# 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  
Serial fs-nanocrystallography (SFX) [74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92] has been one of the first methods implemented at XFELs. The approach is illustrated in Figure 6a. Here, a \(\upmu\)m-sized crystal is intercepted by the intense X-ray pulse to generate a diffraction pattern which encodes the relative orientation between the crystal and the X-ray beam. The process is repeated a number of times until sufficient amount of data is accumulated for structural refinement. The success of this technique relies on an approach called self-terminating diffraction gates, i.e., obtaining sufficient diffraction information when a sample crystal falls apart while an ultra-intense X-ray pulse propagates through it. Experiments at LCLS and simulations involving intense X-ray pulses with energies of up to 3 mJ [1 \(\times\) 10\({}^{13}\) photons/pulse @ 2 keV (\(\uplambda\) = 6 A)] and durations between 30 fs and 400 fs showed that a unique mechanism to ultrashort pulses allows structural information to be collected from crystalline samples using high radiation doses without the requirement for the pulse to terminate before the onset of sample damage. During exposure to a single X-ray pulse focused into a 10 \(\upmu\)m\({}^{2}\) spot with \(I_{\text{peak}}\)\(\sim\)10\({}^{17}\) W cm\({}^{-2}\) a large fraction of the atoms in a protein crystal absorb a photon. This leads to an ultrafast cascade of secondary energy relaxation processes, lattice temperatures of \(\sim\)500,000 K, high pressure, and the ultimate explosion of the sample. The onset of disorder is illustrated in Figure 6b for a small model molecule. As can be observed, the long-range order is completely gone in about 100 fs, and therefore the sample may lose its crystallinity during the transit time of the X-ray pulse. Figure 7 shows the effect of disorder on the accumulation of Bragg peak intensities for an intense X-ray pulse of duration \(\Delta t\) = 150 fs. Since disorder affects higher-resolution peaks in a stronger manner, the counting turns off sooner for higher-resolution Bragg reflections. The aforementioned experiments and modelling demonstrate that SFX does not require X-ray pulses to be shorter than the onset of long-range structural disorder. Instead, the impulsive heating leading to large-amplitude uncorrelated displacement of atoms destroys the crystalline order and shuts down Bragg diffraction. Therefore, SFX measurements in combination with simulations offer a way to characterize the order-to-disorder dynamics, correct for Bragg termination, find optimal X-ray pulse parameters, and perform structural refinement. The success of SFX is highlighted by the determination of a multitude of protein structures from (typically) sub-10 \(\upmu\)m crystals [74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92]. It should be mentioned that the necessity of fs-X-ray pulses for attaining high-resolution structures of radiation-sensitive specimens is questionable. Serial millisecond crystallography at a synchrotron beamline, for instance, equipped with the same high-viscosity injector and a high frame-rate detector allowed for typical protein crystallographic experiments to be performed at room temperature [94]. In addition, fs-electron diffraction has also enabled the structural determination of radiation labile organic crystals at room temperature [41]. Low-dose micro-electron diffraction [95; 96] (microED) is, in fact, likely to become the most convenient and economical approach for determining the structure of sub-\(\upmu\)m crystals. Samples must, however, withstand the high vacuum conditions in the electron column or otherwise be kept at cryogenic temperatures. The main advantage of fs-X-ray probes relies on their ability to elucidate the dynamics of large protein systems.