# 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
#### 2.4.1 Serial fs-X-ray Crystallography
These large molecules are difficult to resolve with FED owing to the relatively low transverse coherence length (high transverse emittance) of fs-electron sources [4; 5] (vide infra Sections 3.2 and 3.3). It has been argued that the combined use of a large crystal and XFEL pulses should provide inherently better structures than those obtained by SFX. In this technique the crystal must be very large and the distance between successive X-ray shots greater than the diameter of the damaged irradiated spot. This approach has been successfully applied at SACLA to obtain the structure of bovine heart cytochrome c oxidase (CoO) at 1.9 A [97] and photosystem II (PS II) at 1.95 A [98]. However, it has been shown that SFX can indeed deliver sub-1.5 A from crystals of photoactive yellow protein (PYP) at room temperature [81]. Figure 6: **Serial fs-nanocrystallography (SFX) approach. ** (**a**) A suspension of \(\upmu\)m-crystals flows in a liquid jet across the X-ray beam, and diffraction is recorded using a pair of 512 \(\times\) 1024-pixel pnCCD detectors. The lower half detector is placed further from the beam center than the upper half detector to increase the accessible range of scattering angles. Thousands of individual diffraction patterns are recorded from single \(\upmu\)m-crystals with pulse lengths up to 300 fs to a resolution of 0.76 nm. (**b**) Onset of disorder in a crystalline lattice during an ultra-intense 100-fs X-ray pulse. Sample: lysergic acid diethylamide. Atomic displacements were calculated using Cretin [93]. For this small molecule, crystalline order is largely destroyed by 100 fs. Adapted from Reference [92] with permission from Springer Nature. Another possible method to bring \(\upmu\)m-crystals to the X-ray beam path is the implementation of fixed-target crystallography chips [99; 100; 101; 102]. Very recently, Schulz et al. [99] demonstrated a hit-and-return (HARE) approach based on the use of fixed-target crystallography chips mounted on a translation stage system. The HARE method enables time-resolved serial synchrotron crystallography with time resolution from milliseconds to seconds or longer. Figure 8 shows one of employed crystallography chips and some characteristic time-resolved structural results obtained from \(\upmu\)m-crystals of _Rhodopseudomonas palustris_ fluoroacetate dehalogenase (FAcD) soaked with a photocaged substrate as a model system. For more information vide infra reference [99]. Figure 7: Plot of relative accumulation of Bragg signal for \(I_{\text{peak}}=1\times 10^{17}\) W cm\({}^{-2}\) and a pulse duration \(\Delta t=150\) fs. Bragg intensities initially accumulate at the same rate proportional to \(I_{\text{peak}}\). As expected, disorder affects higher-resolution peaks sooner (alike to the Debye–Waller effect) and termination of Bragg intensity accumulation may occur during the transit of the X-ray pulse through the crystal, leading to apparent pulse lengths (\(\Delta t_{\text{app}}\)) shorter than the duration of the incident pulse. In this case, \(\Delta t_{\text{app}}=40\) fs (black vertical dashed line), which is close to the turn-off time for the highest-resolution peaks at 0.76 nm. Figure adapted from Reference [92] with permission from Springer Nature.