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Abstract: A comparison is provided between the use of electrons and X-rays for collecting diffraction data from small protein crystals and imaging data from cells and tissues. The paper contains a review element written to enable an understanding of the relevant properties of electrons by those (including one of the authors) more used to X-ray imaging and diffraction. Radiation-damage mechanisms, sample thickness-dependent dose efficiency and energy-dependent scattering cross sections are discussed, together with contrast mechanisms in electron and X-ray imaging. Crossover points are calculated for diffraction from crystals where electrons and X-rays could yield equivalent data quality in the presence of radiation damage. Increasing the electron energy from 300 to 1000 keV results in a ∼43% rise in the electron/X-ray crossover point. However, the maximum information coefficient (useful signal/absorbed dose) for electrons alone occurs for a 250 nm crystal examined at around 800 keV. The impact of inelastic scattering on electron imaging and diffraction is examined, including its role in coherence loss, Bragg spot broadening and background elevation. The possibilities are investigated for locating regions of interest using X-rays for subsequent higher resolution imaging using electrons. For locating a 30 nm diameter protein or virus, the required X-ray dose would be much less than the tolerable dose for electron imaging at 0.5 or 0.25 nm. Overall, these findings are relevant for imaging at different length scales while minimizing dose-induced structural damage.

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Abstract: We have studied the photodissociation of gas-phase bromocyclopropane by 200 nm wavelength ultraviolet radiation using ultrafast electron diffraction. Bromocyclopropane is a prototypical molecule in the study of organobromides, a class of molecules that have a significant impact on atmospheric ozone depletion through their photochemistry. Previous studies have revealed two possible reaction pathways for the photodissociation of bromine from bromocyclopropane; either the C–Br bond dissociates, leaving behind a cyclopropyl ring, or there is a concerted opening of the cyclopropyl ring along with the C–Br bond dissociation. In this work, both our experimental and simulation results indicate that the majority of the UV-photoexcited BCP molecules (88% ± 11% in the experiment) follow the first reaction pathway, in which the cyclopropyl ring remains closed after homolytic C–Br bond cleavage. This direct bond dissociation occurs within the experimental time resolution of 270 fs. In order to differentiate between the possible reaction end-products, both of which have diffraction signals dominated by the bromine atom, a new analysis method has been employed, which is more sensitive to the structure of the end-products.

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Abstract: X-ray diffraction (XRD) of microcrystals is signal-to-noise limited by the inherently weak diffraction. As such, Electron diffraction (ED) is increasingly used to measure diffraction data from submicron crystals, or those deemed too small for XRD due the stronger interaction of electrons with matter. However, many samples which are too thin for XRD are often too thick for ED using the currently available electron beam energies (<300 keV) and hence require thinning by focussed ion beam milling (FIB) which adds additional sample preparation steps. In addition to determining structures from nanocrystals, ED provides Coulomb potential data which are complementary to that obtained with XRD. As such ED data may be necessary to answer particular scientific questions. The macromolecular crystallography beamline, VMXm, at Diamond Light Source, has been optimised for maximising the S:N in XRD experiments with a variable focus high-energy (>20 KeV) X-ray beam, with in-vacuum endstation and the use of low background cryoTEM grids for crystal mounting [1], [2]. This has allowed VMXm to collect high-resolution rotation data from single crystals measuring ∼1.2 μm which were only previously tractable using an X-ray Free Electron Laser [3]. This has pushed the amenable sample envelope at synchrotrons to new dimensions and perhaps near to the practical limit of XRD. Indeed, simulations have predicted the limit to be ∼0.5 μm thick in the case of lysozyme, assuming photoelectron escape [4]. This XRD beamline opens up the possibilities to directly compare XRD and ED datasets and understand the complementarity of these experiments. In this work we present data from cubic human insulin crystals that have been thinned by FIB milling from ∼10 μm to various submicron thicknesses. 200 kV ED data were then collected from these lamellae before XRD data were measured from the same lamellae using VMXm. It was possible to obtain a complete XRD dataset to 2.45 Å using a 1.68 μm3 illuminated volume and a 2.04 Å ED dataset from the same 0.25 μm lamella. We have demonstrated that the data quality is comparable between ED and VMXm from the same crystal, while giving an opportunity to directly compare X-ray and electron derived maps. This includes the comparison of the radiation damage each experiment imparts on the sample [5] as well as the information content [6]. This work indicates that the usable sample envelope for synchrotron X-rays extends to much thinner samples than had been previously thought. It is also the first demonstration of ED and XRD measured from the same crystal volume enabling direct comparison of X-ray and electron derived data. Ultimately, the work will inform the design and use of high energy (MeV) ED instruments such as HeXI and how those can be complemented by XRD derived information from beamlines such as VMXm.

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Abstract: The High-energy Electron Xtallography Instrument (HeXI), currently under construction at Diamond, aims to investigate the use of Mega-electron-volt (MeV) electrons for macromolecular structure determination, thereby broadening the range of samples suitable for electron diffraction. Funded by the Wellcome Trust “Electrifying Life Sciences” grant and Diamond Light Source, the HeXI project will leverage the increased penetration of Mega-electron-volt (MeV) electrons to bridge the crystal size gap between electron and X-ray scattering, enabling the determination of structures from crystals ranging between 300 nm and 3 μm. A tunable electron source, operating between 100 kV and 1 MeV, will be used to: explore improvements in data quality arising from reduced dynamical scattering at higher incident electron energies, and to investigate the interplay between sample thickness and incident electron energy in damage mechanisms. HeXI will use advanced goniometry developed at Diamond for macromolecular X-ray crystallography to reduce measurement geometry instabilities and enhance overall data fidelity. HeXI, depicted in Figure 1, will offer data collection under three modalities:

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Abstract: The HeXI project, funded by the Wellcome Trust “Electrifying Life Sciences” grant and Diamond Light Source, aims to build a dedicated electron diffractometer to investigate the potential of Mega-electron volt (MeV) electrons for the determination of molecular structures from nanometre sized crystals. The HeXI instrument will leverage the increased penetration of MeV electrons and the high precision goniometry, cryo-sample transfer systems and sample preparation methods developed at Diamond to target crystal thicknesses between 300 nm and ∼1 μm to determine the molecular structures of proteins and pharmacologically relevant molecules. The ability to acquire high-fidelity sweep and serial diffraction data from ≤1-micron thick crystals will bridge the current crystal size gap between samples amenable to electron diffraction performed on commercial Transmission Electron Microscopes (TEMs) using <300 nm crystals and microfocus X-ray diffraction of >3 μm crystals at microfocus beamlines.