Flash X-ray diffraction imaging in 3D: a proposed analysis pipeline

TL;DR

This paper introduces a semi-automatic analysis pipeline for 3D reconstruction in Flash X-ray diffraction Imaging, addressing data volume and stochastic challenges, demonstrated on the PR772 virus.

Contribution

It proposes a comprehensive pipeline with novel steps and uncertainty analysis for efficient 3D electron density retrieval from FXI data.

Findings

Successfully reconstructed the PR772 virus structure

Achieved resolution above detector edge

Demonstrated pipeline's effectiveness in real data

Abstract

Modern Flash X-ray diffraction Imaging (FXI) acquires diffraction signals from single biomolecules at a high repetition rate from X-ray Free Electron Lasers (XFELs), easily obtaining millions of 2D diffraction patterns from a single experiment. Due to the stochastic nature of FXI experiments and the massive volumes of data, retrieving 3D electron densities from raw 2D diffraction patterns is a challenging and time-consuming task. We propose a semi-automatic data analysis pipeline for FXI experiments, which includes four steps: hit finding and preliminary filtering, pattern classification, 3D Fourier reconstruction, and post analysis. We also include a recently developed bootstrap methodology in the post-analysis step for uncertainty analysis and quality control. To achieve the best possible resolution, we further suggest using background subtraction, signal windowing, and convex optimization techniques when retrieving the Fourier phases in the post-analysis step. As an application example, we quantified the 3D electron structure of the PR772 virus using the proposed data-analysis pipeline. The retrieved structure was above the detector-edge resolution and clearly showed the pseudo-icosahedral capsid of the PR772.