# The Linac Coherent Light Source: Recent Developments and Future Plans
## 3 LCLS-II Science Opportunities
### Coherent X-ray Imaging at the Nanoscale--Heterogeneity and Dynamics  
The ability to image individual non-identical particles in three dimensions at the atomic scale via coherent X-ray scattering remains one of the driving scientific visions for XFELs. This is based on the concept of \"diffraction before destruction\" whereby the interpretable scattering patterns are generated from individual X-ray pulses (of sufficient intensity and short duration) before X-ray damage effects degrade the achievable resolution [58]. Initial demonstration experiments and applications of single particle imaging methods at LCLS include single-shot coherent diffraction images of viruses [59], bacteriophages [60], organelles [61], and cyanobacteria [62]. Active ongoing research is refining the optimum conditions for single particle imaging. Biological samples are typically comprised of low-Z elements with low X-ray scattering cross-sections, thus yielding scattering snapshots with low signal-to-background ratios. Studies have suggested that the optimum photon energy for single particle imaging is in the tender X-ray range between 2 keV and 6 keV, which may represent the best compromise between scattering cross-section and resolution [63]. The assembly of complete data sets via single particle imaging is hampered by the low number of snapshots (at current low XFEL repetition rates), and further complicated by sample heterogeneity. In order to advance single particle imagine methods, LCLS has launched the single particle imaging (SPI) initiative to develop a roadmap towards the goal of imaging at 3 A resolution [64]. The initiative consists of over 100 scientists from 20 international institutions and covers all aspects of SPI, from ultrafast X-ray induced damage processes to sample delivery and algorithm development. Recently, the SPI initiative reported scattering data from rice dwarf virus particles out to 3.0 A (with scattering signals significantly above background) [65], and have pushed the state-of-the-art for 3D image reconstruction to below 10 nm [66]. LCLS-II presents some important opportunities for single particle imaging--particularly the possibility to image the dynamics of macromolecules and assemblies in near-native environments at room temperature. The high repetition rate of LCLS-II in the near-optimum tender X-ray range can potentially generate 10\({}^{8}\) to 10\({}^{10}\) scattering snapshots per day, thereby driving a qualitative advance in our ability to map the conformational energy landscapes traversed by biological nanomachines. Cryogenic electron microscopy (cryo-EM) results demonstrate the potential to extract three-dimensional structure [67], conformational movies, and energy landscapes from ultralow-signal snapshots of biological complexes cryo-trapped in random orientations at statistically determined points in their work cycle [68]. In the low-signal regime, the number of available snapshots ultimately determines the information content of a conformational movie and the detail with which an energy landscape can be mapped. Complementing single-particle imaging, fluctuation X-ray scattering (fSAXS) has emerged as a method bridging SPI and crystallography and is a potentially powerful approach for understanding protein interactions in native environments. fSAXS is a multi-particle scattering approach (based on a limited ensemble of particles) and is enabled by the combination of ultrafast X-ray pulses and high repetition rate [69; 70; 71]. It potentially provides ~100 times more information than conventional SAXS--sufficient for 3-D reconstruction. As an example, the dynamic fluctuations and conformational response of enzymes to active substrates or small molecules in physiological environments is central to their biological function, and current structural biology methods provide only limited insight. The repetition rate of LCLS-II combined with advanced microfluidic mixing liquid jets may open entirely new opportunities for investigating enzyme dynamics and their response to rapidly introduced substrates.