Dose, exposure time and resolution in serial X-ray crystallography

Abstract

The resolution of X-ray diffraction microscopy is limited by the maximum dose that can be delivered prior to sample damage. In the proposed Serial Crystallography method, the damage problem is addressed by distributing the total dose over many identical hydrated macromolecules running continuously in a single-file train across a continuous X-ray beam, and resolution is then limited only by the available molecular and X-ray fluxes and molecular alignment. Orientation of the diffracting molecules is achieved by laser alignment. We evaluate the incident X-ray fluence (energy/area) required to obtain a given resolution from (1) an analytical model, giving the count rate at the maximum scattering angle for a model protein, (2) explicit simulation of diffraction patterns for a GroEL-GroES protein complex, and (3) the frequency cut off of the transfer function following iterative solution of the phase problem, and reconstruction of an electron density map in the projection approximation. These calculations include counting shot noise and multiple starts of the phasing algorithm. The results indicate counting time and the number of proteins needed within the beam at any instant for a given resolution and X-ray flux. We confirm an inverse fourth power dependence of exposure time on resolution, with important implications for all coherent Xray imaging. We find that multiple single-file protein beams will be needed for subnanometer resolution on current third generation synchrotrons, but not on fourth generation designs, where reconstruction of secondary protein structure at a resolution of 7 Å should be possible with short (below 100 s) exposures.

Room temperature femtosecond X-ray diffraction of photosystem II microcrystals

“…The average dose was 2 × 10 8 Gy, corresponding to about 1,440 absorbed photons/unit cell, which is an order of magnitude higher than the Henderson/ Garman limit (48,49) and about two orders of magnitude higher than the dose used for the previous SR PS II diffraction experiments. The diffraction measurement at these doses, well above the Henderson/Garman limit and at room temperature, is only possible because the short X-ray pulses (<50 fs) outrun the X-ray induced damage and the Coulomb explosion (42,51).…”

Variability of In Vivo Fluid Dose Distribution in Mouse Airways Is Visualized by High-Speed Synchrotron X-Ray Imaging

“…We believe that this research tool has the ability to answer many questions related to fluid dosing, pulmonary drug delivery, and, it is hoped in the future, aerosol delivery to the airways of various animal models. However, in its current form and at the resolution used, it is unlikely to have application in humans, primarily due to the radiation level present (the amount of radiation delivered increases to the 4th power of the resolution 22 ). Nevertheless, some synchrotron facilities have begun to implement human clinical programs for other types of analyses, including mammographic imaging 23 and synchrotron stereotactic radiation therapy for cancer treatment.…”

Current Status of Single Particle Imaging with X-ray Lasers

“…On the contrary, for higher photon energy, the wavelength is shorter and the coherent scattering cross-section is weaker. In order to estimate the scattering intensity per shot, a sphere model with uniform electron density can be used [149],…”