Sobolev, EgorZolotarev, SergeiGiewekemeyer, KlausBielecki, JohanOkamoto, KentoReddy, Hemanth K. N.Andreasson, JakobAyyer, KartikBarak, ImrichBari, SadiaBarty, ABean, RBobkov, SergeyChapman, Henry N.Chojnowski, GrzegorzDaurer, Benedikt J.Rode, Andrei2022-07-202022-07-202399-3650http://hdl.handle.net/1885/269813The emergence of high repetition-rate X-ray free-electron lasers (XFELs) powered by superconducting accelerator technology enables the measurement of significantly more experimental data per day than was previously possible. The European XFEL is expected to provide 27,000 pulses per second, over two orders of magnitude more than any other XFEL. The increased pulse rate is a key enabling factor for single-particle X-ray diffractive imaging, which relies on averaging the weak diffraction signal from single biological particles. Taking full advantage of this new capability requires that all experimental steps, from sample preparation and delivery to the acquisition of diffraction patterns, are compatible with the increased pulse repetition rate. Here, we show that single-particle imaging can be performed using X-ray pulses at megahertz repetition rates. The results obtained pave the way towards exploiting high repetition-rate X-ray free-electron lasers for single-particle imaging at their full repetition rate.We acknowledge European XFEL in Schenefeld, Germany, for provision of X-ray free-electron laser beamtime at Scientific Instrument SPB/SFX and would like to thank the instrument group and facility staff for their assistance. We acknowledge the use of the XBI biological sample preparation laboratory, enabled by the XBI User Consortium. The results of the work were obtained using Maxwell computational resources operated at Deutsches Elektronen-Synchrotron (DESY), Hamburg, Germany, and computational resources of MCC NRC “Kurchatov Institute.” This research used resources of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. We acknowledge the support of funding from: the Swedish Foundation for International Cooperation in Research and Higher Education (STINT); Helmholtz Associations Initiative and Networking Fund and the Russian Science Foundation grant HRSF-0002/18-41-0600; the Russian Science Foundation grant 18-14-00321; European Research Council, “Frontiers in Attosecond X-ray Science: Imaging and Spectroscopy (AXSIS),” ERC-2013-SyG 609920; Fellowship from the Joachim Herz Stiftung; Singapore National Research Foundation Grant number NRF-CRP16-2015-05; Ministry of Education, Science, Research and Sport of the Slovak Republic and by grant APVV-18-0104; the project CZ.02.1.01/0.0/0.0/16_019/0000789 (ADONIS) from European Regional Development Fund, Chalmers Area of Advance; Material Science and the Ministry of Education, Youth and Sports as part of targeted support from the National Programme of Sustainability II; US National Science Foundation (NSF) Science and Technology Center BioXFEL Award 1231306; Helmholtz Initiative and Networking Fund through the Young Investigators Group Program and Deutsche Forschungsgemeinschaft, project B03/SFB755; VR starting grant (2018-03387); FORMAS future research leader (2018-00421); KVA Biosciences 2018 (BS2018-0053); NSF 1231306; German Ministry for Education and Research, BMBF (grant No. 05K2016—Visavix); the Heinrich Pette Institute, Leibniz Institute for Experimental Virology is supported by the Free and Hanseatic City of Hamburg and the Federal Ministry of Health; NSF STC BioXFEL grant 1231306; The National Research Foundation (NRF) of Korea (Grant No. 2017K1A3A7A09016380); the Röntgen-Ångström Cluster; the Swedish Research Council; the Swedish Foundation for Strategic Research. We thank Arwen Pearson for critical reading of the manuscript. Open access funding provided by Uppsala University.application/pdfen-AU© 2020 The authorshttp://creativecommons.org/licenses/by/4.0/202010.1038/s42005-020-0362-y2021-08-01Creative Commons