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The integration optimization module, in conjunction with image pre-processing and analysis modules, has been implemented in the program IOTA, which is distributed as part of the cctbx.xfel package (http://cci.lbl.gov/xfel) under an open-source license. Acknowledgments The authors acknowledge the assistance of S. Michael Soltis and Aina E. Cohen (Structural Molecular Biology Group, SLAC National Laboratory) and Craig M. Ogata (Advanced Photon Source) in collecting the serial diffraction data. We also thank Qianjun Zhou and Minglei Zhao for providing the diffraction data for the synaptotagmin-1/SNARE complex, as well as helpful discussion of data-processing approaches as they were being developed. NKS acknowledges support from NIH/NIGMS grant No. GM102520 for data-processing methods. ATB, WIW and JMB were supported in part by a Hughes Collaborative [http://www.selleckchem.com/products/AC-220.html selleck compound] Innovation Award (HCIA) for the development of new XFEL methods. Footnotes 1This article will form part of a virtual special issue of the journal on free-electron laser software.""More than a decade ago, it was first suggested that the very short and intense pulses of X-ray free-electron lasers (XFELs) have the potential to determine the structure of noncrystalline bioparticles from a large collection [http://en.wikipedia.org/wiki/Thymidine_kinase Thymidine kinase] of single-shot diffraction patterns (Neutze et?al., 2000 ?). This flash X-ray imaging (FXI) concept has been used to reconstruct two-dimensional projections of mimivirus (Seibert et?al., 2011 ?), whole cells (van?der Schot et?al., 2015 ?), cell organelles (Hantke et?al., 2014 ?) and soot particles (Loh et?al., 2012 ?). More recent advances include the characterization of the three-dimensional structure of silver particles (Barke et?al., 2015 ?) and even a full three-dimensional reconstruction of mimivirus (Ekeberg et?al., 2015 ?). In a typical FXI experiment, a stream of biological particles is injected into [http://www.selleckchem.com/products/sch-900776.html selleck chemical] the focus of a pulsed X-ray source. Far-field diffraction data are collected downstream of the interaction region. A more detailed description of the experimental setup, using an aerosol particle injector, has been given for example by Hantke et?al. (2014 ?). Currently, a considerable amount of time during FXI experiments is spent on alignment and optimization of the X-ray source, background reduction and finding the intersection between the particle stream and the X-ray beam. These tasks get increasingly harder as we move towards harder X-rays (>2?keV) in a smaller focus (