Extending the methodology of X-ray crystallography to allow imaging of micrometre-sized non-crystalline specimens

The sensitivity of oceanic thermohaline circulation to freshwater perturbations is a critical issue for understanding abrupt climate change1 Abrupt climate fluctuations that occurred during both Holocene and Late Pleistocene times have been linked to changes in ocean circulation2,3,4,5,6, but their causes remain uncertain One of the largest such events in the Holocene occurred between 8,400 and 8,000 calendar years ago2,7,8 (7,650–7,200 14C years ago), when the temperature dropped by 4–8 °C in central Greenland2 and 15–3 °C at marine4,7 and terrestrial7,8 sites around the northeastern North Atlantic Ocean The pattern of cooling implies that heat transfer from the ocean to the atmosphere was reduced in the North Atlantic Here we argue that this cooling event was forced by a massive outflow of fresh water from the Hudson Strait This conclusion is based on our estimates of the marine 14C reservoir for Hudson Bay which, in combination with other regional data, indicate that the glacial lakes Agassiz and Ojibway9,10,11,12, (originally dammed by a remnant of the Laurentide ice sheet) drained catastrophically ∼8,470 calendar years ago; this would have released >1014 m3 of fresh water into the Labrador Sea This finding supports the hypothesis2,7,8 that a sudden increase in freshwater flux from the waning Laurentide ice sheet reduced sea surface salinity and altered ocean circulation, thereby initiating the most abrupt and widespread cold event to have occurred in the past 10,000 years

Forcing of the cold event of 8,200 years ago by catastrophic drainage of Laurentide lakes

Imaging with visible light today uses numerous contrast mechanisms, including bright- and dark-field contrast, phase-contrast schemes and confocal and fluorescence-based methods. X-ray imaging, on the other hand, has only recently seen the development of an analogous variety of contrast modalities. Although X-ray phase-contrast imaging could successfully be implemented at a relatively early stage with several techniques, dark-field imaging, or more generally scattering-based imaging, with hard X-rays and good signal-to-noise ratio, in practice still remains a challenging task even at highly brilliant synchrotron sources. In this letter, we report a new approach on the basis of a grating interferometer that can efficiently yield dark-field scatter images of high quality, even with conventional X-ray tube sources. Because the image contrast is formed through the mechanism of small-angle scattering, it provides complementary and otherwise inaccessible structural information about the specimen at the micrometre and submicrometre length scale. Our approach is fully compatible with conventional transmission radiography and a recently developed hard-X-ray phase-contrast imaging scheme. Applications to X-ray medical imaging, industrial non-destructive testing and security screening are discussed.

Hard-X-ray dark-field imaging using a grating interferometer

Radiation damage is the main problem which prevents the determination of the structure of a single biological macromolecule at atomic resolution using any kind of microscopy. This is true whether neutrons, electrons or X-rays are used as the illumination. For neutrons, the cross-section for nuclear capture and the associated energy deposition and radiation damage could be reduced by using samples that are fully deuterated and 15N-labelled and by using fast neutrons, but single molecule biological microscopy is still not feasible. For naturally occurring biological material, electrons at present provide the most information for a given amount of radiation damage. Using phase contrast electron microscopy on biological molecules and macromolecular assemblies of approximately 10(5) molecular weight and above, there is in theory enough information present in the image to allow determination of the position and orientation of individual particles: the application of averaging methods can then be used to provide an atomic resolution structure. The images of approximately 10,000 particles are required. Below 10(5) molecular weight, some kind of crystal or other geometrically ordered aggregate is necessary to provide a sufficiently high combined molecular weight to allow for the alignment. In practice, the present quality of the best images still falls short of that attainable in theory and this means that a greater number of particles must be averaged and that the molecular weight limitation is somewhat larger than the predicted limit. For X-rays, the amount of damage per useful elastic scattering event is several hundred times greater than for electrons at all wavelengths and energies and therefore the requirements on specimen size and number of particles are correspondingly larger. Because of the lack of sufficiently bright neutron sources in the foreseeable future, electron microscopy in practice provides the greatest potential for immediate progress.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/S003358350000305X

The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules.

X-ray computer tomography (CT) is fast becoming an accepted tool within the materials science community for the acquisition of 3D images. Here the authors review the current state of the art as CT transforms from a qualitative diagnostic tool to a quantitative one. Our review considers first the image acquisition process, including the use of iterative reconstruction strategies suited to specific segmentation tasks and emerging methods that provide more insight (e.g. fast and high resolution imaging, crystallite (grain) imaging) than conventional attenuation based tomography. Methods and shortcomings of CT are examined for the quantification of 3D volumetric data to extract key topological parameters such as phase fractions, phase contiguity, and damage levels as well as density variations. As a non-destructive technique, CT is an ideal means of following structural development over time via time lapse sequences of 3D images (sometimes called 3D movies or 4D imaging). This includes information nee...

https://www.tandfonline.com/doi/pdf/10.1179/1743280413Y.0000000023

Quantitative X-ray tomography

Diffraction-limited imaging in a scanning transmission x-ray microscope

Summary

Radiation damage to Vicia faba chromosome structure, as measured by the mass loss, was determined in the scanning transmission X-ray microscope for unstained specimens in both the wet and dry states. Dried specimens remain undamaged after either single or multiple images at doses up to 2400 Mrad at wavelengths of 3·15 or 3·64nm. In contrast, wet specimens are damaged irrespective of the imaging protocol. The damage induced by multiple exposures is greater than that seen in a single exposure of the same total dose. Thus, the rate of data collection is greater than or equal to the rate of damage. The damage during multiple exposures of wet chromosomes is influenced by several factors. First, the fixative used influences the extent of radiation damage. Wet chromosomes fixed with glutaraldehyde are more resistant than those fixed with formaldehyde or osmium tetroxide. A second factor is ionic strength. Damage to wet chromosomes increases if the ionic strength decreases below that at which chromatin undergoes a conformational transition. The mass of wet and dry chromosomes is the same, and consequently quantitative measurements can be made on wet specimens. Such measurements give a DNA mass fraction of 39 ± 8% for V. faba chromosomes.

Measurements of wet metaphase chromosomes in the scanning transmission X-ray microscope

http://xrm.phys.northwestern.edu/research/pdf_papers/1992/jacobsen_ultramic92.pdf

Resolution in soft X-ray microscopes

We have observed the chemical changes in PMMA irradiated by x rays in situ. The chemical changes are monitored by micro‐x‐ray absorption near edge structure spectra at the carbon absorption edge. The loss of the ester group (C=O) and formation of C=C bonds have been determined quantitatively from changes in the intensities of the respective π* resonant peaks as a function of dose. Samples prepared under different conditions were examined. From the dose dependence of bond formation, scission and linking, the performance of the resist can be predicted, so that the preparation strategy, dose, and development can be optimized.

/pdf/exposure-strategies-for-polymethyl-methacrylate-from-in-situ-2k6u2hg6gh.pdf

Exposure strategies for polymethyl methacrylate from in situ x-ray absorption near edge structure spectroscopy

Scanning luminescence X‐ray microscopy is based on the use of the very small focused probe of a scanning X‐ray microscope to stimulate visible light emission from phosphors and dyes. Using an undulator X‐ray source and a Fresnel zone plate to produce a focused X‐ray probe, images of P31 phosphor grains with a resolution of 50–75 nm have been obtained, and luminescence from polystyrene spheres loaded with 50–100 μmol/g of fluorescent dye has been imaged. The resolution was not limited by the focused X‐ray probe (the microscope has imaged features at 36‐nm spacing in transmission mode) but by dark noise and the low net efficiency of the luminescence detection system used for this investigation. This technique may make it possible to image dye‐tagged sites of biochemical activity at the resolution of the X‐ray microscope in wet, unsectioned, and unfixed cells, especially with soft X‐ray optimized dyes. Because the image is formed from the detection of signal against a dark background, calculations suggest that the radiation dose for luminescence imaging of dye‐tagged features should be 2–22 times lower than it is in transmission X‐ray microscopy. A possible extension of the technique for three‐dimensional imaging at the transverse resolution of the X‐ray microscope is described, where visible light collection optics might be used to obtain submicrometre axial resolution.

S. Williams

Papers

Diffraction-limited imaging in a scanning transmission x-ray microscope

Measurements of wet metaphase chromosomes in the scanning transmission X-ray microscope

Resolution in soft X-ray microscopes

Exposure strategies for polymethyl methacrylate from in situ x-ray absorption near edge structure spectroscopy

Scanning luminescence X‐ray microscopy: Imaging fluorescence dyes at suboptical resolution