# Recent Advances in Ultrafast Structural Techniques
## 2 Structural Techniques Involving Ultrashort X-ray Pulses
### Introduction to Fourth-Generation Fs-Hard X-ray Sources: X-ray Free Electron Lasers
#### 2.4.2 Time-Resolved Fs-X-ray Crystallography and Scattering  
Since the very first ultrafast structural experiments at SPPS [43; 103; 104], the brightness of XFELs has increased by about one million fold, allowing for the study of more complex systems and photoinduced phenomena such as protein dynamics in crystalline [105; 106; 107; 108; 109; 110; 111; 112] and liquid phases, [113] bond formation in the [Au(CN)\({}_{2}\)-]\({}_{3}\) trimer in solution, [114; 115] lattice dynamics in individual gold nanoparticles, [116] ultrafast melting of charge and orbital order in PCMO, [117] non-linear lattice dynamics [118] and CWD order in YBCO, [119] visualization of breathing modes in nanocrystals at extreme photoexcitation conditions, [120] photoinduced insulator-to-metal (IM) transitions, [121] warm dense matter physics, [122] and ultrafast crystallization [123] and melting [124] in shock-compressed crystals. Figure 9a-d shows time-resolved X-ray diffraction results obtained by Barends et al. [107] at LCLS in the investigation of the ultrafast structural dynamics that follows CO photodissociation in Myoglobin-CO (MbCO). Early time-dependent structural changes are clearly visible. In this work, horse heart MbCO \(\upmu\)m-crystals were injected in random orientations within a thin liquid microjet to be intercepted by the XFEL beam, i.e., the same sample delivery method commonly implemented in the aforementioned SFX experiments. Photolyzing 150-fs optical laser pulses (centered at \(\lambda\) = 532 nm) were used to trigger the structural changes, while time-delayed 80-fs XFEL pulses (\(\lambda\) = 1.8 A) probed the structural rearrangements. A time stamping tool was implemented in order to bring the timing error from \(\sim\)300 fs (intrinsic uncorrected shot-to-shot timing noise) to \(\sim\)50 fs (see Section 2.5 below). Differential electron density maps were calculated from the differential structure factors (light minus  
Figure 8: **Hit-and-return (HARE) method. ** (**a**) An empty chip is shown as a control. The yellow arrows point out underdeveloped features, which appear semi-transparent under infrared (IR) light illumination. (**b**) Loaded chip with yellow arrows representing crystals sitting in the features of the chip. Crystals sitting inside of the features can be clearly distinguished from crystals on its surface and from empty features. (**c**) Changes in fluoroacetate dehalogenase (FAcD) electron density as a function of \(t\) for the active site subunits A and B. Electron density maps are at a cutoff of 2\(\sigma\) (green, protein; blue, ligand (highlighted with red circle)). Figure adapted from Reference [99] with permission from Springer Nature. dark). Barends et al. [107] found that their results are consistent with the conceptual view of Mb dynamics, in which ultrafast modes of the heme couple with collective lower-frequency protein modes [125,126] and subsequent large-scale protein motions. Figure 9e-h illustrates a very similar experimental layout implemented by Arnlund et al. [113] to perform time-resolved (TR) wide-angle X-ray scattering (WAXS) measurements. Here, Arnlund et al. [113] used multiphoton excitation of the photosynthetic reaction center of _B. viridis_ (RCvir) to demonstrate that TR-WAXS data recorded using XFEL radiation can capture ultrafast protein conformational changes in solution. The probe beam consisted of 40-fs X-ray pulses containing 2.6 \(\times\) 10\({}^{12}\) photons/pulse (\(\uplambda\) = 2.1 A) focused into a 10-\(\upmu\)m\({}^{2}\) spot. Detergent-solubilized RCvir samples were photoactivated using 500-fs optical pulses (\(\uplambda\) = 800-nm) with a fluence of 530 mJ cm\({}^{-2}\); this level of excitation leads to considerable multiphoton absorption since it is very close to  
Figure 9: **Time-resolved fs-X-ray diffraction and scattering. ** Left panel: (**a**) stereo image showing the structure of Mb, as well as the electron density difference following photoexcitation for \(t\) = +0.5 ps (contoured at +3\(\sigma\) (green) and -3\(\sigma\) (red)).