The possibility of imaging single proteins constitutes an exciting challenge for x-ray lasers. Despite encouraging results on large particles, imaging small particles has proven to be difficult for two reasons: not quite high enough pulse intensity from currently available x-ray lasers and, as we demonstrate here, contamination of the aerosolized molecules by nonvolatile contaminants in the solution. The amount of contamination on the sample depends on the initial droplet size during aerosolization. Here, we show that, with our electrospray injector, we can decrease the size of aerosol droplets and demonstrate virtually contaminant-free sample delivery of organelles, small virions, and proteins. The results presented here, together with the increased performance of next-generation x-ray lasers, constitute an important stepping stone toward the ultimate goal of protein structure determination from imaging at room temperature and high temporal resolution.

ARC Centre of Advanced Molecular Imaging Department of Chemistry and Physics La Trobe University Melbourne Victoria 3086 Australia

Biomedical and 10 ray Physics Department of Applied Physics AlbaNova University Center KTH Royal Institute of Technology SE 10691 Stockholm Sweden

Centre for BioImaging Sciences Department of Biological Sciences National University of Singapore 14 Science Drive 4 Singapore 117557 Singapore

Chemical Sciences and Engineering Division Argonne National Laboratory Argonne IL 60439 USA

Chemistry Research Laboratory Department of Chemistry Oxford University 12 Mansfield Rd Oxford OX1 3TA UK

Condensed Matter Physics Department of Physics Chalmers University of Technology Gothenburg Sweden

Cryo Electron Microscopy Bijvoet Center for Biomolecular Research Utrecht University 3584 CH Utrecht Netherlands

Department of Physics and Astronomy Northwestern University Evanston IL 60208 USA

Department of Physics and Astronomy Uppsala University Box 516 SE 75120 Uppsala Sweden

Department of Physics National University of Singapore Singapore Singapore

Department of Virology Graduate School of Biomedical and Health Sciences Hiroshima University Hiroshima 734 8551 Japan

Division of Scientific Computing Department of Information Technology Science for Life Laboratory Uppsala University Lägerhyddsvägen 2 SE 751 05 Uppsala Sweden

European XFEL GmbH Holzkoppel 4 22869 Schenefeld Germany

Institut für Optik und Atomare Physik Technische Universität Berlin 10623 Berlin Germany

Institut für Quantenoptik Leibniz Universität Hannover Welfengarten 1 30167 Hannover Germany

Institute for Protein Research Osaka University Suita Osaka 565 0871 Japan

Institute of Physics ELI Beamlines Academy of Sciences of the Czech Republic Na Slovance 2 CZ 18221 Prague Czech Republic

Laboratory of Molecular Biophysics Department of Cell and Molecular Biology Uppsala University Husargatan 3 SE 75124 Uppsala Sweden

Linac Coherent Light Source SLAC National Accelerator Laboratory Menlo Park CA 94025 USA

NERSC Lawrence Berkeley National Laboratory Berkeley CA 94720 USA

Stanford PULSE Institute SLAC National Accelerator Laboratory Menlo Park CA 94025 USA

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Observation of a single protein by ultrafast X-ray diffraction

Effects of radiation damage and inelastic scattering on single-particle imaging of hydrated proteins with an X-ray Free-Electron Laser

The XBI BioLab for life science experiments at the European XFEL

Plasma channel formation in NIR laser-irradiated carrier gas from an aerosol nanoparticle injector