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Flash Diffractive Imaging in Three Dimensions
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics. (Molecular biophysics)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

During the last years we have seen the birth of free-electron lasers, a new type of light source ten billion times brighter than syncrotrons and able to produce pulses only a few femtoseconds long. One of the main motivations for building these multi-million dollar machines was the prospect of imaging biological samples such as proteins and viruses in 3D without the need for crystallization or staining. This thesis contains some of the first biological results from free-electron lasers.

These results include 2D images, both of whole cells and of the giant mimivirus and also con- tains a 3D density map of the mimivirus assembled from diffraction patterns from many virus particles. These are important proof-of-concept experiments but they also mark the point where free-electron lasers start to produce biologically relevant results. The most noteworthy of these results is the unexpectedly non-uniform density distribution of the internals of the mimivirus.

We also present Hawk, the only open-source software toolkit for analysing single particle diffraction data. The Uppsala-developed program suite supports a wide range fo algorithms and takes advantage of Graphics Processing Units which makes it very computationally efficient.

Last, the problem introduced by structural variability in samples is discussed. This includes a description of the problem and how it can be overcome, and also how it could be turned into an advantage that allows us to image samples in all of their conformational states.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , 68 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 960
Keyword [en]
X-ray, diffraction, mimivirus, three dimensional, phase retrieval, EMC, manifold embedding, CXI, FEL, free-electron laser, single particle
National Category
Biophysics
Research subject
Physics with specialization in Biophysics
Identifiers
URN: urn:nbn:se:uu:diva-179643ISBN: 978-91-554-8439-2 (print)OAI: oai:DiVA.org:uu-179643DiVA: diva2:545704
Public defence
2012-10-05, B22, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2012-09-14 Created: 2012-08-21 Last updated: 2013-01-22Bibliographically approved
List of papers
1. Three-dimensional structure determination with an X-ray laser
Open this publication in new window or tab >>Three-dimensional structure determination with an X-ray laser
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Three-dimensional structure determination of a non-crystalline virus has been achieved from a set of randomly oriented continuous diffraction patterns captured with an X-ray laser. Intense, ultra-short X-ray pulses intercepted a beam of single mimivirus particles, producing single particle X-ray diffraction patterns that are assembled into a three-dimensional amplitude distribution based on statistical consistency. Phases are directly retrieved from the assembled Fourier distribution to synthesize a three-dimensional image. The resulting electron density reveals a pseudo-icosahedral asymmetric virion structure with a compartmentalized interior, within which the DNA genome occupies only about a fifth of the volume enclosed by the capsid. Additional electron microscopy data indicate the genome has a chromatin-like fiber structure that has not previously been observed in a virus. 

Keyword
Mimivirus, flash diffraction, three dimensional, imaging, CXI
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-179597 (URN)
Funder
EU, European Research Council
Available from: 2012-08-20 Created: 2012-08-20 Last updated: 2014-09-26
2. Single mimivirus particles intercepted and imaged with an X-ray laser
Open this publication in new window or tab >>Single mimivirus particles intercepted and imaged with an X-ray laser
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2011 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 470, no 7332, 78-81 p.Article in journal (Refereed) Published
Abstract [en]

X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions(1-4). Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma(1). The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval(2). Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a noncrystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source(5). Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-146069 (URN)10.1038/nature09748 (DOI)000286886400037 ()21293374 (PubMedID)
Available from: 2011-02-15 Created: 2011-02-15 Last updated: 2016-05-10
3. Data requirements for single-particle diffractive imaging
Open this publication in new window or tab >>Data requirements for single-particle diffractive imaging
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Single-shot diffractive imaging with ultra-short and very intense coherent X-ray pulses has become a routine experimental technique at new free-electron-laser facilities. Extension to three-dimensional imaging requires many diffraction pat- terns from identical objects captured in different orientations. These can then be combined into a full three-dimensional Fourier transform of the object. The ori- entation of the particle intercepted by the pulsed X-ray beam is usually unknown. This makes it hard to predict the number of patterns required to fully cover the Fourier space. In this paper we provide formulae to estimate the number of expo- sures required to achieve a given coverage of Fourier space as a function of parti- cle size, resolution and shot noise. 

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-179594 (URN)
Funder
EU, European Research Council
Available from: 2012-08-20 Created: 2012-08-20 Last updated: 2014-09-26
4. Structural variability and the incoherent addition of scattered intensities in single-particle diffraction
Open this publication in new window or tab >>Structural variability and the incoherent addition of scattered intensities in single-particle diffraction
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2009 (English)In: Physical Review E. Statistical, Nonlinear, and Soft Matter Physics: Statistical Physics, Plasmas, Fluids, and Related Interdisciplinary Topics, ISSN 1063-651X, E-ISSN 1095-3787, Vol. 80, no 3, 031905- p.Article in journal (Refereed) Published
Abstract [en]

X-ray lasers may allow structural studies on single particles and biomolecules without crystalline periodicity in the samples. We examine here the effect of sample dynamics as a source of structural heterogeneity on the resolution of the reconstructed image of a small protein molecule. Structures from molecular-dynamics simulations of lysozyme were sampled and aligned. These structures were then used to calculate diffraction patterns corresponding to different dynamic states. The patterns were incoherently summed and the resulting data set was phased using the oversampling method. Reconstructed images of hydrated and dehydrated lysozyme gave resolutions of 3.7 angstrom and 7.6 angstrom, respectively. These are significantly worse than the root-mean-square deviation of the hydrated (2.7 angstrom for all atoms and 1.45 angstrom for C-alpha positions) or dehydrated (3.7 angstrom for all atoms and 2.5 angstrom for C-alpha positions) structures. The noise introduced by structural dynamics and incoherent addition of dissimilar structures restricts the maximum resolution to be expected from direct image reconstruction of dynamic systems. A way of potentially reducing this effect is by grouping dynamic structures into distinct structural substates and solving them separately.

Keyword
x-ray-diffraction, electron cascades, proteins, crystallography, resolution, pulses
National Category
Physical Sciences
Identifiers
urn:nbn:se:uu:diva-111993 (URN)10.1103/PhysRevE.80.031905 (DOI)000270383400104 ()1539-3755 (ISBN)
Note

Part 1 501LM Times Cited:0 Cited References Count:32

Available from: 2010-01-05 Created: 2010-01-05 Last updated: 2013-03-01Bibliographically approved
5. Hawk: the image reconstruction package for coherent X-ray diffractive imaging
Open this publication in new window or tab >>Hawk: the image reconstruction package for coherent X-ray diffractive imaging
2010 (English)In: Journal of applied crystallography, ISSN 0021-8898, E-ISSN 1600-5767, Vol. 43, no 6, 1535-1539 p.Article in journal (Other academic) Published
Abstract [en]

The past few years have seen a tremendous growth in the field of coherent X-ray diffractive imaging, in large part due to X-ray free-electron lasers which provide a peak brilliance billions of times higher than that of synchrotrons. However, this rapid development in terms of hardware has not been matched on the software side. The release of Hawk is intended to close this gap. To the authors knowledge Hawk is the first publicly available and fully open source software program for reconstructing images from continuous diffraction patterns. The software handles all steps leading from a raw diffraction pattern to a reconstructed two-dimensional image including geometry determination, background correction, masking and phasing. It also includes preliminary three-dimensional support and support for graphics processing units using the Compute Unified Device Architecture, which speeds up processing by orders of magnitude compared to a single central processing unit. Hawk implements numerous algorithms and is easily extended. This, in combination with its open-source licence, provides a platform for other groups to test, develop and distribute their own algorithms.

Keyword
computer programs, diffractive imaging, free-electron lasers, Hawk, open-source software, phasing, reconstruction
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-121927 (URN)10.1107/S0021889810036083 (DOI)000284550900033 ()
Available from: 2010-03-31 Created: 2010-03-31 Last updated: 2013-01-22Bibliographically approved
6. Femtosecond diffractive imaging of biological cells
Open this publication in new window or tab >>Femtosecond diffractive imaging of biological cells
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2010 (English)In: Journal of Physics B: Atomic, Molecular and Optical Physics, ISSN 0953-4075, E-ISSN 1361-6455, Vol. 43, no 19, 194015- p.Article in journal (Refereed) Published
Abstract [en]

In a flash diffraction experiment, a short and extremely intense x-ray pulse illuminates the sample to obtain a diffraction pattern before the onset of significant radiation damage. The over-sampled diffraction pattern permits phase retrieval by iterative phasing methods. Flash diffractive imaging was first demonstrated on an inorganic test object (Chapman et al 2006 Nat. Phys. 2 839-43). We report here experiments on biological systems where individual cells were imaged, using single, 10-15 fs soft x-ray pulses at 13.5 nm wavelength from the FLASH free-electron laser in Hamburg. Simulations show that the pulse heated the sample to about 160 000 K but not before an interpretable diffraction pattern could be obtained. The reconstructed projection images return the structures of the intact cells. The simulations suggest that the average displacement of ions and atoms in the hottest surface layers remained below 3 angstrom during the pulse.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-147259 (URN)10.1088/0953-4075/43/19/194015 (DOI)000281958100016 ()
Available from: 2011-02-25 Created: 2011-02-24 Last updated: 2016-04-12Bibliographically approved

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