Imaging Living Cells with an X-ray Laser - DiVA 1159152/   Imaging living cells at a

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Text of Imaging Living Cells with an X-ray Laser - DiVA 1159152/   Imaging living cells at a

  • ACTAUNIVERSITATIS

    UPSALIENSISUPPSALA

    2018

    Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1625

    Imaging Living Cells with an X-rayLaser

    GIJS VAN DER SCHOT

    ISSN 1651-6214ISBN 978-91-513-0217-1urn:nbn:se:uu:diva-334219

  • Dissertation presented at Uppsala University to be publicly examined in B/A1:111a, BMC,Husargatan 3, Uppsala, Monday, 12 March 2018 at 09:00 for the degree of Doctor ofPhilosophy. The examination will be conducted in English. Faculty examiner: Professor PierreThibault (University of Southampton).

    Abstractvan der Schot, G. 2018. Imaging Living Cells with an X-ray Laser. Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Science and Technology 1625. 79 pp.Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0217-1.

    Imaging living cells at a resolution higher than the resolution of optical microscopy is asignificant challenge. Fluorescence microscopy can achieve a degree of super-resolution vialabeling cellular components with a fluorescent dye. Reaching nanometer or sub-nanometerresolution requires high-energy radiation with significantly shorter wavelength than that ofoptical light. X-rays and electrons have the requisite wavelengths and could be suitable for suchstudies; however, these probes also cause significant radiation damage. A dose in excess of100,000,000 Gray (Gy, J/kg) would be required to reach nanometer resolution on a cell, and nocell can survive this amount of radiation. As a consequence, much of what we know about cellsat high resolution today comes from dead material.

    Theory predicts that an ultra-short and extremely bright coherent X-ray pulse from an X-rayfree-electron laser can outrun key damage processes to deliver a molecular-level snapshot of acell that is alive at the time of image formation. The principle of diffraction before destructionexploits the difference between the speed of light (the X-ray pulse) and the much slower speedof damage formation. The femtosecond pulse freezes motion in the cell at physiologicaltemperatures on the time scale of atomic vibrations, offering unprecedented time resolution anda plethora of new experimental possibilities.

    This thesis describes the first test experiments on imaging living cells with an X-ray laser. Ipresent results in three essential areas of live cell imaging. (i) We have used an aerosol injectorto introduce live cyanobacteria into the X-ray focus, and recorded diffraction patterns withextremely low background at very high hit rates. (ii) We demonstrated scattered signal beyond 4nm resolution in some of these experiments. (iii) The thesis also describes image reconstruction,using a new fully automated pipeline that I developed during my studies. The reconstruction ofdiffraction patterns was successful for all patterns that did not have saturated pixels. The newsoftware suite, called RedFlamingo, selects exposures with desired properties, can sort themaccording to sample size, shape, orientation, exposure, the number and type of objects in thebeam during the exposure, their distance from each other, and so forth. The software includesvalidation tools to assess the quality of the reconstructions.

    Keywords: Coherent diffractive X-ray imaging, flash X-ray imaging, lensless imaging, singleparticle imaging, cyanobacteria, phasing, image classification, substrate-free sample delivery,X-ray free-electron laser, XFEL, FEL, CDXI, CDI, CXI, FXI

    Gijs van der Schot, Department of Cell and Molecular Biology, Molecular biophysics, Box596, Uppsala University, SE-75124 Uppsala, Sweden.

    Gijs van der Schot 2018

    ISSN 1651-6214ISBN 978-91-513-0217-1urn:nbn:se:uu:diva-334219 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-334219)

  • Dedicated to new Life: Emmanuel and Mathis

  • List of papers

    This thesis is based on the following papers, which are referred to in the textby their Roman numerals.

    I van der Schot, G et al. Imaging single cells in a beam of livecyanobacteria. Nature Communications 6, 5704 (2015).

    II van der Schot, G et al. Open dataset of live cyanobacterial cellsimaged using an X-ray laser. Nature Scientific Data 3, 160064 (2016)

    III Munke, A., van der Schot, G et al. Open dataset of RDV particles. inpreparation

    Reprints were made with permission from the publishers.

  • Supporting papers

    IV G. van der Schot and A.M.J.J. Bonvin. Performance of the WeNMR CS-Rosetta3 web server in CASD-NMR. Journal of Biomolecular NMR,62(4),497-502, 2015.

    V G. van der Schot et al. Improving 3D structure prediction from chemicalshift data. Journal of biomolecular NMR, 57(1):27-35,2013.

    VI T.A. Wassenaar et al. WeNMR: Structural Biology on the Grid. Journalof Grid Computing, 10(4):747-767, 2012.

    VII A. Rosato et al. Blind testing of routine, fully automated determinationof protein structures from NMR data. Structure, 20(2):227-236, 2012.

    VIII M. Vendruscolo et al. Protein structure determination using chemicalshifts, NMR in Mechanistic Systems Biology, Chapter 10, 107-110,2009.

    IX A. Rosato et al. CASD-NMR: critical assessment of automated struc-ture determination by NMR. Nature Methods, 6(9):625-626, 2009.

    X T. Gorkhover Femtosecond X-ray Fourier holography imaging of free-flying nanoparticles. Nature Photonics Accepted

    XI R.P. Kurta et al. Correlations in scattered x-ray laser pulses revealnanoscale structural features of viruses. Phys. Rev. Lett. 119, 158102,2017.

    XII H.K.N. Reddy et al. Coherent soft Xray diffraction imaging of col-iphage PR772 at the Linac coherent light source. Scientific Data, 4:170079,2017.

    XIII B.J. Daurer et al. Experimental strategies for imaging bioparticles withfemtosecond hard X-ray pulses. IUCrJ, 4(3):252-262, 2017.

    XIV M.F. Hantke et al. A data set from flash X-ray imaging of carboxysomes.Scientific data, 3:160061, aug 2016.

    XV A. Munke et al. Coherent diffraction of single Rice Dwarf virus parti-cles using hard X-rays at the Linac Coherent Light Source. Scientificdata, 3:160064, 2016.

    XVI T. Ekeberg et al. Three-Dimensional Reconstruction of the Giant MimivirusParticle with an X-Ray Free-Electron Laser. Physical Review Letters,114(9):098102, 2015.

    XVII M.F. Hantke et al. High-throughput imaging of heterogeneous cell or-ganelles with an X-ray laser. Nature Photonics, 8(12):943-949, 2014.

    XVIII A.V. Martin et al. Noise-robust coherent diffractive imaging with asingle diffraction pattern. Optics Express, 20(15):16650, 2012.

  • Contents

    1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

    2 Interaction of light and matter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142.1 Light as electromagnetic radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142.2 Photon-material interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152.3 Scattering by a single free electron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162.4 Two-body scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.5 Scattering from multiple electrons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192.6 The ewald sphere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212.7 Properties of the Fourier transform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222.8 Radiation damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232.9 Diffraction before destruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

    3 Pulse generation by an XFEL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253.1 The linear accelerator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253.2 Undulator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253.3 Self-amplified stimulated emission (SASE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

    4 Substrate-free sample delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304.1 Gas dynamic virtual nozzle (GDVN) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314.2 Aerodynamic lens stack . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

    5 Data recording . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345.1 Missing data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355.2 Saturation . . . . . . . . . . . . . . . . . .

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