Pattern orientation despite water-induced contrast reduction: a theoretical baseline for ultrafast single-particle X-ray imaging

Single-particle imaging with X-ray free-electron lasers can, in principle, be used to study macromolecules with high spatial and temporal resolution, in a near-native state. A significant challenge, however, is the substantial background scattering from the solvent and reduced contrast when imaging proteins in solution. This study investigates the feasibility of overcoming this limitation by leveraging two recent technological breakthroughs: the generation of stable, ultra-thin liquid sheets and the availability of intense, nanometer-sized X-ray beams. Through simulations using the 15 nm GroEL protein complex as a model system, we demonstrate that a structure can be obtained despite the reduced contrast and the presence of a significant water-induced background. While experimental realization will be challenged by radiation damage and positional jitter, our idealized model isolates the fundamental challenge of contrast reduction. We demonstrate that the substantial water background does not inherently prevent algorithms from successfully orienting faint diffraction patterns, allowing the subsequent phase retrieval pipeline to resolve structures.