznetsova, Y., Neumann, A. & Brueck, S. R. J. Solid-immersion imaging interferometric nanoscopy to the limits of available frequency space. J. Opt. Soc. Am. A 29, 772–781 (2012).

    ADS  Article  Google Scholar 

  19. 19

    Devaney, A. A filtered backpropagation algorithm for diffraction tomography. Ultrason. Imaging 4, 336–350 (1982).

    Article  Google Scholar 

  20. 20

    Cotte, Y., Toy, M. F., Pavillon, N. & Depeursinge, C. Microscopy image resolution improvement by deconvolution of complex fields. Opt. Express 18, 19462–19478 (2010).

    ADS  Article  Google Scholar 

  21. 21

    Cotte, Y. et al. Realistic 3D coherent transfer function inverse filtering of complex fields. Biomed. Opt. Express 2, 2216–2230 (2011).

    Article  Google Scholar 

  22. 22

    Sheppard, C. J. R. & Gu, M. Imaging by a high aperture optical-system. J. Mod. Opt. 40, 1631–1651 (1993).

    ADS  Article  Google Scholar 

  23. 23

    Vertu, S., Flagge, J., Delaunay, J.-J. & Haeberle, O. Improved and isotropic resolution in tomographic diffractive microscopy combining sample and illumination rotation. Cent. Eur. J. Phys. 9, 969–974 (2011).

    Google Scholar 

  24. 24

    den Dekker, A. J. & van den Bos, A. Resolution: a survey. J. Opt. Soc. Am. A 14, 547–557 (1997).

    ADS  Article  Google Scholar 

  25. 25

    Cotte, Y., Toy, M. F. & Depeursinge, C. Beyond the lateral resolution limit by phase imaging. J. Biomed. Opt. 16, 106007 (2011).

    ADS  Article  Google Scholar 

  26. 26

    Montfort, F. et al. Purely numerical compensation for microscope objective phase curvature in digital holographic microscopy: influence of digital phase mask position. J. Opt. Soc. Am. A 23, 2944–2953 (2006).

    ADS  Article  Google Scholar 

  27. 27

    Choi, Y., Yang, T. D., Lee, K. J. & Choi, W. Full-field and single-shot quantitative phase microscopy using dynamic speckle illumination. Opt. Lett. 36, 2465–2467 (2011).

    ADS  Article  Google Scholar 

  28. 28

    Hildebrand, M. et al. Nanoscale control of silica morphology and three-dimensional structure during diatom cell wall formation. J. Mater. Res. 21, 2689–2698 (2006).

    ADS  Article  Google Scholar 

  29. 29

    Sheppard, C. J. R., Kou, S. S. & Depeursinge, C. Reconstruction in interferometric synthetic aperture microscopy: comparison with optical coherence tomography and digital holographic microscopy. J. Opt. Soc. Am. A 29, 244–250 (2012).

    ADS  Article  Google Scholar 

  30. 30

    Portera-Cailliau, C., Pan, D. T. & Yuste, R. Activity-regulated dynamic behavior of early dendritic protrusions: evidence for different types of dendritic filopodia. J. Neurosci. 23, 7129–7142 (2003).

    Article  Google Scholar 

  31. 31

    Tao, H.-z. et al. Selective presynaptic propagation of long-term potentiation in defined neural networks. J. Neurosci. 20, 3233–3243 (2000).

    Article  Google Scholar 

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Acknowledgements

This work was funded by the Swiss National Science Foundation (SNSF, grant no. 205 320–130 543) and EPFL (innovation grant INNO 12-14). The authors acknowledge the Center of MicroNano-Technology (CMI) for cooperation regarding its research facilities. The authors give special thanks to M. Hildebrand for providing diatom samples, and to L. Pollaro for preparing the E. coli samples. The authors also thank P. Lara Rodrigo, C-M. Cotte and Cemico GmbH for assistance with graphics. Finally, the authors thank E. Cuche, CTO of Lyncée Tec, for helpful discussions and suggestions.

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Y.C., F.T., N.P. and C.D. designed the experiments. Y.C. and F.T. performed the experiments and carried out the main data analysis. P.J. and D.B. prepared the biological samples. C.D. and P.Mar. provided overall guidance to the project. All authors discussed the results and contributed to the manuscript.

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Correspondence to Yann Cotte.

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Competing interests

Yann Cotte, Nicolas Pavillon and Christian Depeursinge are named inventors on international patent WO/2011/121523 (publication date 06.10.2011, international filing date 28.03.2011), which is related to the techniques described in this Letter.

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Cotte, Y., Toy, F., Jourdain, P. et al. Marker-free phase nanoscopy. Nature Photon 7, 113–117 (2013). https://doi.org/10.1038/nphoton.2012.329

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