Ultrafast X-ray imaging on individual fragile specimens such as aerosols1, metastable particles2, superfluid quantum systems3 and live biospecimens4 provides high-resolution information that is inaccessible with conventional imaging techniques. Coherent X-ray diffractive imaging, however, suffers from intrinsic loss of phase, and therefore structure recovery is often complicated and not always uniquely defined4,5. Here, we introduce the method of in-flight holography, where we use nanoclusters as reference X-ray scatterers to encode relative phase information into diffraction patterns of a virus. The resulting hologram contains an unambiguous three-dimensional map of a virus and two nanoclusters with the highest lateral resolution so far achieved via single shot X-ray holography. Our approach unlocks the benefits of holography for ultrafast X-ray imaging of nanoscale, non-periodic systems and paves the way to direct observation of complex electron dynamics down to the attosecond timescale.

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We would like to thank J. Geilhufe, E. Guehrs, A. Schropp and S. Eisebitt for many helpful discussions. T.G. acknowledges the P. Ewald fellowship from the Volkswagen Foundation and the Panofsky fellowship from SLAC National Accelerator Laboratory. We would like to thank J. Segal and A. Tomada from SLAC for providing high-resistivity Si wafers. Parts of this research were carried out at the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory. LCLS is an Office of Science User Facility operated for the US Department of Energy Office of Science by Stanford University. This work is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences under contract no. DE-AC02-06CH11357 and contract no. DE-AC02-76SF00515. T.M. acknowledges financial support from BMBF (German Federal Ministry of Education and Research) projects 05K10KT2 and 05K13KT2 as well as DFG (German Research Foundation) BO3169/2-2. This work was supported by the Swedish Research Council, the Knut and Alice Wallenberg Foundation, the European Research Council, the Röntgen-Angström Cluster, ELI Extreme Light Infrastructure Phase 2 (CZ.02.1.01/0.0/0.0/15 008/0000162), ELIBIO (CZ.02.1.01/0.0/0.0/15 003/0000447) from the European Regional Development Fund, Material science and the Chalmers Area of Advance. F.R.N.C.M. acknowledges the Swedish Foundation for Strategic Research. Portions of this research were carried out at Brookhaven National Laboratory, operated under contract no. DE-SC0012704 from the US Department of Energy Office of Science. G.F. acknowledges the support of NKFIH K115504.

Author information


  1. Institut für Optik und Atomare Physik, Technische Universität Berlin, Berlin, Germany

    • Tais Gorkhover
    • , Anatoli Ulmer
    • , Max Bucher
    • , Maria Müller
    • , Daniela Rupp
    • , Mario Sauppe
    •  & Thomas Möller
  2. Linac Coherent Light Source, SLAC National Accelerator Laboratory, Stanford, CA, USA

    • Tais Gorkhover
    • , Ken Ferguson
    • , Max Bucher
    • , Sebastian Carron
    • , Jacek Krzywinski
    • , Michelle Swiggers
    • , Garth Williams
    •  & Christoph Bostedt
  3. Stanford PULSE Institute, SLAC National Laboratory, Menlo Park, CA, USA

    • Tais Gorkhover
    • , Ken Ferguson
    •  & Christoph Bostedt
  4. Chemical Sciences and Engineering Division, Argonne National Laboratory, Lemont, IL, USA

    • Max Bucher
    •  & Christoph Bostedt
  5. Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden

    • Filipe R. N. C. Maia
    • , Johan Bielecki
    • , Max F. Hantke
    • , Benedikt J. Daurer
    • , Carl Nettelblad
    • , Jakob Andreasson
    • , Dirk Hasse
    • , Daniel S. D. Larsson
    • , Kerstin Mühlig
    • , Kenta Okamoto
    • , Alberto Pietrini
    • , Gijs van der Schot
    • , Marvin Seibert
    • , Jonas A. Sellberg
    • , Martin Svenda
    • , Nicusor Timneanu
    • , Daniel Westphal
    • , Alessandro Zani
    •  & Janos Hajdu
  6. NERSC, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    • Filipe R. N. C. Maia
  7. European XFEL GmbH, Schenefeld, Germany

    • Johan Bielecki
    •  & Janos Hajdu
  8. Center for Free-Electron Laser Science, DESY, Hamburg, Germany

    • Tomas Ekeberg
    • , Anton Barty
    • , Andrew Morgan
    •  & Henry N. Chapman
  9. Division of Scientific Computing, Department of Information Technology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden

    • Carl Nettelblad
  10. ELI Beamlines, Institute of Physics, Czech Academy of Science, Prague, Czech Republic

    • Jakob Andreasson
    • , Petr Bruza
    •  & Janos Hajdu
  11. Condensed Matter Physics, Department of Physics, Chalmers University of Technology, Gothenburg, Sweden

    • Jakob Andreasson
  12. Biomedical and X-ray Physics, Department of Applied Physics, AlbaNova University Center, KTH Royal Institute of Technology, Stockholm, Sweden

    • Jonas A. Sellberg
  13. Department of Physics and Astronomy, Uppsala University, Uppsala, Sweden

    • Nicusor Timneanu
  14. NSLS-II, Brookhaven National Laboratory, Upton, NY, USA

    • Garth Williams
  15. Wigner RCP, Institute for Solid State Physics and Optics, Budapest, Hungary

    • Gyula Faigel
  16. Department of Physics, Northwestern University, Evanston, IL, USA

    • Christoph Bostedt


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T.G. conceived the concept of 'in-flight' holography with two sources with support from C.B., T.M. and J.H. The project was led by T.G., C.B., F.R.N.C.M. and J.H. The experimental setup was designed and the experiment was performed by all authors. The bioparticle injector was operated by J.B., M.Sei. and K.M.. The cluster source was operated by K.F., M.B., C.B. and T.G. The biological samples were prepared by D.H., D.S.D.L., K.O. and M.Sve. The online and offline data analysis was carried out by F.M., T.E., M.F.H., B.J.D., C.N., A.M., G.v.S., M.B. and K.F. The images were analysed and processed by A.U. and T.G. The results were interpreted by A.U. and T.G. with input from C.B., G.F., T.M., F.R.N.C.M. and J.H. The manuscript was written by T.G. and A.U. with contributions from C.B., G.F., T.M., F.M. and J.H. and input from all authors.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Tais Gorkhover.