Which resolution?

The relationship between the contrast to noise ratio and intensity based cross-correlation coefficients for both protein crystallography and X-ray imaging are compared. It is concluded that, for protein crystallography at near atomic resolution, the intensity based cross-correlation coefficients give a reasonable indication of the quality of the corresponding electron density. For X-ray imaging of biological materials such as cells and soft tissue, the wide range of contrast of the features means that intensity based correlation coefficients can give a poor indication of the interpretability of an image. Rather than the term resolution, it is the contrast to noise ratio for a feature of interest at the relevant spatial frequency that is more relevant. Additional metrics are required to describe the quality of an image, and these are discussed.

The achievable resolution for X-ray imaging of cells and other soft biological material.

X-ray imaging of soft materials is often difficult because of the low contrast of the components. This particularly applies to frozen hydrated biological cells where the feature of interest can have a similar density to the surroundings. As a consequence, a high dose is often required to achieve the desired resolution. However, the maximum dose that a specimen can tolerate is limited by radiation damage. Results from 3D coherent diffraction imaging (CDI) of frozen hydrated specimens have given resolutions of ∼80 nm compared with the expected resolution of 10 nm predicted from theoretical considerations for identifying a protein embedded in water. Possible explanations for this include the inapplicability of the dose-fractionation theorem, the difficulty of phase determination, an overall object-size dependence on the required fluence and dose, a low contrast within the biological cell, insufficient exposure, and a variety of practical difficulties such as scattering from surrounding material. A recent article [Villaneuva-Perez et al. (2018), Optica, 5, 450-457] concluded that imaging by Compton scattering gave a large dose advantage compared with CDI because of the object-size dependence for CDI. An object-size dependence would severely limit the applicability of CDI and perhaps related coherence-based methods for structural studies. This article specifically includes the overall object size in the analysis of the fluence and dose requirements for coherent imaging in order to investigate whether there is a dependence on object size. The applicability of the dose-fractionation theorem is also discussed. The analysis is extended to absorption-based imaging and imaging by incoherent scattering (Compton) and fluorescence. This article includes analysis of the dose required for imaging specific low-contrast cellular organelles as well as for protein against water. This article concludes that for both absorption-based and coherent diffraction imaging, the dose-fractionation theorem applies and the required dose is independent of the overall size of the object. For incoherent-imaging methods such as Compton scattering, the required dose depends on the X-ray path length through the specimen. For all three types of imaging, the dependence of fluence and dose on a resolution d goes as 1/d 4 when imaging uniform-density voxels. The independence of CDI on object size means that there is no advantage for Compton scattering over coherent-based imaging methods. The most optimistic estimate of achievable resolution is 3 nm for imaging protein molecules in water/ice using lensless imaging methods in the water window. However, the attainable resolution depends on a variety of assumptions including the model for radiation damage as a function of resolution, the efficiency of any phase-retrieval process, the actual contrast of the feature of interest within the cell and the definition of resolution itself. There is insufficient observational information available regarding the most appropriate model for radiation damage in frozen hydrated biological material. It is advocated that, in order to compare theory with experiment, standard methods of reporting results covering parameters such as the feature examined (e.g. which cellular organelle), resolution, contrast, depth of the material (for 2D), estimate of noise and dose should be adopted.

A comparison of absorption and phase contrast for X-ray imaging of biological cells. Erratum.

An error in the calculation for X-ray absorption imaging has been identified in the paper by Nave (2018) [J. Synchrotron Rad. 25, 1490-1504]. The required fluence and dose in the paper are a factor of ten too low for this mode of imaging.

A comparison of absorption and phase contrast for X-ray imaging of biological cells.

X-ray imaging allows biological cells to be examined at a higher resolution than possible with visible light and without some of the preparation difficulties associated with electron microscopy of thick samples. The most used and developed technique is absorption contrast imaging in the water window which exploits the contrast between carbon and oxygen at an energy of around 500 eV. A variety of phase contrast techniques are also being developed. In general these operate at a higher energy, enabling thicker cells to be examined and, in some cases, can be combined with X-ray fluorescence imaging to locate specific metals. The various methods are based on the differences between the complex refractive indices of the cellular components and the surrounding cytosol or nucleosol, the fluids present in the cellular cytoplasm and nucleus. The refractive indices can be calculated from the atomic composition and density of the components. These in turn can be obtained from published measurements using techniques such as chemical analysis, scanning electron microscopy and X-ray imaging at selected energies. As examples, the refractive indices of heterochromatin, inner mitochondrial membranes, the neutral core of lipid droplets, starch granules, cytosol and nucleosol are calculated. The refractive index calculations enable the required doses and fluences to be obtained to provide images with sufficient statistical significance, for X-ray energies between 200 and 4000 eV. The statistical significance (e.g. the Rose criterion) for various requirements is discussed. The calculations reveal why some cellular components are more visible by absorption contrast and why much greater exposure times are required to see some cellular components. A comparison of phase contrast as a function of photon energy with absorption contrast in the water window is provided and it is shown that much higher doses are generally required for the phase contrast measurements. This particularly applies to those components with a high carbon content but with a mass density similar to the surrounding cytosol or nucleosol. The results provide guidance for the most appropriate conditions for X-ray imaging of individual cellular components within cells of various thicknesses.

Imperfection and radiation damage in protein crystals studied with coherent radiation.

Fringes and speckles occur within diffraction spots when a crystal is illuminated with coherent radiation during X-ray diffraction. The additional information in these features provides insight into the imperfections in the crystal at the sub-micrometre scale. In addition, these features can provide more accurate intensity measurements (e.g. by model-based profile fitting), detwinning (by distinguishing the various components), phasing (by exploiting sampling of the molecular transform) and refinement (by distinguishing regions with different unit-cell parameters). In order to exploit these potential benefits, the features due to coherent diffraction have to be recorded and any change due to radiation damage properly modelled. Initial results from recording coherent diffraction at cryotemperatures from polyhedrin crystals of approximately 2 µm in size are described. These measurements allowed information about the type of crystal imperfections to be obtained at the sub-micrometre level, together with the changes due to radiation damage.

Matching X-ray beam and detector properties to protein crystals of different perfection.

An analysis is given of the effect of different beam and detector parameters on the sharpness of recorded diffraction features for macromolecular crystals of different quality. The crystal quality parameters include crystal strain, crystal or mosaic block size and mosaic block misorientation. Calculations are given for instrument parameters such as angular resolution of the detector, beam divergence and wavelength bandpass to be matched to the intrinsic diffraction properties from these crystals with the aim of obtaining the best possible data out of each crystal. Examples are given using typical crystal imperfections obtained from the literature for both room-temperature and cryo-cooled crystals. Possible implications for the choice of X-ray source, beamline design, detector specifications, instrument set-up and data processing are discussed, together with the limitations of the approach.

The Protein Information Management System (PiMS): a generic tool for any structural biology research laboratory.

The techniques used in protein production and structural biology have been developing rapidly, but techniques for recording the laboratory information produced have not kept pace. One approach is the development of laboratory information-management systems (LIMS), which typically use a relational database schema to model and store results from a laboratory workflow. The underlying philosophy and implementation of the Protein Information Management System (PiMS), a LIMS development specifically targeted at the flexible and unpredictable workflows of protein-production research laboratories of all scales, is described. PiMS is a web-based Java application that uses either Postgres or Oracle as the underlying relational database-management system. PiMS is available under a free licence to all academic laboratories either for local installation or for use as a managed service.

Coherent X-ray diffraction investigation of twinned microcrystals.

Coherent X-ray diffraction has been used to study pseudo-merohedrally twinned manganite microcrystals. The analyzed compositions were Pr(5/8)Ca(3/8)MnO(3) and La(0.275)Pr(0.35)Ca(3/8)MnO(3). The prepared loose powder was thermally attached to glass (and quartz) capillary walls by gentle heating to ensure positional stability during data collection. Many diffraction data sets were recorded and some of them were split as expected from the main observed twin law: 180° rotation around [101]. The peak splitting was measured with very high precision owing to the high-resolution nature of the diffraction data, with a resolution (Δd/d) better than 2.0 × 10(-4). Furthermore, when these microcrystals are illuminated coherently, the different crystallographic phases of the structure factors induce interference in the form of a speckle pattern. The three-dimensional speckled Bragg peak intensity distribution has been measured providing information about the twin domains within the microcrystals. Research is ongoing to invert the measured patterns. Successful phase retrieval will allow mapping out the twin domains and twin boundaries which play a key role in the physical properties.

Radiation damage in protein crystals examined under various conditions by different methods.

Investigation of radiation damage in protein crystals has progressed in several directions over the past couple of years. There have been improvements in the basic procedures such as calibration of the incident X-ray intensity and calculation of the dose likely to be deposited in a crystal of known size and composition with this intensity. There has been increased emphasis on using additional techniques such as optical, Raman or X-ray spectroscopy to complement X-ray diffraction. Apparent discrepancies between the results of different techniques can be explained by the fact that they are sensitive to different length scales or to changes in the electronic state rather than to movement of atoms. Investigations have been carried out at room temperature as well as cryo-temperatures and, in both cases, with the introduction of potential scavenger molecules. These and other studies are leading to an overall description of the changes which can occur when a protein crystal is irradiated with X-rays at both cryo- and room temperatures. Results from crystallographic and spectroscopic radiation-damage experiments can be reconciled with other studies in the field of radiation physics and chemistry.

The optimum conditions to collect X-ray data from very small samples.

A previous paper [Nave & Hill (2005). J. Synchrotron Rad. 12, 299-303] examined the possibility of reduced radiation damage for small crystals (10 microm and below in size) under conditions where the photoelectrons could escape from the sample. The conclusion of this paper was that higher-energy radiation (e.g. 40 keV) could offer an advantage as the photoelectron path length was greater and less energy would be deposited in the crystal. This paper refines these calculations further by including the effects of energy deposited owing to Compton scattering and the energy difference between the incident photon and the emitted photoelectron. An estimate is given for the optimum wavelength for collecting data from a protein crystal of a given size and composition. Another way of reducing radiation damage from a protein crystal is to collect data with a very short pulsed X-ray source where a single image can be obtained before subsequent radiation damage occurs. A comparison of this approach compared with the use of shorter wavelengths is made.

A high-throughput structural biology/proteomics beamline at the SRS on a new multipole wiggler.

The North West Structural Genomics Centre's beamline, MAD10, at the SRS receives the central part of the radiation fan (0.5 mrad vertically, 4 mrad horizontally) produced by a new 2.46 T ten-pole wiggler. The optical arrangement of the beamline consists of a Rh-coated collimating Si mirror, a fixed-exit-beam double-crystal monochromator with sagittal bending for horizontal focusing and a second Rh-coated Si mirror for vertical focusing. The double-crystal Si (111) monochromator allows data collection in the 5-13.5 keV photon energy range with rapid (subsecond) tunability and high energy resolution. The monochromatic beam is optimized through a 200 microm collimator. The beamline end station has been designed around a Mar desktop beamline with high-throughput cryogenic sample changer, Mar225 CCD detector, liquid-N(2) autofill system and an ORTEC C-TRAIN-04 energy-resolving high-count-rate X-ray fluorescence detector. The instrument is optimized for MAD/SAD applications in protein crystallography with the additional mode of operation of online single-crystal EXAFS studies on the same crystals. Thus, screening of metals/Se in the crystal can be performed quickly prior to MAD/SAD data collection by exciting the crystal with X-rays of appropriate energy and recording an energy-dispersive fluorescence spectrum. In addition, this experimental set-up allows for parallel XAFS measurements on the same crystal to monitor 'radiation-induced' changes, if any, in e.g. the redox state of metal centres to be detected for a 'metallic' functional group during crystallographic data collection. Moreover, careful minimization of the thickness of the Be window maximizes the intensity performance for the 2.0-2.5 A softer wavelength range. This range also covers the K-edges of a number of important 3d transition metals as well as the L-edges of xenon and iodine and enhanced sulfur f ''.

Towards an understanding of radiation damage in cryocooled macromolecular crystals.

Interest in radiation damage is growing rapidly owing to the surge in macromolecular crystallography experiments carried out at modern brilliant synchrotron macromolecular crystallography beamlines. Work on the characterization of radiation damage in cryocooled protein crystals is starting to have some impact on our understanding of the problem and of how damage might be affecting both the process of structure solution and the actual structure obtained. A brief review of the most recent developments is given together with an assessment of the remaining problems. Although progress is being made, the understanding of radiation damage is far from complete. Methods for recognizing the damage and treating the data are being made available but they are still at an early stage of development.

Will reduced radiation damage occur with very small crystals?

The primary event which occurs when an X-ray photon of energy less than 30 keV is absorbed in a protein crystal (or other organic material) is the production of a photoelectron with a similar energy to that of the absorbed photon. The electron then scatters inelastically off the surrounding material losing energy in the process. This reduction in energy takes place over track lengths of a few microm for 20 keV electrons. The vector distances between the initial and final positions of the photoelectrons are less than the track lengths owing to the non-linear tracks followed by the electrons. For crystals with smaller dimensions than the vector distances, a significant proportion of the energy could leave the crystal with the high-energy electrons. This could provide an advantage in terms of reduced radiation damage. In order to estimate the possible benefits, calculations of the electron tracks are given, initially using the continuous slowing-down approximation. A Monte Carlo approach is then used to provide more accurate values of the vector distance travelled by electrons inside a protein crystal. The calculations indicate that significant reductions in radiation damage could occur for crystals of a few microm in size. The benefits would be greater when operating at higher energies. In addition, a scheme for realising the possible benefits in a practical situation is described. This could then form the basis of trial experiments.