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Imaging single cells in a beam of live cyanobacteria with an X-ray laser
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
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2015 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 5704Article in journal (Refereed) Published
Abstract [en]

There exists a conspicuous gap of knowledge about the organization of life at mesoscopic levels. Ultra-fast coherent diffractive imaging with X-ray free-electron lasers can probe structures at the relevant length scales and may reach sub-nanometer resolution on micron-sized living cells. Here we show that we can introduce a beam of aerosolised cyanobacteria into the focus of the Linac Coherent Light Source and record diffraction patterns from individual living cells at very low noise levels and at high hit ratios. We obtain two-dimensional projection images directly from the diffraction patterns, and present the results as synthetic X-ray Nomarski images calculated from the complex-valued reconstructions. We further demonstrate that it is possible to record diffraction data to nanometer resolution on live cells with X-ray lasers. Extension to sub-nanometer resolution is within reach, although improvements in pulse parameters and X-ray area detectors will be necessary to unlock this potential.

Place, publisher, year, edition, pages
2015. Vol. 6, article id 5704
National Category
Structural Biology
Identifiers
URN: urn:nbn:se:uu:diva-245040DOI: 10.1038/ncomms6704ISI: 000350034400002PubMedID: 25669616OAI: oai:DiVA.org:uu-245040DiVA, id: diva2:790292
Available from: 2015-02-24 Created: 2015-02-24 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Imaging Living Cells with an X-ray Laser
Open this publication in new window or tab >>Imaging Living Cells with an X-ray Laser
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Imaging living cells at a resolution higher than the resolution of optical microscopy is a significant challenge. Fluorescence microscopy can achieve a degree of super-resolution via labeling cellular components with a fluorescent dye. Reaching nanometer or sub-nanometer resolution requires high-energy radiation with significantly shorter wavelength than that of optical light. X-rays and electrons have the requisite wavelengths and could be suitable for such studies; however, these probes also cause significant radiation damage. A dose in excess of 100,000,000 Gray (Gy, J/kg) would be required to reach nanometer resolution on a cell, and no cell can survive this amount of radiation. As a consequence, much of what we know about cells at high resolution today comes from dead material.

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

This thesis describes the first test experiments on imaging living cells with an X-ray laser. I present results in three essential areas of live cell imaging. (i) We have used an aerosol injector to introduce live cyanobacteria into the X-ray focus, and recorded diffraction patterns with extremely low background at very high hit rates. (ii) We demonstrated scattered signal beyond 4 nm 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 of diffraction patterns was successful for all patterns that did not have saturated pixels. The new software suite, called RedFlamingo, selects exposures with desired properties, can sort them according to sample size, shape, orientation, exposure, the number and type of objects in the beam during the exposure, their distance from each other, and so forth. The software includes validation tools to assess the quality of the reconstructions.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 79
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1625
Keyword
Coherent diffractive X-ray imaging, flash X-ray imaging, lensless imaging, single particle imaging, cyanobacteria, phasing, image classification, substrate-free sample delivery, X-ray free-electron laser, XFEL, FEL, CDXI, CDI, CXI, FXI
National Category
Biophysics Atom and Molecular Physics and Optics Cell Biology
Research subject
Chemistry with specialization in Biophysics
Identifiers
urn:nbn:se:uu:diva-334219 (URN)978-91-513-0217-1 (ISBN)
Public defence
2018-03-12, B/A1:111a, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2018-02-19 Created: 2017-11-21 Last updated: 2018-03-07

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van der Schot, GijsSvenda, MartinMaia, Filipe R. N. C.Hantke, MaxSeibert, M. MarvinAndreasson, JakobTimneanu, NicusorWestphal, DanielHasse, DirkCarlsson, Gunilla H.Larsson, Daniel S. D.Andersson, IngerHajdu, JanosEkeberg, Tomas

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van der Schot, GijsSvenda, MartinMaia, Filipe R. N. C.Hantke, MaxSeibert, M. MarvinAndreasson, JakobTimneanu, NicusorWestphal, DanielHasse, DirkCarlsson, Gunilla H.Larsson, Daniel S. D.Andersson, IngerHajdu, JanosEkeberg, Tomas
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