Some practical considerations about the effects of radiation damage on hydrated cells imaged by X-ray fluorescence microscopy

Radiation damage is the general curse of structural biologists who use synchrotron small-angle X-ray scattering (SAXS) to investigate biological macromolecules in solution. The EMBL-P12 biological SAXS beamline located at the PETRAIII storage ring (DESY, Hamburg, Germany) caters to an extensive user community who integrate SAXS into their diverse structural biology programs. The high brilliance of the beamline [5.1 × 1012 photons s−1, 10 keV, 500 (H) µm × 250 (V) µm beam size at the sample position], combined with automated sample handling and data acquisition protocols, enable the high-throughput structural characterization of macromolecules in solution. However, considering the often-significant resources users invest to prepare samples, it is crucial that simple and effective protocols are in place to limit the effects of radiation damage once it has been detected. Here various practical approaches are evaluated that users can implement to limit radiation damage at the P12 beamline to maximize the chances of collecting quality data from radiation sensitive samples.

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A Perspective on Molecular Structure and Bond-Breaking in Radiation Damage in Serial Femtosecond Crystallography

Crystals ◽

10.3390/cryst10070585 ◽

2020 ◽

Vol 10 (7) ◽

pp. 585 ◽

Cited By ~ 2

Author(s):

Carl Caleman ◽

Francisco Jares Junior ◽

Oscar Grånäs ◽

Andrew V. Martin

Keyword(s):

Radiation Damage ◽

Protein Dynamics ◽

Conformational Changes ◽

Bond Breaking ◽

Time Resolved ◽

Metal Centers ◽

Disulfide Bonding ◽

X Ray ◽

Sample Structure ◽

Effects Of Radiation

X-ray free-electron lasers (XFELs) have a unique capability for time-resolved studies of protein dynamics and conformational changes on femto- and pico-second time scales. The extreme intensity of X-ray pulses can potentially cause significant modifications to the sample structure during exposure. Successful time-resolved XFEL crystallography depends on the unambiguous interpretation of the protein dynamics of interest from the effects of radiation damage. Proteins containing relatively heavy elements, such as sulfur or metals, have a higher risk for radiation damage. In metaloenzymes, for example, the dynamics of interest usually occur at the metal centers, which are also hotspots for damage due to the higher atomic number of the elements they contain. An ongoing challenge with such local damage is to understand the residual bonding in these locally ionized systems and bond-breaking dynamics. Here, we present a perspective on radiation damage in XFEL experiments with a particular focus on the impacts for time-resolved protein crystallography. We discuss recent experimental and modelling results of bond-breaking and ion motion at disulfide bonding sites in protein crystals.

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Radiation damage and derivatization in macromolecular crystallography: a structure factor's perspective

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798315021555 ◽

2016 ◽

Vol 72 (3) ◽

pp. 388-394 ◽

Cited By ~ 5

Author(s):

Robin L. Owen ◽

Darren A. Sherrell

Keyword(s):

Data Collection ◽

Radiation Damage ◽

Relative Intensity ◽

Macromolecular Crystallography ◽

Site Specific ◽

X Ray ◽

Effects Of Radiation ◽

Short Time ◽

Approximate Rate ◽

Time Dose

During, or even after, data collection the presence and effects of radiation damage in macromolecular crystallography may not always be immediately obvious. Despite this, radiation damage is almost always present, with site-specific damage occurring on very short time (dose) scales well before global damage becomes apparent. A result of both site-specific radiation damage and derivatization is a change in the relative intensity of reflections. The size and approximate rate of onset of X-ray-induced transformations is compared with the changes expected from derivatization, and strategies for minimizing radiation damage are discussed.

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Comparison of helical scan and standard rotation methods in single-crystal X-ray data collection strategies

Journal of Synchrotron Radiation ◽

10.1107/s1600577516018488 ◽

2017 ◽

Vol 24 (1) ◽

pp. 42-52 ◽

Cited By ~ 12

Author(s):

Ivan Polsinelli ◽

Martin Savko ◽

Cecile Rouanet-Mehouas ◽

Lidia Ciccone ◽

Susanna Nencetti ◽

...

Keyword(s):

Radiation Damage ◽

Disulfide Bonds ◽

X Ray ◽

Rotation Method ◽

Rotation Methods ◽

Anomalous Signal ◽

Effects Of Radiation ◽

Helical Scan ◽

Collection Strategies ◽

Meaningful Data

X-ray radiation in macromolecular crystallography can chemically alter the biological material and deteriorate the integrity of the crystal lattice with concomitant loss of resolution. Typical alterations include decarboxylation of glutamic and aspartic residues, breaking of disulfide bonds and the reduction of metal centres. Helical scans add a small translation to the crystal in the rotation method, so that for every image the crystal is shifted to expose a fresh part. On beamline PROXIMA 2A at Synchrotron SOLEIL, this procedure has been tested with various parameters in an attempt to understand how to mitigate the effects of radiation damage. Here, the strategies used and the crystallographic metrics for various scenarios are reported. Among these, the loss of bromine from bromophenyl moieties appears to be a useful monitor of radiation damage as the carbon–bromine bond is very sensitive to X-ray irradiation. Two cases are focused on where helical scans are shown to be superior in obtaining meaningful data compared with conventional methods. In one case the initial resolution of the crystal is extended over time, and in the second case the anomalous signal is preserved to provide greater effective multiplicity and easier phasing.

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Phase transition in an organic ferroelectric: glycinium phosphite, with and without X-ray radiation damage

Acta Crystallographica Section B Structural Science Crystal Engineering and Materials ◽

10.1107/s2052520621003127 ◽

2021 ◽

Vol 77 (3) ◽

Author(s):

Nikita E. Bogdanov ◽

Boris A. Zakharov ◽

Dmitry Chernyshov ◽

Philip Pattison ◽

Elena V. Boldyreva

Keyword(s):

Radiation Damage ◽

Thermal Evolution ◽

Laboratory Data ◽

Ferroelectric State ◽

Volume Increase ◽

X Ray ◽

Cell Parameters ◽

Anisotropic Displacement Parameters ◽

Effects Of Radiation ◽

Data Collections

Thermal evolution of an organic ferroelectric, namely, glycinium phosphite, was probed by multi-temperature single-crystal diffraction using synchrotron radiation and also by a similar experiment with a laboratory X-ray diffractometer. Both series of measurements showed a transition from the paraelectric to the ferroelectric state at nearly the same temperature, T c = 225 K. Temperature evolution of the unit-cell parameters and volume are drastically different for the synchrotron and laboratory data. The latter case corresponds to previous reports and shows an expected contraction of the cell on cooling. The data collected with the synchrotron beam show an abnormal nonlinear increase in volume on cooling. Structure analysis shows that this volume increase is accompanied by a suppression of scattering at high angles and an apparent increase of the anisotropic displacement parameters for all atoms; we therefore link these effects to radiation damage accumulated during consecutive data collections. The effects of radiation on the formation of the polar structure of ferroelectric glycinium phosphite is discussed together with the advantages and drawbacks of synchrotron experimentation with fine temperature sampling.

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The effects of radiation damage on the spectral resolution of the Chandrayaan-1 x-ray spectrometer

10.1117/12.858648 ◽

2010 ◽

Author(s):

T. E. Walker ◽

D. R. Smith ◽

C. J. Howe ◽

B. J. Kellett ◽

P. Sreekumar ◽

...

Keyword(s):

Radiation Damage ◽

Spectral Resolution ◽

X Ray ◽

Effects Of Radiation

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The effects of radiation damage on the spectral resolution of the Chandrayaan-1 x-ray spectrometer over the full mission duration

10.1117/12.926983 ◽

2012 ◽

Author(s):

T. E. Walker ◽

D. R. Smith

Keyword(s):

Radiation Damage ◽

Spectral Resolution ◽

X Ray ◽

Mission Duration ◽

Effects Of Radiation

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Studies of metaphase chromosomes in the scanning transmission x-ray microscope: Image fidelity, radiation damage and specimen preparation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100129826 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1038-1039

Author(s):

Shawn Williams ◽

Xiaodong Zhang ◽

Susan Lamm ◽

Jack Van’t Hof

Keyword(s):

Visible Light ◽

Radiation Damage ◽

Cross Section ◽

Imaging Techniques ◽

Dose Range ◽

Specimen Preparation ◽

X Rays ◽

Metaphase Chromosomes ◽

X Ray ◽

Scanning Transmission

The Scanning Transmission X-ray Microscope (STXM) is well suited for investigating metaphase chromosome structure. The absorption cross-section of soft x-rays having energies between the carbon and oxygen K edges (284 - 531 eV) is 6 - 9.5 times greater for organic specimens than for water, which permits one to examine unstained, wet biological specimens with resolution superior to that attainable using visible light. The attenuation length of the x-rays is suitable for imaging micron thick specimens without sectioning. This large difference in cross-section yields good specimen contrast, so that fewer soft x-rays than electrons are required to image wet biological specimens at a given resolution. But most imaging techniques delivering better resolution than visible light produce radiation damage. Soft x-rays are known to be very effective in damaging biological specimens. The STXM is constructed to minimize specimen dose, but it is important to measure the actual damage induced as a function of dose in order to determine the dose range within which radiation damage does not compromise image quality.

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Scanning x-ray fluorescence microscopy and principal component analysis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100133394 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1752-1753

Author(s):

Brian Cross

Keyword(s):

Principal Component Analysis ◽

Fluorescence Microscopy ◽

Spatial Resolution ◽

High Speed ◽

Image Acquisition ◽

Principal Component ◽

Fluorescence Microscope ◽

Acquisition Rate ◽

X Ray ◽

Speed Up

A relatively new entry, in the field of microscopy, is the Scanning X-Ray Fluorescence Microscope (SXRFM). Using this type of instrument (e.g. Kevex Omicron X-ray Microprobe), one can obtain multiple elemental x-ray images, from the analysis of materials which show heterogeneity. The SXRFM obtains images by collimating an x-ray beam (e.g. 100 μm diameter), and then scanning the sample with a high-speed x-y stage. To speed up the image acquisition, data is acquired "on-the-fly" by slew-scanning the stage along the x-axis, like a TV or SEM scan. To reduce the overhead from "fly-back," the images can be acquired by bi-directional scanning of the x-axis. This results in very little overhead with the re-positioning of the sample stage. The image acquisition rate is dominated by the x-ray acquisition rate. Therefore, the total x-ray image acquisition rate, using the SXRFM, is very comparable to an SEM. Although the x-ray spatial resolution of the SXRFM is worse than an SEM (say 100 vs. 2 μm), there are several other advantages.

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Characterization of defects in tabular silver halide grains

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100178367 ◽

1990 ◽

Vol 48 (4) ◽

pp. 1046-1047

Author(s):

C. Goessens ◽

D. Schryvers ◽

J. Van Landuyt ◽

A. Verbeeck ◽

R. De Keyzer

Keyword(s):

Radiation Damage ◽

Silver Halide ◽

Chemical Characteristics ◽

X Ray ◽

Free Region ◽

Central Defect ◽

Crystallographic Defects ◽

Physical And Chemical ◽

Two Sides

Silver halide grains (AgX, X=Cl,Br,I) are commonly recognized as important entities in photographic applications. Depending on the preparation specifications one can grow cubic, octahedral, tabular a.o. morphologies, each with its own physical and chemical characteristics. In the present study crystallographic defects introduced by the mixing of 5-20% iodide in a growing AgBr tabular grain are investigated. X-ray diffractometry reveals the existence of a homogeneous Ag(Br1-xIx) region, expected to be formed around the AgBr kernel. In fig. 1 a two-beam BF image, taken at T≈100 K to diminish radiation damage, of a triangular tabular grain is presented, clearly showing defect contrast fringes along four of the six directions; the remaining two sides show similar contrast under relevant diffraction conditions. The width of the central defect free region corresponds with the pure AgBr kernel grown before the mixing with I. The thickness of a given grain lies between 0.15 and 0.3 μm: as indicated in fig. 2 triangular (resp. hexagonal) grains exhibit an uneven (resp. even) number of twin interfaces (i.e., between + and - twin variants) parallel with the (111) surfaces. The thickness of the grains and the existence of the twin variants was confirmed from CTEM images of perpendicular cuts.

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