New features in the dose estimation program RADDOSE-3D are summarised. They include the facility to enter a diffraction intensity decay model which modifies the "Diffraction Weighted Dose" output from a "Fluence Weighted Dose" to a "Diffraction-Decay Weighted Dose", a description of RADDOSE-ED for use in electron diffraction experiments, where dose is historically quoted in electrons/Å2 rather than in gray (Gy), and finally the development of a RADDOSE-3D GUI, enabling easy access to all the options available in the program.

Determining the correct magnified pixel size of single-particle cryoEM micrographs is necessary to maximize resolution and enable accurate model building. Here we describe a simple and rapid procedure for determining the absolute magnification in an electron cryomicroscope to a precision of <0.5%. We show how to use the atomic lattice spacings of crystals of thin and readily available test specimens, such as gold, as an absolute reference to determine magnification for both room temperature and cryogenic imaging. We compare this method to other commonly used methods, and show that it provides comparable accuracy in spite of its simplicity. This magnification calibration method provides a definitive reference quantity for data analysis and processing, simplifies the combination of multiple datasets from different microscopes and detectors, and improves the accuracy with which the contrast transfer function of the microscope can be determined. We also provide an open source program, magCalEM, which can be used to accurately estimate the magnified pixel size of a cryoEM dataset ex post facto.

Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose-built instrument operating at 100 keV-including advances in electron optics, detection, and processing-that makes structure determination fast and simple at a fraction of current costs. The instrument attains its theoretical performance limits, allowing atomic resolution imaging of gold test specimens and biological molecular structure determination in hours. We demonstrate its capabilities by determining the structures of eleven different specimens, ranging in size from 140 kDa to 2 MDa, using a fraction of the data normally required. CryoEM with a microscope designed specifically for high-efficiency, on-the-spot imaging of biological molecules will expand structural biology to a wide range of previously intractable problems.

We investigate potential improvements in using electron cryomicroscopy to image thick specimens with high-resolution phase contrast imaging. In particular, using model experiments, electron scattering theory, Monte Carlo and multislice simulations, we determine the potential for improving electron cryomicrographs of proteins within a cell using chromatic aberration (Cc) correction. We show that inelastically scattered electrons lose a quantifiable amount of spatial coherence as they transit the specimen, yet can be used to enhance the signal from thick biological specimens (in the 1000 to 5000 Å range) provided they are imaged close to focus with an achromatic lens. This loss of information quantified here, which we call “specimen induced decoherence”, is a fundamental limit on imaging biological molecules in situ. We further show that with foreseeable advances in transmission electron microscope technology, it should be possible to directly locate and uniquely identify sub-100 kDa proteins without the need for labels, in a vitrified specimen taken from a cell.

Electron cryomicroscopy (cryoEM) has made great strides in the last decade, such that the atomic structure of most biological macromolecules can, at least in principle, be determined. Major technological advances - in electron imaging hardware, data analysis software, and cryogenic specimen preparation technology - continue at pace and contribute to the exponential growth in the number of atomic structures determined by cryoEM. It is now conceivable that within the next decade we will have structures for hundreds of thousands of unique protein and nucleic acid molecular complexes. But the answers to many important questions in biology would become obvious if we could identify these structures precisely inside cells with quantifiable error. In the context of an abundance of known structures, it is appropriate to consider the current state of electron cryomicroscopy for frozen specimens prepared directly from cells, and try to answer to the question of the title, both now and in the foreseeable future.

Journal Article Using Inelastically Scattered Electrons to Enhance Imaging of Biological Macromolecules from Any Layer of a Thick Specimen Get access Joshua L Dickerson, Joshua L Dickerson MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK Search for other works by this author on: Oxford Academic Google Scholar Peng-Han Lu, Peng-Han Lu Ernst Ruska-Centrum für Mikroskopie und Spektroskopie mit Elektronen, Forschungszentrum Jülich GmbH, Jülich, Germany Search for other works by this author on: Oxford Academic Google Scholar Dilyan Hristov, Dilyan Hristov MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK Search for other works by this author on: Oxford Academic Google Scholar Rafal E Dunin-Borkowski, Rafal E Dunin-Borkowski Ernst Ruska-Centrum für Mikroskopie und Spektroskopie mit Elektronen, Forschungszentrum Jülich GmbH, Jülich, Germany Search for other works by this author on: Oxford Academic Google Scholar Christopher J Russo Christopher J Russo MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK Corresponding author: crusso@mrc-lmb.cam.ac.uk Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 28, Issue S1, 1 August 2022, Pages 1280–1282, https://doi.org/10.1017/S1431927622005268 Published: 01 August 2022

X-ray characterisation methods have undoubtedly enabled cutting-edge advances in all aspects of materials research. Despite the enormous breadth of information that can be extracted from these techniques, the challenge of radiation-induced sample change and damage remains prevalent. This is largely due to the emergence of modern, high-intensity X-ray source technologies and the growing potential to carry out more complex, longer duration in situ or in operando studies. The tunability of synchrotron beamlines enables the routine application of photon energy-dependent experiments. This work explores the structural stability of [Rh(COD)Cl](2), a widely used catalyst and precursor in the chemical industry, across a range of beamline parameters that target X-ray energies of 8 keV, 15 keV, 18 keV and 25 keV, on a powder X-ray diffraction synchrotron beamline at room temperature. Structural changes are discussed with respect to absorbed X-ray dose at each experimental setting associated with the respective photon energy. In addition, the X-ray radiation hardness of the catalyst is discussed, by utilising the diffraction data collected at the different energies to determine a dose limit, which is often considered in protein crystallography and typically overlooked in small molecule crystallography. This work not only gives fundamental insight into how damage manifests in this organometallic catalyst, but will encourage careful consideration of experimental X-ray parameters before conducting diffraction on similar radiation-sensitive organometallic materials.

A controversy exists as to whether the signal in a high resolution phase contrast electron micrograph of a particle in a thick specimen is the same irrespective of the particle's position along the beam axis. Different conceptions of inelastic scattering and its effects on wave interference have led to radically different expectations about the degree of phase contrast vs. depth. Here we examine the information available from bright field phase contrast images of small crystalline particles on the top or bottom of a thick support. The support is an aluminium foil which has strong plasmon resonances that cause a large proportion of the electron beam to lose energy in transit. Phase contrast micrographs of the atomic lattice of two ensembles of platinum particles were measured in an energy loss window corresponding to the first plasmon resonance. The signal measured for particles on top was equal to that for particles on the bottom of the foil to within a 99% confidence interval, and the measurements exclude other models of depth dependent phase contrast in the literature to >5σ. These observations are consistent with quantum theory which considers dynamical effects as independent of event sequence and is distinct from the "top-bottom effect" observed in amplitude contrast. We thus confirm that phase contrast using inelastically scattered electrons can be obtained equally well from particles within any layer of a thick specimen.

Electron cryotomography (cryoET) is an application of transmission electron cryomicroscopy (cryoEM) used to determine three-dimensional structures of cells and macromolecules [1]. The samples used for cryoET are often thicker than 1000 Å, meaning many 300 keV electrons suffer at least one inelastic scattering event in transit through the specimen (Figure 1). Electrons that inelastically scatter have, by definition, lost significant energy and are thus incorrectly focused in the image due to the chromatic aberration ( C c ) in the objective lens. As a result, elastically scattered electrons that have also inelastically scattered do not provide high resolution information in phase contrast images and will instead contribute noise. The current practice is to remove these electrons with an electron energy filter to reduce the noise. Given the advent of practical chromatic aberration correction [2], there is a potential improvement in signal by incorporating these inelastically scattered electrons in the image in the context of an achromatic objective lens. Unfortunately, inelastic scattering not only changes the energy but also the direction of electrons, which can be thought of as reducing their spatial coherence. The aim of this study is to evaluate the conditions necessary to best use inelastically scattered electrons for biological imaging and determine the potential improvement in signal afforded by achromatic imaging of the

X-ray characterisation techniques are invaluable for probing material characteristics and properties, and have been instrumental in discoveries across materials research. However, there is a current lack of understanding of how X-ray induced effects manifest in small molecular crystals. This is of particular concern as new X-ray sources with ever increasing brilliance are developed. In this paper, systematic studies of X-ray-matter interactions are reported on two industrially important catalysts, [Ir(COD)Cl]2 and [Rh(COD)Cl]2, exposed to radiation in X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) experiments. From these complimentary techniques, changes to structure, chemical environments, and electronic structure are observed as a function of X-ray exposure, allowing comparisons of stability to be made between the two catalysts. Radiation dose is estimated using recent developments to the RADDOSE-3D software for small molecules and applied to powder XRD and XPS experiments. Further insights into the electronic structure of the catalysts and changes occurring as a result of the irradiation are drawn from density functional theory (DFT). The techniques combined here offer much needed insight into the X-ray induced effects in transition metal catalysts and consequently, their intrinsic stabilities. There is enormous potential to extend the application of these methods to other small molecular systems of scientific or industrial relevance.

Increasingly, microbeams and microcrystals are being used for macromolecular crystallography (MX) experiments at synchrotrons. However, radiation damage remains a major concern since it is a fundamental limiting factor affecting the success of macromolecular structure determination. The rate of radiation damage at cryotemperatures is known to be proportional to the absorbed dose, so to optimize experimental outcomes, accurate dose calculations are required which take into account the physics of the interactions of the crystal constituents. The program RADDOSE-3D estimates the dose absorbed by samples during MX data collection at synchrotron sources, allowing direct comparison of radiation damage between experiments carried out with different samples and beam parameters. This has aided the study of MX radiation damage and enabled prediction of approximately when it will manifest in diffraction patterns so it can potentially be avoided. However, the probability of photoelectron escape from the sample and entry from the surrounding material has not previously been included in RADDOSE-3D, leading to potentially inaccurate does estimates for experiments using microbeams or microcrystals. We present an extension to RADDOSE-3D which performs Monte Carlo simulations of a rotating crystal during MX data collection, taking into account the redistribution of photoelectrons produced both in the sample and the material surrounding the crystal. As well as providing more accurate dose estimates, the Monte Carlo simulations highlight the importance of the size and composition of the surrounding material on the dose and thus the rate of radiation damage to the sample. Minimizing irradiation of the surrounding material or removing it almost completely will be key to extending the lifetime of microcrystals and enhancing the potential benefits of using higher incident X-ray energies.

For macromolecular structure determination at synchrotron sources, radiation damage remains a major limiting factor. Estimation of the absorbed dose (J kg −1 ) during data collection at these sources by programs such as RADDOSE-3D has allowed direct comparison of radiation damage between experiments carried out with different samples and beam parameters. This has enabled prediction of roughly when radiation damage will manifest so it can potentially be avoided. X-ray free-electron lasers (XFELs), which produce intense X-ray pulses only a few femtoseconds in duration, can be used to generate diffraction patterns before most of the radiation damage processes have occurred and hence hypothetically they enable the determination of damage-free atomic resolution structures. In spite of this, several experimental and theoretical studies have suggested that structures from XFELs are not always free of radiation damage. There are currently no freely available programs designed to calculate the dose absorbed during XFEL data collection. This article presents an extension to RADDOSE-3D called RADDOSE-XFEL , which calculates the time-resolved dose during XFEL experiments. It is anticipated that RADDOSE-XFEL could be used to facilitate the study of radiation damage at XFELs and ultimately be used prior to data collection so that experimenters can plan their experiments to avoid radiation damage manifesting in their structures.

Traditionally small-molecule crystallographers have not usually observed or recognized significant radiation damage to their samples during diffraction experiments. However, the increased flux densities provided by third-generation synchrotrons have resulted in increasing numbers of observations of this phenomenon. The diversity of types of small-molecule systems means it is not yet possible to propose a general mechanism for their radiation-induced sample decay, however characterization of the effects will permit attempts to understand and mitigate it. Here, systematic experiments are reported on the effects that sample temperature and beam attenuation have on radiation damage progression, allowing qualitative and quantitative assessment of their impact on crystals of a small-molecule test sample. To allow inter-comparison of different measurements, radiation-damage metrics (diffraction-intensity decline, resolution fall-off, scaling B-factor increase) are plotted against the absorbed dose. For ease-of-dose calculations, the software developed for protein crystallography, RADDOSE-3D, has been modified for use in small-molecule crystallography. It is intended that these initial experiments will assist in establishing protocols for small-molecule crystallographers to optimize the diffraction signal from their samples prior to the onset of the deleterious effects of radiation damage.

Using X-ray energies higher than those normally used (5–15 keV) for macromolecular X-ray crystallography (MX) at synchrotron sources can theoretically increase the achievable signal as a function of dose and reduce the rate of radiation damage. In practice, a major stumbling block to the use of higher X-ray energy has been the reduced quantum efficiency of silicon detectors as the X-ray energy increases, but hybrid photon-counting CdTe detectors are optimized for higher X-ray energies, and their performance has been steadily improving. Here the potential advantages of using higher incident beam energy together with a CdTe detector for MX are explored, with a particular focus on the advantages that higher beam energies may have for MX experiments with microbeams or microcrystals. Monte Carlo simulations are presented here which for the first time include the efficiency responses of some available X-ray detectors, as well as the possible escape of photoelectrons from the sample and their entry from surrounding material. The results reveal a `sweet spot' at an incident X-ray energy of 26 keV, and show a greater than factor of two improvement in diffraction efficiency at this energy when using microbeams and microcrystals of 5 µm or less.