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.

High-throughput imaging of heterogeneous cell organelles with an X-ray laser

Nature Photonics volume 8, pages 943–949 (2014)Cite this article

Subjects

Abstract

We overcome two of the most daunting challenges in single-particle diffractive imaging: collecting many high-quality diffraction patterns on a small amount of sample and separating components from mixed samples. We demonstrate this on carboxysomes, which are polyhedral cell organelles that vary in size and facilitate up to 40% of Earth's carbon fixation. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min with the Linac Coherent Light Source running at 120 Hz. We separate different structures directly from the diffraction data and show that the size distribution is preserved during sample delivery. We automate phase retrieval and avoid reconstruction artefacts caused by missing modes. We attain the highest-resolution reconstructions on the smallest single biological objects imaged with an X-ray laser to date. These advances lay the foundations for accurate, high-throughput structure determination by flash-diffractive imaging and offer a means to study structure and structural heterogeneity in biology and elsewhere.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: The experimental set-up to image aerosolized carboxysomes.
Figure 2: Sample injection.
Figure 3: Computational purification of carboxysomes using low-resolution fit parameters and the autocorrelation function.
Figure 4: Measured size distribution of single carboxysome particles in the gas phase.
Figure 5: Computational purification of carboxysomes on the basis of size and shape.
Figure 6: Reconstructed image of a single carboxysome compared to the image of an icosahedron with uniform density.

References

  1. Neutze, R., Wouts, R., van der Spoel, D., Weckert, E. & Hajdu, J. Potential for biomolecular imaging with femtosecond X-ray pulses. Nature 406, 752–757 (2000).

    Article  ADS  Google Scholar 

  2. Bergh, M., Huldt, G., Timneanu, N., Maia, F. R. N. C. & Hajdu, J. Feasibility of imaging living cells at subnanometer resolutions by ultrafast X-ray diffraction. Q. Rev. Biophys. 41, 181–204 (2008).

    Article  Google Scholar 

  3. Chapman, H. N. et al. Femtosecond diffractive imaging with a soft-X-ray free-electron laser. Nature Phys. 2, 839–843 (2006).

    Article  ADS  Google Scholar 

  4. Chapman, H. N. et al. Femtosecond X-ray protein nanocrystallography. Nature 470, 73–77 (2011).

    Article  ADS  Google Scholar 

  5. Seibert, M. M. et al. Single mimivirus particles intercepted and imaged with an X-ray laser. Nature 470, 78–81 (2011).

    Article  ADS  Google Scholar 

  6. Bernal, J. D., Fankuchen, I. & Perutz, M. F. An X-ray study of chymotrypsin and haemoglobin. Nature 141, 523–524 (1938).

    Article  ADS  Google Scholar 

  7. Shannon, C. E. Communication in the presence of noise. Proc. Inst. Radio Eng. 37, 10–21 (1949).

    MathSciNet  Google Scholar 

  8. Sayre, D. On the implication of a theorem due to Shannon. Acta Crystallogr. 5, 834 (1952).

    Google Scholar 

  9. Fienup, J. R. Reconstruction of an object from the modulus. Opt. Lett. 3, 27–29 (1978).

    Article  ADS  Google Scholar 

  10. Gerchberg, R. W. & Saxton, W. O. A practical algorithm for the determination of the phase from image and diffraction plane pictures. Optik 35, 237–246 (1972).

    Google Scholar 

  11. Miao, J., Charalambous, P., Kirz, J. & Sayre, D. Extending the methodology of X-ray crystallography to allow imaging of micrometre-sized non-crystalline specimens. Nature 400, 342–344 (1999).

    Article  ADS  Google Scholar 

  12. Marchesini, S. et al. X-ray image reconstruction from a diffraction pattern alone. Phys. Rev. B 68, 140101 (2003).

    Article  ADS  Google Scholar 

  13. Luke, D. R. Relaxed averaged alternating reflections for diffraction imaging. Inverse Prob. 21, 37–50 (2005).

    Article  ADS  MathSciNet  Google Scholar 

  14. Huldt, G., Szöke, A. & Hajdu, J. Diffraction imaging of single particles and biomolecules. J. Struct. Biol. 144, 219–227 (2003).

    Article  Google Scholar 

  15. Loh, D. & Elser, V. Reconstruction algorithm for single-particle diffraction imaging experiments. Phys. Rev. E 80, 1–20 (2009).

    Article  Google Scholar 

  16. Maia, F. R. N. C., Ekeberg, T., Tımneanu, N., van der Spoel, D. & Hajdu, J. Structural variability and the incoherent addition of scattered intensities in single-particle diffraction. Phys. Rev. E 80, 031905 (2009).

    Article  ADS  Google Scholar 

  17. Fischer, N., Konevega, A. L., Wintermeyer, W., Rodnina, M. V. & Stark, H. Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy. Nature 466, 329–333 (2010).

    Article  ADS  Google Scholar 

  18. Sander, B., Golas, M. M., Luhrmann, R. & Stark, H. An approach for de novo structure determination of dynamic molecular assemblies by electron cryomicroscopy. Structure 18, 667–676 (2010).

    Article  Google Scholar 

  19. Elmlund, D. & Elmlund, H. SIMPLE: Software for ab initio reconstruction of heterogeneous single-particles. J. Struct. Biol. 180, 420–427 (2012).

    Article  Google Scholar 

  20. Emma, P. et al. First lasing and operation of an ångstrom-wavelength free-electron laser. Nature Photon. 4, 641–647 (2010).

    Article  ADS  Google Scholar 

  21. Shively, J. M., Ball, F., Brown, D. H. & Saunders, R. E. Functional organelles in prokaryotes: polyhedral inclusions (carboxysomes) of Thiobacillus neapolitanus. Science 182, 584–586 (1973).

    Article  ADS  Google Scholar 

  22. Rae, B. D., Long, B. M., Badger M. R. & Price, G. D. Functions, compositions, and evolution of the two types of carboxysomes: polyhedral microcompartments that facilitate CO2 fixation in cyanobacteria and some proteobacteria. Microbiol. Mol. Biol. Rev. 77, 357–379 (2013).

    Article  Google Scholar 

  23. Iancu, C. V. et al. The structure of isolated Synechococcus strain WH8102 carboxysomes as revealed by electron cryotomography. J. Mol. Biol. 372, 764–773 (2007).

    Article  Google Scholar 

  24. Espie, G. S. & Kimber, M. S. Carboxysomes: cyanobacterial Rubisco comes in small packages. Photosynth. Res. 109, 7–20 (2011).

    Article  Google Scholar 

  25. Bozek, J. D. AMO instrumentation for the LCLS X-ray FEL. Eur. Phys. J. Spec. Top. 169, 129–132 (2009).

    Article  Google Scholar 

  26. Bostedt, C. et al. Ultra-fast and ultra-intense X-ray sciences: first results from the Linac Coherent Light Source free-electron laser. J. Phys. B 46, 164003 (2013).

    Article  ADS  Google Scholar 

  27. DePonte, D. P. Gas dynamic virtual nozzle for generation of microscopic droplet streams. J. Phys. D 41, 195505 (2008).

    Article  ADS  Google Scholar 

  28. Murphy, W. K. & Sears, G. W. Production of particulate beams. J. Appl. Phys. 35,1986–1987 (1964).

    Article  ADS  Google Scholar 

  29. Bogan, M. J. et al. Single particle X-ray diffractive imaging. Nano Lett. 8, 310–316 (2008).

    Article  ADS  Google Scholar 

  30. Strüder, L. et al. Large-format, high-speed, X-ray pnCCDs combined with electron and ion imaging spectrometers in a multipurpose chamber for experiments at 4th generation light sources. Nucl. Instrum. Meth. Phys. Res. A 614, 483–496 (2010).

    Article  ADS  Google Scholar 

  31. Miao, J., Sayre, D. & Chapman, H. N. Phase retrieval from the magnitude of the Fourier transforms of nonperiodic objects. J. Opt. Soc. Am. A 15, 1662–1669 (1998).

    Article  ADS  Google Scholar 

  32. Miao, J. et al. Quantitative image reconstruction of GaN quantum dots from oversampled diffraction intensities alone. Phys. Rev. Lett. 95, 085503 (2005).

    Article  ADS  Google Scholar 

  33. Thibault, P., Elser, V., Jacobsen, C., Shapiro, D. & Sayre, D. Reconstruction of a yeast cell from X-ray diffraction data. Acta Crystallogr. A 62, 248–261 (2006).

    Article  ADS  Google Scholar 

  34. Kassemeyer, S. et al. Femtosecond free-electron laser x-ray diffraction data sets for algorithm development. Opt. Express 20, 4149–4158 (2012).

    Article  ADS  Google Scholar 

  35. Van der Spoel, D., Marklund, E. G., Larsson, D. S. D. & Caleman, C. Proteins, lipids, and water in the gas phase. Macromol. Biosci. 11, 50–59 (2011).

    Article  Google Scholar 

  36. Rouse, S. L., Marcoux, J., Robinson, C. V. & Sansom, M. S. P. Dodecyl maltoside protects membrane proteins in vacuo. Biophys. J. 105, 648–656 (2013).

    Article  ADS  Google Scholar 

  37. Tito, M. A., Tars, K., Valegård, K., Hajdu, J. & Robinson, C. V. Electrospray time-of-flight mass spectrometry of the intact MS2 virus capsid. J. Am. Chem. Soc. 122, 3550–3551 (2000).

    Article  Google Scholar 

  38. Maia, F. R. N. C., Ekeberg, T., van der Spoel, D. & Hajdu, J. Hawk: the image reconstruction package for coherent X-ray diffractive imaging. J. Appl. Crystallogr. 43, 1535–1539 (2010).

    Article  Google Scholar 

  39. Chapman, H. N. et al. High-resolution ab initio three-dimensional X-ray diffraction microscopy. J. Opt. Soc. Am. A 23, 1179–1200 (2006).

    Article  ADS  Google Scholar 

  40. Saxton, W. O. & Baumeister, W. The correlation averaging of a regularly arranged bacterial cell envelope protein. J. Microsc. 127, 127–138 (1982).

    Article  Google Scholar 

  41. Van Heel, M., Keegstra, W., Schutter, W. & van Brüggen E. F. J. in Structure and Function of Invertebrate Respiratory Proteins, EMBO Workshop 1982, Life Chemistry Reports (ed. Wood, E. J.) Suppl. 1, 69–73 (1982).

  42. Van Heel, M. & Schatz, M. Fourier shell correlation threshold criteria. J. Struct. Biol. 151, 250–262 (2005).

    Article  Google Scholar 

  43. Barty, A. et al. A new resource for processing serial X-ray diffraction data. J. Appl. Cryst. 47, 1118–1131 (2014).

    Article  Google Scholar 

  44. Thibault, P. Algorithmic Methods in Diffraction Microscopy PhD thesis, Cornell Univ. (2007).

  45. Maia, F. R. N. C. The coherent X-ray imaging data bank. Nature Methods 9, 854–855 (2012).

    Article  Google Scholar 

  46. Hamzeh, F. M. & Bragg, R. H. Small angle scattering of X-rays from groups of nonrandomly oriented ellipsoids of revolution of low concentration. J. Appl. Phys. 45, 3189–3195 (1974).

    Article  ADS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Swedish Research Council, the Knut and Alice Wallenberg Foundation, the European Research Council, the Röntgen-Ångström Cluster and Stiftelsen Olle Engkvist Byggmästare. Portions of this research were carried out at the Linac Coherent Light Source, a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The authors thank the scientific and technical staff of the LCLS for support. The authors thank the CAMP collaboration for giving access to their experimental set-up and for supporting the experiment at the LCLS. The authors also acknowledge the Max Planck Society for funding the development and operation of the CAMP instrument.

Author information

Author notes

  1. Max F. Hantke and Dirk Hasse: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Cell and Molecular Biology, Laboratory of Molecular Biophysics, Uppsala University, Husargatan 3 (Box 596), Uppsala, SE-751 24, Sweden

    Max F. Hantke, Dirk Hasse, Filipe R. N. C. Maia, Tomas Ekeberg, Katja John, Martin Svenda, Nicusor Timneanu, Daniel S. D. Larsson, Gijs van der Schot, Gunilla H. Carlsson, Margareta Ingelman, Jakob Andreasson, Daniel Westphal, M. Marvin Seibert, Kerstin Mühlig, Janos Hajdu & Inger Andersson

  2. NERSC, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Filipe R. N. C. Maia

  3. Centre for BioImaging Sciences, National University of Singapore, 14 Science Drive 4, 117557, Singapore, Singapore

    N. Duane Loh

  4. ARC Centre of Excellence for Coherent X-ray Science, School of Physics, The University of Melbourne, Victoria, 3010, Australia

    Andrew V. Martin

  5. Center for Free-Electron Laser Science, DESY, Notkestrasse 85, Hamburg, 22607, Germany

    Mengning Liang, Francesco Stellato, Richard A. Kirian, Daniel Rolles, Sascha Epp, Henry N. Chapman & Anton Barty

  6. INFN and Physics Department, University of Rome ‘Tor Vergata’, Via della Ricerca Scientifica 1, Rome, 00133, Italy

    Francesco Stellato

  7. LCLS, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, 94025, California, USA

    Daniel P. DePonte, M. Marvin Seibert, Sebastian Schorb, Ken Ferguson, Christoph Bostedt, Sebastian Carron & John D. Bozek

  8. PNSensor GmbH, Römerstrasse 28, München, 80803, Germany

    Robert Hartmann

  9. Max Planck Institute for Extraterrestrial Physics, Giessenbachstrasse, Garching, 85741, Germany

    Nils Kimmel

  10. Department of Physics, Kansas State University, 331 Cardwell Hall, Kansas, 66506, Manhattan, USA

    Artem Rudenko

  11. European XFEL GmbH, Albert-Einstein-Ring 19, Hamburg, 22761, Germany

    Janos Hajdu

Authors
  1. Max F. Hantke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dirk Hasse
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Filipe R. N. C. Maia
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tomas Ekeberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Katja John
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Martin Svenda
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. N. Duane Loh
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Andrew V. Martin
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Nicusor Timneanu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Daniel S. D. Larsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Gijs van der Schot
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Gunilla H. Carlsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Margareta Ingelman
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Jakob Andreasson
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Daniel Westphal
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Mengning Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Francesco Stellato
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Daniel P. DePonte
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Robert Hartmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Nils Kimmel
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Richard A. Kirian
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. M. Marvin Seibert
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Kerstin Mühlig
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Sebastian Schorb
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Ken Ferguson
    View author publications

    You can also search for this author in PubMed Google Scholar

  26. Christoph Bostedt
    View author publications

    You can also search for this author in PubMed Google Scholar

  27. Sebastian Carron
    View author publications

    You can also search for this author in PubMed Google Scholar

  28. John D. Bozek
    View author publications

    You can also search for this author in PubMed Google Scholar

  29. Daniel Rolles
    View author publications

    You can also search for this author in PubMed Google Scholar

  30. Artem Rudenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  31. Sascha Epp
    View author publications

    You can also search for this author in PubMed Google Scholar

  32. Henry N. Chapman
    View author publications

    You can also search for this author in PubMed Google Scholar

  33. Anton Barty
    View author publications

    You can also search for this author in PubMed Google Scholar

  34. Janos Hajdu
    View author publications

    You can also search for this author in PubMed Google Scholar

  35. Inger Andersson
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.H., I.A., M.F.H., F.R.N.C.M. and T.E. developed the imaging concept and conceived the experiment. M.F.H., F.R.N.C.M., T.E., A.B., N.D.L., A.M., G.V.D.S. and D.L. developed ideas and software to process the diffraction data. D.H., K.J., G.H.C., M.S., M.I. and I.A. prepared and characterized carboxysomes for the study. J.H., B.I., D.P.D., R.A.K., M.S., J.A., M.M.S. and D.W. developed and operated the sample injector. J.D.B., C.B., S.C., N.T., M.S. and M.M.S. operated the beamline at the LCLS. R.H. and N.K. operated the pnCCD detectors. M.F.H., F.R.N.C.M., T.E., N.D.L., G.V.D.S., A.B., J.A., M.M.S., M.S., M.L., F.S., D.R., A.R., S.E., H.N.C. and J.H. characterized the imaging apparatus and carried out the experiment. M.F.H., F.R.N.C.M. and T.E. processed the data. M.F.H., F.R.N.C.M., I.A. and J.H. analysed the results and wrote the manuscript with input from the others.

Corresponding author

Correspondence to Janos Hajdu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 618 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hantke, M., Hasse, D., Maia, F. et al. High-throughput imaging of heterogeneous cell organelles with an X-ray laser. Nature Photon 8, 943–949 (2014). https://doi.org/10.1038/nphoton.2014.270

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nphoton.2014.270

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