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Imaging Living Cells with an X-ray Laser
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics. (Laboratory of Molecular Biophysics)
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
Keywords [en]
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: urn:nbn:se:uu:diva-334219ISBN: 978-91-513-0217-1 (print)OAI: oai:DiVA.org:uu-334219DiVA, id: diva2:1159152
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
List of papers
1. Imaging single cells in a beam of live cyanobacteria with an X-ray laser
Open this publication in new window or tab >>Imaging single cells in a beam of live cyanobacteria with an X-ray laser
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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.

National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-245040 (URN)10.1038/ncomms6704 (DOI)000350034400002 ()25669616 (PubMedID)
Available from: 2015-02-24 Created: 2015-02-24 Last updated: 2017-12-04Bibliographically approved
2. Open data set of live cyanobacterial cells imaged using an X-ray laser
Open this publication in new window or tab >>Open data set of live cyanobacterial cells imaged using an X-ray laser
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2016 (English)In: Scientific Data, E-ISSN 2052-4463, Vol. 3, article id 160058Article in journal (Refereed) Published
Abstract [en]

Structural studies on living cells by conventional methods are limited to low resolution because radiation damage kills cells long before the necessary dose for high resolution can be delivered. X-ray free-electron lasers circumvent this problem by outrunning key damage processes with an ultra-short and extremely bright coherent X-ray pulse. Diffraction-before-destruction experiments provide high-resolution data from cells that are alive when the femtosecond X-ray pulse traverses the sample. This paper presents two data sets from micron-sized cyanobacteria obtained at the Linac Coherent Light Source, containing a total of 199,000 diffraction patterns. Utilizing this type of diffraction data will require the development of new analysis methods and algorithms for studying structure and structural variability in large populations of cells and to create abstract models. Such studies will allow us to understand living cells and populations of cells in new ways. New X-ray lasers, like the European XFEL, will produce billions of pulses per day, and could open new areas in structural sciences.

National Category
Biophysics
Identifiers
urn:nbn:se:uu:diva-300201 (URN)10.1038/sdata.2016.58 (DOI)000390225400003 ()
Note

Data Descriptor

Available from: 2016-08-05 Created: 2016-08-05 Last updated: 2017-11-28Bibliographically approved

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