Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

White-light diffraction tomography of unlabelled live cells

Nature Photonics volume 8, pages 256–263 (2014)Cite this article

Subjects

Abstract

We present a technique called white-light diffraction tomography (WDT) for imaging microscopic transparent objects such as live unlabelled cells. The approach extends diffraction tomography to white-light illumination and imaging rather than scattering plane measurements. Our experiments were performed using a conventional phase contrast microscope upgraded with a module to measure quantitative phase images. The axial dimension of the object was reconstructed by scanning the focus through the object and acquiring a stack of phase-resolved images. We reconstructed the three-dimensional structures of live, unlabelled, red blood cells and compared the results with confocal and scanning electron microscopy images. The 350 nm transverse and 900 nm axial resolution achieved reveals subcellular structures at high resolution in Escherichia coli cells. The results establish WDT as a means for measuring three-dimensional subcellular structures in a non-invasive and label-free manner.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The scattering problem.
Figure 2: WDT of RBCs.
Figure 3: WDT of E. coli cells.
Figure 4: WDT of HT29 cells.
Figure 5: Illustration of data acquisition.

Similar content being viewed by others

References

  1. Als-Nielsen, J. & McMorrow, D. Elements of Modern X-ray Physics (Wiley, 2001).

    Google Scholar 

  2. Jiang, H. et al. Quantitative 3D imaging of whole, unstained cells by using X-ray diffraction microscopy. Proc. Natl Acad. Sci. USA 107, 11234–11239 (2010).

    Article  ADS  Google Scholar 

  3. Raines, K. S. et al. Three-dimensional structure determination from a single view. Nature 463, 214–217 (2010).

    Article  ADS  Google Scholar 

  4. Born, M. & Wolf, E. Principles of Optics: Electromagnetic Theory of Propagation, Interference and Diffraction of Light 7th expanded edn (Cambridge Univ. Press, 1999).

    Book  Google Scholar 

  5. Devaney, A. J. Inverse-scattering theory within the Rytov approximation. Opt. Lett. 6, 374–376 (1981).

    Article  ADS  Google Scholar 

  6. Wolf, E. in Advances in Imaging and Electron Physics Vol. 165 (ed. Hawkes, P. W.) Ch. 7 (Academic, 2011).

    Google Scholar 

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

    Article  ADS  Google Scholar 

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

    Article  ADS  Google Scholar 

  9. Popescu, G. Quantitative Phase Imaging of Cells and Tissues (McGraw-Hill, 2011).

    Google Scholar 

  10. Paganin, D. & Nugent, K. A. Noninterferometric phase imaging with partially coherent light. Phys. Rev. Lett. 80, 2586–2589 (1998).

    Article  ADS  Google Scholar 

  11. Marquet, P. et al. Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with subwavelength axial accuracy. Opt. Lett. 30, 468–470 (2005).

    Article  ADS  Google Scholar 

  12. Chalut, K. J., Brown, W. J. & Wax, A. Quantitative phase microscopy with asynchronous digital holography. Opt. Express 15, 3047–3052 (2007).

    Article  ADS  Google Scholar 

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

    Article  ADS  Google Scholar 

  14. Ikeda, T., Popescu, G., Dasari, R. R. & Feld, M. S. Hilbert phase microscopy for investigating fast dynamics in transparent systems. Opt. Lett. 30, 1165–1168 (2005).

    Article  ADS  Google Scholar 

  15. Popescu, G., Ikeda, T., Dasari, R. R. & Feld, M. S. Diffraction phase microscopy for quantifying cell structure and dynamics. Opt. Lett. 31, 775–777 (2006).

    Article  ADS  Google Scholar 

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

    Article  ADS  Google Scholar 

  17. Rappaz, B. et al. Comparative study of human erythrocytes by digital holographic microscopy, confocal microscopy, and impedance volume analyzer. Cytometry A 73A, 895–903 (2008).

    Article  Google Scholar 

  18. Khmaladze, A., Kim, M. & Lo, C. M. Phase imaging of cells by simultaneous dual-wavelength reflection digital holography. Opt. Express 16, 10900–10911 (2008).

    Article  ADS  Google Scholar 

  19. Shaked, N. T., Rinehart, M. T. & Wax, A. Dual-interference-channel quantitative-phase microscopy of live cell dynamics. Opt. Lett. 34, 767–769 (2009).

    Article  ADS  Google Scholar 

  20. Park, Y. K. et al. Refractive index maps and membrane dynamics of human red blood cells parasitized by plasmodium falciparum. Proc. Natl Acad. Sci. USA 105, 13730–13735 (2008).

    Article  ADS  Google Scholar 

  21. Park, Y. K. et al. Measurement of red blood cell mechanics during morphological changes. Proc. Natl Acad. Sci. USA 107, 6731–6736 (2010).

    Article  ADS  Google Scholar 

  22. Mir, M. et al. Optical measurement of cycle-dependent cell growth. Proc. Natl Acad. Sci. USA 108, 13124–13129 (2011).

    Article  ADS  Google Scholar 

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

    Article  ADS  Google Scholar 

  24. Chen, B. Q. & Stamnes, J. J. Validity of diffraction tomography based on the first Born and the first Rytov approximations. Appl. Opt. 37, 2996–3006 (1998).

    Article  ADS  Google Scholar 

  25. Carney, P. S., Wolf, E. & Agarwal, G. S. Diffraction tomography using power extinction measurements. J. Opt. Soc. Am. A 16, 2643–2648 (1999).

    Article  ADS  MathSciNet  Google Scholar 

  26. 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).

    Article  MathSciNet  Google Scholar 

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

    Article  ADS  Google Scholar 

  28. Charriere, F. et al. Cell refractive index tomography by digital holographic microscopy. Opt. Lett. 31, 178–180 (2006).

    Article  ADS  Google Scholar 

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

    Article  Google Scholar 

  30. Cotte, Y. et al. Marker-free phase nanoscopy. Nature Photon. 7, 113–117 (2013).

    Article  ADS  Google Scholar 

  31. Choi, W. S., Fang-Yen, C., Badizadegan, K., Dasari, R. R. & Feld, M. S. Extended depth of focus in tomographic phase microscopy using a propagation algorithm. Opt. Lett. 33, 171–173 (2008).

    Article  ADS  Google Scholar 

  32. Zhou, R., Edwards, C., Arbabi, A., Popescu, G. & Goddard, L. L. Detecting 20 nm wide defects in large area nanopatterns using optical interferometric microscopy. Nano Lett. 13, 3716–3721 (2013).

    Article  ADS  Google Scholar 

  33. Goodman, J. W. Speckle Phenomena in Optics: Theory and Applications (Roberts & Co., 2007).

    Google Scholar 

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

    Article  ADS  Google Scholar 

  35. Wang, Z. et al. Spatial light interference tomography (SLIT). Opt. Express 19, 19907–19918 (2011).

    Article  ADS  Google Scholar 

  36. Bon, P., Aknoun, S., Savatier, J., Wattellier, B. & Monneret, S. in Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XX (eds Cogswell, C. J., Brown, T. G., Conchello, J.-A. & Wilson, T.) 858–918 SPIE (2013).

  37. Pawley, J. B. Handbook of Biological Confocal Microscopy 3rd edn (Springer, 2006).

    Book  Google Scholar 

  38. Ding, H. F., Wang, Z., Nguyen, F., Boppart, S. A. & Popescu, G. Fourier transform light scattering of inhomogeneous and dynamic structures. Phys. Rev. Lett. 101, 238102 (2008).

    Article  ADS  Google Scholar 

  39. Chew, W. C. Waves and Fields in Inhomogeneous Media (IEEE Press, 1995).

    Google Scholar 

  40. Kim, T., Sridharan, S. & Popescu, G. in Handbook of Coherent-Domain Optical Methods Vol. 1 (ed. Tuchin, V. V.) Ch. 7, 259–290 (Springer, 2013).

    Book  Google Scholar 

  41. Huang, D. et al. Optical coherence tomography. Science 254, 1178–1181 (1991).

    Article  ADS  Google Scholar 

  42. Ralston, T. S., Marks, D. L., Carney, P. S. & Boppart, S. A. Interferometric synthetic aperture microscopy. Nature Phys. 3, 129–134 (2007).

    Article  ADS  Google Scholar 

  43. Bain, B. J. A Beginner's Guide to Blood Cells 2nd edn (Blackwell, 2004).

    Book  Google Scholar 

  44. Khairy, K., Foo, J. & Howard, J. Shapes of red blood cells: comparison of 3D confocal images with the bilayer-couple model. Cell. Mol. Bioeng. 1, 173–181 (2008).

    Article  Google Scholar 

  45. Babacan, S. D., Wang, Z., Do, M. & Popescu, G. Cell imaging beyond the diffraction limit using sparse deconvolution spatial light interference microscopy. Biomed. Opt. Exp. 2, 1815–1827 (2011).

    Article  Google Scholar 

  46. Donachie, W. D. Co-ordinate regulation of the Escherichia coli cell cycle or the cloud of unknowing. Mol. Microbiol. 40, 779–785 (2001).

    Article  Google Scholar 

  47. Raskin, D. M. & De Boer, P. A. J. Rapid pole-to-pole oscillation of a protein required for directing division to the middle of Escherichia coli. Proc. Natl Acad. Sci. USA 96, 4971–4976 (1999).

    Article  ADS  Google Scholar 

  48. Ghosh, A. S. & Young, K. D. Helical disposition of proteins and lipopolysaccharide in the outer membrane of Escherichia coli. J. Bacteriol. 187, 1913–1922 (2005).

    Article  Google Scholar 

  49. Wiener, N. Generalized harmonic analysis. Acta Mathematica 55, 117–258 (1930).

    Article  MathSciNet  Google Scholar 

  50. Khintchine, A. Eine verschärfung des poincaréschen ‘wiederkehrsatzes’. Comp. Math 1, 177–179 (1935).

Download references

Acknowledgements

This research was supported in part by the National Science Foundation (grants CBET-1040462 MRI, CBET 08-46660 CAREER) and the Science and Technology Center for Emergent Behaviors of Integrated Cellular Systems (EBICS, CBET-0939511). The authors thank R. Bashir and K. Park for providing HT29 cells, I. Golding and M. Bednarz for providing E. coli cells and S. Robinson for assistance with SEM imaging of RBCs. The authors also thank J. Howard, K. Khairy and J.-J. Foo for providing confocal images of RBCs. R.Z. acknowledges support from the Beckman Foundation through a Beckman Graduate Fellowship. For more information, visit http://light.ece.illinois.edu

Author information

Author notes

  1. Taewoo Kim and Renjie Zhou: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Electrical and Computer Engineering, Quantitative Light Imaging Laboratory, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    Taewoo Kim, Renjie Zhou, Mustafa Mir & Gabriel Popescu

  2. Department of Electrical and Computer Engineering, Photonic Systems Laboratory, Micro and Nanotechnology Laboratory, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    Renjie Zhou & Lynford L. Goddard

  3. Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    S. Derin Babacan

  4. Department of Electrical and Computer Engineering, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    P. Scott Carney

Authors
  1. Taewoo Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Renjie Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mustafa Mir
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. S. Derin Babacan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. P. Scott Carney
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lynford L. Goddard
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gabriel Popescu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.P., R.Z. and S.D.B. proposed the idea. G.P., R.Z., T.K. and P.S.C. developed the theoretical description of the method. T.K. and R.Z. performed three-dimensional PSF calculations. T.K. and M.M. performed quantitative phase imaging. S.D.B. and M.M. developed the sparse deconvolution method. T.K. and R.Z. performed data analysis and three-dimensional reconstruction. G.P. and L.L.G. supervised the research. All authors contributed to writing the manuscript.

Corresponding author

Correspondence to Gabriel Popescu.

Ethics declarations

Competing interests

G.P. has financial interest in Phi Optics, Inc., a company developing quantitative phase imaging technology for materials and life science applications, which, however, did not sponsor the research.

Supplementary information

Supplementary information

Supplementary information (PDF 2058 kb)

Supplementary information

Supplementary movie (MP4 867 kb)

Supplementary information

Supplementary movie (MP4 1164 kb)

Supplementary information

Supplementary movie (MP4 1115 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kim, T., Zhou, R., Mir, M. et al. White-light diffraction tomography of unlabelled live cells. Nature Photon 8, 256–263 (2014). https://doi.org/10.1038/nphoton.2013.350

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nphoton.2013.350

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing