Radiation damage represents a fundamental limit in the determination of protein structures via macromolecular crystallography (MX) at third-generation synchrotron sources. Over the past decade, improvements in both source and detector technology have led to MX experiments being performed with smaller and smaller crystals (on the order of a few microns), often using microfocus beams. Under these conditions, photoelectrons (PEs), the primary agents of radiation-damage in MX, may escape the diffraction volume prior to depositing all of their energy. The impact of PE escape is more significant at higher beam energies (>20 keV) as the electron inelastic mean free path (IMFP) is longer, allowing the electrons to deposit their energy over a larger area, extending further from their point of origin. Software such as RADDOSE-3D has been used extensively to predict the dose (energy absorbed per unit mass) that a crystal will absorb under a given set of experimental parameters and is an important component in planning a successful MX experiment. At the time this study was undertaken, dose predictions made using RADDOSE-3D were spatially-resolved, but did not yet account for the propagation of PEs through the diffraction volume. Hence, in the case of microfocus crystallography, it is anticipated that deviations may occur between the predicted and actual dose absorbed due to the influence of PEs. To explore this effect, we conducted a series of simulations of the dose absorbed by micron-sized crystals during microfocus MX experiments. Our simulations spanned beam and crystal sizes ranging from 1 µm to 5 µm for beam energies between 9 keV and 30 keV. Our simulations were spatially and temporarily resolved and accounted for the escape of PEs from the diffraction volume. The spatially-resolved dose maps produced by these simulations were used to predict the rate of intensity loss in a Bragg spot, a key metric for tracking global radiation damage. Our results were compared to predictions obtained using a recent version of RADDOSE-3D that did not account for PE escape; the predicted crystal lifetimes are shown to differ significantly for the smallest crystals and for high-energy beams, when PE escape is included in the simulations.

Serial Synchrotron Crystallography (SSX) is rapidly emerging as a promising technique for collecting data for time-resolved structural studies or for performing room temperature micro-crystallography measurements using micro-focused beamlines. SSX is often performed using high frame rate detectors in combination with continuous sample scanning or high-viscosity or liquid jet injectors. When performed using ultra-bright X-ray Free Electron Laser (XFEL) sources serial crystallography typically involves a process known as ’diffract-and-destroy’ where each crystal is measured just once before it is destroyed by the intense XFEL pulse. In SSX, however, particularly when using high-viscosity injectors (HVIs) such as Lipidico, the crystal can be intercepted multiple times by the X-ray beam prior to exiting the interaction region. This has a number of important consequences for SSX including whether these multiple-hits can be incorporated into the data analysis or whether they need to be excluded due to the potential impact of radiation damage. Here, we investigate the occurrence and characteristics of multiple hits on single crystals using SSX with lipidico. SSX data are collected from crystals as they tumble within a high viscous stream of silicone grease flowing through a micro-focused X-ray beam. We confirmed that, using the Eiger 16M, we are able to collect up to 42 frames of data from the same single crystal prior to it leaving the X-ray interaction region. The frequency and occurrence of multiple hits may be controlled by varying the sample flow rate and X-ray beam size. Calculations of the absorbed dose confirm that these crystals are likely to undergo radiation damage but that nonetheless incorporating multiple hits into damage-free data should lead to a significant reduction in the number of crystals required for structural analysis when compared to just looking at a single diffraction pattern from each crystal.